INSTITUTO OSWALDO CRUZ Doutorado em Biologia Celular e Molecular Interação Trypanosoma cruzi-célula hospedeira: danos moleculares e celulares e o efeito de amiodarona na recuperação de cardiomiócitos infectados DANIEL ADESSE PEDRA MARTINS Rio de Janeiro 2010 ii INSTITUTO OSWALDO CRUZ Pós-Graduação em Biologia Celular e Molecular DANIEL ADESSE PEDRA MARTINS Interação Trypanosoma cruzi-célula hospedeira: danos moleculares e celulares e o efeito de amiodarona na recuperação de cardiomiócitos infectados Tese apresentada ao Instituto Oswaldo Cruz como parte dos requisitos para obtenção do título de Doutor em Biologia Celular e Molecular Orientação: Dra. Maria de Nazareth S. L. Meirelles Dra. Luciana Ribeiro Garzoni Prof. Dr. Herbert B. Tanowitz (Orientador Colaborador) RIO DE JANEIRO 2010 iii INSTITUTO OSWALDO CRUZ Pós-Graduação em Biologia Celular e Molecular Daniel Adesse Pedra Martins Interação Trypanosoma cruzi-célula hospedeira: danos moleculares e celulares e o efeito de amiodarona na recuperação de cardiomiócitos infectados Orientação: Dra. Maria de Nazareth S. L. Meirelles Dra. Luciana R. Garzoni Prof. Dr. Herbert B. Tanowitz (Orientador Colaborador) Apresentada e aprovada em 18 de Outubro de 2010 EXAMINADORES: Profa. Dra. Joseli Lannes Vieira (Presidente) – IOC-FIOCRUZ Profa. Dra. Constança Felícia De Paoli de Carvalho Britto – IOC-FIOCRUZ Prof. Dr. Antônio Carlos Campos de Carvalho – IBCCF-UFRJ Profa. Dra. Mariana Caldas Waghabi – IOC-FIOCRUZ Profa. Dra. Solange Lisboa de Castro – IOC-FIOCRUZ REVISÃO: Prof. Dra. Solange Lisboa de Castro – IOC-FIOCRUZ Rio de Janeiro, 18 de Outubro de 2010 Ficha catalográfica elaborada pela Biblioteca de Ciências Biomédicas/ ICICT / FIOCRUZ - RJ M386 Martins, Daniel Adesse Pedra Interação Trypanosoma Cruzi-célula hospedeira: danos moleculares e celulares e o efeito de amiodarona na recuperação de cardiomiócitos infectados / Daniel Adesse Pedra Martins. – Rio de Janeiro, 2010. xii, 113 f. : il. ; 30 cm. Tese (doutorado) – Instituto Oswaldo Cruz, Pós-Graduação em Biologia Celular e Molecular, 2010. Bibliografia: f. 89-113 1. Trypanosoma Cruzi. 2. Microarrays. 3. Caveolina-3. 4. Hipertrofia. 5. Amiodarona. I. Título. CDD 616.9363 iv “É preciso estar atento e forte, Não temos tempo de temer a morte” “Se eu tivesse mais alma pra dar, eu daria. Isso pra mim é viver.” (Caetano Veloso) vAGRADECIMENTOS A finalização desta tese é, sem dúvida, não só a compilação de resultados experimentais obtidos nos últimos três anos. É também o encerramento de mais um dos muitos ciclos que iniciamos na vida e que eu tive a sorte, ou privilégio, de poder iniciar. Incontáveis pessoas, amigos, pesquisadores, colaboradores participaram de certa forma da minha formação e por isso gostaria de agradecer a algumas delas. Primeiramente agradeço à vidae a D’s, por me darem tantas alegrias, tantas situações inusitadas e desafios. Agradeço aos meus pais Chico e Leila e meus irmãos Bruno e Dora por terem me ensinado e apoiado tanto; por terem ultrapassado esse vínculo e se tornado grandes amigos e companheiros; À minha família pelo carinho e por ser um grande exemplo de diversidade, por ter tradições tão fortes que estão já enraizadas em mim. Em especial, agradeço à vó Sara pela vivacidade e alegria tão grandes que parecem infinitas; À Diana por ser um grande amor, por me entender tão bem e por ser sempre a melhor companhia; À Maria de Nazareth Meirelles por me receber de braços abertos na pesquisa. Pela sua paixão pela ciência e pelos constantes conselhos; por vibrar com cada conquista minha e sempre incentivar a irmos além; À Luciana Ribeiro Garzoni, a Lú, por ser um exemplo de pesquisadora. Obrigado por ser ao mesmo tempo orientadora, amiga, dupla de trabalho e pelos nossos longos momentos de discussões e troca de idéias; Às alunas de iniciação científica Julie, Juliana, Priscila, Larissa, Laura, Janaína, Líndice e Nathália, por me ensinarem a ensinar, pela paciência e pela dedicação ao trabalho no laboratório; vi Aos professores Herbert B. Tanowitz e David C. Spray pelo convite para trabalhar com suas equipes em NY; pelas excelentes discussões e seus valiosos ensinamentos; agradeço em especial a Débora, André, Mia, Márcia, Sylvia, Fran, Vicki e Alfie pela amizade sincera começada em NY e mantida até hoje; Aos estudantes, pesquisadores e técnicos do antigo DUBC (LUC, LBCM, LBE e LBC) pelos bons momentos na bancada e confraternizações. A Elen Melo e Mariana Waghabi pelos puxões de orelha e pelas risadas em Caxambú; A Dra. Solange Lisboa de Castro pela revisão impecável e sagaz desta tese; aos membros da banca avaliadora por serem pesquisadores que eu admiro muito e por aceitarem prontamente meu convite; Aos grandes amigos que me acompanham desde a época da escola, Shomer e dança: Gabi, Lúcia, Bianco, Danilo, Michel, Migalha, Davi, Adolfo, Amanda, Abu, Ilana, Camila(s), Kiki, Kiko, e tantos outros, por agüentarem muitas maluquices, pelas viagens, projetos, conquistas e muitas risadas. À Juliana, Marialice, Tatiana e Kelly pelo apoio constante, pelas trocas de e-mail tão engraçadas, por uma amizade que começou na iniciação científica e se tornou indispensável; À Edna pelo grande apoio à nossa casa, pela enorme paciência e carinho para cuidar de todos nós; Por último agradeço ao Kineret, por ser outra empreitada que me traz tantas alegrias e realizações; por encher minha semana de música e dança e me forçar a aproveitar e otimizar ao máximo as poucas 24 horas de cada dia. vii Esta tese é composta de quatro publicações, três artigos publicados em revistas indexadas e um capítulo de livro. Outros três trabalhos foram publicados durante o curso de Doutorado em Biologia Celular e Molecular: 1) Trypanosoma cruzi induces changes in cardiac connexin43 expression. Adesse D, Garzoni LR, Huang H, Tanowitz HB, de Nazareth Meirelles M, Spray DC. Microbes Infect. 2008. 10(1):21-8. 2) Fibrosis and hypertrophy induced by Trypanosoma cruzi in a three- dimensional cardiomyocyte-culture system. Garzoni LR, Adesse D, Soares MJ, Rossi MI, Borojevic R, de Meirelles Mde N. J Infect Dis. 2008. 15;197(6):906- 15. 3) Dissecting coronary angiogenesis: 3D co-culture of cardiomyocytes with endothelial or mesenchymal cells. Garzoni LR, Rossi MI, de Barros AP, Guarani V, Keramidas M, Balottin LB, Adesse D, Takiya CM, Manso PP, Otazú IB, Meirelles Mde N, Borojevic R. Exp Cell Res. 2009. 15;315(19):3406-18. Data de matrícula no programa de Doutorado: 18 de Julho de 2007. Neste período o aluno participou de seis congressos internacionais e ganhou cinco prêmios por apresentação de trabalhos ou travel grants. Resumo viii RESUMO A doença de Chagas é causada pelo protozoário Trypanosoma cruzi sendo a principal causa de morbi-mortalidade entre as cardiopatias na América Latina, onde é endêmica. No presente trabalho nós utilizamos modelos experimentais tais como cultivo celular e infecção de camundongos albinos para avaliar aspectos relacionados à patogênese desta doença. Primeiramente utilizamos a técnica de microarranjos de oligonucleotídeos para determinar as diferentes assinaturas transcriptômicas induzidas por quatro diferentes cepas de T. cruzi em uma linhagem de células musculares. Neste trabalho, verificamos que os genes alterados significativamente nas células infectadas (p<0,05) foramdiferentes para cada cepa e apenas 21 sofreram a mesma alteração nas quatro cepas estudadas. No entanto, mioblastos apresentaram assinaturas transcriptômicas únicas após infecção pelas quatro cepas de T. cruzi utilizadas. Estes resultados indicam que a infecção com diferentes cepas do parasito modula vias similares, porém não idênticas, nas células hospedeiras. Em seguida analisamos o impacto da infecção de cardiomiócitos murinos na expressão de caveolina-3 (Cav-3), proteína formadora das cavéolas, importantes para a manutenção da homeostase de cálcio intracelular e envolvida em processos de hipertrofia cardíaca. A infecção in vitro e in vivo por T. cruzi induziu redução significativa (p<0,05)nos níveis de Cav-3. Esta redução foi acompanhada pela ativação da quinase regulada por sinal extracelular (ERK), majoritariamente envolvida no processo de remodelamento e hipertrofia cardíacos, sugerindo a participação desta via de sinalização na patogênese da doença de Chagas. Por último, avaliamos a capacidade tripanocida do composto antiarrítmico amiodarona em culturas de cardiomiócitos infectadas. Este composto apresentou atividade seletiva anti-T. cruzi, promovendo danos ultra-estruturais às formas amastigotas intracelulares e a recuperação da célula hospedeira, incluindo restabelecimento do citoesqueleto de actina e junções comunicantes, além da contratilidade espontânea das culturas. O conjunto de dados gerado com esta tese traz importantes avanços para o entendimento do estabelecimento da doença de Chagas cardíaca e novas opções para o tratamento etiológico desta doença negligenciada. Abstract ix ABSTRACT Chagas’ disease is caused by the protozoanTrypanosoma cruzi and is the main cause of morbidity and mortality by heart problems in endemic countries in Latin America. In the present work we employed as experimental models cell cultures and murine experimental infection to evaluate aspects related to the pathogenesis of this disease. In the first part we determined through oligonucleotide microarrays the transcriptomic signatures induced in a myoblast cell line by four reference T. cruzi strains. We observed that the significantly altered (p<0.05) genes differed for each strain studied and only 21 suffered changes in the same direction in all four strains. However, infected myoblasts presented proportional alterations in the the overall transcriptome. These results indicate that the infection with distinct strains modulate similar, but not identical, pathways in the host cell. Next we analyzed the impact of T. cruzi infection on cardiac caveolin-3 (Cav-3) expression, which has an important role in the maintenance of intracellular calcium homeostasis and is also involved in cardiac hypertrophy pathways. Both in vitro and in vivo infection induced a significant reduction of Cav-3 levels. This reduction was followed by the activation of extracellular signal regulated kinase (ERK), which plays a major role in cardiac remodeling and hypertrophy, suggesting that this pathway may be involved in the pathogenesis of Chagas’ heart disease. Finally we tested the tripanocidal potential of the anti-arrhythmic drug amiodarone in infected cultures of cardiac myocytes. This compound displayed a selective anti-T. cruzi activity, inducing ultrastructural alterations in intracellular amastigotes and promoted host cell recovery with actin cytoskeleton and gap junction reassembly, followed by restoration of the spontaneous contractility. The data generated in this work bring important advances into the understanding of Chagas heart disease establishment and also a new option for the etiological treatment of this neglected disease. Lista de Abreviaturas x LISTA DE ABREVIATURAS AIDS: Ssíndrome da Iimunodeficiência Aadquirida CCL3: quimiocina do tipo CC- ligante-3 (MIP1) CCL5: quimiocina do tipo CC ligante-5 (RANTES) ERK: quinase regulada por sinal extracelular GPI: glicosilfosfatidilinositol IFN-gama-interferon IgG: imunoglobulina G IgM: imunoglobulina M IL-12: interleucina-12 IL-4: interleucina-4 IL-6: interleucina-6 IP3: fofatidilinositol 1,4,5-trifosfato KDa: quilodálton MAPK: proteína quinase ativada por mitógeno MMP9: metalopeptidase de matriz-9 mRNA: ácido ribonucléico mensageiro PCR: reação em cadeia da polimerase PDGF: fator de crescimento derivado de plaquetas PKB: proteína quinase B PKC: proteína quinase C TGF-: fator de crescimento de transformação- TIMP-1: inibidor de metalopeptidase de tecido-1 TNF-fator de necrose tumoral- VEGF: fator de crescimento de endotélio vascular xii ÍNDICE RESUMO.........................................................................................................................viii ABSTRACT. .....................................................................................................................ix LISTA DE ABREVIATURAS.................................................................................... ..........x INTRODUÇÃO..................................................................................................................1 1. A doença de Chagas..............................................................................................2 1.1 Aspectos epidemiológicos......................................................................................2 1.2 Trypanosoma cruzi - aspectos biológicos..............................................................4 1.3 Fisiopatogenia........................................................................................................6 1.4 Tratamento..........................................................................................................12 2. Interação T. cruzi-célula hospedeira.....................................................................15 2.1. Estrutura e função das cavéolas no tecido cardíaco..........................................18 2.2. Efeito da infecção sobre junções comunicantes e eletrofisiologia......................22 2.2.1 Estrutura e funcionamento das junções comunicantes.....................................23 2.2.2 Impacto da infecção na expressão de conexina43...........................................25 JUSTIFICATIVA..............................................................................................................27 OBJETIVOS....................................................................................................................29 RESULTADOS................................................................................................................30 Trabalho #1.....................................................................................................................32 Trabalho #2.....................................................................................................................42 Trabalho #3.....................................................................................................................51 Trabalho #4....................................................................................................................70 DISCUSSÃO...................................................................................................................79 CONCLUSÕES...............................................................................................................88 REFERÊNCIAS BIBLIOGRÁFICAS................................................................................89 Introdução 1 INTRODUÇÃO Introdução 2 1. A doença de Chagas 1.1. Aspectos epidemiológicos A doença de Chagas ou tripanossomíase americana (CID 10: B57) é uma doença endêmica na América Latina e atualmente atinge 9 a 12 milhões de pessoas (de Souza, 2007). A doença é causada por formas infectivas do protozoário Trypanosoma cruzi, um parasito intracelular obrigatório, da família Trypanosomatidae que é transmitido por triatomíneos. Carlos Chagas observou pela primeira vez a presença de protozoários flagelados no trato intestinal de insetos barbeiros (Panstrogylus megistus) e também em mamíferos, incluindo humanos que apresentavam estes flagelados em seu sangue periférico (Chagas, 1909). Existem mais de 120 espécies conhecidas de vetores para o T. cruzi, sendo as mais importantes: Triatoma infestans (Argentina, Bolívia, Chile, Peru e Uruguai), Rhodnius prolixus (Colômbia, México, Venezuela e América Central) e Triatoma dimidiata (Equador, México e América Central). A transmissão da doença pelo T. infestans no Brasil, seu principal vetor, foi decretada erradicada no 15°Encontro da Comissão Intergovernamental do Cone Sul contra doença de Chagas (Intergovernment Commission of the Southern Cone Initiative against Chagas’ disease) em 2006 (Schofield et al., 2006). A transmissão também foi eliminada no Uruguai (1997) e Chile (1999) e em algumas áreas da Argentina, Bolívia, Paraguai e América Central. Apesar desta vitória, ainda há preocupação com outros possíveis vetores e reservatórios para o T. cruzi como: (a) Panstrongylus megistus infectados que já foram encontrados em residências no estado do Rio de Janeiro (Lisboa et al., 2004); (b) marsupiais (Didelphis marsupialis) que podem ser reservatórios de formas tripomastigota e epimastigota (Deane et al., 1984; Pinho et al., 2000); e (c) os surtos de infecção de alimentos contaminados com fezes de Triatomas ou com o próprio triatomíneo, cada vez mais freqüentes no Brasil (Benchimol Barbosa, 2006). Introdução 3 Formas epimastigotas de T. cruzi se desenvolvem no lúmen intestinal do inseto vetor, elas são não-infectivas, e se multiplicam extracelularmente por divisão binária A diferenciação para forma infectiva, tripomastigota metacíclica, se dá na porção final do reto do inseto, num processo chamado metaciclogênese, iniciado quando epimastigotas aderem à dermepor uma zona de adesão do flagelo (Rocha et al., 2006). Durante o repasto sanguíneo o barbeiro deixa no local da picada suas fezes, que contêm as formas metacíclicas. Estas formas penetram a pele do hospedeiro vertebrado, através de lesão ocorridda durante a picada. Após sete a dez dias de incubação, pode-se desenvolver no local um edema periorbital local chamado chagoma de inoculação (Mazza, 1940) ou um edema ocular bipalpebral ou unipalpebral chamado sinal de Romaña, provavelmente resultante de uma resposta imunológica localizada dependente de IFN- (Chessler et al., 2009). O ciclo de transmissão do parasito é completado quando um vetor invertebrado alimenta-se de sangue de um hospedeiro Figura 1: Esquema do ciclo de transmissão do Trypanosoma cruzi. As setas azuis correspondem aos estágios da presença do parasita no hospedeiro vertebrado (humano) e as vermelhas, no hospedeiro invertebrado. Fonte: Centro de Controle de Doenças (Center for Disease Control). www.dpd.cdc.gov/dpdx Estágios no Humano Tripomastigota metacíclico penetram varias células no local da picada. Dentro da célula transformam-se em amastigota Estágios no Triatomineo Repasto sanguíneo (passa tripomastigota metacíclicas pelas fezes, parasitas entram pela ferida ou membranas mucosas) Tripomastigotas podem infectar outras células em outros tecidos. As manifestações clinicas resultam deste ciclo infectivo Repasto sanguíneo (ingere tripomastigotas) Fase infectiva Fase de diagnostico Formas epimastigota Divisões binárias Tripomastigota metacíclico Amastigotas se multiplicam por fissão binária nas células de tecidos infectados Introdução 4 vertebrado que contém formas tripomastigota sanguíneas, que ao atingirem o intestino do inseto se diferenciam em forma epimastigota (Figura 1). Apesar de a transmissão vetorial ser predominante na América Latina (90%), há uma parcela de transmissão congênita (0,5-8%) e por transfusão de sangue (5-20%) (Dias, 2000), que também têm sido observadas em áreas não-endêmicas como nos Estados Unidos, Europa, Austrália e Japão (Pellegrino, 1949; Grant et al., 1989; Coura & Viñas, 2010). Epidemiologicamente podem ser considerados três ciclos de transmissão, sendo o de maior importância o doméstico (que perpetua a infecção em humanos). No ciclo silvestre participam roedores, marsupiais e outros animais silvestres. No ciclo peridoméstico participam roedores, marsupiais, gatos e cães, servindo de ligação entre os ciclos doméstico e silvestre (Rey, 2008). Diversas abordagens tentaram classificar a estrutura das populações de T. cruzi, tentando definir o número de sub-grupos existentes, que, dependendo da técnica empregada, podem ser chamados de zimodema, schizodema, biodema, clones e linhagens. Atualmente estas diferentes populações são divididas em seis unidades de tipificação discreta (DTUs) distintas, de acordo com seu conteúdo genético, designados T. cruzi (TC) I a VI (revisto por Zingales et al., 2009). A distribuição geográfica destes grupos indica que as cepas TC II a VI são os principais agentes causais da doença de Chagas no sul da América do Sul. TC I estaria presente apenas no ciclo silvestre, apesar de alguns estudos demonstrarem que este grupo é o mais encontrado em infecções na Colômbia, Venezuela e América Central (Añez et al., 2004, Espinoza et al., 2010). 1.2. Trypanosoma cruzi - Aspectos biológicos O Trypanosoma cruzi é um protozoário digenético, que pertence ao filo Protozoa, subfilo Sarcomastigophora, superclasse Mastigophora, classe Zoomastigophorea, ordem Kinetoplastida, subordem Trypanosomatina, família Trypanosomatidae, gênero Trypanosoma e subgênero Schizotrypanum (Hoare, 1966). Dentro da subordem Trypanosomatina estão também Trypanosoma brucei e Leishmania spp., agentes causadores da tripanossomíase africana e Leishmaniose, respectivamente. O T. cruzi tem como característica a presença de corpúsculo basal Introdução 5 nas três formas evolutivas (amastigota, epimastigota e tripomastigota), de onde se origina o flagelo. O flagelo de T. cruzi segue o mesmo padrão de microtúbulos de outros flagelos, porém o tamanho desta estrutura pode variar de acordo com a forma evolutiva do parasito. As formas amastigotas apresentam flagelo de aproximadamente 1 m de comprimento, enquanto que em tripomastigotas e epimastigotas esta estrutura pode atingir 20 m. Além disso, em epimastigotas e tripomastigotas, o flagelo está aderido lateralmente ao corpo celular por uma junção considerada similar a desmossomos (de Souza et al., 1978a). O cinetoplasto, presente nos tripanossomatídeos, é encontrado em todos os membros da ordem Kinetoplastida. Esta estrutura faz parte da mitocôndria única, cujo DNA é correspondente a 20-25% do DNA total de epimastigotas (Benard et al., 1979). Em epimastigotas, o cinetoplasto encontra-se anterior ao núcleo e nas formas amastigota e tripomastigota, na região posterior ao núcleo (Figura 2). Os tripanossomatídeos apresentam também uma camada de microtúbulos associados fortemente à membrana plasmática, chamados microtúbulos subpeliculares, que estão associados à rigidez da membrana e ao tamanho destes parasitos (revisto em de Souza, 2002). Figura 2: Representação esquemática da ultraestrutura de epimastigota de Trypanosoma cruzi e suas organelas, descritas por microscopia eletrônica de transmissão. Adaptado de Souza, 2008. Introdução 6 O T. cruzi apresenta outras organelas peculiares, tais como os acidocalcisomos, descritos como organelas eletrondensas de aproximadamente 200 nm, responsáveis pelo estoque intracelular de cálcio e outros íons (Docampo et al., 1995). Os acidocalcisomas estão presentes nas três formas de T. cruzi e são organelas acídicas, assim como os reservossomos, que são encontrados apenas em epimastigotas (Correa et al., 2002). Os reservossomos foram os primeiros compartimentos endocíticos descritos em epimastigotas de T. cruzi (de Souza et al., 1978b), caracterizados como grandes vesículas desprovidas de membranas internas e contendo inclusões lipídicas. Estas organelas estão localizadas na parte posterior do corpo do parasito, ocorrendo uma progressiva redução na quantidade destas estruturas durante o processo de metaciclogênese, não sendo encontradas em tripomastigotas (revisto em de Souza et al., 2002). Os reservosomos têm pH luminal igual a 6,0, semelhante a compartimentos tardios da via endocítica e foram descritos como sítios de acúmulo de proteínas endocitadas, como observado em estudos utilizando-se transferrina, albumina e peroxidase (Soares & de Souza, 1991). Apesar disso, não foi observada marcação positiva para proteínas de lisossomos nestas organelas (Cunha-e- Silva et al., 2002). Os reservosomos são responsáveis por acúmulo de proteínas, entre elas uma cisteína peptidase (cruzipaína), principal protease de T. cruzi (Soares, 1999). As formas amastigota e epimastigota possuem o citóstoma, uma profunda invaginação da membrana plasmática, que gera uma depressão que pode chegar até a região próxima ao núcleo. O citóstoma está ligado ao flagelo e é associado à endocitose de fase fluida e mediada por receptores (Soares, 1999). 1.3. Fisiopatogenia Após a inoculação das formas metacíclicas no hospedeiro vertebrado, inicia-se o processo de diferenciação para formas amastigotas no citoplasma das células, que sofrem diversos ciclos de divisão binária, e se diferenciam novamente para tripomastigota. Em seguida os parasitos rompem a célula hospedeira e podem atingir a circulação sanguínea e também órgãos distantes do local da infecção primária. Esta Introdução 7 fase inicial da doença é chamada de fase aguda, e é caracterizada pela infecção de diferentes tipos celulares, inclusive macrófagos, células musculares lisas e estriadas e fibroblastos. O diagnóstico clínico é obtido em menos de 10% dos casos devido à sintomatologia não específica (Punukollu et al., 2007; Marin-Neto et al., 2007), apesar de ser possível observar linfadenopatia, hepatoesplenomegalia, náusea, vômito, diarréia, anorexia, irritação de meninge e aumento nos níveis de enzimas cardíacas no sangue (revisto em Tanowitz et al., 1992). A primeira reação à infecção é uma inflamação mononuclear local, devido à ruptura das células parasitadas. Há uma resposta humoral composta de anticorpos IgM, proteínas do complemento e também migração de células mononucleares para o sítio de infecção (Andrade , 1999). Introdução 8 Esta fase dura de um a três meses e após este período há uma diminuição na produção de imunoglobulinas de classe IgM e aumento naquelas de classe IgG, além de uma drástica redução na parasitemia. Neste momento há a transição para fase crônica, na qual 20-50% dos pacientes podem permanecer em uma forma indeterminada da doença por 10 a 20 anos e 25-35% irão desenvolver a forma cardíaca (Dias, 2000). O diagnóstico fica limitado a métodos parasitológicos indiretos tais como Figura 3: Esquema hipotético dos principais fatores envolvidos na cardiopatia chagásica crônica e suas possíveis interações que podem levar a diferentes graus de lesões cardíacas. Traduzido de Higuchi et al., 2003. SA: nódulo sino-atrial; AV: nódulo átrio-ventricular. Infecção aguda por T. cruzi Resposta imune inadequada Resposta imuneadequada Fase crônica da doença de Chagas Persistência de parasitas (imunossupressão e apoptose das células inflamatórias) Recirculação periódica do parasita Resposta por hipersensibilidade tardia ou autoimune Microcirculação dilatada, lesões isquêmicas em zonas de vascularização periférica Fibrose no miocárdio ventricular esquerdo e em nódulos SA e AV Arritmia Morte Súbita Baixo número de parasitas remanescentes Baixa resposta por hipersensibilidade tardia Pouca inflamação, fibrose ou hipertrofia do miocárdio Forma indeterminada Ativação do sistema complemento, liberação de PDGF Miocardite e fibrose Alterações de matriz extracelular Dilatação ventricular Insuficiência cardíaca crônica Introdução 9 xenodiagnóstico, hemocultivo ou sorologia e o paciente em geral não apresenta sintomas graves durante o período indeterminado. Apesar de as formas digestivas (megaesôfago e megacólon) não serem incomuns, a mais importante manifestação é a cardiomiopatia (Higuchi et al., 2003), que será descrita a seguir. Durante a fase aguda da doença, é observado parasitismo em quase todos os tecidos do hospedeiro. Apesar disso, em cerca de 90% dos casos há envolvimento ou comprometimento do tecido cardíaco, incluindo necrose miocitolítica focal e fibrose reparadora causadas pela destruição dos miócitos e por células inflamatórias, como macrófagos e linfócitos (Tanowitz et al., 1992). O alto tropismo de algumas cepas de T. cruzi por tecido muscular estriado (Andrade et al., 1974) resulta em acentuado parasitismo das células miocárdicas. A infecção de cardiomiócitos estimula a secreção de citocinas pró-inflamatórias (ex: IL-12, IFN-, TNF-), quimiocinas (CCL3, CCL5) (Marino et al., 2004; Roffe et al., 2010) e óxido nítrico (Borges et al., 2009). Estas moléculas estimulam a migração e ativação de células do sistema mononuclear tais como macrófagos e linfócitos T CD4+ e principalmente CD8+ (revisto em Martin & Tarleton, 2004). A correta expressão destas moléculas pró-inflamatórias é crítica para o controle da infecção por T. cruzi, incluindo a distribuição, a composição de células mononucleares e o estado de ativação destes infiltrados inflamatórios (Paiva et al., 2009). Devido ao dano tecidual, a miocardite pode ocasionar taquicardia, insuficiência cardíaca congestiva e cardiomegalia. Em alguns pacientes pode ocorrer prolongamento do intervalo P-R (nódulo átrio-ventricular), alterações inespecíficas da onda T (repolarização do ventrículo) e baixa voltagem (Higuchi et al., 1987). Estes fatores são determinantes para a progressão da doença durante a fase crônica e a manifestação ou não da forma cardíaca da doença, a cardiomiopatia chagásica crônica (CCC) (Figura 3). Baseadas em evidências reunidas através de modelos experimentais e estudos em humanos, existem atualmente quatro principais hipóteses para explicar os mecanismos de patogênese da CCC (revisto por Marin-Neto et al., 2007): (a) Mecanismos Imunológicos: apesar da presença de antígenos e DNA de T. cruzi juntamente com a presença de linfócitos T CD8+ ativados no coração durante a fase crônica, há escassez de parasitos detectáveis (Fonseca et al., 2005) promovendo dano tecidual. Alguns cientistas hipotetizaram que algumas proteínas de T. cruzi, incluindo a cruzipaína possuem homologia a proteínas cardíacas, tais como a cadeia Introdução 10 pesada de miosina, assim gerando uma resposta auto-imune humoral ou celular (revisto em Kierszenbaum, 2005). Esta hipótese afastou por muitas décadas o desenvolvimento de novas abordagens quimioterápicas específicas, inclusive para a fase crônica (revisto em Urbina, 2010). Além disso, não foi criado nenhum programa de vacinação para doença de Chagas, com a justificativa de que aumentando a resposta imune contra o T. cruzi poderia causar uma maior severidade da doença auto-imune na fase crônica. A maior evidência para a teoria da autoimunidade é a observação de que danos teciduais são observados mesmo quando da ausência de parasitos e a presença de auto- anticorpos no tecido cardíaco (revisto em Higuchi et al., 2003). (b) Desenervação Parassimpática (Teoria Neurogênica): A observação de que a infecção experimental ou humana por T. cruzi induz uma redução na população de neurônios no tecido cardíaco, associada à inflamação periganglionar e a uma reação autoimune antineural (Ribeiro dos Santos et al., 1979; Soares & Santos, 1999), gerou a hipótese de que CCC estaria principalmente relacionada a danos no sistema nervoso parassimpático. No entanto, esta hipótese perdeu força após a constatação de que a patogênese da doença de Chagas é muito complexa e encontrou alguns obstáculos conceituais, tais como o fato de que a CCC pode ser causada aparentemente por qualquer cepa de T. cruzi, inclusive por aquelas não-neurotrópicas (Miles et al., 1981). Mesmo assim a teoria neurogênica é relevante principalmente porque os danos ao sistema parassimpático podem ser um dos mecanismos que induzem a morte súbita nos pacientes chagásicos (revisto em Marin-Neto et al, 2007). (c) Alterações na Microcirculação: Diversos trabalhos vêm demonstrando que a infecção por T. cruzi, e o consequente desenvolvimento da CCC podem estar relacionados a graves alterações na microcirculação do tecido cardíaco, incluindo aumento da viscosidade do sangue (Berra e cols., 2005), diminuição da velocidade de hemácias (Tanowitz et al., 1996) e aumento da agregação plaquetária (Tanowitz et al., 1990), durante infecção experimental pelo parasito. Este efeito foi revertido com o uso experimental de verapamil, que inibe a agregação plaquetária (Morris et al., 1989). Baseado em investigações clínicas (Hiss et al., 2009), seria possível hipotetizar que a hipoperfusão crônica no miocárdio pode contribuir como um mecanismo patogênico através da geração de regiões de micro-isquemias, com o desenvolvimento de miocitólise e fibrose reparadora no ventrículo esquerdo observadas na doença de Introdução 11 Chagas, independente do parasitismo. No entanto esta hipótese ainda necessita de comprovação clínica, tais como o estudo mostrando o efeito benéfico do tratamento em longo prazo de vasodilatadores ou anti-agregação plaquetária no curso clínico da CCC. (d) Persistência do parasito: No início da década de 1990, alguns grupos descreveram que, apesar da drástica redução na parasitemia após a fase aguda, persiste o parasitismo por T. cruzi na fase crônica no tecido cardíaco, e que este parasitismo é acompanhado de uma reação inflamatória (Higuchi et al, 1993; Jones et al., 1993). Mais recentemente, com o desenvolvimento de métodos mais modernos de detecção como PCR quantitativo, tornou-se evidente que durante a fase crônica é possível detectar a presença de T. cruzi em sítios específicos do hospedeiro tais como tecido cardíaco e adiposo (Combs et al., 2005), resultando numa reação inflamatória multi-focal. Esta teoria está apoiada em várias evidências experimentais e clínicas, dentre elas (i) a presença de parasitos no local ou nas proximidades ao local de dano tecidual, (ii) o desenvolvimento de métodos moleculares de diagnóstico, com maior sensibilidade de detecção do parasito (Britto et al., 1995; Galvão et al., 2003) e (iii) as evidências de que a imunossupressão experimental (Taniwaki et al., 2005) ou em pacientes, como no caso de transplante de órgãos (Almeida et al., 1996) ou pacientes de AIDS (Rivera et al., 2004; Vaidian et al., 2004), com reduzido número de linfócitos T CD4+ (Del Castillo et al., 1990), há reativação da infecção (Simões et al., 1995). Nestes casos, uma melhora na resposta imune resultaria numa menor gravidade da doença de Chagas crônica (Tarleton, 2001). Além disso, segundo Higuchi e colaboradores (2003) apenas a autoimunidade não seria suficiente para explicar o aspecto multifocal da miocardite e a localização de fibrose preferencialmente em certas regiões como as paredes apical e posterior esquerda durante a CCC. A fisiopatologia da CCC consiste principalmente da prevalência de miocardite, acompanhado de acúmulo de densas fibras de colágeno ao redor de fibras ou grupos de fibras cardíacas, que em geral estão hipertrofiadas (Higuchi et al., 1999). Este fenômeno pode ocorrer em resposta a um aumento na demanda por trabalho cardíaco causado por vários estresses patológicos (incluindo, possivelmente, a miocitólise observada na infecção pelo T. cruzi). O coração se adapta através da hipertrofia compensatória dos miócitos em doenças cardiovasculares, tais como hipertensão, doença vascular e infarto do miocárdio, e é um fator de risco independente Introdução 12 para morbidade cardíaca e mortalidade. Um estímulo hipertrófico induz aumento no tamanho celular, com ausência de divisão celular através de sinalização de cálcio e ativação de PKC, MAPK e PKB/Akt (Dorn & Force, 2005) e é acompanhado por aumento na síntese de proteínas com reprogramação da expressão gênica (Takeo et al., 2000). A hipertrofia cardíaca é induzida por uma variedade de fatores, tais como peptídeos vasoativos, fatores de crescimento, citocinas e hormônios (revisto em Jeong et al., 2009). Na doença de Chagas, além da lise dos cardiomiócitos induzida pelo parasitismo e também pela ação de células inflamatórias, há um processo de fibrose reparadora que interfere na propagação do impulso elétrico cardíaco, gerando uma insuficiência cardíaca progressiva (Andrade, 1999). Por este motivo, há hipertrofia das fibras cardíacas num mecanismo que compensa a deficiência causada pela perda destas fibras e mantém a dinâmica circulatória. O desenvolvimento da fibrose pode ser favorecido pela produção de citocinas e fatores de crescimento secretados por macrófagos e linfócitos, tais como IFN-IL-4, IL- 6 e TNF- (Reis et al., 1997), apesar de estudos in vitro apontarem para a contribuição dos cardiomiócitos na produção de citocinas e quimiocinas durante infecção pelo T. cruzi (Machado et al., 2000; Waghabi et al., 2005). A exacerbação da expressão de proteínas de matriz extracelular (ex: fibronectina e colágeno) ocorreria como consequência da redução nos níveis cardíacos de MMP9 (Soares et al., 2004) e de aumento na expressão de TGF- e de TIMP-1 (Araujo-Jorge et al., 2002; Soares et al., 2010). O esquema na Figura 3 resume os principais fatores envolvidos na CCC e as interações que podem levar às diferentes lesões cardíacas. Essas lesões podem causar alterações diversas no funcionamento do coração como taquicardia (taquiarritmia) ventricular sustentada, que pode incluir palpitações e dispnéia (Leite et al., 2001); alterações na sístole e diástole do ventrículo esquerdo (Nunes et al., 2006); fibrilação atrial; bloqueio átrio-ventricular e morte súbita (Valente et al., 2006). 1.4. Tratamento Do fim da década de 60 até a década de 80 o Nifutimox (Lampit®, Bayer; 3- metil-4-[5’-nitrofurfurilideneamina] tetrahidro-4H-1,4-tiazina-1,1-dióxido) (Figura 4) era Introdução 13 utilizado como quimioterápico para o tratamento anti-T. cruzi. Devido aos efeitos colaterais causados aos pacientes a droga foi descontinuada no Brasil, Argentina, Chile e Uruguai. Atualmente, o tratamento para doença de Chagas é feito principalmente com o composto Benzonidazol (Rochagan®no Brasil ou Radanil® na Argentina, Roche; N- benzil-2-nitroimidazol acetamida) (Figura 4). Esta droga atua por ligações covalentes ou outras interações de intermediários de nitroredução com componentes do parasito ou se ligando a DNA, lipídeos e proteínas do parasito (revisto em Coura & de Castro, 2002). Benzonidazol (Bz) tem um índice de eficácia média de 60% para casos de infecção aguda e infecções recentes (Silva et al., 1974). Não se sabe ao certo por que estes compostos nitro-heterocíclicos têm eficácia tão baixa, principalmente durante a fase crônica, mas acredita-se que isto ocorra devido às propriedades farmacocinéticas desfavoráveis e sua baixa penetração tecidual (revisto em Urbina, 2010). No entanto, alguns estudos recentes mostraram que o uso de Bz tanto na infecção crônica experimental (Garcia et al., 2005) e humana (Viotti et al., 2006) reduziu o parasitismo, miocardite, alterações na condução cardíaca, extrassístole ventricular e sorologia. Estes resultados promissores estimularam, por exemplo, a criação de um amplo estudo para avaliar a eficácia do uso de Bz em pacientes portadores de doença de Chagas crônica cardíaca, chamado BENEFIT (Benzonidazole Evaluation for Interrupting Trypanosomiasis) (Marin-Neto et al., 2009), mas até o momento não há nenhum estudo que demonstre total cura parasitológica com uso deste composto durante a fase crônica. Outro aspecto importante a respeito do tratamento é que existe uma importante variabilidade biológica entre as cepas de T. cruzi em relação à virulência, patogenicidade, tropismo tissular, estresse oxidativo e resistência à quimioterapia (Sanchez et al., 1990; Veloso et al., 2001; Mielniczki-Pereira et al., 2007). A cepa Colombiana, por exemplo, é destacada por sua resistência a Bz (Filardi & Brener, 1987). Por esta razão, o Programa Integrado de Doença de Chagas (PIDC), iniciativa da Fundação Oswaldo Cruz, elaborou um protocolo para validação de novos compostos como possíveis candidatos para tratamento de doença de Chagas, que inclui o teste in vitro e in vivo da ação destes compostos contra cepa Y e Colombiana (Romanha et al., 2010). Introdução 14 As novas classes de compostos que vêm sendo testados são direcionadas para inibir vias metabólicas exclusivas dos tripanosomatídeos. Dentre essas moléculas, destacam-se: (a) cisteína peptidase (cruzipaína), principal enzima proteolítica de T. cruzi. O composto K-777 inibe a proliferação de amastigotas e epimastigotas (Engel et al., 1998), porém sem induzir cura parasitológica; (b) síntese e metabolismo de tripanotiona, uma via metabólica exclusiva de tripanossomatídeos, importante para o combate a radicais livres (Urbina & Docampo, 2003). Alguns compostos testados como a tioridazina e cloripramina induziram redução da mortalidade de camundongos, mas não promoveram a cura parasitológica nem apresentaram ação seletiva contra T. cruzi (revisto em Wilkison & Kelly, 2009); (c) metabolismo de pirofosfato, responsável pela resposta a estresse, homoregulação e energia dos tripanossomatídeos. Os bisfofonatos foram testados contra T. cruzi in vitro e in vivo (Garzoni et al., 2004a,b, Bouzahzah et al., 2005) e mostraram efeito anti-proliferativo de formas amastigotas e epimastigotas porém sem causar cura parasitológica; (d) biossíntese de ergosterol, principal fonte de esteróis de fungos e tripanossomatídeos foi validada quimicamente como alvo para quimioterapia (de Souza & Rodrigues, 2009). Alguns compostos como cetoconazol, itraconazol e terbinafina, utilizados como anti-fúngicos, tem relevante atividade tripanocida em modelos murinos, mas não apresentaram resultados similares em Figura 4: Estrutura química de Benzonidazol e nifurtimox Introdução 15 humanos (revisto em Urbina, 2002). Um novo composto, derivado dos triazóis, Posaconazol (SCH 56592), induz cura parasitológica em modelos animais e também em pacientes crônicos, causando profundas alterações ultraestruturais nas três formas evolutivas de T. cruzi (Lazardi et al., 1990), inclusive em cepas resistentes a Bz e mais eficaz em prevenir dano cardíaco do que esta droga padrão (Olivieri et al., 2010). Apesar de promissores, estes compostos ainda não estão disponíveis ou aprovados para o tratamento etiológico da doença de Chagas. Dessa forma, o tratamento para CCC é o mesmo que para insuficiência cardíaca congestiva, que em geral inclui -bloqueadores, diuréticos, inibidores da angiotensina e do receptor para angiotensina e antiarrítmicos como a amiodarona. À medida que a doença progride, as opções terapêuticas ficam ainda mais restritas e o transplante cardíaco surge como alternativa para as terapias padrão. Devido ao número limitado de doadores e o risco de re-agudização em pacientes que fazem uso de imunossupressores, alguns estudos vêm desenvolvendo modelos de terapia celular direcionado para doença de Chagas (revisto em Campos de Carvalho et al., 2009). Esta abordagem se baseia em estudos pioneiros utilizando transplante de células-tronco em modelos de infarto do miocárdio (Orlic et al., 2001). A injeção sistêmica de células mononucleares de medula óssea de camundongos chagásicos crônicos resultou em aumento de apoptose em células inflamatórias, redução de área fibrótica, consequente da redução dos níveis de MMP9 (Soares et al., 2004) e reversão da dilatação do ventrículo direito (Goldenberg et al., 2008). 2. Interação T. cruzi-célula hospedeira. Dado o envolvimento do tecido cardíaco durante a patogênese da doença de Chagas, nosso laboratório desenvolveu um modelo de cultivo de células cardíacas em monocamadas adaptado do método da gota pendente (Meyer & Oliveira, 1948). Neste modelo, as células são isoladas por método de dissociação enzimática e sedimentadas em substrato de gelatina e apresentam estruturas e funcionalidade similares às características dos cardiomiócitos in vivo (Meirelles et al., 1986). Resultados do nosso grupo demonstraram que as células em cultura passam por um processo miogênico, que inclui fusão celular (Meirelles et al., 1986) e aumento Introdução 16 no mRNA para -actina cardíaca (Pereira et al., 2000). As análises ultraestruturais demonstraram que as células apresentavam estruturas contráteis (miofibrilas), sistema sarcotubular, junções celulares, mitocôndrias próximas à miofibrilas (Meirelles et al., 1986; Barbosa & Meirelles, 1995) e estruturas endocíticas (Soeiro et al., 2002). Como muitos outros microorganismos intracelulares obrigatórios, o T. cruzi desenvolveu mecanismos que permitem a invasão de praticamente qualquer tipo celular, sejam células fagocíticas profissionais ou não (de Meirelles et al., 1980; Barbosa & Meirelles, 1995; Nagajyothi et al., 2008). Esses mecanismos envolvem o reconhecimento de moléculas na superfície da célula hospedeira e do parasito, que resultam na incorporação das mesmas. O estudo da interação T. cruzi-célula hospedeira ajudou a esclarecer aspectos do reconhecimento e invasão da célula hospedeira pelo T. cruzi (Meirelles et al., 1986; Barbosa & Meirelles, 1992; Araujo-Jorge et al., 1992; Barbosa et al., 1993; Carvalho et al., 1999); e para testar quimioterápicos anti-T. cruzi (de Castro & Meirelles, 1987; de Castro et al., 1992; Garzoni et al., 2004a; Silva et al., 2006). Cardiomiócitos embrionários de camundongos infectados pelo T. cruzi, sofreram alterações eletrofisiológicas (Aprigliano et al., 1993) e estruturais, tais como quebra de filamentos de citoesqueleto (Pereira et al., 1993, 2000), incluindo miofibrilas (Taniwaki et al., 2006; Melo et al., 2006) e perda de adesão focal (Melo et al., 2004). O modelo de cultivo in vitro de células cardíacas também revelou o possível papel destas no remodelamento cardíaco observado durante o curso da doença de Chagas. Estes estudos revelaram que a infecção de cardiomiócitos, células endoteliais e fibroblastos (Costales et al., 2009) induz a produção de moléculas efetoras microbicidas como o óxido nítrico (NO), citocinas e quimiocinas tais como CCL-5/RANTES, TNF, IL-1 (Machado et al., 2000) e TGF- (Waghabi et al.. 2005), bem como um aumento na taxa de proliferação celular (Hassan et al., 2006; Nagajyothi et al., 2006). . Recentemente desenvolvemos um novo modelo de cultivo de células cardíacas tri-dimensional para estudar a infecção por T. cruzi (Garzoni et al., 2008). Neste modelo as células são cultivadas em frascos de cultura nos quais não é possível a adesão ao substrato, formando agregados multicelulares, que expressam proteínas características do tecido cardíaco como tropomiosina sarcomérica, conexina43, fibronectina e VEGF (Garzoni et al., 2009). Quando infectados por T. cruzi (cepa Y), estes microtecidos Introdução 17 sofreram hipertrofia e fibrose, com aumento da imunorreatividade para laminina, colágeno tipo IV e fibronectina (Garzoni et al., 2008). Este é um modelo promissor para estudar migração de células inflamatórias e mecanismos envolvidos na gênese de fibrose e hipertrofia cardíacos durante a infecção. Os diferentes modelos de cultivo celular existentes atualmente são ferramentas altamente necessárias e confiáveis para a elucidação dos mais variados aspectos envolvidos no desenvolvimento da doença de Chagas no hospedeiro vertebrado e sua importância no contexto da infecção in vivo. Abaixo descrevemos alguns destes aspectos com maior detalhamento. O íon cálcio é um importante segundo-mensageiro em células eucarióticas e diversos trabalhos já descreveram os efeitos da infecção pelo T. cruzi nos níveis e na dinâmica do cálcio na célula hospedeira. Um estudo pioneiro de infecção de células HeLa pelo T. cruzi demonstrou que houve um aumento nos níveis de cálcio intracelular, e que este aumento está relacionado ao processo de invasão da célula (Osuna et al., 1990). Este influxo de Ca+2 intracelular parece ser induzido por fatores solúveis secretado pelas formas tripomastigotas, criando um cenário favorável para a entrada do parasito na célula hospedeira: um aumento no cálcio citoplasmático libera mais cálcio estocado no retículo endoplasmático via IP3 e induz a despolimerização de filamentos de actina, facilitando a migração de lisossomos necessária para a invasão da célula pelo parasito, (Tardieux et al., 1992, 1994; Rodriguez, et al., 1995). Utilizando lisados protéicos de tripomastigotas, Barr e colaboradores (1996) verificaram um aumento nos níveis de cálcio em cardiomiócitos obtidos de cães, induzido por uma enzima processadora do agonista de cálcio, mais tarde chamada de oligopeptidase B (Caler et al., 1998). Quando ativado, este agonista se liga ao seu receptor na célula hospedeira, ativando fosfolipase C e gerando inositol 1,4,5-trifosfato (IP3). O IP3 se ligaria ao seu receptor na membrana do retículo endoplasmático promovendo um aumento nos níveis de cálcio intracelular (Caler et al., 1998). Por outro lado, em cardiomiócitos murinos, apenas a adesão da forma tripomastigota à membrana do miócito induziu aumentos sustentados no cálcio intracelular, também essencial para a invasão da célula hospedeira (Garzoni et al., 2003). Além de ter um papel na invasão do parasito, os níveis citoplasmáticos de cálcio Introdução 18 parecem ter relevância para a multiplicação das formas amastigotas, conforme demonstrado num modelo in vitro de infecção de mioblastos (Schettino et al., 1995). Nesta etapa do ciclo intracelular do T. cruzi, nosso grupo observou uma drástica redução nas variações cíclicas de Ca+2 intracelular em cardiomiócitos altamente parasitados (L.R.G., dados não publicados), concomitantemente com uma diminuição significativa da carga de superfície dos cardiomiócitos (Soeiro et al., 1995), provavelmente graças a uma enzima presente na membrana plasmática das formas tripomastigotas chamada transialidase, que remove o ácido siálico da superfície da célula hospedeira (Schekman et al., 1994) Estudos prévios demonstraram que alterações na permeabilidade celular ao cálcio foram induzidas após a remoção de ácido siálico de cardiomiócitos com tratamento com neuraminidase (Frank et al., 1977). Subsequentemente, foi demonstrado que este tratamento altera as correntes de cálcio tipo T- e tipo L- e leva a alterações eletrofisiológicas na célula (Yee et al., 1991). No músculo cardíaco, os sparks de cálcio são induzidos pela entrada de cálcio dependente de potencial de membrana através de canais de cálcio ativados por voltagem presentes na membrana plasmática em domínios de túbulos T e cavéolas (Furstenau et al., 2000; Lohn et al., 2000). 2.1. Estrutura e função das cavéolas no tecido cardíaco. As cavéolas foram identificadas em 1953 através de análises por microscopia eletrônica de células endoteliais de capilar de rato (Palade, 1953) e possui esse nome devido a seu formato, que se assemelha a pequenas “cavernas”.Cavéolas são compartimentos móveis de 50-100 nm, envolvidos em eventos de endocitose, que possuem forma característica de vaso, estando presentes na membrana plasmática. Além da morfologia clássica, invaginada, as cavéolas podem apresentar variadas formas na célula incluindo vesículas intracelulares, que podem se fundir e formar túbulos e estruturas similares e “cachos de uva”, com tamanhos superiores a 100nm (revisto por Smart et al., 1999). Uma importante característica das cavéolas é a presença de caveolina, principal componente protéico destas estruturas, juntamente com colesterol e Introdução 19 esfingolipídeos, que interagem e se oligomerizam, dando o formato característico das cavéolas (Chang et al., 1992). Estas podem estar tanto abertas para comunicação direta com o espaço extracelular como fechadas para processar moléculas ou estocá- las (Suzuki & Sugi, 1989), em um processo conhecido como “potocitose”: as cavéolas possuem grandes quantidades de receptores específicos (por exemplo: receptores para ácido fólico ou HDL) e a ligação destas moléculas a seus receptores promove uma incorporação independente de um processo endocítico (Anderson et al., 1992). As cavéolas podem também ser um sítio de entrada de cálcio, similar aos túbulos T das células musculares, uma vez que altas concentrações deste íon estão presentes nesses sítios. A localização destas estruturas na periferia celular pode também controlar a distribuição espacial de cálcio, regulando sua entrada e saída da célula. As cavéolas são plataformas de sinalização, por compartimentalizar e concentrar moléculas sinalizadoras como subunidades de proteína G e óxido nítrico sintase endotelial (eNOS). As caveolinas inibem também a ativação e a sinalização de muitas proteínas, incluindo c-Src, H-Ras e as quinases ativadas por mitógenos (MAPK) e eNOS (revisto por Williams & Lisanti, 2005; Tourkina et al., 2008). Caveolina é a proteína formadora das cavéolas e pode também funcionar como molécula sinalizadora a partir de proteínas ligadas a âncoras de GPI até uma quinase residente (Sargiacomo et al., 1993; Chang et al., 1994). A família de genes de caveolina consiste de três membros (revisto em Krajewska & Maslowska, 2004):  caveolina-1: presente em humanos no cromossomo 7, possui 3 exons que codificam duas isoformas (Cav-1 e Cav-1), sendo a classe mais abundante, expressa constitutivamente em diferentes tipos celulares tais como células epiteliais e endoteliais, adipócitos, fibroblastos e células musculares lisas. Cav- 1 apresenta 24 KDa e 178 resíduos e é traduzida de um RNAm diferente de Cav-1, que apresenta 21 KDa e 147 resíduos. A caveolina-1 é uma proteína transmembrana e sua estrutura parece um grampo de cabelo, com as porções N- e C- terminais voltadas para a face citoplasmática e uma região hidrofóbica inserida na bicamada lipídica. A porção N-terminal é a região mais variável entre as isoformas de caveolina e é responsável por permitir a interação de Cav-1 com outras proteínas através de um domínio de oligomerização, composto por 40 aminoácidos. O domínio transmembrana desta proteína está envolvido Introdução 20 principalmente na formação de oligômeros de caveolina-1 e -2 de até 350KDa (Das et al., 1999);  caveolina-2: é comumente co-expressa com Cav-1 apresentando 38% de identidade e 58% de similaridade com a mesma (Scherer et al., 1997). Caveolina-2 apresenta três isoformas: Cav-2 (162 aminoácidos), Cav-2 (149 aminoácidos) e Cav-2 (menor e menos abundante). Cav-2 é encontrada apenas como monômeros ou homodímeros, sendo incapaz de gerar grandes oligômeros sem a presença de Cav-1 (revisto por Krajewska & Maslowska, 2004). Apesar de ser aparentemente uma proteína acessória para Cav-1, Cav-2 possui um papel seletivo para a fisiologia dos mamíferos, uma vez que animais deficientes nesta proteína apresentam disfunções pulmonares (Razani et al., 2002);  caveolina-3: pode ser chamada também de M-caveolina, por ser encontrada praticamente apenas em células diferenciadas de músculo liso, cardíaco e esquelético (Song et al., 1996), enquanto que outros trabalhos também descreveram a expressão de Cav-3 por astrócitos e condrócitos (Schwab et al., 1999). O gene CAV3 está presente no cromossomo 3 humano e traduz uma proteína de 151 aminoácidos com massa molecular de 23 KDa e 85% de similaridade com Cav-1 (Tang et al., 1996). Assim como Cav-1, Cav-3 forma homo-oligômeros de 14-16 monômeros que são capazes de formar cavéolas independentemente da co-expressão de Cav-1 ou -2. A expressão de Cav-3 em células musculares está restrita a células completamente diferenciadas tais como miotubos multinucleares ou cardiomiócitos in vitro, os quais apresentam Cav-3 em grandes quantidades na membrana plasmática, sendo indetectável em mioblastos indiferenciados (Volonte et al., 2003). Além disso, Cav-3 está localizada nos túbulos-T da membrana de células musculares maduras ou durante o desenvolvimento do músculo esquelético em camundongos (Parton et al., 1997), sugerindo um papel na formação dos túbulos-T durante o desenvolvimento muscular. Introdução 21 Essa idéia é reforçada pela verificação de que camundongos knock-out (KO) para Cav-3 (-/-) apresentam anormalidades nos túbulos-T (Galbiati et al., 2001). De maneira similar, camundongos KO duplo (dKO) para Cav-1/3 são viáveis (Park et al., 2002), porém apresentam uma progressiva cardiomiopatia dilatada. Este trabalho demonstrou que, apesar de haver diminuição no número de cavéolas no coração de Figura 5: Visão esquemática da interação entre caveolina-1 e outras proteínas, incluindo canais de cálcio e receptor de IP3 para gerar um influxo de cálcio citoplasmático. IP3: inositol trifosfato; IP3R3: receptor de IP3; SERCA: Ca2+ ATPase de retículo endoplasmático; NCX: Na+/Ca2+ ATPase; GPCR: receptor ligado a proteína G; TRPC1: canal de Ca2+ acionado por voltagem; ER/SR: retículo endoplasmático ou retículo sarcoplasmático (em células musculares); MP: Membrana Plasmática. Traduzido de Hardin & Vallejo, 2008. Introdução 22 camundongos Cav-1 e -3 KO, nos animais dKO Cav-1/-3 não foi possível detectar a presença de cavéolas no sarcolema (Park et al., 2002). Além dos danos à formação de túbulos T em cardiomiócitos pela ausência de caveolinas, as cavéolas são abundantes em canais iônicos e transportadores tais como o trocador Na+/Ca2+, canais de cálcio ativados por voltagem tipo L (Cav1.2), canais de K+ e de Na+, criando complexos de sinalização celular importantes para a fisiologia da célula (Figura 5) (Balijepalli et al., 2006; Hardin & Vallejo, 2008). Dada a variedade de diferentes canais iônicos localizados nas cavéolas, é provável que estas contribuam para a gênese de arritmias. Tanto as arritmias congênitas quanto as adquiridas, como observado em pacientes com insuficiência cardíaca, podem envolver alterações nas funções das cavéolas (revisto por Balijepalli & Kamp, 2009). Estes danos congênitos podem incluir mutações no gene CAV3, gerando a Síndrome do QT longo tipo 9 (Vatta et al., 2006) e a Síndrome da Morte Súbita Infantil (Cronk et al., 2007). A importância da correta expressão de caveolinas para a fisiologia cardiovascular fica ainda mais evidenciada com os modelos animais KO para caveolina- 1, -3 e -1/-3, uma vez que estes animais apresentaram severo espessamento da parede do ventrículo esquerdo, fibrose intersticial, miocitólise e ativação da via de p42/44 MAPK, também chamada ERK (quinase regulada por sinal extracelular) (Woodman et al., 2002). Esta quinase apresenta um papel importante como efetora de resposta hipertrófica cardíaca e proliferação celular no sistema cardiovascular (Gillepsie-Brown et al., 1995). Dados obtidos por experimentos in vitro também indicam um papel das Cav-1 e -3 como reguladores negativos da sinalização por ERK 1/2 (Engelman et al., 1998). Durante a fase aguda da doença de Chagas em modelo murino, Nagajyothi e colaboradores (2006) descreveram uma redução nos níveis de Cav-1, -2 e -3 e aumento na forma fosforilada de ERK nos corações de animais infectados por T. cruzi. Este efeito também foi observado na infecção in vitro de adipócitos, nos quais houve redução da expressão de Cav-1 e aumento da expressão de citocinas, quimiocinas e ativação de p38 MAPK e ERK (Nagajyothi et al., 2008). Estes dados apontam para um papel importante desempenhado pelas caveolinas cardíacas durante a infecção por T. cruzi. 2.2. Efeito da infecção sobre junções comunicantes e eletrofisiologia Introdução 23 Devido às alterações elétricas no tecido cardíaco durante a doença de Chagas e à importância da correta expressão das conexinas para manutenção da homeostase do coração, alguns trabalhos tentaram esclarecer que efeito a infecção de cardiomiócitos por T. cruzi geraria nas junções comunicantes. O acoplamento entre duas células é feito através de junções comunicantes (gap junctions), que são canais intercelulares, onde ocorre livre troca de íons e pequenas moléculas. No tecido cardíaco, por exemplo, a presença dessas junções permite a formação de um sincício entre os cardiomiócitos, auxiliando na propagação da corrente elétrica e, assim, a manutenção da sincronia da contração do tecido. 2.2.1. Estrutura e funcionamento das junções comunicantes A unidade funcional de uma junção comunicante é o conexon, um hemi-canal formado por seis subunidades protéicas. Para ocorrer o acoplamento elétrco entre duas células é preciso haver interação entre seus conexons para formação de canais. Apesar de haver um espaço (gap) entre as duas membranas plasmáticas, os dois conexons interagem e se ligam no espaço extracelular para formar um canal intercelular bimembranar fortemente selado (Unger et al., 1999). Cada conexon é formado por seis unidades da proteína transmembrana, não-glicosilada, conexina (Cx) (Figura 6). Estas proteínas fazem parte de uma família de genes que, em humanos, possui aproximadamente 20 isoformas, nomeadas de acordo com o peso molecular predito pelo produto de tradução do RNAm (ex: Cx43 tem 43KDa). As conexinas são dividas em dois grupos principais: tipo  (Cx33, Cx37, Cx40, Cx43, Cx45, Cx46, Cx50, Cx57) e tipo  (Cx26, Cx30, Cx31, Cx32) (Urban et al., 1999). As conexinas possuem uma porção N-terminal citoplasmática, quatro domínios trans-membrana, que formam duas alças extracelulares e uma volta (loop) citoplasmática. Introdução 24 Para a contração do coração, o impulso elétrico é iniciado por células auto- excitatórias no nódulo sinoatrial e é transferido com alta velocidade até o nódulo átrio- ventricular, onde a condução é atrasada. Em seguida, os impulsos elétricos se propagam rapidamente através do sistema His-Purkinje aos miócitos ventriculares que contraem simultaneamente (Kreuzberg et al., 2006). A ativação elétrica do miocárdio requer a transferência intercelular de corrente, que é feita pelas junções comunicantes, já que apenas nestas junções é possível haver a transferência da corrente de despolarização. A Cx43 é a principal forma encontrada no tecido cardíaco, além de Cx40 e Cx45, localizadas nos discos intercalares, regiões de contato entre dois cardiomiócitos onde são também encontradas junções aderentes e desmossomos. As células endoteliais presentes no coração também expressam Cx37 além de Cx40 e Figura 6: Representação esquemática da estrutura e composição das junções comunicantes em A, evidenciando o conexon formado pelo hexâmero de conexinas. A estrutura da conexina está representada no esquema em B, mostrando as duas alças extracelulares, o loop intracelular e as porções C- e N- terminal, citoplasmáticas. Na cauda C-terminal estão apontados os sítios de interação de Cx com outras proteínas. Adaptado de Cottrel & Burt, 2005. Introdução 25 Cx43 (Kanno & Saffitz, 2001). As conexinas estão distribuídas pelo coração de forma que no ventrículo a proteína majoritária é a Cx43; no átrio e sistema de condução (fibras de Purkinje), a Cx40 (alta condutância) e no sistema de condução atrioventricular, a Cx45 (baixa condutância). Durante uma injúria, o tecido cardíaco sofre remodelamento de Cx43 que em alguns casos pode ter direta relação com o dano elétrico resultante. O remodelamento de Cx43 pode ser dividido em duas modalidades: distribuição de placas de Cx43 e alterações na expressão de Cx43 (proteína ou mRNA). Em zonas do miocárdio próximas àcicatriz do infarto, há perda da ordem normal de distribuição de Cx43, cuja imunomarcação passa a ser localizada nas laterais das fibras cardíacas. Da mesma forma, em casos de hipertrofia cardíaca e morte súbita, é possivel observar lateralização, desorganização e internalização de Cx43. Baixos níveis de expressão de proteína Cx43 ou mRNA para Cx43 foram detectados em pacientes com insuficiência cardíaca terminal, cardiomiopatia dilatada idiopática e doença cardíaca isquêmica (Severs et al., 2004;Gourdie et al., 2006). No entanto, apenas danos que causem uma redução de mais de 50% na quantidade de junções comunicantes no coração são capazes de gerar efeitos na velocidade de condução (Jongsma & Wilders, 2000). 2.2.2. Impacto da infecção por T. cruzi na expressão deconexina43 A infecção de cardiomiócitos obtidos de neonatos de rato pela cepa Tulahuen de T. cruzi (TC I) por 48 h resultou na perda de comunicação celular das células parasitadas como revelado por microinjecção de corante (dye transfer). Além disso, as células infectadas apresentaram redução na imunomarcação para Cx43 e redução na automaticidade da contração. Curiosamente, as formas amastigotas intracelulares também apresentaram forte marcação quando o anticorpo anti-Cx43 (181A) foi utilizado (de Carvalho et al., 1992). Este trabalho indicou que as alterações na distribuição e funcionalidade de Cx43 estão relacionadas com a presença do parasito na célula, independente de fatores solúveis envolvidos na infecção das culturas. Além disso, astrócitos e células da meninge (pia-máter e aracnóide) quando infectados in vitro e in vivo por T. cruzi (Tulahuen) e Toxoplasma gondii, também apresentaram redução no acoplamento celular e na marcação para Cx43 e Cx26. No entanto, culturas infectadas Introdução 26 após 72h não apresentaram variação nos níveis de conexina ou nos níveis de fosforilação de Cx43 (Campos de Carvalho et al., 1998). Os autores argumentaram que estes resultados conflitantes poderiam ser explicados por uma possível alteração no transporte de Cx43 para a membrana plasmática ou porque a região de Cx43 que o anticorpo reconhece estaria mascarada devido a alterações conformacionais da proteína. Curiosamente o modelo de infecção experimental de cardiomiócitos de feto de camundongo Swiss Webster pela cepa Y de T. cruzi (TC II) resultou no aumento da contratilidade dos miócitos (Aprigliano et al., 1993) e em alterações nos níveis de Cx43 in vitro e in vivo (Adesse et al., 2008). Outra observação importante foi a de que o uso de soro de pacientes chagásicos crônicos em culturas de miócitos murinos, resultou em uma redução no acoplamento celular (Costa et al., 2000). Este resultado pode indicar um papel de fatores solúveis presentes na cardiopatia chagásica na regulação da expressão de conexina durante a infecção in vivo. Os níveis séricos de TGF- são aumentados durante a doença de Chagas e estão diretamente relacionados com a geração de resposta fibrótica no tecido cardíaco (revisto em Araújo-Jorge et al., 2008). TGF-parece estar diretamente relacionado à redução de Cx43 observada na infecção in vitro de cardiomiócitos de camundongo e humana por T. cruzi (Waghabi et al., 2009). Justificativa 27 JUSTIFICATIVA A doença de Chagas afeta aproximadamente 11 milhões de pessoas na América Latina e em 30% dos casos crônicos há comprometimento da função cardíaca com hipertrofia das fibras cardíacas e fibrose, gerando arritmias, bloqueio átrio-ventricular e morte súbita. Sabe-se que os vários isolados de T. cruzi existentes apresentam diferenças quanto àinfectividade, tropismo tissular, resistência à quimioterapia e ao estresse oxidativo. Outras diferenças também são observadas quando comparadas diferentes combinações de cepa de T. cruzi-hospedeiro, como demonstrado em estudos que s investigaram o impacto da infecção na miogênese (Rowin et al., 1983; Meirelles et al., 1986); eletrofisiologia e expressão de conexinas (Campos de Carvalho et al., 1998; Adesse et al., 2008) e homeostase de cálcio intracelular (Barr et al., 1992; Garzoni et al., 2003). A correta expressão de conexinas e caveolinas é essencial para correta sincronia e sinalização celular para contração do tecido cardíaco. Nosso grupo descreveu recentemente que a infecção por T. cruzi induz redução na expressão de Cx43 em cardiomiócitos infectados in vitro e no coração de camundongos infectados durante a fase aguda (Adesse et al., 2008). Cx43 forma canais intercelulares e depende de diversas interações proteína-proteína para manter o acoplamento célula-célula (revisto em Herve et al., 2004). A família das caveolinas tem papel importante na regulação de Cx43, uma vez que Cx43 interage diretamente com Cav-1 e -2 em queratinócitos humanos (Langlois et al., 2008) e com av-3 em células musculares cardíacas (Liu et al., 2010; Adesse et al., resultados não publicados). As cavéolas são estruturas formadas por caveolina e estão presentes em domínios da membrana plasmática que participam da regulação de cálcio intracelular em cardiomiócitos (Rothberg et al., 1992). No coração de camundongos infectados por T. cruzi foi descrita uma redução no RNAm de Cav-1, -2 e -3 (Nagajyothi et al., 2006). Esta redução pode ter relevância para geração de um fenótipo hipertrófico na doença de Chagas, como observado em camundongos KO para Cav-1, Cav-3 e duplo KO Cav- Justificativa 28 1/-3 (Woodman et al., 2002), que apresentaram inclusive ativação da via de MAPK/ERK. Neste trabalho nos propusemos a esclarecer alguns aspectos relacionados à doença de Chagas. Primeiramente, comparamos a infecção de células musculares por quatro cepas de T. cruzi distintas, com intuito de identificar grupos de genes comumente afetados nos quatro modelos, visando a busca de biomarcadores. Em seguida investigamos o efeito da infecção de cardiomiócitos murinos na expressão de Cav-3 e a conseqüente ativação de 1/2 ERK (quinase regulada por sinal extracelular), além de transpor estas observações para um modelo in vivo de infecção experimental murina, nas fases aguda e crônica. Por último, testamos a capacidade tripanocida de amiodarona em culturas de cardiomiócito. Este composto é um conhecido antiarrítmico, que também possui a capacidade de inibir a síntese de esteróis de fungos e protozoários como T. cruzi e Leishmania mexicana. Neste estudo avaliamos também o potencial de recuperação das células hospedeiras após o tratamento, validando este composto como potencial candidato para o tratamento etiológico da doença de Chagas. Objetivos 29 OBJETIVOS Objetivo geral:Utilizar modelos experimentais de infecção in vitro e in vivo por Trypanosoma cruzi para estudar mecanismos envolvidos na patogênese da doença de Chagas. Objetivos específicos: 1) Comparar as assinaturas transcriptômicas induzidas por diferentes cepas de T. cruzi em uma linhagem de célula muscular esquelética de rato; 2) Avaliar o efeito da infecção in vitro e in vivo pelo T. cruzi na expressão de Cav-3 cardíaca e a possível ativação da via de uma quinase ativada por mitógeno (MAPK/ERK); 3) Testar a ação tripanocida do composto amiodarona em culturas de cardiomiócito infectadas e verificar a possível recuperação da fisiologia das células através da análise do citoesqueleto de actina, expressão de Cx43 e eletrocontratilidade espontânea. Resultados 30 RESULTADOS Resultados 31 Os resultados gerados nesta tese foram divididos em três artigos e um capítulo de livro. No Trabalho #1 investigamos as assinaturas transcriptômicas que quatro cepas de Trypanosoma cruzi induzem em uma mesma linhagem de célula mioblástica esquelética (L6E9). Para este fim, utilizamos a técnica de microarranjos de cDNA (“microarray”), que permite analisar simultaneamente 27.000 transcritos de forma imparcial nas diferentes condições experimentais. Neste trabalho nós apresentamos as categorias de genes que foram alteradas após a infecção com as cepas Y, CL, Brazil e Tulahuen e apontamos alguns genes como possíveis candidatos a biomarcadores da doença. O impacto da infecção por T. cruzi na expressão de Cav-3 e sua relevância na patogênese da de algumas doenças infecciosas foi abordado no Trabalho #2 e em um capítulo de livro (Trabalho #3). Estas contribuições não só mostram as alterações na expressão de Cav-3, mas também a concomitante ativação da via de sinalização da quinase regulada por sinal extracelular (ERK 1/2). Na última parte desta tese (Trabalho #4), aprofundamos nosso estudo sobre o impacto da infecção por T. cruzi na expressão de Cx43 cardíaca. Nossos dados prévios mostraram que a infecção de cardiomiócitos murinos por T. cruzi (cepa Y) reduziu significativamente a expressão desta proteína. Neste trabalho, nós testamos o potencial tripanocida do composto amiodarona em culturas infectadas e verificamos recuperação da célula hospedeira, incluindo a expressão de Cx43 e F-actina após erradicação do parasitismo. Resultados 32 Trabalho #1: Daniel Adesse, Dumitru A. Iacobas, Sanda Iacobas, Luciana R. Garzoni, Maria de Nazareth Meirelles, Herbert B. Tanowitz and David C. Spray. “Transcriptomic Signatures of Alterations in a Myoblast Cell Line Infected with Four Distinct Strains of Trypanosoma cruzi” Publicado na American Journal of Tropical Medicine and Hygiene (Maio de 2010) 1Am. J. Trop. Med. Hyg., 00(0), 2010, pp. 000–000 doi:10.4269/ajtmh.2010.09-0399 Copyright © 2010 by The American Society of Tropical Medicine and Hygiene INTRODUCTION Chagas disease, caused by infection with the flagellate pro- tozoan parasite Trypanosoma cruzi , is a widespread disease in Latin America affecting millions of people. 1 Infective trypo- mastigotes invade peripheral cells and transform into multipli- cative amastigote forms. The initial (acute) phase of the disease is characterized by intense tissue parasitism involving the heart, skeletal and smooth muscle cells, liver, fat, and brain that is accompanied by intense focal inflammation and necrosis. 2 Some patients can evolve to a chronic phase of the disease that can include cardiac and/or digestive forms. The severity of the chronic phase may be related to the efficiency of the host immune response in resolving the infection during the acute phase, 3 but this has never been proven. Moreover, there are several reports of differences of tropism of T. cruzi to host tissue, which is also associated with the pathogenesis of chronic Chagas disease. 4 , 5 Differences in the pathogenesis of the disease among patients may vary according to differences in both hosts and parasite strain. 6 Among differences in T. cruzi strains are their resistance to chemotherapy, oxidative stress, and infectivity in the mouse. 7 – 9 Although previous in vivo and in vitro microar- ray analyses using cultured cells 10 , 11 and hearts of mice 12 – 14 infected with T. cruzi showed that this infection results in pro- found alterations in the host cells, the degree to which these results are applicable to all T. cruzi strains found in infected individuals has not been explored previously. Since host immune response, tissue parasitism, and para- site strains may be important factors in the pathogenesis of chronic Chagas disease, we have used gene-array analysis to compare the alterations in host cells caused by four different stocks of T. cruzi . The present study characterizes the transcrip- tomic changes in cultured rat myoblasts that result from infec- tion with each strain and highlights common genes that were similarly or differentially modulated by each strain. Analysis reveals host cell changes that might lead to an understanding of previously observed differences in pathogenesis in vivo . METHODS Cells and parasites. The L 6 E 9 rat myoblast cell line was maintained in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and 1% penicillin/streptomycin at 37°C with 5% CO 2 atmosphere. 15 Cells were dissociated with trypsin/ ethylenediaminetetracetic acid (EDTA) solution (Gibco), and 10 6 cells were plated in 100-mm 2 cell-culture dishes. After 24 hours of plating, cells were washed with Phosphate Buffered Saline (PBS) containing Ca 2+ and Mg 2+ (Gibco) and infected with 2 × 10 6 trypomastigote forms of T. cruzi in DMEM. Parasites of the Y, CL Brener , 16 Tulahuen, and Brazil strains were obtained from supernatants of infected L 6 E 9 cultures. Forty-eight hours post-infection, cells were washed twice with Ca 2+ /Mg 2+ PBS to remove free trypomastigotes in the supernatant, and they were re-fed with fresh supplemented DMEM. Total RNA was harvested at 72 hours post-infection using guanidinium thiocyanate-phenol-chloroform extraction (TRIZOL) reagent (Invitrogen, Carlsbad, CA ), following the protocol indicated by the manufacturer, when at least 25% of the cultured cells were infected, presented only intracellular amastigotes, and had no release of trypomastigotes, which would lead to re-infection of culture. T. cruzi genotyping. The different isolates of T. cruzi used in this work were identified using the method described by Fernandes and others 17 according to their phylogenetic lineage. Briefly, genomic DNA from 5 × 10 8 epimastigote forms of the Y, CL, Brazil, and Tulahuen strains was extracted using the DNeasy kit (Qiagen, Hilden, Germany ). Multiplex polymerase chain reaction (PCR) was performed using 150 ng of DNA, and the primers were designed to recognize the mini-exon gene of the parasites using a pool of five nucleotides: three were derived from a hypervariable region of the T. cruzi mini-exon repeat ( T. cruzi 1 [TC1], 5¢ ACA CTT TCT GTG GCG CTG ATC G; TC 2, 5¢ TTG CTC GCA CAC TCG GCT GCA T; TC 3, 5¢ CCG CGW ACA ACC CCT MAT AAA AAT G) and an oligonucleotide from a specific region of the Trypanosoma rangelii non-transcribed spacer (TR; 5¢ CCT ATT GTG ATC CCC ATC TTC G). A common downstream oligonucleotide corresponds to sequences present in the most conserved region of the mini-exon gene (ME; Transcriptomic Signatures of Alterations in a Myoblast Cell Line Infected with Four Distinct Strains of Trypanosoma cruzi Daniel Adesse, * Dumitru A. Iacobas , Sanda Iacobas , Luciana R. Garzoni , Maria de Nazareth Meirelles , Herbert B. Tanowitz , and David C. Spray Laboratorio de Ultra-estrutura Celular, Instituto Oswaldo Cruz–Fiocruz, Rio de Janeiro, Brazil; Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York ; Department of Pathology, Albert Einstein College of Medicine, Bronx, New York Abstract. We examined the extent to which different Trypanosoma cruzi strains induce transcriptomic changes in cultured L 6 E 9 myoblasts 72 hours after infection with Brazil (TC I), Y (TC II), CL (TC II), and Tulahuen (TC II) strains. Expression of 6,289 distinct, fully annotated unigenes was quantified with 27,000 rat oligonucleotide arrays in each of the four replicas of all control and infected RNA samples. Considering changes greater than 1.5-fold and P values < 0.05, the Tulahuen strain was the most disruptive to host transcriptome (17% significantly altered genes), whereas the Y strain altered only 6% of the genes. The significantly altered genes in the infected cells were largely different among the strains, and only 21 genes were similarly changed by all four strains. However, myoblasts infected with different strains showed proportional overall gene-expression alterations. These results indicate that infection with different parasite strains mod- ulates similar but not identical pathways in the host cells. * Address correspondence to Daniel Adesse, Avenida Brasil 4365, Pavilhão Carlos Chagas, Rio de Janeiro, Rio de Janeiro , Brazil. E-mail: daniel.a@ioc.fiocruz.br 2 ADESSE AND OTHERS 5¢ TAC CAA TAT AGT ACA GAA ACT G). PCR reaction was performed using the Multiplex PCR kit (Qiagen) with the initial denaturing cycle of 95°C (15 seconds) followed by 30 cycles of 94°C (30 seconds, denaturing), 60°C (30 seconds, annealing), and 72°C (30 seconds, extension); a final extension cycle of 72°C lasts for 10 minutes and is followed by a soak cycle (4°C) using a PTC-100 Thermocycler (M.J. Research Inc ., Massachusetts, (USA)). PCR fragments were loaded into a 2% agarose/ Tris base, boric acid, EDTA (TBE) gel with 0.008% ethidium bromide, and images were acquired using Kodak 1D Scientific Imaging Systems. Light microscopy. Cells (6 × 10 4 ) were plated into glass cover slips in 24-well plates. After 24 hours, 10 6 trypomastigotes were added to cultures in fresh supplemented DMEM, and infection was followed as described above. After 72 hours of infection, cells were washed three times and fixed with glutaraldehyde for Giemsa staining. 18 Cover slips were digitally photographed using a Zeiss Axioplan microscope. Microarray analysis. We used the protocol optimized in our laboratory 19 according to the standards of the Microarray Gene Expression Data Society. Briefly, 20 µg Trizol extracted total RNA from each culture dish was reverse transcribed in the presence of fluorescent Alexa Fluor 555-aha-dUTP (green fluorescent emission) or Alexa 647-aha-dUTP (red emission; Invitrogen) to label cDNAs. Differently labeled RNA samples from biological replicas of control (uninfected cells cultured for the same duration) or infected with one strain at a time were co-hybridized (“multiple yellow” strategy 20 ) overnight at 50°C with rat 27k oligonucleotide arrays printed by Duke University (full technical information available at http://www.ncbi.nlm .nih.gov/geo/query/acc.cgi?acc=GSE18175 ). All spots affected by local corruption, with saturated pixels, or with foreground fluorescence less than twice the background fluorescence (where noise may obscure the quantity) were removed from the analysis. The background subtracted signals were normalized iteratively, 19 alternating red/green, interblock, lowess and scale intraslide and interslide normalization until the fluctuation of the ratio between the spot median and the corresponding block median of valid spots became less than 5% between successive iteration steps. Normalized expression levels were organized into redundancy groups (each group composed of all spots probing the same gene) and were represented by the weighted average of the values of individual spots. The abundance of host cell transcripts was considered as significantly altered after infection if the absolute fold-change was greater than 1.5- fold and the P value of the heteroscedastic t test (two-sample, unequal variance), applied to the means of the background- subtracted normalized fluorescence values in the four biological replicas of the compared transcriptomes, was greater than 0.05. This composite criterion to identify the significantly altered gene expression minimizes the number of false hits without eliminating too many true hits. The 50% change cut-off (1.5-fold) was selected to be significantly larger than the overall less than 10% interslide technical noise determined for the bacterial controls. Gene categories. GenMapp and MappFinder programs ( www.genmapp.org; Gladstone Institute, University of California, Figure 1. Characterization of L 6 E 9 cell infection by four T. cruzi strains. Giemsa staining of L 6 E 9 cells after 72 hours of infection with ( A ) Y, ( B ) CL, ( C ) Brazil, and ( D ) Tulahuen strains of T. cruzi was performed. All strains studied successfully infected the myoblasts, and at 72 hours post infec- tion (hpi) , it was possible to observe amastigote forms in the host cell’s cytoplasm as shown in details in A inset. (Bars = 50 µm.) To confirm genetic background of the parasite stocks used for infection, genomic DNA from epimastigote forms was isolated and amplified with Multiplex PCR with primers derived from a hypervariable region of the T. cruzi mini exon. ( E ) The Y, CL, and Tulahuen strains had a PCR product of 250 bp, indicating that they belong to the T. cruzi (TC) II family, and the Brazil strain had a 200-bp product, indicating that it belongs to TC I family. 3GENE EXPRESSION CHANGES IN T. CRUZI INFECTED MYOBLASTS: STRAIN MATTERS San Francisco, CA) were used to determine whether or not altered gene expressions differed significantly from chance for the overlapping functional and structural Gene Ontology (GO) categories. RESULTS Multiplex PCR was performed to confirm correspondence of the strains of T. cruzi used in this work to the phylogenetic classification as described in the literature. We verified that the Y, CL, and Tulahuen parasites displayed a 250-bp PCR product, indicating that they belong to the T. cruzi II (TC II) group. The Brazil strain, whose effects in host cells and animals have been characterized previously by our group, 15 , 21 belongs to the TC I group, because it displays a 200-bp PCR product ( Figure 1E ). Data complying with the Minimum Information about Microarray Experiments (MIAME) were deposited in the National Center for Biotechnology Information (NCBI) data- base ( http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE 18175 ) as series GSE18175. In this experiment, we quan tified 6,289 distinct, well-annotated unigenes in all samples. The impact of infection with each of the four strains of T. cruzi on the transcriptome changes of the same immortal- ized cell line was strikingly different ( Figure 2 ). Thus, as shown in Table 5 , there were only two (0.03%) genes significantly decreased, and 19 (0.3%) increased by all four T. cruzi strains. However, 4,340 (69%) genes were not significantly altered by any of the strains. The Venn diagrams in Figure 2 illustrate this observation by showing the number of genes equally increased ( Figure 2B ) or decreased ( Figure 2C ) by two, three, or all four strains of T. cruzi . Analysis of functional classes of genes modulated by the infection with different T. cruzi strains was performed with GenMapp software, and it revealed a very small number of gene functional categories with an abundance that was simi- larly affected by each pair of parasite strains ( Table 7 ). Below, we describe in more detail the differences that were observed. Y strain. We observed that the Y strain altered expression of 426 (6%) of the 6,996 quantified host cell genes. Among these genes, 150 were decreased, and 276 were increased after 72 hours of infection. When we identified individual genes whose expression was altered exclusively by the Y strain, we found that 87 genes were increased, such as α-1-integrin (1.76- fold), intercellular adhesion molecule (1.79-fold), presenilin 2 (3.81-fold), transforming growth-factor beta regulated gene (1.73-fold), and vascular cell-adhesion molecule 1 (1.91-fold). Sixty-five genes were decreased, such as cardiac Ca 2+ ATPase (−1.6-fold), cadherin (−2.88-fold), and Cyp26b1 (cytochrome P450, family 26, subfamily b, polypeptide 1; −2.83-fold) ( Table 1 ). The Y strain affected the expression of 11 gene categories including calcium and metal binding, regulation of transcription, and complexes of protein ( Table 7 ). CL strain. When the rat myoblasts were infected with the CL strain, we observed that 763 of 7,133 (11%) genes were significantly altered (53 decreased and 710 increased). The CL strain uniquely modulated the expression of 517 genes (494 were increased and 23 decreased), which corresponded to alterations in 14 gene categories including protein, intracellular protein, proton and ion transport, and ubiquitin cycle ( Table 7 ). Some of the genes that the CL strain modulated were adenylate Figure 2. Profiles of transcriptomic changes caused by each strain of T. cruzi in host cells. The percentage of genes modulated in L 6 E 9 cells by each T. cruzi strain is shown in A . Genes with fold-change value ³ 1.5 and P value £ 0.05 were quantified and plotted in this histogram, in which bars represent a percentage of the valid genes. Bars in white and black represent genes whose expression was decreased or increased exclusively by each strain, respectively. Bars in gray and light gray rep- resent genes that were increased and decreased, respectively, and shared this modulation by at least two strains. The Venn diagrams represent the number of genes significantly (fold change ³ 1.5 and P value < 0.05) ( B ) increased and ( C ) decreased in common by all four strains, by three strains, by pairs of strains, and by individual strains of the parasite. The number of genes equally altered by CL + Y and Brazil + Tulahuen strains is indicated below the diagrams. Note the low number of genes altered in the same direction by all strains studied (19 increased and 2 decreased). cyclase 2 (1.56-fold), α-spectrin (1.7-fold), annexin A1 (1.7-fold), caspase 7 (1.76-fold), epsin 2 (1.97), glutamate receptor (3.38), matrix metallopeptidase (MMP) 3 (2.51-fold), and syntaxin (−1.56-fold) ( Table 2 ). 4 ADESSE AND OTHERS Brazil strain. The Brazil strain induced significant alteration of 7.31% genes of L 6 E 9 by more than 1.5-fold. Among these 494 modulated genes, 117 were decreased, and 377 were increased. Some interesting genes that had their transcription altered by infection with the Brazil strain were cardiomyopathy associated gene (3.37-fold), MMP 1B (2.44-fold), mitogen- activated protein kinase 9 (2.12-fold), fibroblast growth factor (1.94-fold), desmin (1.63-fold), and tropomyosin 4 (−2.45-fold) ( Table 3 ). Ten functional categories of genes were highly modulated in L 6 E 9 by Brazil strain, including metallopeptidase Table 1 Y Strain Gene name Gene symbol Fold change Alanyl-tRNA synthetase Aars 1.85 Actin, gamma, cytoplasmic 1 Actg1 1.55 Chloride intracellular channel 2 Clic2 1.55 Cyclin-dependent kinase inhibitor 2B (p15; inhibits CDK4) Cdkn2b 1.65 Glycogen synthase kinase 3 beta Gsk3b 1.52 Integrin alpha 1 Itga1 1.76 Intercellular adhesion molecule 1 Icam1 1.79 Lectin, galactose binding, soluble 3 Lgals3 1.52 Presenilin 2 Psen2 3.814 Protein C receptor, endothelial Procr 2.04 Pyruvate kinase, muscle Pkm2 1.70 Syndecan -protein Sdcbp 1.854 Transforming growth-factor beta-regulated gene 1 Tbrg1 1.73 Vascular cell-adhesion molecule 1 Vcam1 1.91 X-linked myotubular myopathy gene 1 Mtm1 1.53 Actin, alpha 1, skeletal muscle Acta1 –4.84 Acyl-CoA synthetase long-chain family member 3 Acsl3 –2.63 ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 Atp2a2 –1.60 Cadherin 15 Cdh15 –2.88 Calsenilin, presenilin-binding protein, EF hand transcription factor Csen –1.62 Cytochrome P450, family 26, subfamily b, polypeptide 1 Cyp26b1 –2.83 Guanine monphosphate synthetase Gmps –1.94 Myosin light chain, phosphorylatable, fast skeletal muscle Mylpf –1.68 Rho family GTPase 2 Rnd2 –1.62 Table 2 CL strain Gene name Gene symbol Fold change Adenylate cyclase 2 Adcy3 1.56 Alpha-spectrin 2 Spna2 1.70 Angiopoietin-like 2 AF159049 1.56 Annexin A1 Anxa1 1.56 Casein kinase 1, gamma 1 Csnk1g1 1.95 Caspase 7 Casp7 1.76 Chemokine-like factor Cklf 1.76 Chondroitin sulfate proteoglycan 6 Cspg6 1.64 Desmuslin Dmn 1.60 Epsin 2 Epn2 1.97 Glutamate receptor, ionotropic, N-methyl D-aspartate 2B Grin2b 3.38 Integrin alpha 5 Itga5 1.84 Interleukin 1 receptor, type I Il1r1 2.27 Matrix metallopeptidase 10 Mmp10 2.40 Matrix metallopeptidase 3 Mmp3 2.52 Phosphatidylethanolamine N-methyltransferase Pemt 2.03 Phospholipase D1 Pld1 1.58 Plasminogen activator, urokinase receptor Plaur 1.76 Proteasome (prosome, macropain) 26S subunit, non-ATPase, 2 Psmd2 1.99 Chaperonin subunit 4 (delta) Cct4 –1.57 Syntaxin 8 Stx8 –1.56 T-cell immunomodulatory protein Cda08 –2.61 Table 3 Brazil strain Gene name Gene symbol Fold change H2A histone family, member Y H2afy 3.70 Cardiomyopathy associated 3 Cmya3 3.37 Protein kinase C, gamma Prkcc 2.70 Myocyte enhancer factor 2D Mef2d 2.43 Mitogen-activated protein kinase 9 Mapk9 2.12 Fibroblast growth factor 21 Fgf21 1.95 ATPase, H+/K+ exchanging, beta polypeptide Atp4b 1.90 Phosphatidylinositol 3-kinase, C2 domain containing, gamma polypeptide Pik3c2g 1.78 Paladin Pald 1.67 Desmin Des 1.63 Phosphatidylinositol 3 kinase, regulatory subunit, polypeptide 3 Pik3r3 –1.53 Coronin, actin-binding protein, 1B Coro1b –1.91 Synaptotagmin XI Syt11 –1.91 Proteasome (prosome, macropain) 28 subunit, beta Psme2 –2.35 Keratin 25D Krt25d –2.38 Tropomyosin 4 Tpm4 –2.46 Table 4 Tulahuen strain Gene name Gene symbol Fold change Activin A receptor type II-like 1 Acvrl1 7.60 Cadherin 3, type 1, P-cadherin (placental) Cdh3 4.65 Cardiac ankyrin repeat kinase Cark 2.54 CDK5 regulatory subunit-associated protein 1 Cdk5rap1 2.40 Chymotrypsinogen B Ctrb 2.64 Glucose 6 phosphatase, catalytic, 3 G6pc3 1.65 Inositol 1,4,5-trisphosphate 3-kinase C Itpkc 2.37 Interleukin 11 Il11 1.68 Kinesin family member C1 Kifc1 2.04 Myotrophin Mtpn 1.51 ATPase, Ca++ transporting, plasma membrane 1 Atp2b1 –2.76 Bcl2 modifying factor Bmf –2.93 Calcium channel, voltage-dependent, beta 3 subunit Cacnb3 –1.88 Calreticulin Calr –2.04 Caspase 9 Casp9 –1.75 Cytochrome c oxidase, subunit Va Cox5a –1.62 Cytokine-induced apoptosis inhibitor 1 Ciapin1 –1.67 Dynein cytoplasmic 1 heavy chain 1 Dync1h1 –1.86 Ectonucleoside triphosphate diphosphohydrolase 1 Entpd1 –2.90 Farnesyltransferase, CAAX box, alpha Fnta –1.66 Hypoxia inducible factor 1, alpha subunit Hif1a –1.52 Inositol 1,4,5-triphosphate receptor 3 Itpr3 –3.70 Janus kinase 3 Jak3 –1.64 Junctional adhesion molecule 3 Jam3 –2.25 Laminin, beta 2 Lamb2 –2.21 Matrix metallopeptidase 11 Mmp11 –3.46 Muscle, skeletal, receptor tyrosine kinase Musk –2.73 Phospholipase D2 Pld2 –2.88 Protein kinase C, nu Prkcn –2.57 Synaptojanin 2 Synj2 –2.35 Troponin T2, cardiac Tnnt2 –2.89 Tumor protein p53 Tp53 –1.92 5GENE EXPRESSION CHANGES IN T. CRUZI INFECTED MYOBLASTS: STRAIN MATTERS activity, small GTPase-mediated signal transduction, and ubiquitin cycle ( Table 7 ). Tulahuen strain. The Tulahuen strain of T. cruzi induced the highest percentage of altered gene expression in the myoblasts (17.35%) with 761 decreased genes and 383 increased genes, of which, 617 and 139, respectively, were uniquely observed in Tulahuen-infected dishes. Additionally, infection with this strain significantly modulated the expression of 18 gene categories, including cholesterol biosynthesis, immune response, lipid metabolism, and receptor activity ( Table 7 ). Some relevant examples of host cell genes significantly modu- lated during infection were p-cadherin (4.64-fold), cardiac ankyrin repeat kinase (2.54-fold), chymotrypsinogen B (2.64- fold), H2A histone family member Z (3.18-fold), interleukin 11 (1.68-fold), myotrophin (1.51-fold), caspase 9 (−1.75- fold), cytochrome c oxidase, subunit Va (−1.61-fold), heavy and light chain dynein (−1.86- and −1.81-fold, respectively), farnesyltransferase (−1.65-fold), hypoxia-inducible factor 1α subunit (−1.51-fold), janus kinase 3 (−1.64-fold), matrix metallopeptidase 11 (−3.46-fold), and cardiac troponin T2 (−2.89-fold) ( Table 4 ). Genes modulated equally by all T. cruzi strains as possible disease biomarkers. We observed that 13 (0.18%) host cell genes had the same pattern of significant modulation by all four strains of T. cruzi studied. These genes, thus, may represent disease biomarkers that could be useful in detecting the disease independent of the parasite strain. Only two (0.027%) were decreased, MMP-14 (−1.9-fold) and C1q and tumor necrosis factor-related protein 1 (−2.0-fold), and 11 (0.15%) increased, such as solute carrier family 1 (glutamate/neutral amino acid transporter; 1.9-fold), G protein-coupled receptor (2.1-fold), cardiotrophin-like cytokine factor 1 (2.13-fold), pericentriolar material 1 (3.22-fold), and DNA damage-inducible transcript 3 (4.37-fold). Table 5 shows the list of these thirteen genes and their modulation by each strain of the parasite. GenMapp analysis revealed that only nine gene categories were equally expressed by some pairs of strains such as receptor activity (Brazil and Tulahuen) and ubiquitin cycle (Brazil and CL) ( Table 7 ). Oppositely modulated genes. Surprisingly, our arrays revealed that some transcripts increased by one specific strain were decreased by another strain. Table 6 contains all the 24 genes that behaved in this manner. Some genes of interest were cytochrome P450, family 2, subfamily d, polypeptide 22 (3-fold by Brazil strain and −2.9-fold by Y strain), neuropathy target esterase-like 1 (2.1-fold by Y strain and −1.8-fold by CL strain), platelet-derived growth-factor receptor, β polypeptide (1.7-fold by CL strain and −1.6-fold by Tulahuen strain), protein kinase D2 (1.5-fold by Brazil strain and −1.6-fold by Y strain), and RNA polymerase 1-1 (1.6-fold by CL strain and −1.6-fold by Tulahuen strain). Table 5 Similar results Gene name Gene symbol Fold change (strains) Brazil CL Tulahuen Y C1q and tumor necrosis factor-related protein 1 C1qTNF1 –1.90 –1.80 –2.70 –1.80 Matrix metallopeptidase 14 MMP14 –2.00 –1.90 –2.00 –1.70 Cardiotrophin-like cytokine factor 1 Clcf1 2.20 2.30 1.80 2.30 Cut-like 1 (Drosophila) Cutl1 7.72 2.58 5.36 1.59 DNA-damage inducible transcript 3 Ddit3 9.30 3.08 2.41 2.64 Excision repair cross-complementing rodent repair deficiency, complementation group 3 Ercc3 2.19 2.67 1.59 2.36 G protein-coupled receptor, family C, group 5, member A Gprc5a 1.94 2.53 1.87 1.99 GrpE-like 1, mitochondrial Grpel1 1.60 1.95 1.66 1.91 Pericentriolar material 1 Pcm1 2.70 4.25 2.23 3.70 Proprotein convertase subtilisin/kexin type 7 Pcsk7 6.93 2.87 2.38 2.15 Serologically defined colon cancer antigen 3 Sdccag3 3.36 2.71 2.67 2.50 Solute carrier family 1 (glutamate/neutral amino acid transporter) Slc1a4 1.59 2.50 1.82 1.68 Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein Ywhaq 2.84 2.85 1.77 2.56 Table 6 Opposite results Gene name Gene symbol Fold change (strains) Brazil CL Tulahuen Y ADP-ribosylation factor guanine nucleotide-exchange factor 2 Arfgef2 – 1.56 –1.54 – CDC -like kinase 2 LOC365842 – 1.52 –1.64 – Cytochrome P450, family 2, subfamily d, polypeptide 22 Cyp2d22 2.97 – – –2.92 NAD synthetase 1 Nadsyn1 – 1.56 –1.63 – Neuropathy target esterase-like 1 Ntel1 – –1.78 – 2.11 Platelet-derived growth-factor receptor, beta polypeptide Pdgfrb – 1.68 –1.64 – Pleckstrin homology, Sec7, and coiled-coil domains 1 Pscd1 – 1.53 –1.53 – Preoptic regulatory factor-2 PORF-2 – 1.62 –1.56 – Protein kinase D2 Prkd2 1.50 – – –1.62 Regenerating islet-derived 1 Reg1 1.67 –2.31 – – RNA polymerase 1-1 Rpo1-1 – 1.56 –1.56 – Son of sevenless homolog 1 Sos1 –1.53 1.82 – – Sulfotransferase family 1A, phenol-preferring, member 1 Sult1a1 – – 1.90 –1. 53 Thymoma viral proto-oncogene 2 Akt2 – 1.54 –1.64 – Transducer of ErbB-2.1 Tob1 – – 1.58 –1.72 6 ADESSE AND OTHERS Correlations between infections with four strains. Our studies identifying individual genes that were significantly altered by the four strains of T. cruzi revealed a surprising diversity with only a few genes similarly changed by infection with all strains. However, because this analysis selects only individuals, it does not compare subtle global changes throughout the transcriptome. To compare global trancriptomic alteration patterns in L 6 E 9 cells infected by the four strains used in the current investigation, we compared the entire gene-array datasets obtained from the infection with each strain against each of the other strains. With Origin software (OriginLab, Northampton, MA), we plotted results of these pairs as log 2 values of their expression ratios. In all six of the comparisons of expression changes induced by infection with separate Table 7 Gene categories Gene Ontology (GO) ID GO Name No. changed No. measured No. in GO Percent changed P value Y strain 5634 Nucleus 22 557 1,643 3.95000 0.004 45449 Regulation of transcription 12 264 884 4.55000 0.010 6355 Regulation of transcription, DNA-dependent 11 237 790 4.64000 0.012 5509 Calcium-ion binding 8 149 538 5.37000 0.013 6350 Transcription 12 278 920 4.32000 0.014 46872 Metal-ion binding 20 593 1,913 3.37000 0.049 5743 Mitochondrial inner membrane 4 22 63 18.18000 0.006 19866 Organelle inner membrane 4 29 79 13.79000 0.026 16757 Transferase activity, transferring glycosyl groups 5 48 144 10.42000 0.042 43234 Protein complex 9 396 1,280 2.27000 0.052 16853 Isomerase activity 4 34 74 11.77000 0.052 CL strain 5829 Cytosol 4 68 121 5.88000 0.002 5622 Intracellular 18 1,331 3,766 1.35000 0.004 15031 Protein transport 5 154 348 3.25000 0.011 6512 Ubiquitin cycle 3 102 281 2.94000 0.049 3824 Catalytic activity 8 1,611 5,073 0.50000 0.054 6886 Intracellular protein transport 3 103 237 2.91000 0.054 16757 Transferase activity, transferring glycosyl groups 10 48 144 20.83000 0.011 15992 Proton transport 6 24 64 25.00000 0.023 4197 Cysteine-type endopeptidase activity 7 34 91 20.59000 0.032 6811 Ion transport 18 118 555 15.25000 0.038 3924 Gtpase activity 5 22 45 22.73000 0.040 16491 Oxidoreductase activity 25 185 539 13.51000 0.045 6812 Cation transport 14 86 375 16.28000 0.046 Brazil strain 8237 Metallopeptidase activity 3 38 133 7.89000 0.020 4984 Olfactory receptor activity 9 24 1,034 37.50000 0.000 1584 Rhodopsin-like receptor activity 9 46 1,391 19.57000 0.001 4872 Receptor activity 20 195 2,013 10.26000 0.002 7186 G-protein–coupled receptor protein-signaling pathway 11 82 1,581 13.41000 0.005 43234 Protein complex 12 396 1,280 3.03000 0.022 7264 Small gtpase mediated signal transduction 8 66 179 12.12000 0.027 5615 Extracellular space 4 23 136 17.39000 0.029 6512 Ubiquitin cycle 1 102 281 0.98000 0.046 5525 GTP binding 10 99 237 10.10000 0.050 Tulahuen strain 6457 Protein folding 16 68 138 23.53000 0.001 5488 Binding 219 1,786 5,708 12.26000 0.010 3743 Translation initiation factor activity 7 25 37 28.00000 0.019 6695 Cholesterol biosynthesis 4 11 15 36.36000 0.021 6955 Immune response 10 46 258 21.73913 0.033 6629 Lipid metabolism 20 120 304 16.67000 0.053 4984 Olfactory receptor activity 7 24 1,034 29.17000 0.000 1584 Rhodopsin-like receptor activity 9 46 1,391 19.57000 0.001 16020 Membrane 63 740 3,798 8.51000 0.003 19866 Organelle inner membrane 7 29 79 24.14000 0.003 5743 Mitochondrial inner membrane 5 22 63 22.73000 0.012 4872 Receptor activity 20 195 2,013 10.26000 0.014 16021 Integral to membrane 41 494 2,888 8.30000 0.018 5856 Cytoskeleton 13 114 337 11.40000 0.021 7186 G-protein–coupled receptor protein-signaling pathway 10 82 1,581 12.20000 0.029 9058 Biosynthesis 9 284 923 3.17000 0.036 16874 Ligase activity 2 124 329 1.61000 0.046 5739 Mitochondrion 15 149 290 10.07000 0.048 7GENE EXPRESSION CHANGES IN T. CRUZI INFECTED MYOBLASTS: STRAIN MATTERS Figure 3. Correlation of datasets obtained by microarray analyses of the infection of L 6 E 9 cell line with Y, CL, Brazil, and Tulahuen strains of T. cruzi . The datasets of each strain were plotted against each other in pairs of parasite strains. The histograms show how the transcriptomic changes induced by all strains have a highly significant correlation (all have P values < 0.0001). R 2 values were ( A ) 0.5, ( B ) 0.51, ( C ) 0.67, ( D ) 0.52, ( E ) 0.47, and ( F ) 0.53. T. cruzi strains ( Figure 3 ), the regression coefficients (r 2 values) for these linear relations were highly significant, and P values in all cases were less than 0.0001. This finding indicates that although infection with each of the parasite strains leads to only partially overlapping alterations in the genes that are most affected, there is an overall similarity in the pattern of gene-expression alterations resulting from infection with all the strains. DISCUSSION Chagas disease represents a spectrum of pathogenesis, varying both in its severity and the organ systems afflicted. Although various host factors, such as competence to launch immune response, may account in part for differences in the pathogenesis of the disease, parasite strain is also an important variable, 6 resulting in differences in viability, infectivity, and tissue tropism. 7 – 9 The present study was undertaken to evalu- ate the extent to which global gene-expression alteration was similarly altered after in vitro infection of a myoblast cell line with four distinct T. cruzi strains. The microarray analyses described in this study were per- formed on the myoblast cell line L 6 E 9 during infection with four reference strains of T. cruzi , each with well-characterized rates of in vivo and in vitro infectivity, resistance to che- motherapy, and pathogenesis in vivo. We used the Y and CL strains as representatives of the TC II group of T. cruzi , known to be found in central and eastern Brazil, which is commonly associated with the “mega” syndromes (cardio- megaly, megacolon, and megaesophagous). 22 The Tulahuen strain was chosen to represent the TC I group, however, the Multiplex PCR performed with genomic DNA of parasites of this strain revealed that it was actually a strain belonging to the TC II family. The Brazil strain was selected, because 8 ADESSE AND OTHERS it has been shown to cause a dilated cardiomyopathy associ- ated with a reduction in fractional shortening and myocardial wall thinning in mice. 13 Genotyping revealed that the Brazil strain belongs to the TC I group, the predominant group in Venezuela and central Brazil, which is usually associated with electrocardiographic (ECG) abnormalities . 22 We have compared the datasets obtained from the microar- ray analyses in two ways, resulting in different but comple- mentary conclusions regarding the pathogenesis of T. cruzi infection in vitro and perhaps, applicable to in vivo infection as well. First, through identification of the genes that had altered expression after infection, we identified a large num- ber of gene expression changes in the infected cells, but only a very small number of these were common to infection with each parasite strain. We concluded from this analysis that each strain induces a particular fingerprint of pathology. One such fingerprint may include genes encoding cell-junction proteins, which was evidenced by our finding that both Tulahuen and Y strains down-regulated several cellular junction genes such as junction plakoglobin, junctional adhesion molecule 3, and adipocyte-specific adhesion molecule as well as cadherin 15 (Y strain). T. cruzi infection was also shown to alter adhe- sion molecules of the host such as connexin43 23 and cadherin- catenin. 11 , 24 These findings highlight an evolving concept that many types of cardiomyopathy target expression or involve mutations in molecular components of the intercalated disk (see reference 25 for review and reference 26 for changes in sepsis). Thus, as pointed out in a recent review, 27 cardiomyopa- thies, including chronic chagasic cardiomyopathy, may be con- sidered to be junctionopathies. An additional conclusion from the small number of genes found to be commonly regulated in infections with all strains (e.g., cut-like 1, DNA-damage inducible transcript 3, propro- tein convertase subtilisin/kexin type 7) may provide a subset of biomarkers that could be potentially useful for diagnosis of acute T. cruzi infection and possibly, also reliably determine both indeterminate phase and chronic disease. The second type of analysis that we performed on the gene-expression datasets was to compare overall transcrip- tomic changes in myoblasts infected with each T. cruzi strain through regression analysis of relative expression levels of each gene. For this, we compared each of the six pair-wise combinations of parasite strains, in each case finding a highly significant positive slope. This analysis emphasizes the con- cept that although Chagas disease shows a spectrum of mani- festations, it does indeed represent a syndrome of common phenotypic alterations. The shallow slopes of the regression lines indicate that the changes induced by the parasite strains, although similar, are on average very low in amplitude; this emphasizes again the potential utility of the few commonly altered biomarkers. The significant alteration in few overlapping genes among myoblasts infected with all strains poses a formidable chal- lenge to the development of disease biomarkers that can be used to detect disease endemic areas where endogenous infective strains differ, although the few genes that we have detected with strong and significant expression changes offer such a possibility. Nevertheless, the overall transcriptomic sig- nature of the disease that is revealed in the strong correlations between subtle expression alteration of all genes may provide a source whereby additional biomarkers may be discovered as common principal components of the acute response. Received July 17, 2009. Accepted for publication October 25, 2009. Acknowledgments: The authors thank Vicki L. Braunstein (AECOM) and Angela Santos (LUC–Fiocruz) for the technical support with maintenance of the T. cruzi strains and Nadia Nehme and Dr. Octavio Fernandes (Fiocruz) and Aisha Cordero (AECOM) for the assistance with T. cruzi genotyping. We also thank Ethan Mackenzie (AECOM) for the help with GenMapp analysis. D.A. was supported in part by a grant from the Fogarty International Center-NIH D43 W007129 (HBT) and grants from The US National Institutes of Health AI-076248(HBT), HL-73732(HBT, DCS) and from CNPq, CNPq, PAPES IV-FIOCRUZ. Authors’ addresses: Daniel Adesse, Luciana Ribeiro Garzoni, and Maria de Nazareth Meirelles, Laboratório de Ultraestrutura Celular, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil. Dumitru A. Iacobas, Sanda Iacobas, and David C. Spray, Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY. Herbert B. Tanowitz, Department of Pathology, Albert Einstein College of Medicine, Bronx, NY. REFERENCES 1. de Souza W , 2007 . Chagas’ disease: facts and reality . Microbes Infect 9: 544 – 545 . 2. Tanowitz HB , Kirchhoff LV , Simon D , Morris SA , Weiss LM , Wittner M , 1992 . Chagas’ disease . Clin Microbiol Rev 5 : 400 – 419 . 3. Higuchi Mde L , Benvenuti LA , Martins Reis M , Metzger M , 2003 . Pathophysiology of the heart in Chagas’ disease: current status and new developments . Cardiovasc Res 60: 96–107 . 4. Carneiro M , Romanha AJ , Chiari E , 1991 . Biological characteriza- tion of Trypanosoma cruzi strains from different zymodemes and schizodemes . Mem Inst Oswaldo Cruz 86: 387 – 393 . 5. Andrade SG , 1999 . Trypanosoma cruzi : clonal structure of para- site strains and the importance of principal clones . Mem Inst Oswaldo Cruz 94 (Suppl 1) : 185 – 187 . 6. Andrade SG , Magalhães JB , 1996 . Biodemes and zymodemes of Trypanosoma cruzi strains: correlations with clinical data and experimental pathology . Rev Soc Bras Med Trop 30 : 27 – 35 . 7. Veloso VM , Carneiro CM , Toledo MJ , Lana M , Chiari E , Tafuri WL , Bahia MT , 2001 . Variation in susceptibility to benznidazole in isolates derived from Trypanosoma cruzi parental strains . Mem Inst Oswaldo Cruz 96: 1005 – 1011 . 8. Mielniczki-Pereira AA , Chiavegatto CM , López JA , Colli W , Alves MJ , Gadelha FR , 2007 . Trypanosoma cruzi strains, Tulahuen 2 and Y, besides the difference in resistance to oxi- dative stress, display differential glucose-6-phosphate and 6-phosphogluconate dehydrogenases activities . Acta Trop 101 : 54 – 60 . 9. Sanchez G , Wallace A , Olivares M , Diaz N , Aguilera X , Apt W , Solari A , 1990 . Biological characterization of Trypanosoma cruzi zymodemes: in vitro differentiation of epimastigotes and infectivity of culture metacyclic trypomastigotes to mice . Exp Parasitol 71: 125 – 133 . 10. Shigihara T , Hashimoto M , Shindo N , Aoki T , 2008 . Transcriptome profile of Trypanosoma cruzi -infected cells: simultaneous up- and down-regulation of proliferation inhibitors and promoters . Parasitol Res 102 : 715 – 722 . 11. Imai K , Mimori T , Kawai M , Koga H , 2005 . Microarray analysis of host gene-expression during intracellular nests formation of Trypanosoma cruzi amastigotes . Microbiol Immunol 49: 623 – 631 . 12. Mukherjee S , Belbin TJ , Spray DC , Iacobas DA , Weiss LM , Kitsis RN , Wittner M , Jelicks LA , Scherer PE , Ding A , Tanowitz HB , 2003 . Microarray analysis of changes in gene expression in a murine model of chronic chagasic cardiomyopathy . Parasitol Res 91 : 187 – 196 . 13. Mukherjee S , Nagajyothi F , Mukhopadhyay A , Machado FS , Belbin TJ , Campos de Carvalho A , Guan F , Albanese C , Jelicks LA , Lisanti MP , Silva JS , Spray DC , Weiss LM , Tanowitz HB , 2008 . Alterations in myocardial gene expression associated with experimental Trypanosoma cruzi infection . Genomics 91 : 423 – 432 . 14. Garg N , Popov VL , Papaconstantinou J , 2003 . Profiling gene transcription reveals a deficiency of mitochondrial oxidative 9GENE EXPRESSION CHANGES IN T. CRUZI INFECTED MYOBLASTS: STRAIN MATTERS phosphorylation in Trypanosoma cruzi -infected murine hearts: implications in chagasic myocarditis development . Biochim Biophys Acta 1638: 106–120 . 15. Rowin KS , Tanowitz HB , Wittner M , Nguyen HT , Nadal-Ginard B , 1983 . Inhibition of muscle differentiation by Trypanosoma cruzi . Proc Natl Acad Sci USA 80: 6390 – 6394 . 16. Bertelli MS , Brener Z , 1980 . Infection of tissue culture cells with bloodstream trypomastigotes of Trypanosoma cruzi . J Parasitol 66: 992 – 997 . 17. Fernandes O , Santos SS , Cupolillo E , Mendonça B , Derre R , Junqueira AC , Santos LC , Sturm NR , Naiff RD , Barret TV , Campbell DA , Coura JR , 2001 . A mini-exon multiplex poly- merase chain reaction to distinguish the major groups of Trypanosoma cruzi and T. rangeli in the Brazilian Amazon . Trans R Soc Trop Med Hyg 95: 97 – 99 . 18. Garzoni LR , Caldera A , Meirelles Mde N , de Castro SL , Docampo R , Meints GA , Oldfield E , Urbina JA , 2004 . Selective in vitro effects of the farnesyl pyrophosphate synthase inhibitor rise- dronate on Trypanosoma cruzi . Int J Antimicrob Agents 23: 273 – 285 . 19. Iacobas DA , Iacobas S , Li WE , Zoidl G , Dermietzel R , Spray DC , 2005 . Genes controlling multiple functional pathways are tran- scriptionally regulated in connexin43 null mouse heart . Physiol Genomics 20: 211–223 . 20. Iacobas DA , Fan C , Iacobas S , Spray DC , Haddad GG , 2006 Transcriptomic changes in developing kidney exposed to chronic hypoxia . Biochem Biophys Res Commun 13: 329–338 . 21. Combs TP , Nagajyothi F , Mukherjee S , de Almeida CJ , Jelicks LA , Schubert W , Lin Y , Jayabalan DS , Zhao D , Braunstein VL , Landskroner-Eiger S , Cordero A , Factor SM , Weiss LM , Lisanti MP , Tanowitz HB , Scherer PE , 2005 . The adipocyte as an important target cell for Trypanosoma cruzi infection . J Biol Chem 24: 24085–24094 . 22. Miles MA , Cedillos RA , Póvoa MM , de Souza AA , Prata A , Macedo V , 1981 Do radically dissimilar Trypanosoma cruzi strains (zymodemes) cause Venezuelan and Brazilian forms of Chagas’ disease ? Lancet 20: 1338–1340 . 23. Adesse D , Garzoni LR , Huang H , Tanowitz HB , de Nazareth Meirelles M , Spray DC , 2008 . Trypanosoma cruzi induces changes in cardiac connexin43 expression . Microbes Infect 10: 21 – 28 . 24. de Melo TG , Meirelles Mde N , Pereira MC , 2008 . Trypanosoma cruzi alters adherens junctions in cardiomyocytes . Microbes Infect 10: 1405 – 1410 . 25. Saffitz JE , Hames KY , Kanno S , 2007 . Remodeling of gap junc- tions in ischemic and nonischemic forms of heart disease . J Membr Biol 218 : 65 – 71 . 26. Celes MR , Torres-Dueñas D , Alves-Filho JC , Duarte DB , Cunha FQ , Rossi MA , 2007 . Reduction of gap and adherens junction pro- teins and intercalated disc structural remodeling in the hearts of mice submitted to severe cecal ligation and puncture sepsis . Crit Care Med 35: 2176 – 2185 . 27. Spray DC , Tanowitz HB , 2007 . Pathology of mechanical and gap junctional co-coupling at the intercalated disc: is sepsis a junc- tionopathy ? Crit Care Med 35: 2231 – 2232 . Resultados 42 Trabalho #2 Daniel Adesse, Michael P. Lisanti, David C. Spray, Fabiana S. Machado, Maria de Nazareth Meirelles, Herbert B. Tanowitz and Luciana Ribeiro Garzoni Trypanosoma cruzi infection results in the reduced expression of caveolin-3 in the heart Publicado na Cell Cycle (Abril de 2010) www.landesbioscience.com Cell Cycle 1 Cell Cycle 9:8, 1-8; April 15, 2010; © 2010 Landes Bioscience RepoRt RepoRt *Correspondence to: Michael P. Lisanti and Herbert B. Tanowitz; Email: michael.lisanti@kimmelcancercenter.org and herbert.tanowitz@einstein.yu.edu Submitted: 02/08/10; Accepted: 02/10/10 Previously published online: www.landesbioscience.com/journals/cc/article/11509 Introduction Chagas disease is caused by the protozoan parasite Trypanosoma cruzi,1 and results in heart disease in endemic areas of Latin America.2 Acute infection is usually associated with intense myo- cardial inflammation characterized by an upregulation of cytok- ines and chemokines.3,4 The ensuing cardiovascular remodeling may result in cardiomyopathy. The dilated cardiomyopathy is associated with congestive heart failure, arrhythmias, conduction abnormalities and thrombo-embolic events.2,5 The mouse model of T. cruzi infection has been intensely studied because it recapit- ulates many of the pathological, functional and immunological features of the human disease. In addition, primary cultures of rodent neonatal cardiac myocytes have been utilized as a tool to investigate the effect of T. cruzi on the heart.6-13 For example, T. cruzi alters cardiac myocyte Ca2+ homeostasis.6-13 and gap junc- tional communication.14,15 These infection-associated changes may contribute to the arrhythmias observed in this infection. Caveolae are motile, membrane-bound compartments con- taining molecules that participate in cell signaling such as enzymes that generate messengers from substrates in the envi- ronment, substrates that are enzymatically converted into mes- sengers and high-affinity binding sites that concentrate chemical signals. Caveolins are protein components of caveolae that are important in modulating a variety of biological functions includ- ing the modifications of signaling systems.18 Caveolae and caveo- Trypanosoma cruzi infection results in the reduced expression of caveolin-3 in the heart Daniel Adesse,1,2,4 Michael p. Lisanti,6,7,* David C. Spray,3,4 Fabiana S. Machado,5 Maria de Nazareth Meirelles,1 Herbert B. tanowitz2,3,* and Luciana Ribeiro Garzoni1 1Laboratório de Ultra-estrutura Celular; Instituto oswaldo Cruz/FIoCRUZ; Rio de Janeiro, RJ Brazil; Departments of 2pathology; 3Medicine; and 4the Dominick purpura Department of Neuroscience; Albert einstein College of Medicine; Bronx, NY USA; 5Department of Biochemistry and Immunology; Institute of Biological Sciences; Federal University of Mina Gerais; Belo Horizonte, Brazil; 6Department of Stem Cell Biology & Regenerative Medicine and the Kimmel Cancer Center; thomas Jefferson University; philadelphia, pA USA; and 7the Muscular and Neurodegenerative Disease Unit; University of Genoa and G. Gaslini pediatric Institute; Genoa, Italy Key words: Trypanosoma cruzi, cardiac myocytes, caveolin-3, caveolae, ERK T hi s m an us cr ip t ha s be en p ub lis he d on lin e, p ri or t o pr in ti n g. O n ce t he is su e is c om pl et e an d pa ge n um be rs h av e be en a ss ig n ed , t he c it at io n w ill c ha n ge a cc or di n gl y. lins play critical roles in calcium homeostasis in the heart16 and are disrupted by T. cruzi infection.17 Caveolae are signaling platforms that compartmentalize and concentrate signaling molecules such as G-protein subunits and endothelial nitric oxide synthase. Caveolins inhibit the down- stream activation and signaling of many proteins, including c-Src, H-Ras, mitogen-activated protein (MAP) kinases, and eNOS.19,20 Caveolin-1 (Cav-1) is expressed ubiquitously, although at differ- ent levels in different tissues; Cav-2 is tightly co-expressed with Cav-1, whereas Cav-3 is expressed predominantly in striated muscle cells.21 Importantly, Cav-1 and Cav-3 null mice display a cardiomyopathy, with left ventricle wall thickening, fibrosis and p42/44 MAPK (ERK 1/2) activation.22 The activation of ERK1/2 plays an important role in the pathogenesis of cardiac hypertrophic responses and cellular pro- liferation in the cardiovascular system.23 In vitro data support a role for Cav-1 and Cav-3 as negative regulators of ERK sig- naling.24 During acute infection with the Tulahuen strain of T. cruzi Nagajyothi et al.17 described reduced levels of Cav-1, -2 and -3 and increased ERK phosphorylation in hearts from infected mice. T. cruzi infection of mice and of cultured endothelial and smooth muscle cells resulted in activation of AP-1 which participates in cardiovascular remodeling through ERK 1/2 phosphorylation.25,26 We now report that the infection of cultured cardiac myo- cytes with T. cruzi resulted in a reduction of Cav-3 expression Caveolae are motile, membrane-bound compartments that contain a number of molecules that participate in cell signaling. Caveolins are protein markers of caveolae and function in a variety of biological processes. Caveolin-3 (Cav-3) is expressed in muscle cells and Cav-3 null mice display a cardiomyopathic phenotype. Ultrastructural cytochemistry, confocal microscopy and immunoblotting revealed a reduction in Cav-3 expression and an activation of eRK (extracellular- signal-regulated kinase) 48 hours after Trypanosoma cruzi infection of cultured cardiac myocytes. CD-1 mice infected with the Brazil strain of T. cruzi displayed reduced expression of Cav-3 and activation of eRK 66 days post infection (dpi). By 180 dpi there was a normalization of these values. these data suggest that the reduction in Cav-3 expression and the activation of eRK during the early phase of infection may contribute to the pathogenesis of chagasic cardiomyopathy. 2 Cell Cycle Volume 9 Issue 8 T. cruzi and ERK phosphorylation. Since an inverse rela- tionship has been reported between the activation of ERK and the reduction in Cav-3 expression24 we examined the impact of infection on ERK activation in cardiac myocytes at various time-points post infection, from one to 72 hours. The protein lysates were subjected to immunoblot analysis and probed with specific antibodies. We verified that at all time points studied, the infection did not alter total ERK expression (Fig. 4A and B). However, there was a progressive activation of ERK, as evidenced by increased phosphorylation in the cardiac myocytes; starting at 2 hours post infection (40%) and a 3-fold increase 72 hours post infection (Fig. 4A and B). We next analyzed ERK expression activation in hearts of infected mice at 66 and 180 dpi. While total ERK expression remained unaltered, phosphorylated forms of ERK at 66 dpi (when Cav-3 levels were decreased) had a 2.3- fold increase when compared to age-matched controls. At 180 dpi we observed that phosphoERK expression was 68% decreased in infected heart tissue (Fig. 4C and D). Discussion The effects of T. cruzi infection and its progression to heart dis- ease is an ongoing interest by our groups and of others, since chagasic cardiomyopathy is the main cause of heart disease in Latin America. The parasite alters many of the host cell pro- cesses such as intracellular calcium homeostasis,9,27 cell-cell communication14,15 and cytoskeleton stability.28,29 Alterations caused to host cells in the heart during acute infection where tis- sue parasitism is high and is associated with an intense inflamma- tory response and myonecrosis, may determine the development of the chronic disease. In the present study, we have focused on caveolae, which are membrane-bound signaling complexes involved in Ca2+ homeo- stasis as well as other cell functions.19 We evaluated the distribu- tion of caveolae on infected cardiac myocytes by ultra-structural cytochemistry using lanthanum nitrate (La). La displaces Ca2+ from its binding sites on sarcolemma and can be used as a tracer for surface Ca2+ binding sites, abundantly found in caveolae.30,31 The La staining permits the observation of caveolae distribu- tion and intracellular vesicles, with positive La staining indicat- ing sites of transport and storage of Ca2+. In the current study we observed that T. cruzi-infected cardiac myocytes displayed a reduction in La staining on sarcolemma when compared with non infected cells after 48 hours of infection.32 This is in agree- ment with previous results showing (a) that T. cruzi could alter cardiac myocyte endocytotic uptake of small molecules,32 and (b) that caveolin (Cav)-1 null mice also display impaired endo- cytosis.33 We confirmed the alterations in caveolae distribution during infection with T. cruzi by immunostaining and immun- blot analysis demonstrating a significant reduction in the expres- sion of Cav-3 48 and 72 hours post infection. Since caveolae are involved in Ca2+ regulation in cardiac myocytes and T. cruzi infection alters these structures, it is possible that caveolae dis- ruption could be involved with the changes on Ca2+ homeostasis in cardiac myocytes. By depleting caveolae with cyclodextrin34 a dose-dependent decrease in frequency, amplitude and spatial and activation of ERK 1/2. This was also observed in infected mice. These observations strongly suggest that infection with T. cruzi results in the reduced expression of Cav-3 and the activa- tion of ERK. The cardiomyopathic phenotype of Cav-3 null mice suggests that a reduction in Cav-3 expression contributes to the cardiomyopathy of chagasic cardiomyopathy. ERK likely plays an important role in cardiac remodeling and arrhythmogenesis that accompany chagasic cardiomyopathy. Results Caveolae and caveolin-3 (Cav-3) expression are reduced as a result of T. cruzi infection. Ultrastructural analysis revealed that uninfected cardiac myocytes displayed features of differentiated myocytes such as myofibrils near mitochondria, sarcoplasmic reticulum, T-tubules and cell-cell junctions (Fig. 1A–C). Use of the lanthanum (La) as an electron opaque tracer for divalent ion (Ca2+) binding sites revealed numerous caveolae on the surface of cultured cardiac myocytes and subsurface vesicles containing the tracer (Fig. 1B and C). After 24 hours of infection, trypo- mastigotes were identified by the presence of basket shaped kine- toplasts and subpellicular microtubules (Fig. 1D). We observed intracellular amastigotes in the cytoplasm of host cells present- ing typical structures such as reservosomes and acidocalcisomes at 48 h (Fig. 1E). Beginning at 24 hr post infection, there was both reduced surface staining with La and a smaller number of caveolae in infected cells (Fig. 1D). These differences persisted at longer time-points, with surface La labeling virtually absent at 72 hours post infection (Fig. 1E and F). To quantitate alterations in caveolae and Cav-3 abundance and distribution, we performed immunostaining and immuno- blotting during the course of infection. Cultured cardiac myo- cytes displayed specific reactivity to anti-Cav-3 antibody, since no staining was detected in endothelial cells or fibroblasts (not shown). Cav-3 expression was abundant at and beneath the surface membrane of cardiac myocytes cultured for 96 hours (Fig. 2). However, 72 hours post infection, highly infected cells showed a striking reduction in Cav-3 immunoreactivity (Fig. 2B). Immunoblot analyses of the myocytes were consistent with both La cytochemistry and Cav-3 immunostaining. We observed that infected cardiac myocytes displayed Cav-3 levels similar to controls at 24 hours post infection and that there was a significant reduction at 48 (74%) and 72 (51%) hours post infec- tion (p < 0,05, ANOVA) (Fig. 2C and D). T. cruzi in vivo infection and caveolin-3 (Cav-3) expres- sion. We further analyzed the changes in Cav-3 in infected mice 66 and 180 days post infection (dpi). Immunoblot analy- sis of the heart tissue at these time points revealed that at 66 dpi there was a 46% decrease in Cav-3 levels, which were fully restored at 180 dpi (Fig. 3). Immunohistochemistry of the heart tissue at 66 dpi revealed that non-infected animals displayed abundant Cav-3 amongst cardiac myocytes, with T-tubule pat- terns (Fig. 3 and arrows). This pattern was altered in infected hearts, where we observed areas of tissue damage (Fig. 3B) and areas in which cardiac myocytes displayed mostly intracellular staining. www.landesbioscience.com Cell Cycle 3 contribute to the occurrence of arrhythmias observed in cha- gasic heart disease. One major mechanism by which expression of caveolins is downregulated is activation of the mitogen-activated protein kinase family (MAPK), including the p42/44 MAPK, also extent of Ca2+ sparks was observed in smooth muscle cells and in cardiac myocytes: These observations suggest that alterations in the molecular assembly and ultra-structure of caveolae may lead to pathophysiological changes in Ca2+ signaling.16 Through disruption of Ca2+ homeostasis, alteration in caveolae may Figure 1. Ultrastructural cytochemistry. the tracer Lanthanum (La) Nitrate was used to reveal Ca2+ binding sites in cardiac myocytes. (A–C) Represen- tative uninfected cardiac myocytes displayed longitudinal (LM) and transversal (tM) myofibrils, abundant mitochondria (M) and t-tubules (tt). Note in (C) the cell-cell junctions are depicted by arrowheads. La staining reveals intense electron dense staining in the plasma membrane with many cave- olae (arrows), which can be observed in more detail in the inserts. (C) Some vesicles close to the membrane show staining with the tracer. (D) After 24 hours of infection a parasite is observed with subpellicular microtubules and a round kinetoplast (K). (e) A representative infected cardiac myocyte 48 hours post infection containing an intracellular amastigote with a reservosome (R) and an acidocalcisome (a). (N = Nucleus). (F) there was a reduction in La staining and alterations in caveolae abundance 72 hours after infection. trypomastigotes (p) are observed in the cell cytoplasm and a decrease in surface staining by La. original magnification: (A and F) = 26,000X; (B, C and e) = 41,000X; (D) = 47,350X. 4 Cell Cycle Volume 9 Issue 8 immunohistochemistry and immunoblot; and a 3-fold increase in ERK phosphorylation was observed. This in accordance with previous studies that shown reduced Cav-1, Cav-2 and Cav-3 during acute murine T. cruzi infection followed by ERK phos- phorylation.3,17 Cav-1 null mice display hyperactivation of ERK in the heart and these mice also display cardiac hypertrophy with normal substrate utilization and expression of genes involved in energy metabolism.36-38 In addition, Cav-3 null mice display increased expression of phosphorylated ERK and a cardiomyo- pathic phenotype.22 The observations in the current report demonstrate that T. cruzi infection alters Cav-3 expression in cardiac myocytes. known as extracellular signal regulated kinase (ERK 1/2).19,35 The infected myocytes displayed activation of this protein kinase starting at 2 hours post infection as determined by immunoblot analysis using phospho-specific antibodies, without any signifi- cant alteration in total protein expression. This observation is consistent with our previous demonstration of ERK activation in cultured endothelial and smooth muscle cells after infection with the Tulahuen strain of T. cruzi.25 Since ERK phosphorylation is involved in cardiac remodel- ing, we studied Cav-3 and phospho-ERK expression in heart lysates of infected mice 66 days and 180 days post infection. Cav-3 levels were drastically reduced at 66 dpi as observed by Figure 2. Caveolin-3 (Cav-3) expression is decreased after T. cruzi infection. Cultured cardiac myocytes were infected for 3 days. (A) Representative confocal Laser Scanning analysis of Cav-3 distribution in uninfected cardiac myocytes fixed and stained after 96 hours in culture. Cells presented abundant caveosomes (arrowheads) and peripheral staining (white arrows). the inset depicts higher magnification of the Cav-3 staining. (B) After 72 hours of infection, Cav-3 signal was reduced among highly parasitized cardiac myocytes, present mostly at cell periphery and with reduced presence of caveosomes. (C) Representative immunoblot of Cav-3 (23 KDa) expression in infected and uninfected cardiac myocytes. Anti-GApDH (36 KDa) anti- body was used as a loading control. (D) Bar graph demonstrating quantification of Cav-3 expression as normalized by GApDH. there was a significant decrease of Cav-3 expression 48 (74%) and 72 (51%) hours post infection (n = 4) **p < 0.01; *p < 0.05, ANoVA. Bars = 20 µm. www.landesbioscience.com Cell Cycle 5 mice. For mouse experiments 6–8 week old male CD-1 mice were injected with 5 x 104 trypomastigotes of the Brazil strain and parasitemia was evaluated by counting in a hemocytometer. All experiments were approved by the Institutional Animal Care Committee of the Albert Einstein College of Medicine. For in vitro experiments the Y strain was utilized. These trypomastig- otes were maintained in cultured cardiac myocytes as previously described until used.39 CD-1 mice infected with the Brazil strain had a mortality of 50% by 45 days post infection (dpi). The parasitemia peaked at 30 to 35 dpi at 7.5 x 105 trypomastigotes/ml and then waned. There was no mortality after 45 dpi. Hearts were collected at 66 The ERK 1/2 activation indicates that Cav-3 downregulation is an important contributor in the cardiac injury observed during chagasic cardiomyopathy, including the hypertrophy, inflamma- tion, fibrosis and arrhythmias, as observed in Cav-1, Cav-3 and the double knockout mice.22,36,37 These observations also suggest that the caveolins may provide a novel target for potential thera- peutic agents. Material and Methods Parasites and mice. The Brazil strain of T. cruzi was main- tained in C3H/Hej (Jackson Laboratories, Bar Harbor, ME) Figure 3. Caveolin-3 (Cav-3) in infected hearts. Hearts obtained from infected (Brazil strain) and uninfected CD-1 mice were embedded in paraffin and processed for immunohistochemistry. Sections were stained with anti-Cav-3 antibody (green) and DApI (blue) and observed by confocal microscopy. (A) Representative uninfected cardiac tissue displayed intense Cav-3 staining of cardiac myocytes, especially in pericardial area with t-tubule patterns (arrows). the inset shows higher magnification of the Cav-3 staining. (B) this staining pattern was altered in the hearts of infected mice at 66 days post infection (dpi) where areas of tissue damage were evident (*). In other areas Cav-3 staining was observed mainly in the cytoplasm of cardiac myocytes (arrowheads). Bars = 20 µm. (C) Representative immunoblot lot analysis of Cav-3 expression in cardiac tissue. Hearts of mice 66 and 180 dpi were harvested and protein lysates probed with anti-Cav-3 antibody. GApDH (36 KDa) was used as a loading control. At 66 dpi, infection induced 46% reduction in Cav-3 expression, which was restored to control levels at 180 dpi (n = 4 for each group). (*p < 0.05, ANoVA). 6 Cell Cycle Volume 9 Issue 8 Protease Inhibitor Cocktail was obtained from Roche Molecular Biochemicals (Indianapolis, IN.) Goat anti-rabbit IgG and goat anti-mouse IgG HRP-labeled antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-BIP antibodies were obtained from Affinity Bioreagents (Rockford, IL). Experiments with cardiac myocytes. Hearts were obtained from 18 day old embryos of mice and disassociated by mechani- cal and enzymatic dissociation methods using 0.05% trypsin and 0.01% collagenase in phosphate buffered saline (PBS, pH 7.2) at 37°C, following methods previously described.5 Briefly, ven- tricular heart muscle cells were plated on 0.02% gelatin-coated glass cover slips maintained in 24-well plates for immunostaining assays or on plastic dishes for electron microscopy and immuno- blotting. The cells were maintained at 37°C in 5% CO 2 atmo- sphere in DMEM supplemented with 5% FBS, 1 mM CaCl 2 , 1 mM L-glutamine, 2% chick embryo extract, 1,000 U/mL peni- cillin and streptomycin 50 µg/ml-1 (complete Eagle medium). Cardiac myocytes were infected with the Y strain trypomastig- otes at a multiplicity of infection of 10:1. Ultra-structural cytochemistry. Uninfected and T. cruzi- infected cardiac myocytes cultured in plastic culture dishes were dpi and 180 dpi. Samples were either processed for immunohis- tochemistry or quick frozen in liquid nitrogen, crushed using a mortar and pestle (Humboldt, Schiller Park, IL), resuspended in lysis buffer and sonicated for immunoblotting. Reagents and antibodies. Trypsin was obtained from Difco Laboratories (Detroit, Michigan), Type II collagenase was obtained from Worthington Biochemical Corporation (Lakewood, NJ). Fetal bovine serum (FBS), L-glutamine, penicillin, streptomycin, CaCl 2 , Dulbecco’s Modified Eagle’s Medium (DMEM), RPMI, glutaraldehyde, sodium cacodylate, osmium tetroxide,acetoneand bovine serum albumin (BSA) were obtained from Sigma-Aldrich (St. Louis, MO). Lanthanum nitrate was obtained from Merk KGaA, (Darmstadt, Germany) and Epon 812 resin from Polysciences Inc., (Warrington, PA). Anti-caveolin-3 antibody was obtained from Affinity Bioreagents (Golden, CO), polyclonal goat anti-rabbit Alexa Fluor 488 antibody was obtained from Invitrogen (Carlsbad, CA), BCA Protein Assay Reagent (bicinchoninic acid) and 4'-6-Diamidino- 2-phenylindole (DAPI) from Thermo Scientific (Rockford, IL). Rabbit polyclonal anti-phosphorylated ERK and total ERK were obtained from Cell Signaling (Beverly, MA) and the Figure 4. Trypanosoma cruzi infection induces activation of eRK 1/2 in cultured cardiac myocytes and in the myocardium of infected mice. (A) Cultured cardiac myocytes were infected with T. cruzi and blots were probed against anti-total eRK and peRK. there was no change in total eRK over a 72 hours infection. However, a progressive increase in peRK (activation) over the same period of time was observed. GApDH (36 KDa) was used as loading control in myocyte samples (n = 3 for each group). (C and D) Hearts obtained from Brazil strain infected mice (66 and 180 days post infection, dpi) were subjected to SDS-pAGe analysis and probed with anti-phosphoeRK antibody. T. cruzi infection induced a 2.3-fold increase in peRK (activation) at 66 dpi and a 68% decrease at 180 dpi as compared with age-matched uninfected controls. BIp (78 KDa) was used as loading control for heart tissue samples (n = 4 for each group) (*p < 0.05, ANoVA). www.landesbioscience.com Cell Cycle 7 10% Roche Protease Inhibitor Cocktail at 1, 2, 6, 24, 48 and 72 hours post infection). Hearts from infected CD1 mice and age-matched control mice were frozen in liquid nitrogen, resus- pended in lysis buffer and sonicated. Samples were frozen at -80°C until used, the lysates were sonicated and protein con- centration was measured using the BCA Protein Assay Reagent. Five to 10 µg of protein was loaded and resolved in 12% SDS- polyacrylamide gels. After resolving, proteins were transferred to nitrocellulose membranes (Whatman) and incubated with rabbit polyclonal anti-caveolin-3 antibody or rabbit polyclonal anti-phosphorylated ERK or total ERK diluted in TBST with 5% skim milk overnight at 4°C. For loading controls mouse anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 36 KDa) monoclonal antibody and anti-BIP antibody were used. Membranes were washed with TBST and incubated with sec- ondary goat anti-rabbit IgG and secondary goat anti-mouse IgG HRP-labeled antibody for 1 h at 25°C, followed by incubation with chemoluminescent kit ECL. Statistical analysis. Statistical analyses were performed using the ANOVA test, with the level of significance set at p < 0.05. The data represent average (±standard error of the mean) and a minimum of three independent experiments. Acknowledgements PAPES (Programa de Apoio a Pesquisa-CNPq); D.A. was sup- ported in part by a Fogarty International Training Grant D43 TW007129 (H.B.T.). This work was also supported by NIH grants AI076248 and AI052739 (H.B.T.) and HL-73732 (H.B.T. and D.C.S.) and by CNPq (M.N.M.) and FAPEMIG and PRPq- UFMG (F.S.M.). The authors thank Ms. Angela Santos and Mr. Dazhi Zhao for the excellent technical support. washed with PBS, fixed with 2.5% glutaraldehyde for 1 hour at 4°C, followed by washes first with Na-cacodylate buffer and then collidine buffer. Cells were post-fixed with 1% osmium tetrox- ide, 1.3% lanthanum nitrate in collidine buffer for 2 hours at 4°C. Cells were then dehydrated in an ascending series of acetone (30–100%) and embedded in Epon 812 resin. Ultra-thin sec- tions were picked up with 300-mesh copper grids, stained with uranyl acetate and lead citrate and examined using a Zeiss EM 10C transmission electron microscope. Immunofluorescence. Cells were washed with PBS (pH 7.2) at 72 hours post infection and fixed in 4% paraformaldehyde for 5 minutes at 20°C. After the washes cells were permeabilized with 0.05% Triton and non-specific staining was blocked with 4% BSA. Anti-Cav-3 antibody was incubated overnight 4°C, after which cells were washed and incubated with secondary polyclonal goat anti-rabbit Alexa Fluor 488 antibody for 1 hour at 37°C. DNA was stained with DAPI. For immunohistochem- istry of heart tissue sections, samples were embedded in paraf- fin, sectioned with a microtome and placed on slides. Paraffin was removed incubating slides at 60°C, then with xylene and descending ethanol series. After washes with PBS, samples were blocked with BSA and incubated with anti-caveolin-3 antibody for 72 hours at 4°C. Secondary anti-rabbit Alexa Fluor 488 anti- body was incubated for 2 hours at 20°C. Auto-fluorescence of the heart tissue was removed by incubating samples with 0.01% Evans blue solution and DNA was stained with DAPI. All slides were mounted with DABCO and images were acquired with a Zeiss 510 Meta Laser Scanning Confocal Microscope. Immunoblotting. At desired times, cells were washed three times with PBS and scraped with 300 µl of lysis buffer (2 mM PMSF, 5 mM EDTA, 1 mM Na 3 VO 4 , 1 mM NaHCO 3 , References 1. Chagas C. Nova espécie morbida do homem, pro- duzida por um Trypanosoma (Trypanosoma cruzi): Nota prévia, Brasil Médico 1909; 23:161. 2. Tanowitz HB, Machado FS, Jelicks LA, Shirani J, de Carvalho AC, Spray DC, et al. Perspectives on Trypanosoma cruzi-induced heart disease (Chagas dis- ease). Prog Cardiovasc Dis 2009; 51:524-39. 3. Huang H, Chan J, Wittner M, Jelicks LA, Morris SA, Factor SM, et al. Expression of cardiac cytokines and inducible form of nitric oxide synthase (NOS2) in Trypanosoma cruzi-infected mice. J Mol Cell Cardiol 1999; 31:75-88. 4. Machado FS, Souto JT, Rossi MA, Esper L, Tanowitz HB, Aliberti J, et al. Nitric oxide synthase-2 modulates chemokine production by Trypanosoma cruzi-infected cardiac myocytes. Microbes Infect 2008; 10:1558-66. 5. Punukollu G, Gowda RM, Khan IA, Navarro VS, Vasavada BC. Clinical aspects of the Chagas’ heart disease. Int J Cardiol 2006. 6. Meirelles MN, de Araujo-Jorge TC, Miranda CF, de Souza W, Barbosa HS. Interaction of Trypanosoma cruzi with heart muscle cells: ultra-structural and cytochemical analysis of endocytic vacuole formation and effect upon myogenesis in vitro. Eur J Cell Biol 1986; 41:198-206. 7. Aprigliano O, Masuda MO, Meirelles MN, Pereira MC, Barbosa HS, Barbosa JC. Heart muscle cells acutely infected with Trypanosoma cruzi: characteriza- tion of electrophysiology and neurotransmitter respons- es. J Mol Cell Cardiol 1993; 25:1265-74. 8. Barbosa HS, Meirelles MN. Evidence of participation of cytoskeleton of heart muscle cells during the inva- sion of Trypanosoma cruzi. Cell Struct Funct 1995; 20:275-84. 9. Garzoni LR, Masuda MO, Capella MM, Lopes AG, de Meirelles Mde N. Characterization of [Ca2+] i responses in primary cultures of mouse cardiomyocytes induced by Trypanosoma cruzi trypomastigotes. Mem Inst Oswaldo Cruz 2003; 98:487-93. 10. Taniwaki NN, Machado FS, Massensini AR, Mortara RA. Trypanosoma cruzi disrupts myofibrillar organiza- tion and intracellular calcium levels in mouse neonatal cardiomyocytes. Cell Tissue Res 2006; 324:489-96. 11. Moreno SN, Silva J, Vercesi AE, Docampo R. Cytosolic- free calcium elevation in Trypanosoma cruzi is required for cell invasion. J Exp Med 1994; 180:1535-40. 12. Rodriguez A, Rioult MG, Ora A, Andrews NW. A trypanosome-soluble factor induces IP3 formation, intracellular Ca2+ mobilization and microfilament rear- rangement in host cells. J Cell Biol 1995; 129:1263- 73. 13. Bergdolt BA, Tanowitz HB, Wittner M, Morris SA, Bilezikian JP, Moreno AP, Spray DC. Trypanosoma cruzi: effects of infection on receptor-mediated chro- notropy and Ca2+ mobilization in rat cardiac myocytes. Exp Parasitol 1994; 78:149-60. 14. de Carvalho AC, Tanowitz HB, Wittner M, Dermietzel R, Roy C, Hertzberg EL et al. Gap junction distribu- tion is altered between cardiac myocytes infected with Trypanosoma cruzi. Circ Res 1992; 70:733-42. 15. Adesse D, Garzoni LR, Huang H, Tanowitz HB, de Nazareth Meirelles M, Spray DC. Trypanosoma cruzi induces changes in cardiac connexin43 expression. Microbes Infect 2008; 10:21-8. 16. Furstenau M, Lohn M, Ried C, Luft FC, Haller H, Gollasch M. Calcium sparks in human coronary artery smooth muscle cells resolved by confocal imaging. J Hypertens 2000; 18:1215-22. 17. Nagajyothi F, Desruisseaux M, Bouzahzah B, Weiss LM, Andrade Ddos S, Factor SM, et al. Cyclin and caveolin expression in an acute model of murine Chagasic myocarditis. Cell Cycle 2006; 5:107-12. 18. Sargiacomo M, Sudol M, Tang Z, Lisanti MP. Signal transducing molecules and glycosyl-phosphatidylinosi- tol-linked proteins form a caveolin-rich insoluble com- plex in MDCK cells. J Cell Biol 1993; 122:789-807. 19. Williams TM, Lisanti MP. The Caveolin genes: from cell biology to medicine. Ann Med 2004; 36:584-95. 20. Tourkina E, Richard M, Gööz P, Bonner M, Pannu J, Harley R, et al. Antifibrotic properties of caveolin-1 scaffolding domain in vitro and in vivo. Am J Physiol Lung Cell Mol Physiol 2008; 294:843-61. 21. Song KS, Scherer PE, Tang Z, Okamoto T, Li S, Chafel M, et al. Expression of caveolin-3 in skeletal, cardiac and smooth muscle cells. Caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins. J Biol Chem 1996; 271:15160-5. 22. Woodman SE, Park DS, Cohen AW, Cheung MW, Chandra M, Shirani J, et al. Caveolin-3 knock-out mice develop a progressive cardiomyopathy and show hyperactivation of the p42/44 MAPK cascade. J Biol Chem 2002; 277:38988-97. 23. Gillespie-Brown J, Fuller SJ, Bogoyevitch MA, Cowley S, Sugden PH. The mitogen-activated protein kinase kinase MEK1 stimulates a pattern of gene expression typical of the hypertrophic phenotype in rat ventricular cardiomyocytes. J Biol Chem 1995; 270:28092-6. 8 Cell Cycle Volume 9 Issue 8 36. Park DS, Woodman SE, Schubert W, Cohen AW, Frank PG, Chandra M, et al. Caveolin-1/3 double- knockout mice are viable, but lack both muscle and non-muscle caveolae, and develop a severe cardiomyo- pathic phenotype. Am J Pathol 2002; 160:2207-17. 37. Cohen AW, Park DS, Woodman SE, Williams TM, Chandra M, Shirani J, et al. Caveolin-1 null mice develop cardiac hypertrophy with hyperactivation of p42/44 MAP kinase in cardiac fibroblasts. Am J Physiol Cell Physiol 2003; 284:457-74. 38. Augustus AS, Buchanan J, Gutman E, Rengo G, Pestell RG, Fortina P, et al. Hearts lacking caveolin-1 develop hypertrophy with normal cardiac substrate metabolism. Cell Cycle 2008; 7:2509-18. 39. Garzoni LR, Caldera A, Meirelles Mde N, de Castro SL, Docampo R, Meints GA, et al. Selective in vitro effects of the farnesyl pyrophosphate synthase inhibitor risedronate on Trypanosoma cruzi. Int J Antimicrob Agents 2004; 23273-85. 30. Langer GA, Frank JS. Lanthanum in heart cell culture. Effect on calcium exchange correlated with its localiza- tion. J Cell Biol 1972; 54:441-55. 31. Miller TW, Tormey JM. Calcium displacement by lanthanum in subcellular compartments of rat ven- tricular myocytes: characterization by electron probe microanalysis. Cardiovasc Res 1993; 27:2106-12. 32. Soeiro MN, Silva-Filho FC, Meirelles MN. The nature of anionic sites and the endocytic pathway in heart muscle cells. J Submicrosc Cytol Pathol 1994; 26:121- 30. 33. Li J, Scherl A, Medina F, Frank PG, Kitsis RN, Tanowitz HB, et al. Impaired phagocytosis in caveolin-1 deficient macrophages. Cell Cycle 2005; 4:1599-607. 34. Lohn M, Furstenau M, Sagach V, Elger M, Schulze W, Luft FC, et al. Ignition of calcium sparks in arterial and cardiac muscle through caveolae. Circ Res 2000; 87:1034-9. 35. Cohen AW, Hnasko R, Schubert W, Lisanti MP. Role of caveolae and caveolins in health and disease. Physiol Rev 2004; 84:1341-79. 24. Engelman JA, Chu C, Lin A, Jo H, Ikezu T, Okamoto T, et al. Caveolin-mediated regulation of signaling along the p42/44 MAP kinase cascade in vivo. A role for the caveolin-scaffolding domain. FEBS Lett 1998; 428:205-11. 25. Mukherjee S, Huang H, Petkova SB, Albanese C, Pestell RG, Braunstein VL, et al. Trypanosoma cruzi infection activates extracellular signal-regulated kinase in cultured endothelial and smooth muscle cells. Infect Immun 2004; 72:5274-82. 26. Huang H, Petkova SB, Cohen AW, Bouzahzah B, Chan J, Zhou J-N, et al. Activation of Transcription factors (AP-1 and NFκB) in Murine Chagasic Myocarditis. Activation of transcription factors AP-1 and NFκB in murine Chagasic myocarditis. Infect Immun 2003; 71:2859-67. 27. Tardieux I, Webster P, Ravesloot J, Boron W, Lunn JA, Heuser JE, et al. Lysosome recruitment and fusion are early events required for trypanosome invasion of mam- malian cells. Cell 1992; 71:1117-30. 28. Mott A, Lenormand G, Costales J, Fredberg JJ, Burleigh BA. Modulation of host cell mechanics by Trypanosoma cruzi. J Cell Physiol 2009; 218:315-22. 29. Pereira MC, Costa M, Chagas Filho C, de Meirelles MN. Myofibrillar breakdown and cytoskeletal alterations in heart muscle cells during invasion by Trypanosoma cruzi: immunological and ultrastructural study. J Submicrosc Cytol Pathol 1993; 25:559-69. Resultados 51 Trabalho #3 Fabiana S. Machado, Nilda E. Rodriguez, Daniel Adesse, Luciana R. Garzoni, Lisia Esper, Michael P. Lisanti, Robert D. Burk, Chris Albanese, Koenraad Van Doorslaer, Louis M. Weiss, Fnu Nagajyothi, Joshua D. Nosanchuk, Mary E. Wilson and Herbert B. Tanowitz “Recent developments in the interactions between caveolin and pathogens” Caveolins and Caveolae: Role in Signaling and Disease Mechanisms. Editado por Jasmin JF, Frank PG and Lisanti MP. Landes Bioscience. 2010 1CHAPTER RECENT DEVELOPMENTS IN THE INTERACTIONS BETWEEN CAVEOLIN AND PATHOGENS Fabiana S. Machado,*,1 Nilda E. Rodriguez,2 Daniel Adesse,3 Luciana R. Garzoni,3 Lisia Esper,1 Michael P. Lisanti,4 Robert D. Burk,5-8 Chris Albanese,9 Koenraad Van Doorslaer,6 Louis M. Weiss,10,11 Fnu Nagajyothi,10 Joshua D. Nosanchuk,10 Mary E. Wilson2 and Herbert B. Tanowitz*,10,11 1Department of Biochemistry and Immunology, Institute for Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Brazil; 2VA Medical Center, Iowa City and Department of Internal Medicine, University of Iowa, Carver College of Medicine, Iowa City, Iowa, USA; 3Laboratório de Ultra-estrutura Celular; Instituto Oswaldo Cruz/FIoCRUZ; Rio de Janeiro, Brazil; 4Departments of Stem Cell Biology and Regenerative Medicine, Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania, USA; Departments of 5Pediatrics, 6Microbiology and Immunology, 7Obstetrics and Gynecology and Woman’s Health, and 8Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, New York, USA; 9Departments of Pathology and Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA; Departments of 10Medicine and 11Pathology, Albert Einstein College of Medicine, Bronx, New York, USA *Corresponding Authors: Herbert B. Tanowitz and Fabiana S. Machado—Emails:herbert.tanowitz@einstein.yu.edu and machadofs@icb.ufmg.br Abstract: The role of caveolin and caveolae in the pathogenesis of infection has only recently been appreciated. In this chapter, we have highlighted some important new data on the role of caveolin in infections due to bacteria, viruses and fungi but with particular emphasis on the protozoan parasites Leishmania spp., Trypanosoma cruzi and Toxoplasma gondii. This is a continuing area of research and the final chapter has not been written on this topic. Caveolins and Caveolae: Roles in Signaling and Disease Mechanisms, edited by Jean-Francois Jasmin, Philippe G. Frank and Michael P. Lisanti. ©2010 Landes Bioscience and Springer Science+Business Media. © 20 10 C op yr ig ht L an de s B io sc ie nc e an d S pr in ge r. N ot f or D is tr ib ut io n 2 CAVEOLINS AND CAVEOLAE INTRODUCTION The first steps in the initiation of an infection are the attachment and entry of a pathogen into a host cell. It has long been assumed that an understanding of these initial events may result in new methods for control and treatment of infections. A role for caveolae and caveolin proteins in these processes has only recently been investigated. In this chapter, it is not our intent to review all of microbiology and describe how each and every microorganism interacts with caveolae and caveolin proteins, but rather to focus our attention on some recent important new developments. Pathogens enter mammalian cells to escape the immune system of the host and/or as part of their requirement to maintain a replicative cycle. In the past several years, this topic has been reviewed by others.1-5 These articles highlighted the role of caveolae and caveolin proteins, such as caveolin-1 (Cav-1) and lipid rafts in the entry of diverse pathogens into the host cell. As discussed in detail in other chapters in this book, Cav-1 is a critical structural protein in the formation of the flask shaped caveolae lining the plasma membrane. In addition, the role of caveolins in the pathogenesis of infection may be related to an effect on components of the immune system such as lymphocytes and macrophages.6,7 VIRUSES Caveolae are enriched in cholesterol and glycolipids, such as the glycosphingolipid GM1, glycosylphosphatidylinositol-anchored proteins and caveolin. Caveolae–mediated viral entry into human cell lines has been described and viruses that enter cells via caveolae apparently act as signaling ligands, triggering signal transduction events and actin rearrangement in the host cell, resulting in pathogen uptake (Table 1).8,9 The mechanisms by which a virus gains entry via caveolae are still not completely understood. The SV40 virus may bind to the MHC Class I complex, which recruits Cav-1 to the site of viral attachment from preformed caveolae and the associated lipid rafts are then formed around the virus. Alternatively, the SV40 virus bound to MHC may be associated with preformed caveolae. Regardless of the precise mechanism, SV40 containing caveolae pinch off from the plasma membrane and are transported to the endoplasmic reticulum via a caveosome.8 Polyoma virus, Echovirus, Respiratory Syncitial Virus (RSV) and the filoviruses (Ebola and Marburg viruses) are additional examples of viruses that are associated with Cav-1. The effects of lipid raft disrupting agents on Ebola infection indicate that membrane lipid rafts are important in the entry of filoviruses.1 The HIV-1 receptors are associated with lipid rafts in T-cells and the disruption of the integrity of these lipid rafts likely inhibits HIV infection. The blood brain barrier (BBB) integrity is maintained by tight junctions,10,11 and an intact BBB is crucial for preventing the trafficking of HIV into the brain. Early in the course of HIV infection, virus crosses the BBB via HIV-1 infected monocytes in macrophages and microglial cells in the brain. During the subsequent inflammatory response, leukocytes enter into the central nervous system through breaches in the BBB. Weiss et al 12 demonstrated that HIV‑1‑Tat protein is a powerful pro‑inflammatory agent that causes transendothelial cell migration of monocytes. Recently, Zhong et al13 demonstrated that Tat-mediated activation of Ras signaling is regulated by Cav-1 in brain endothelial cells and that © 20 10 C op yr ig ht L an de s B io sc ie nc e an d S pr in ge r. N ot f or D is tr ib ut io n 3INTERACTIONS BETWEEN CAVEOLIN AND PATHOGENS Table 1. Summary of papillomavirus entry pathways HPV Type Pathway Identified Methods Used Ref. BPV1 clathrin mediated entry, biochemical inhibitors, shuttling from endosomes co-localization studies, to caveosomes caveolinl shRNA, dominant negative cavl, 293 cells, pseudovirions 27 BPV1/HPV16 clathrin mediated entry biochemical inhibitors, co-localization studies, C127 cells, virions and VLPs 19 HPV16 clathrin/caveolae siRNA KO of clathrin, cavl, independent, lipid raft dynamin and tetraspanins, independent, dynamin biochemical inhibitors, independent, tetraspanins caveolin ‑/‑ cells, dominant involved negative inhibitors 293TT and HELA cells, pseudovirions 23 HPV16 clathrin mediated entry, biochemical inhibitors, shuttling from endosomes to co-localization studies, caveosomes caveolinl shRNA, HaCaT cells, pseudovirions 26 HPV16/HPV31 clathrin mediated entry biochemical inhibitors, (HPV16), caveolar uptake dominant negative inhibitors, (HPV31) HaCaT cells, pseudovirions 22 HPV16/HPV31 clathrin mediated entry biochemical inhibitors, 293TT and COS7 cells, pseudovirions 20 HPV31 Caveolar mediated uptake, biochemical inhibitors, Rab5 mediated shuttling to co-localization studies, endosome dominant negative Rab5, HaCaT cells, pseudovirions 28 HPV16/31 clathrin mediated entry biochemical inhibitors and 58 (HPV16 and 58) caveolar co-localization studies, COS7 uptake (HPV31) cells, VLPs and pseudovirions 18 HPV33 non-caveolar uptake biochemical inhibitors, COS7 and HELA cells, pseudovirions 21 © 20 10 C op yr ig ht L an de s B io sc ie nc e an d S pr in ge r. N ot f or D is tr ib ut io n 4 CAVEOLINS AND CAVEOLAE inhibition of Cav-1 and Ras signaling attenuates Tat-induced disruption of the tight junction proteins. Papillomaviruses (PVs) infect the mucosal and cutaneous stratified squamous epithelia. These infections are associated with both benign and malignant neoplasias and the human papillomaviruses (HPVs) cause virtually all cases of cervical cancer.14 The 8 kb, circular viral genome is encapsulated by a complex of L1 (major) and L2 (minor) structural proteins.15 Upon binding to heparan sulfate proteoglycans, the PV capsid undergoes a series of conformational changes resulting in the N-terminus of L2 becoming sensitive to cleavage by furin.16 A recent study suggested that these conformational changes may occur on the extracellular matrix prior to transfer to the cell.17 Earlier work employed chemical inhibitors and/or microscopic localization to suggest that HPV16, HPV33, HPV58 and Bovine PV1 (BPV1) use a clathrin-dependent pathway, whereas similar studies suggest the use of a caveolae-dependent pathway for HPV31.18-22 However, more recent work suggests that HPV31 may also use a clathrin-dependent pathway.20 Other authors have suggested that PVs can enter cells independent of either pathway.23 The lack of in vitro culture methods for the production of infectious virus has limited the study of PV entry. Most studies have used viral-like particles (VLPs) produced in insect cells or pseudovirions in which the L1/L2 capsid carries a reporter gene. It is possible that the use of these laboratory-made particles and nontarget cell lines could explain the observed differences between entry mechanisms. For example, it has been demonstrated that HPV16 enters dendritic cells and Langerhans cells via distinct pathways.24 The observations that a simian virus, JC virus (a polyoma virus), enters cells via clathrin-mediated endocytosis before being shuttled to caveolae-derived vesicles25 led to the identification of a similar pathway for HPV16 and BPV1.26,27 Based on recent studies, HPV16 virions first colocalize with markers of early endosomes; then, beginning at around 20 minutes post-entry, increasing amounts of the virons colocalize with Cav-1. Four hours post-entry the HPV16 virions are present in the endoplasmic reticulum. These observations strongly suggest that HPV16 and BPV1 enter the host cell via a clathrin-dependent pathway, after which they get shuttled from endosomes to caveolae. Conversely, HPV31 enters cells through the caveolar pathway followed by Rab5-dependent shuttling towards endosomes.28 Since Rab5 controls the transport from endosomes to caveosomes and vice-versa, it is tempting to speculate that both HPV16 and HPV31 could shuttle back and forth between the caveolar and endosomal pathways following entry. This hypothesis is supported by the ultrastructural observation that both HPV16 and HPV31 end up in similar looking vesicles.18 However, studies in which cells have been co-infected with HPV16 and HPV31 have not shown colocalization of both viral types.23 It is possible that the differences in entry half-time (4 hours for HPV16 vs 14 hours for HPV31) could explain these observations. Thus, it appears that HPV31 has evolved to (predominantly) enter cells through a uniquely different pathway from the other tested PV types. This is surprising since HPV16 and HPV31 are evolutionary more closely related to each other than HPV16 is to HPV58 or BPV1 (HPV16 and HPV31 share approximately 83% amino acid similarity (PAM250 matrix) across the L1 structural protein). One hypothesis is that these two highly related viruses evolved to use different entry mechanisms to avoid competition. However, the much higher prevalence of HPV16 may suggest otherwise. One key observation is that it appears that the HPV16-E5 protein up-regulates Cav-1 at the plasma-membrane of © 20 10 C op yr ig ht L an de s B io sc ie nc e an d S pr in ge r. N ot f or D is tr ib ut io n 5INTERACTIONS BETWEEN CAVEOLIN AND PATHOGENS cervical cells. Taken together, these findings suggest that caveolin proteins might play a role in PV entry, but it will be necessary to sort out discrepant results and inter-type differences in order to adequately assess their role. BACTERIA There is limited information regarding the role of caveolins in bacterial infection. However, early studies suggested that caveolae may be an important alternative pathway for endocytosis of bacteria.1,2,4,5 Chemical agents have been used to study the function of caveolae including nystatin, filipin and methyl‑b-cyclodextrin (MbCD). These agents disrupt the cholesterol enriched lipid rafts. Many pathogens require lipid rafts for entry and colocalize with markers of caveolae to invade host cells. However, only a small number of bacterial pathogens have actually been shown to require caveolin expression for host cell entry. Escherichia coli is an important cause of human infection including those of the urinary and gastrointestinal tracts. Studies involving E. coli invasion into mast and bladder epithelial cells have revealed that caveolae-dependent endocytosis is a mechanism for bacteria to invade both phagocytic and nonphagocytic cells. Importantly, intracellular bacteria can colocalize with Cav-1 and compounds that cause disruption of caveolae by removing membrane cholesterol inhibited bacterial invasion. E. coli uptake and invasion is dependent on the organization of lipid rafts and Cav-1 expression.29 Furthermore, Campylobacter jejuni, is an important cause of diarrhea world-wide whose invasion of intestinal epithelial cells is dependent on Cav-1 expression.30 In an intraperitoneal model of sepsis using lipopolysaccharide injection, Cav-1 null mice were observed to be resistant to lung injury and their mortality was reduced due to a reduction in inflammation.31 However, in a Cav-1 null mouse model of Salmonella typhimirium, an important cause of human diarrhea and systemic illness, Medina et al32 found that higher levels of proinflammatory cytokines, chemokines and nitric oxide accompanied increased bacterial burden in the spleen. Surprisingly, no differences in S. typhimurium invasion of macrophages between Cav-1 null or wild type macrophages were observed. Infections with Pseudomonas aeruginosa are observed most commonly in hospitalized immunocompromised individuals, resulting in urinary tract infections, pneumonia and sepsis. This is especially true of those individuals on mechanical ventilation and those suffering from severe burns. Also, over 80% of individuals with the genetic disorder Cystic fibrosis (CF) have pulmonary infection with P. aeruginosa.33 Individuals with CF have a defect in the transmembrane conductance regulator (CFTR).34,35 In normal individuals, infection with this bacteria stimulates the formation of lipid rafts that contain the CFTR thus allowing the organism to invade the respiratory epithelium and initiate inflammatory and apoptotic processes leading to shedding of bacteria‑containing epithelial cells.35-37 However, in CF patients these responses are absent and P. aeruginosa becomes established as a chronic infection.38 Mechanistically, knockdown of either Cav-1, which also eliminates Cav-2, or Cav-2 alone reduces the uptake of P. aeruginosa into rat bronchial epithelial cells.39 Importantly, Bajmoczi et al40 demonstrated that following entry into host cells, P. aeruginosa colocalizes with Cav-1 and CFTR. More recently, increased mortality, bacterial burden and inflammation was demonstrated in Cav-1 KO compared with wild type mice, which correlated with a decreased ability of © 20 10 C op yr ig ht L an de s B io sc ie nc e an d S pr in ge r. N ot f or D is tr ib ut io n 6 CAVEOLINS AND CAVEOLAE Cav‑1‑deficient neutrophils to phagocytose P. aeruginosa. Additionally, the colonization of P. aeruginosa was more efficient in Cav‑1 KO mice. Taken together, these preclinical observations strongly suggest that Cav-1 contributes to innate immunity to P. aeruginosa infection, which may have clinical implications for human CF patients.41 PROTOZOAN PARASITES Leishmania Depending on the species, infection with members of the genus Leishmania may result in cutaneous, mucocutaneous, or visceral leishmaniasis. These diseases are found wide-spread in tropical and sub-tropical areas of the world and are major causes of morbidity and mortality. Additionally, leishmaniasis is an opportunistic infection in patients with HIV/AIDS.42 Leishmania spp. have a life cycle consisting of two stages, the promastigote and the amastigote. The extracellular promastigote develops in the gut of the sand fly vector until it becomes a fully virulent metacyclic promastigote. Metacyclogenesis is a process during which surface molecules associated with virulence, such as lipophosphoglycan (LPG) and MSP (called GP63), are modulated in their expression and/or posttranslational modifications.43-45 Attainment of full promastigote virulence coincides with the feeding cycle of the insect vector.43-45 During a blood meal, the sand fly inoculates the parasite in the skin whereupon it is phagocytosed first by neutrophils and then by macrophages, the ultimate host cell.46 Inside the macrophage, parasites transform from promastigotes to amastigotes over two to five days. Thereafter, amastigotes are the only form found in the mammalian host. Amastigote replication leads to the release of amastigotes from infected macrophages; amastigotes in turn are taken up by non‑infected macrophages, thus spreading the infection.47,48 Leishmania enter macrophage phagosomes that ultimately fuse with lysosomes. The survival of Leishmania spp. in this hostile intracellular environment has been primarily attributed to the capacity of amastigotes to withstand the phagolysosomal compartment and Figure 1. Intracellular L. i. chagasi colocalizes with caveolin-1. Bone marrow macrophages were infected with carboxy‑fluorescein diacetate succinimidyl (CFSE) labeled L. i. chagasi promastigotes (green). Macrophage and parasite DNA was stained with TO-PRO-3 (blue). Confocal microscopy was used to assess colocalization of markers at serial time points. Caveolin-1 (red) clustered at the entry site of L. i. chagasi promastigotes and remained associated with parasites for 24 to 48 hours after infection. Shown is a picture taken at 24 hours of infection. Scale bar: 5 mm. A color version of this image is available at www.landesbioscience.com/curie. © 20 10 C op yr ig ht L an de s B io sc ie nc e an d S pr in ge r. N ot f or D is tr ib ut io n 7INTERACTIONS BETWEEN CAVEOLIN AND PATHOGENS to the ability of the parasite to down-modulate macrophage activation.49-54 Accumulating evidence demonstrates that different external stimuli induce distinctive types of macrophage activation with divergent pro and/or anti‑inflammatory profiles.55-57 L. infantum chagasi is a cause of visceral leishmaniasis. Infection of BALB/c mouse macrophages with L.i chagasi Figure 2. Caveolin-3 (Cav-3) expression is diminished after Trypanosoma cruzi infection: Cardiac myocytes were isolated from mouse embryos and infected with trypomastigotes. Confocal microscopy showed that uninfected cultures displayed abundant Cav-3 immunoreactivity (A), including peripheral staining. Cav-3 signal was reduced among highly parasitized myocytes (B) and was present predominantly at the cell periphery. DNA staining by DAPI permitted visualization of host cell nucleus and the amastigote kinetoplastid DNA. Bars = 20 mm. Reproduced with permission from: Adesse D et al. Cell Cycle 2010; 9:1639‑164.81 © 20 10 C op yr ig ht L an de s B io sc ie nc e an d S pr in ge r. N ot f or D is tr ib ut io n 8 CAVEOLINS AND CAVEOLAE promastigotes initiates a pattern of gene expression that is neither classically activated nor alternatively activated, but which demonstrates a novel type of macrophage activation characterized by an anti‑inflammatory profile and an increase in the caveolae‑related molecules dynamin-2, Cav-1 and Cav-3.58 This increase in caveolae components upon L. i. chagasi infection suggests the presence of a feedback mechanism that may be triggered by the depletion of surface caveolae upon parasite uptake. Employing confocal microscopy, virulent L. i. chagasi promastigotes were found to colocalize with the caveolae markers GM1 and Cav-1 both during entry and up to 24 hours after murine macrophage infection (Fig. 1). Entry of promastigotes via Cav-1 correlated with a delay in lysosome fusion of approximately 24 to 48 hours; a time coinciding with their promastigote-to-amastigote conversion.48,59 Co-localization of promastigotes with Cav-1 also correlated with increased parasite survival. In contrast, serum opsonization of attenuated (avirulent) L. i. chagasi promastigotes precluded parasites to enter macrophages through lipid rafts/caveolae. Unlike virulent L. i. chagasi, avirulent parasites entered compartments that fused early with lysosomes usually within the initial three hours of infection and failed to survive in macrophages. Because caveolae are enriched in cholesterol, transient treatment of macrophages with the cholesterol chelating agent MbCD was used to investigate the role of lipid rafts/caveolae in Leishmania infection. MbCD treatment does not affect macrophage viability if the cell membrane cholesterol is extracted while preserving the intracellular cholesterol pools.59-61 Under these conditions, pretreatment with MbCD significantly impaired parasite entry and inhibited replication for up to 72 hours after phagocytosis, even though surface cholesterol was restored by 4 hours after treatment. Furthermore, macrophage pretreatment with MbCD accelerated the rate of lysosome fusion and led to rapid intracellular killing. Side by side comparisons showed that transient disruption of lipid rafts/caveolae caused virulent metacyclic L. i. chagasi to enter macrophages through a phagocytic pathway, leading to early lysosome fusion and intracellular death, resembling the entry of attenuated parasites.59 Surface LPG and MSP on metacyclic promastigotes are able to bind and inactivate the serum protein C3b to C3bi, facilitating parasite uptake through the macrophage receptor CR3, which localizes in caveolae.62-65 A comparison of the receptors used by avirulent log phase versus virulent metacyclic L. i. chagasi in human macrophages demonstrated that metacyclic parasites preferentially enter through CR3 but not the mannose receptor and that the metacyclic parasites colocalize with Cav-1 during the initial hour of infection. In contrast, log-phase parasites ligated both the mannose receptor and CR3 and failed to colocalize with Cav-1. The ability of metacyclic, but not log phase promastigotes to associate with CR3 and Cav-1 also correlated with a slower kinetics of lysosome fusion and increased parasite survival.66 The amastigote form of the parasite expresses low levels of MSP and no LPG; the latter of which inhibits lysosome fusion with promastigotes. Amastigotes can survive in the phagolysosomes and it has been suggested that the unique nutritional requirements of the amastigote can only be met in this degradative compartment.67-69 As such, amastigotes might not benefit from entering macrophages through a cholesterol‑rich/caveolae uptake mechanism that delays lysosome fusion. Indeed, side by side experiments with metacyclic promastigotes and hamster-derived amastigotes of L. i. chagasi supported this hypothesis. Transient depletion of cholesterol from BALB/c mouse macrophages did not affect the entry of amastigotes or their kinetics of lysosome fusion (submitted manuscript, Rodriguez, Gaur, Allen and Wilson), suggesting that the entry and survival of © 20 10 C op yr ig ht L an de s B io sc ie nc e an d S pr in ge r. N ot f or D is tr ib ut io n 9INTERACTIONS BETWEEN CAVEOLIN AND PATHOGENS amastigotes is independent of cholesterol-rich microdomains including caveolae. Taken together, these results suggest that virulent, but not attenuated or log phase promastigotes, are able to exploit a caveolae-mediated pathway to facilitate their entry and intracellular survival by a mechanism that includes delayed lysosome fusion until their conversion into amastigotes and the establishment of infection. Trypanosoma cruzi Trypanosoma cruzi causes Chagas disease. It is an important cause of acute myocarditis, chronic cardiomyopathy and gastrointestinal disorders in endemic areas.70 Chagas disease is found in endemic areas of Mexico, Central and South America. In recent years, there has been an increased recognition of Chagas disease among immigrants from endemic areas into North America and Europe. Chagas disease is also an opportunistic infection in patients with HIV/AIDS.71-73 The parasite has a complex life cycle. During a blood meal from an infected mammalian host, the insect vector ingests blood-form trypomastigotes, which undergo transformations and after 3 to 4 weeks, infective, nondividing metacyclic trypomastigotes are present in the hindgut of the vector and are deposited with the feces of the vector during subsequent blood meals. Transmission to the new host occurs when the parasite-laden feces contaminate oral or nasal mucous membranes, the conjunctivas, or other vulnerable surfaces. Other modes of transmission include blood transfusion, organ donation, congenital, breast milk, ingestion of contaminated food or drink and laboratory accident. When the trypomastigotes enter a host cell they transform into amastigotes where they multiply by binary fission and again transform to trypomastigotes. Trypomastigotes are released as the host cell ruptures and disseminate through the lymphatics and the bloodstream to find new cells to invade. The precise mechanism(s) by which the parasite enters the host cell is not entirely understood, however several receptors have been implicated in this process. Although any nucleated mammalian cell can be parasitized, those of the cardiovascular system, including cardiac myocytes, cardiac fibroblasts, endothelial cells and vascular smooth muscle cells, as well as cells of the reticuloendothelial, nervous and muscle systems and adipose tissue, appear to be favored. T. cruzi infection is characterized by an intense inflammatory reaction accompanied by an upregulation of cytokines and chemokines.73,74 Pathological examination of the cardiovascular system in both human samples and experimental acute chagasic myocarditis reveals inflammation, myonecrosis, vasculitis and numerous parasite pseudocysts. In chronic chagasic cardiomyopathy, inflammation, fibrosis, myocytolysis and vasculitis persist but there are few parasites in the infected tissues. In many chronically infected individuals there is a dilated cardiomyopathy. In order to understand the mechanisms involved in progression of disease and the development of the resulting cardiomyopathy, many groups have focused their research either on the in vitro infection of cardiac cells with T. cruzi or through the use of murine models of infection. Caveolin is a negative regulator of extracellular signal-regulated kinases (ERK) and cyclin D1.75 Thus, a reduction in the expression of Cav-1 and Cav-3 generally results in the upregulation of ERK activity and an induction of cyclin D1 expression, which contribute to cardiac myocyte hypertrophy and ultimately cardiomyopathy. Interestingly, Cav-1 and Cav-3 null mice as well as the Cav-1/Cav-3 double null mice display a cardiomyopathic phenotype associated with cardiac myocyte hypertrophy and interstitial fibrosis.76-78 Thus, © 20 10 C op yr ig ht L an de s B io sc ie nc e an d S pr in ge r. N ot f or D is tr ib ut io n 10 CAVEOLINS AND CAVEOLAE it was of great interest that during the acute phase of T. cruzi infection a reduction in the expression of Cav-1, Cav-2 and Cav-3 was observed. This was accompanied by activation of ERK, activator protein 1 (AP-1), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and increased cyclin D1 expression.79,80 The change in Cav-1 expression in infected mice was the result in part of infection of the cardiac fibroblasts since cardiac myocytes do not express Cav-1. At 60 days post-infection, which is considered the sub-acute/chronic phase, there was a reduction in Cav-3 expression which normalized by day 180 post-infection,81 though the increase in the expression of ERK persisted. Thus, it would appear that the initial reduction in Cav-3 expression may trigger an increase in ERK, leading to cardiac myocyte hypertrophy. Chagas disease is also a vasculopathy and in carotid arteries obtained from T. cruzi infected mice there was a reduction in the expression of Cav-1 and Cav-3 and activation of ERK, cyclin D1 and endothelin-1(ET-1).82 These findings may in part explain the vasoconstriction observed as a result of this infection. T. cruzi infection also results in a reduction of Cav-1 and Cav-2 expression and an increase in activated ERK, cyclin D1 and ET-1.82,83 In addition, infection of cardiac myocytes results in a reduction of Cav-3 expression and a concomitant increase in activated ERK (Fig. 2).81 Taken together T. cruzi infection results in cardiomyopathy and a vasculopathy in which caveolin, ERK, cyclin D1 and ET-1 contribute to cardiovascular remodeling and the pathogenesis of chagasic heart disease. Employing the highly virulent Tulahuen strain of T. cruzi infection, Medina et al84 infected both Cav-1 wild type and Cav-1 null mice and while the resulting mortality was 100% in both genetic backgrounds, death was slightly delayed in the wild type mice. The parasitemia in the Cav‑1 null mice was significantly reduced compared with wild type mice. In both groups there were numerous pseudocysts, myonecrosis and marked inflammation. Interestingly, infection of cultured cardiac fibroblasts obtained from Cav‑1 null and wild type mice revealed no differences in infectivity. Determination of serum levels of several inflammatory mediators revealed a significant reduction in IFN‑g, TNF-a and components of the nitric oxide pathway in infected Cav-1 null mice, while infection of wild type mice resulted in an increase in these inflammatory mediators. The defective production of chemokines and cytokines observed in vivo is, in part, attributed to Cav-1 null macrophages. These results suggest that Cav-1 may play an important role in the normal development of immune responses. Recently, Barrias et al demonstrated that infection of mouse peritoneal macrophages with the Y strain of T. cruzi was impaired when cells were treated with MbCD.85 The contributions of caveolin in Chagas disease are not entirely understood and investigators are continuing to explore these interactions. Toxoplasma gondii Toxoplasma gondii is a ubiquitous Apicomplexan obligate intracellular protozoan parasite of mammals and birds. It has long been recognized as being an important cause of congenital infection with chorioretinitis and central nervous system manifestations and has also emerged as an opportunistic pathogen in immune compromised hosts where it primarily causes encephalitis.86-88 Although overwhelming disseminated toxoplasmosis has been reported, the predilection of this parasite for the central nervous system causing necrotizing encephalitis and the eye causing chorioretinitis constitutes its major threat to patients. The development of these diseases is a consequence of the transition of the resting or latent bradyzoite stage to the active rapidly replicating form, the tachyzoite © 20 10 C op yr ig ht L an de s B io sc ie nc e an d S pr in ge r. N ot f or D is tr ib ut io n 11INTERACTIONS BETWEEN CAVEOLIN AND PATHOGENS stage.86-87,89-93 It is likely that in chronic toxoplasmosis tissue cysts (bradyzoites) regularly transform to tachyzoites and that these active forms are removed or sequestered by the immune system, while some invade host cells differentiating to new tissue cysts.86,89,90,93 T. gondii replicates within a parasitophorous vacuole, isolated from host vesicular traffic.94 T. gondii are capable of actively invading host cells95,96 and these invasion processes require parasite motility, orientation toward the host cell and sequential discharge of three secretory organelles termed micronemes, rhoptries and dense granules.97,98 Host cell cholesterol is required for entry and intracellular replication of this and other pathogens.99-102 Central roles for cholesterol are suggested at the attachment, penetration and intracellular multiplication stages. Depletion of membrane cholesterol leads to a loss of invaginated caveolae.103 The predominant sterol in T. gondii membranes is cholesterol and it has been unequivocally demonstrated that this parasite is auxotrophic for this major lipid.101 T. gondii actively intercepts low-density lipoprotein (LDL)-derived cholesterol that has transited through host lysosomes by a yet undefined mechanism. Cholesterol trafficking from mammalian lysosomes to intravacuolar T. gondii requires functional host Niemann–Pick type cholesterol proteins (NPC), which are known to mediate cholesterol egress across the endosomal (mainly NPC1) and lysosomal (mainly NPC2) membranes,104 and trafficking is independent of pathways involving the host Golgi and endoplasmic reticulum.101 The uncoupling between LDL uptake and cholesterol biosynthesis occurring during T. gondii infection101 leads to the assumption that these pathways are dramatically perturbed in infected cells. Caveolae/caveolins have been proposed to function in a number of cholesterol- trafficking steps. These steps include selective cholesterol uptake from HDL via the scavenger receptor SR‑B1 located in caveolae (cholesterol influx) and the delivery of newly synthesized cholesterol from the endoplasmic reticulum membrane caveolae, where it is delivered to HDL (cholesterol efflux).105 However, studies have shown that high levels of host caveolae vesicles are not needed for trafficking of LDL‑derived lysosomal cholesterol to Toxoplasma in mammalian cells.101 It has been suggested that a probable scenario during T. gondii infection is that following LDL receptor-mediated endocytosis, cholesterol is liberated from LDL cholesteryl ester in early hydrolytic compartments containing the enzyme acid lipase. The cholesterol then effluxes from the NPC‑containing late endosome/lysosome before trafficking to the parasitophorous vacuole. The post-endolysosomal movement of cholesterol to the parasitophorous vacuole is blocked by inhibitors of vesicular transport but does not require vesicle fusion or host endolysosome fusion with the parasitophorous vacuole. Transit to the parasitophorous vacuole is direct, rather than across host organelles, e.g., endoplasmic reticulum, Golgi101,106 or through the host plasma membranes.107 However, the parasitophorous vacuole is accessible to sterol acceptors in the medium by an unknown mechanism. Neither the host sterol carriers SCP-2 nor caveolins are involved and the process is independent of vimentin intermediate filaments. Host cholesterol is delivered to the parasitophorous vacuole via lipid extractor-or transporter-like proteins on the parasitophorous vacuole membrane, then trafficked within the vacuolar space, perhaps in association with the tubulo-vesicular network (TVN) secreted by T. gondii before internalization into the parasite interior via parasite plasma membrane proteins and storage as cholesteryl esters in lipid bodies. Rhoptries are elongated club-shaped organelles containing a densely packed granular material in their basal bulbous portion and are related to secretory lysosomes or exosomes.108 T. gondii rhoptries are formed via the endocytic pathway109 and contribute to the formation © 20 10 C op yr ig ht L an de s B io sc ie nc e an d S pr in ge r. N ot f or D is tr ib ut io n 12 CAVEOLINS AND CAVEOLAE of the Toxoplasma and Plasmodium parasitophorous vacuole membranes (PVMs) by releasing their contents from the anterior end of the parasite during invasion.98,110,111 Toxoplasma rhoptries also contain lipids, including large amounts of cholesterol and phosphatidylcholine.112,113 In these organelles, the cholesterol/phospholipid molar ratio (1.5/1) is too high for lipid bilayer stability, suggesting that some rhoptry cholesterol molecules may be organized in a crystalline array inside the organelle. Although it is plausible that rhoptry cholesterol is incorporated into the PVM during invasion and that rhoptry discharge can effectively compensate for the absence of caveolae in host cells, this possibility requires testing. The biogenesis of the PVM surrounding Toxoplasma is only partially understood. Capacitance measurements in patch-clamped host cells indicate that at least 80% of the membrane in the nascent vacuolar membrane is host cell derived.114 This is consistent with observations showing that fluorescent tracers inserted into the host plasma membrane before infection were incorporated into the nascent PVM of Toxoplasma110,115,116 as well as into the Plasmodium PVM.117 Additionally, multiple lines of evidence suggest that rhoptry contents contribute directly to formation of the vacuolar membrane.109,110,118-120 These apparently conflicting observations can be resolved by postulating a two‑step process of invasion. Initial discharge of the rhoptry contents directly into the host cytoplasm leads to coalescence of multivesicular structures, which then fuse with the nascent vacuole that is derived primarily from the host cell plasma membrane.121 Whether and how cholesterol contributes to this unusual process of cell invasion by T. gondii is not clear. Studies have evaluated the ability of cholesterol-depleted parasites to invade normal cells, of cholesterol-depleted cells to be invaded by untreated parasites, as well as the ability of normal parasites to invade caveolin-minus cells. The results demonstrate that T. gondii is dependent upon host plasma membrane cholesterol to trigger organelle discharge. Neither rhoptry-derived cholesterol nor caveolae microdomains in the host cell plasma membrane are required to complete invasion. These results identify a heretofore unexpected mechanism by which cholesterol regulates microbial entry into mammalian cells. Lisanti and colleagues122,123 discovered that purified caveolae microdomains contained an abundance of signaling molecules, such as Src-like tyrosine kinases and heterotrimeric G proteins and proposed that Cav-1 and caveolae may serve as docking points for numerous cell surface receptors, which, when activated by ligand binding, are recruited to caveolae.122,123 It has been demonstrated that GTPase activity of G protein-subunits could be suppressed by a peptide derived from the NH2 terminus of Cav-1 termed the caveolin scaffolding domain, demonstrating the interdependence of these proteins for functional activity.124 Finally, studies have shown that interaction of the T. gondii-derived molecule cyclophillin-18 (C-18) with a chemokine receptor (CCR5) on dendritic cells is critical for IL‑12 (a pro‑inflammatory mediator) induction in vivo and the subsequent control of parasite growth.125 Since CCR5 is associated with G-protein-subunits, it is interesting to consider the possible participation of caveolin in the signaling pathways and transcriptional events downstream of CCR5 triggered by T. gondii. © 20 10 C op yr ig ht L an de s B io sc ie nc e an d S pr in ge r. N ot f or D is tr ib ut io n 13INTERACTIONS BETWEEN CAVEOLIN AND PATHOGENS FUNGI Although significant efforts have led to an appreciation that sterol‑rich membrane domains significantly impact the function of fungal membranes,126 there is limited information regarding the role of lipid rafts and caveolae in the pathogenesis of fungal disease in mammalian hosts. The most in depth analysis of a fungal interaction with cellular membrane rafts comes from a study on Paracoccidioides brasiliensis, a dimorphic fungus endemic in the environment in Central and South America.125 This fungus causes a spectrum of human disease ranging from mild localized infection to disseminated deep lesions, characterized by granulomatous inflammation. P. brasiliensis is frequently acquired via inhalation. Using epithelial alveolar cells, Maza et al127 demonstrated that host cell lipid rafts are critical for engaging P. brasiliensis cells. They demonstrated that ganglioside GM1 appeared to be recruited to the point of adhesion of P. brasiliensis to this host cell. Furthermore, pretreatment of the alveolar cells with either MbCD to deplete host cell cholesterol, or nystatin, to bind host cholesterol, significantly reduced fungal adhesion. Pneumocystis species are frequent pulmonary pathogens in hosts with compromised cellular immune responses, especially individuals with AIDS and the fungus can intimately engage type I alveolar epithelial cells.128 Although Pneumocystis associates with cells, the fungus is an extracellular pathogen. Nevertheless, the fungus interdigitates with the alveolar cells and activates caveolae of the host cells, resulting in the close proximity of plasmalamellar vesicles in the areas of contact. Presumably, this allows the fungus to parasitize the host cells for nutrients. Beta-glucans are major components of many fungal cell walls including that of Candida albicans, which is the most prevalent cause of systemic mycoses. Beta-glucans from C. albicans can interact with very long fatty acid-containing lactosylceramide lipid rafts on the plasma membrane of human neutrophils, inducing migration of the neutrophil toward the fungus129,130 and, presumably, enhancing phagocytosis of the yeast. Histoplasma capsulatum, a dimorphic fungus endemic to the Mississippi and Ohio River Valleys of the USA and in regions within Central and South America, causes pulmonary and disseminated infections, especially in individuals with compromised immunity. Calcium binding protein (CBP) is a well known virulence factor for the fungus that recently was shown to have structural homology with mammalian saposin B.131 Hence, it has been proposed that CBP may interact with host glycolipids, including those present in caveolae. This finding underscores the probable broad and yet undiscovered import of caveolae in interactions with pathogenic fungi. Future work will continue to elucidate the functional importance of caveolae interactions with fungi during adhesion, internalization and internal processing. CONCLUSION A growing list of pathogens, including viruses, bacteria and their associated toxins, fungi and even prions, can interact with caveolae membrane domains.2 The intracellular trafficking of these agents via caveolae differs dramatically from the usual route of ligands internalized by clathrin-mediated endocytosis. The use of caveolae for cellular entry allows the pathogen to avoid classical endosome‑lysosome trafficking and, consequently, avoid degradative compartments within the cell. © 20 10 C op yr ig ht L an de s B io sc ie nc e an d S pr in ge r. N ot f or D is tr ib ut io n 14 CAVEOLINS AND CAVEOLAE ACKNOWLEDGEMENTS The work was supported by CNPq and FAPEMIG (FSM), NIH grants AI- 076248 (HBT) NIH Grants AI39454 (LMW) and AI045540 (MEW and Veterans Affairs Merit Review (MEW). Daniel Adesse was supported in part by a Fogarty Training Grant D43 TW007129 (HBT). This work was also supported in part by a Career Developmental Award (CDA-2) from the Departments of Veterans Affairs (NER). REFERENCES 1. Duncan MJ, Shin J-S, Abraham SN. Microbial entry through caveolae: variations on a theme. Cellular Microbiol 2002; 4:783‑781. 2. Cohen AW, Hnasko R, Schubert W et al. Role of caveolae and caveolins in health and disease. Physiol Rev 2004; 84:1341‑1379. 3. Suzuki T, Suzuki Y. Viral infection and lipid rafts. Biol Pharmacol Bull 2006; 29:1538‑1541. 4. Zaas D, Swan Z, Brown BJ et al. The expanding role of caveolin proteins in microbial pathogenesis. Communicative & Integrative Biology 2009; 2:535‑537. 5. Shin JS, Abraham SN. Caveolae as portals of entry for microbes. 2001; Microbes Inf 3:755‑761. 6. Li J, Scherl A, Medina F et al. Impaired phagocytosis in Caveolin‑1 Deficient Macrophages. Cell Cycle 2005; 4:1599‑607. 7. Medina FA, Williams TM, Sotgia F et al. A novel role for caveolin-1 in B lymphocyte function and the development of thymus‑independent immune responses. Cell Cycle 2006; 5:1865‑1871. 8. Pelkmans L, Kartenbeck J, Helenius A. Caveolar endocytosis of simian virus 40 reveals a new two-step vesiculartransport pathway to the ER. Nat Cell Biol 2001; 3:473‑483. 9. Norkin LC, Anderson HA, Wolfrom SA et al. Caveolar endocytosis of simian virus 40 is followed by brefeldin A-sensitive transport to the endoplasmic reticulum, where the virus disassembles. J Virol 2002; 76:5156‑5166. 10. Hawkins BT, Davis TP. The blood-brain barrier/neurovascular unit in health and disease. Pharmacol Rev 2005; 57:173‑185. 11. Abbott NJ, Rönnbäck L, Hansson E. Astrocyte-endothelial interactions at the blood-brain barrier. Nat Rv Neurosci 2006; 7:41‑53. 12. Weiss JM, Nath A, Major EO et al. HIV-1 Tat induces monocyte chemoattractant protein-1 mediated monocyte trans migration across a model of the human blood brain barrier and upregulates CCR5 expression on human monocytes. J Immunol 1999; 163:2953‑2959. 13. Zhong Y, Smaet EJ, Weksler B et al. Caveolin-1 regulates HIV-1 Tat–induced alterations of tight junction protein expression via modulation of the RAS signaling. J Neurosci 2008; 28:7788‑7796. 14. Munoz N, Castellsague X, de Gonzalez AB et al. Chapter 1: HPV in the etiology of human cancer. Vaccine 2006; 24S3:S1‑S10. 15. Baker TS, Newcomb WW, Olson NH et al. Structures of bovine and human papillomaviruses. Analysis by cryoelectron microscopy and three‑dimensional image reconstruction. Biophys J 1991; 60:1445-1456. 16. Richards RM, Lowy DR, Schiller JT et al. Cleavage of the papillomavirus minor capsid protein, L2, at a furin consensus site is necessary for infection. Proc Natl Acad Sci U S A 2006; 103:1522‑1527. 17. Kines RC, Thompson CD, Lowy DR et al. The initial steps leading to papillomavirus infection occur on the basement membrane prior to cell surface binding. Proc Natl Acad Sci U S A 2009; 106:20458‑20463. 18. Bousarghin L, Touze A, Sizaret PY et al. Human papillomavirus types 16, 31 and 58 use different endocytosis pathways to enter cells. J Virol 2003; 77:3846‑3850. 19. Day PM, Lowy DR, Schiller JT. Papillomaviruses infect cells via a clathrin-dependent pathway. Virology 2003; 307:1‑11. 20. Hindmarsh PL, Laimins LA. Mechanisms regulating expression of the HPV 31 L1 and L2 capsid proteins and pseudovirion entry. Virol J 2007; 4:19. 21. Selinka HC, Giroglou T, Sapp M. Analysis of the infectious entry pathway of human papillomavirus type 33 pseudovirions. Virology 2002; 299:279‑287. 22. Smith J L, Campos SK, Ozbun MA. Human papillomavirus type 31 uses a caveolin 1- and dynamin 2‑mediated entry pathway for infection of human keratinocytes. J Virol 2007; 81:9922‑9931. 23. Spoden G, Freitag K, Husmann et al. Clathrin- and caveolin-interdependent entry of human papillomavirus type16‑ involvement of tetraspanin‑enriched microdomains (TEMs). Plos One 2008; 3:e3313. © 20 10 C op yr ig ht L an de s B io sc ie nc e an d S pr in ge r. N ot f or D is tr ib ut io n 15INTERACTIONS BETWEEN CAVEOLIN AND PATHOGENS 24. Yan M, Peng J, Jabbar IA et al. Despitedifferences between dendritic and Langerhans cells in the mechanism of papillomavirus‑ like particle antigen update, both cells cross‑prime T‑cells. Virology 2004; 324:297‑310. 25. Querbes W, O’Hara BA, Williams G et al. Invasion of host cells by JC virus identifies a novel role for caveolae in endosomal sorting of noncaveolar ligands. J Virol 2006; 80:9402‑9413. 26. Laniosz V, Dabydeen SA, Havens MA et al. Human papillomavirus type 16 infection of human keratinocytes requires clathrin and caveolin‑1 and is brefeldin a sensitive. J Virol 2009; 83:8221‑32. 27. Laniosz V, Holthusen KA, Meneses PI. Bovine papillomavirus type 1: from clathrin to caveolin. J Virol 2008; 82:6288‑6298. 28. Smith JL, Campos SK, Wandinger-Ness A. Ozbun. Caveolin-1-dependent infectious entry of human papillomavirus type 31 in human keratinocytes proceeds to the endosomal pathway for pH-dependent uncoating. J Virol 2008; 82:9505‑9512. 29. Duncan MJ, Li G, Shin JS et al. Bacterial penetration of bladder epithelium through lipid rafts. J Biol Chem 2004; 279:18944‑18951. 30. Watson RO, Galán JE. Campylobacter jejuni survives within epithelial cells by avoiding delivery to lysosomes. PLoS Pathog 2008; 4:e14. 31. Garrean S, Gao XP, Brovkovych V et al. Caveolin‑1 regulates NF‑kappaB activation and lung inflammatory response to sepsis induced by lipopolysaccharide. J Immunol 2006; 177:4853‑4860. 32. Medina FA, de Almeida CJ, Dew E et al. Caveolin‑1‑deficient mice show defects in innate immunity and inflammatory immune response during Salmonella enterica serovar Typhimurium infection. Infect Immun 2006; 74:6665‑6674. 33. Lyczak JB, cannon CL, Pier GB et al. Lung infections associated with cystic fibrosis. Clin Microbiol rev 2002; 15:194‑222. 34. Zeitlin PI. Pseudomonas aeruginosa: can studies in engineered cells tell us why is it such a problem in people with cystic fibrosis? Focus on “Cystic fibrosis transmembrane conductance regulator and caveolin‑1 regulate epithelial cell internalization of Pseudomonas aeruginosa” Am J Physiol Cell Physiol 2009; 297:C235-C237. 35. Kowalski MP, Pier G. Localization of cystic fibrosis transmembrane conductance regulator to lipid rafts of epithelial cells is required for Pseudomonas aeruginosa‑induced cellular activation. J Immunol 2004; 172:418-425. 36. Grassmé H, Becker KA, Zhang Y et al. Ceramide in bacterial infections and cystic fibrosis. Biol Chem 2008; 389:1371‑1379. 37. Grassmé H, Becker KA, Zhang Y et al. Ceramide in Pseudomonas aeruginosa infections and cystic fibrosis. Cell Physiol Biochem 2010; 26:57‑66. 38. Pier GB, Grout M, Zaidi TS. Cystic fibrosis transmembrane conductance regulator is an epithelial cell receptor for clearance of Pseudomonas aeruginosa from the lung. Proc Natl Acad Sci U S A 1997; 94:12088-12093. 39. Abraham SN, Duncan MJ, LI G et al. Bacterial penetration of the mucosal barrier by targeting lipid rafts. J Investig Med 2005; 53:318‑321. 40. Bajmoczi M, Gadjeva M, Alper SL et al. Cystic fibrosis transmembrane conductance regulator and caveolin-1 regulate epithelial cell internalization of Pseudomonas aeruginosa. Am J Physiol Cell Physiol 2009; 297:C263‑277. 41. Gadjeva M, Paradis‑Bleau C, Priebe GP et al. Caveolin‑1 modifies the immunity to Pseudomonas aeruginosa. J Immunol 2010; 184:296‑302. 42. Alvar J, Aparicio P, Aseffa A et al. The relationship between Leishmaniasis and AIDS: the second 10 years. Clin microbial Rev 2008; 21:334‑359. 43. McConville M J, Turco SJ, Ferguson MA et al. Developmental modification of lipophosphoglycan during the differentiation of Leishmania major promastigotes to an infectious stage. EMBO J 1992; 11:3593-3600. 44. Sacks DL, Pimenta PF, McConville MJ et al. Stage‑specific binding of Leishmania donovani to the sand fly vector midgut is regulated by conformational changes in the abundant surface lipophosphoglycan. J Exp Med 1995; 181:685‑697. 45. Yao C, Chen Y, Sudan B et al. Leishmania chagasi: homogenous metacyclic promastigotes isolated by buoyant density are highly virulent in a mouse model. Exp Parasitol 2008; 118:129‑133. 46. Bogdan C, Rollinghoff M. How do protozoan parasites survive inside macrophages? Parasitol Today 1999; 15:22‑28. 47. Galvao‑Quintao L, Alfieri SC, Ryter A. Intracellular differentiation of Leishmania amazonensis promastigotes to amastigotes: presence of megasomes, cysteine proteinase activity and susceptibility to leucine‑methyl ester. Parasitology 1990; 101 Pt 1:7‑13. 48. Wilson M E, Innes DJ, Sousa AD. Early histopathology of experimental infection with Leishmania donovani in hamsters. J Parasitol 1987; 73:55‑63. © 20 10 C op yr ig ht L an de s B io sc ie nc e an d S pr in ge r. N ot f or D is tr ib ut io n 16 CAVEOLINS AND CAVEOLAE 49. Barral A, Barral-Netto M, Yong EC et al. Transforming growth factor b as a virulence mechanism for Leishmania braziliensis. Proc Natl Acad Sci U S A 1993; 90:3442‑3446. 50. Channon JY, Roberts MB, Blackwell JM. A study of the differential respiratory burst activity elicited by promastigotes and amastigotes of Leishmania donovani in murine resident peritoneal macrophages. Immunology 1984; 53:345‑355. 51. Gantt KR, Schultz-Cherry S, Rodriguez N et al. Activation of TGF-b by Leishmania chagasi: importance for parasite survival in macrophages. J Immunol 2003; 70:2613‑2620. 52. Meier CL, Svensson M, Kaye PM. Leishmania-induced inhibition of macrophage antigen presentation analyzed at the single‑cell level. J Immunol 2003; 171:6706‑6713. 53. Nandan D, Lo R, Reiner NE. Activation of phosphotyrosine phosphatase activity attenuates mitogen-activated protein kinase signaling and inhibits c-FOS and nitric oxide synthase expression in macrophages infected with Leishmania donovani. Infect Immun 1999; 67:4055‑4063. 54. Pearson RD, Harcus JL, Roberts D et al. Differential survival of Leishmania donovani amastigotes in human monocytes. J Immunol 1983; 131:1994‑1999. 55. Gordon S. Alternative activation of macrophages. Nat Rev Immunol 2003; 3:23‑35. 56. Martinez FO, Sica A, Mantovani A et al. Macrophage activation and polarization. Front Biosci 2008; 13:453-461. 57. Mosser DM, Edwards JP. Exploring the full spectrum of macrophage activation. Nat Rev Immunol 2008; 8:958-969. 58. Rodriguez NE, Chang HK, Wilson ME. Novel program of macrophage gene expression induced by phagocytosis of Leishmania chagasi. Infect Immun 2004; 72:2111‑2122. 59. Rodriguez NE, Gaur U, Wilson ME. Role of caveolae in Leishmania chagasi phagocytosis and intracellular survival in macrophages. Cell Microbiol 2006; 8:1106‑1120. 60. Naroeni A, Porte F. Role of cholesterol and the ganglioside GM(1) in entry and short-term survival of Brucella suis in murine macrophages. Infect Immun 2002; 70:1640‑1644. 61. Rohde M, Muller E, Chhatwal GS et al. Host cell caveolae act as an entry-port for group A streptococci. Cell Microbiol 2003; 5:323‑342. 62. Brittingham A, Chen G, McGwire BS et al. Interaction of Leishmania gp63 with cellular receptors for fibronectin. Infect Immun 1999; 67:4477‑4484. 63. Harris J, Werling D, Hope JC et al. Caveolae and caveolin in immune cells: distribution and functions. Trends Immunol 2002; 23:158‑164. 64. Wilson ME, Pearson RD. Roles of CR3 and mannose receptors in the attachment and ingestion of Leishmania donovani by human mononuclear phagocytes. Infect Immun 1988; 56:363‑369. 65. Wozencraft AO, Blackwell JM. Increased infectivity of stationary-phase promastigotes of Leishmania donovani: correlation with enhanced C3 binding capacity and CR3-mediated attachment to host macrophages. Immunology 1987; 60:559‑563. 66. Ueno N, Bratt CL, Rodriguez NE et al. Differences in human macrophage receptor usage, lysosomal fusion kinetics and survival between logarithmic and metacyclic Leishmania infantum chagasi promastigotes. Cell Microbiol 2009; 11:1827‑1841. 67. Kima PE. The amastigote forms of Leishmania are experts at exploiting host cell processes to establish infection and persist. Int J Parasitol 2007; 37:1087‑1096. 68. McConville MJ, de Souza D, Saunders E et al. Living in a phagolysosome; metabolism of Leishmania amastigotes. Trends Parasitol 2007; 23:368‑375. 69. Naderer T, McConville MJ. The Leishmania-macrophage interaction: a metabolic perspective. Cell Microbiol 2008; 10:301‑308. 70. Tanowitz HB, Machado FS, Jelicks LA et al. Perspectives on Trypanosoma cruzi-induced heart disease (Chagas disease). Prog Cardiovasc Dis 2009; 51:524‑539. 71. Vaidian AK, Weiss LM, Tanowitz HB. Chagas’ disease and AIDS. Kinetoplastid Biol Dis 2004; 13;3(1):2. 72. Sartori AM, Ibrahim KY, Nunes Westphalen EV et al. Manifestations of Chagas disease (American trypanosomiasis) in patients with HIV/AIDS. Ann Trop Med Parasitol 2007; 101(1):31‑50. 73. Huang H, Chan J, Wittner M et al. Expression of cardiac cytokines and inducible form of nitric oxide synthase (NOS2) in Trypanosoma cruzi‑infected mice. J Mol Cell Cardiol 1999; 31:75‑88. 74. Machado FS, Souto JT, Rossi MA et al. Nitric oxide synthase-2 modulates chemokine production by Trypanosoma cruzi‑infected cardiac myocytes. Microbes Infect 2008; 10:1558‑1566. 75. Hulit J, Bash T, Fu M et al. The cyclin D1 gene is transcriptionally repressed by caveolin-1. J Biol Chem 2000; 275:21203‑21209. 76. Cohen AW, Park DS, Woodman SE et al. Caveolin-1 null mice develop cardiac hypertrophy with hyperactivation of p42/44 MAP kinase in cardiac fibroblasts. Am J Physiol Cell Physiol 2003; 284:C457-474. © 20 10 C op yr ig ht L an de s B io sc ie nc e an d S pr in ge r. N ot f or D is tr ib ut io n 17INTERACTIONS BETWEEN CAVEOLIN AND PATHOGENS 77. Park DA, Woodman SE, Schubert W et al. Caveoilin-1/3 double knockout mice are viable, but lack both muscle and nonmuscle caveolae and develop a severe cardiomyopathic phenotype. Am J Pathol 2002; 160:2207-2217. 78. Woodman, SE, Park DS, Cohen AW et al. Caveolin-3 Knock-out Mice Develop a Progressive Cardiomyopathy and Show Hyper‑activation of the p42/44 MAP kinase cascade. J Biol Chem 2002; 277:38988‑38997. 79. Nagajyothi F, Desruisseaux M, Bouzahzah B et al. Cyclin and caveolin expression in an acute model of murine Chagasic myocarditis. Cell Cycle 2006; 5:107‑112. 80. Huang H, Petkova SB, Cohen AW et al. Activation of Transcription factors (AP-1 and NF-kB) in Murine Chagasic Myocarditis. Activation of transcription factors AP-1 and NF-kB in murine Chagasic myocarditis. Infect Immun 2003;71:2859‑2567. 81. Adesse D, Lisanti MP, Spray DC et al. Trypanosoma cruzi infection results in the reduced expression of caveolin‑3 in the heart. Cell Cycle 2010; 9:1639‑164. 82. Hassan GS, Mukherjee S, Nagajyothi F et al. Trypanosoma cruzi infection induces proliferation of vascular smooth muscle cells. Infect Immun 2006; 74:152‑159. 83. Mukherjee S, Huang H, Petkova SB et al. Trypanosoma cruzi infection activates extracellular signal-regulated kinase in cultured endothelial and smooth muscle cells. Infect Immun 2004; 72:5274‑5282. 84. Medina FA, Cohen AW, de Almeida CJ et al. Immune dysfunction in caveolin-1 null mice following infection with Trypanosoma cruzi (Tulahuen strain). Microbes Infect 2007; 9:325‑333. 85. Barrias ES, Dutra JM, De Souza W et al. Participation of macrophage membrane rafts in Trypanosoma cruzi invasion process. Biochem Biophys Res Commun 2007; 363:828‑834. 86. Weiss LM, Kim K, ed. Toxoplasma gondii the model Apicomplexan: Perspectives and Methods. 2007, Academic Press. 87. Wong SY, Remington JS. Biology of Toxoplasma gondii. AIDS 1993; 7(3):299‑316. 88. Luft BJ, Hafner R, Korzun AH et al. Toxoplasmic encephalitis in patients with the acquired immunodeficiency syndrome. N Engl J Med 1993; 329:995‑1000. 89. Frenkel JK, Escajadillo A. Cyst ruptureas a pathogenic mechanismof toxoplasmic encephalitis. Am J Trop Med Hyg 1987; 36:517‑522. 90. Ferguson DJ, Hutchison WM, Pettersen E. Tissue cyst rupture in mice chronically infected with Toxoplasma gondii. An immunocytochemical and ultrastructural study. Parasitol Res 1989; 75:599‑603. 91. Weiss LM, Kim K. The development and biology of bradyzoites of Toxoplasma gondii. Front Biosci 2000; 5:D391‑405. 92. Dubey JP. Advances in the life cycle of Toxoplasma gondii. Int J Parasitol 1998; 28:1019‑1024. 93. Dubey JP, Lindsay DS, Speer CA. Structures of Toxoplasma gondii tachyzoites, bradyzoites and sporozoites and biology and development of tissue cysts. Clin Microbiol Rev 1998; 11:267‑99. 94. Joiner KA, Fuhrman SA, Mietinnen H et al. Toxoplasma gondii: fusion competence of parasitophorous vacuoles in Fc receptor transfected fibroblasts. Science 1990; 249:641‑646. 95. Dobrowolski JM, Sibley LD. Toxoplasma invasion of mammalian cells is powered by the actin cytoskeleton of the parasite. Cell 1996; 84:933‑939. 96. Dobrowolsk, JM, Carruthers VB, Sibley LD. Participation of myosin in gliding motility and host cell invasion by Toxoplasma gondii. Mol Microbiol 1997; 26:163‑173. 97. Dubremetz JF, Achbarou A, Bermudes D et al. Kinetics and pattern of organelle exocytosis during Toxoplasma gondii/host‑cell interaction. Parasitol Res 1993; 79:402‑408. 98. Carruthers VB, Sibley LD. Sequential protein secretion from three distinct organelles of Toxoplasma gondii accompanies invasion of human fibroblasts. Eur J Cell Bio 1997; 73:114‑123. 99. NorkinLC. Caveolae in the uptake and targeting of infectious agents and secreted toxins. Adv Drug 0. Deliv Rev 2001; 49:301‑315. 100. Samuel BU, Mohandas N, Harrison T et al. The role of cholesterol and glycosylphosphatidylinositol‑ anchored proteins of erythrocyte rafts in regulating raft protein content and malarial infection. J Biol Chem 2001; 276, 29319‑29329. 101. Coppens I, Sinai AP, Joiner KA. Toxoplasma gondii exploits host lowdensity lipoprotein receptor-mediated endocytosis for cholesterol acquisition. J Cell Biol 2000; 149:167‑180. 102. Charron AJ, Sibley LD. Host cells: mobilizable lipid resources for the intracellular parasite Toxoplasma gondii. J Cell Sci 2002; 115:3049‑3059. 103. Chang WJ, Rothberg KG, Kamen BA et al. Lowering the cholesterol content of MA104 cells inhibits receptor‑mediated transport of folate. J Cell Biol 1992; 118:63‑69. 104. Sleat DE, Wiseman JA, El-Banna M et al. Genetic evidence for nonredundant functional cooperativity between NPC1 and NPC2 in lipid transport. Proc Natl Acad Sci U S A 2004; 101:5886‑5891. 105. Ikonen E, Heino S, Lusa S. Caveolins and membrane cholesterol. Biochem Soc Trans 2004; 32:121‑123. 106. Coppens I, Vielemeyer O. Insights into unique physiological features of neutral lipids in Apicomplexa: from storage to potential mediation in parasite metabolic activities. Int J Parasitol 2005; 35:5. © 20 10 C op yr ig ht L an de s B io sc ie nc e an d S pr in ge r. N ot f or D is tr ib ut io n 18 CAVEOLINS AND CAVEOLAE 107. Sehgal A, Bettio S, Pypaert M et al. Peculiarities of Host Cholesterol Transport to the Unique Intracellular Vacuole Containing Toxoplasma. Traffic 2005; 6:1125‑1141. 97‑615. 108. Que X, Ngo H, Lawton J et al. The cathepsin B of Toxoplasma gondii, toxopain-1, is critical for parasite invasion and rhoptry protein processing. J Biol Chem 2002; 277:25791‑25797. 109. Hoppe H, Ngo HM, Yang M. Targeting to rhoptry organelles of Toxoplasma gondii involves evolutionarily conserved mechanisms. Nat Cell Biol 2001; 2:449‑456. 110. Nichols BA, Chiappino ML, O’Connor GR. Secretion from the rhoptries of Toxoplasma gondii during host‑cell invasion. J Ultrastruct Res 1983; 83:85‑98. 111. Porchet‑Hennere E, Torpier G. Relations entre Toxoplasma et sa cellule‑hote. Protistologica 1983; 19:357-370. 112. Foussard F, Leriche MA, Dubremetz JF. Characterization of the lipid content of Toxoplasma gondii rhoptries. Parasitology 1992; 102;367‑370. 113. Coppens I, Joiner KA. Host but not parasite cholesterol controls Toxoplasma cell entry by modulating organelle discharge. Mol Biol Cell 2003; 14:3804‑3820. 114. Suss-Toby E, Zimmerberg J, Ward GE. Toxoplasma invasion: the parasitophorous vacuole is formed from host cell plasma membrane and pinches off via a fission pore. Proc Natl Acad Sci U S A 1996; 93:8413-8418. 115. Mordue DG, Desai N, Dustin M et al. Invasion by Toxoplasma gondii establishes a moving junction that selectively excludes host cell plasma membrane proteins on the basis of their membrane anchoring. J Exp Med 1999; 190:1783‑1792. 116. Mordue DG, Hakansson S Niesman I et al. Toxoplasma gondii resides in a vacuole that avoids fusion with host cell endocytic and exocytic vesicular trafficking pathways. Exp Parasitol 1999; 92:87‑99. 117. Ward GE, Miller LH, Dvorak JA. The origin of parasitophorous vacuole membrane lipids in malaria-infected erythrocytes. J Cell Sci 1993;106:237‑248. 118. Aikawa M, Komata Y, Asai T et al. Transmission and scanning electron microscopy of host cell entry by Toxoplasma gondii. Am J Pathol 1977; 87:285‑296. 119. Bannister LH, Mitchell GH, Butcher GA et al. Lamellar membranes associated with rhoptries in erythrocytic merozoites of Plasmodium knowlesi: a clue to the mechanism of invasion. Parasitology 1986; 92:291‑303. 120. Beckers CJ, Dubremetz JF, Mercereau-Puijalon O et al. The Toxoplasma gondii rhoptry protein ROP 2 is inserted into the parasitophorous vacuole membrane, surrounding the intracellular parasite and is exposed to the host cell cytoplasm. J Cell Biol 1984; 127:947‑961. 121. Hakansson S, Charron AJ, Sibley LD. Toxoplasma vacuoles: a two-step process of secretion and fusion forms the parasitophorous vacuole. EMBO J 2001; 20:3132‑3144. 122. Lisanti MP, Scherer PE, Vidugiriene J et al. Characterization of caveolin-rich membrane domains isolated from an endothelial‑rich source: implications for human disease. J Cell Bio 1994; 126:111‑126. 123. Sargiacomo M, Sudol M, Tang ZL et al. Signal transducing molecules and GPI-linked proteins form a caveolin‑rich insoluble complex in MDCK cells. J Cell Biol 1993; 122:789‑807. 124. Li S, Okamoto T, Chun M et al. Evidence for a regulated interaction of hetero-trimeric G proteins with caveolin. J Biol Chem 1995; 270:15693‑15701. 125. Wachtler V, Balasubramanian MK.Yeast lipid rafts?—an emerging view. Rends Cell Biol 2006; 6:1‑4. 126. Aliberti J, Reis e Sousa C, Schito M et al. CCR5 provides a signal for microbial induced production of IL-12 by CD8a+ dendritic cells. Nat Immunol 2000; 1:83‑87. 127. Mazza PK, Straus AH, Toledo MS et al. Interaction of epithelial cell membrane rafts with Paracoccidioides brasiliensis leads to fungal adhesion and Src-family kinase activation. Microbes Infect 2008; 10:540‑547. 128. Settnes OP, Nielsen MJ. Host-parasite relationship in Pneumocystis carinii infection: activation of the plasmalemmal vesicular system in type I alveolar epithelial cells. J Protozool 1991; 38:174S‑176S. 129. Iwabuchi K, Prinetti A, Sonnino S et al. Involvement of very long fatty acid-containing lactosylceramide in lactosylceramide‑mediated superoxide generation and migration in neutrophils. Glycoconj J 2008; 25:357-374. 130. Sato T, Iwabuchi K, Nagaoka I et al. Induction of human neutrophil chemotaxis by Candida albicans-derived b-1,6-long glycoside side-chain-branched b‑glucan. J Leukoc Biol 2006; 80:204‑211. 131. Beck MR, Dekoster GT, Cistola DP et al. NMR structure of a fungal virulence factor reveals structural homology with mammalian saposin B. Mol Microbiol 2009; 72:344‑353. © 20 10 C op yr ig ht L an de s B io sc ie nc e an d S pr in ge r. N ot f or D is tr ib ut io n Resultados 70 Trabalho #4 Amiodarone inhibits Trypanosoma cruzi infection and promotes cardiac cell recovery with gap junction and cytoskeleton reassembly in vitro Daniel Adesse, Eduardo M. Azzam, Maria N. Meirelles, Julio A. Urbina, Luciana R. Garzoni Aceito na Antimicrobial Agents and Chemotherapy em Outubro de 2010 e publicado em Janeiro de 2011 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 2011, p. 000 Vol. 55, No. 1 0066-4804/11/$08.000 doi:10.1128/AAC.01129-10 Copyright © 2011, American Society for Microbiology. All Rights Reserved. Amiodarone Inhibits Trypanosoma cruzi Infection and Promotes Cardiac Cell Recovery with Gap Junction and Cytoskeleton Reassembly In Vitro Daniel Adesse,1† Eduardo Meirelles Azzam,1† Maria de Nazareth L. Meirelles,1 Julio A. Urbina,2* and Luciana R. Garzoni1 Laborato´rio de Ultraestrutura Celular, Instituto Oswaldo Cruz, Av. Brasil 4365, Manguinhos, 21045-900 Rio de Janeiro, Brazil,1 and Laboratorio de Química Biolo´gica, Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Cientificas, Apartado Postal 21.627, Caracas 1020A, Venezuela2 Received 16 August 2010/Returned for modification 18 October 2010/Accepted 31 October 2010 We present the results of the first detailed study of the antiproliferative and ultrastructural effects of amiodarone on Trypanosoma cruzi, the causative agent of Chagas’ disease. Moreover, we report the effects of this compound on the recovery of F-actin fibrils, connexin43, and contractility in T. cruzi-infected cardiac myocytes. Amiodarone is the most prescribed class III antiarrhythmic agent and is frequently used for the symptomatic treatment of Chagas’ disease patients with cardiac compromise. In addition, recent studies identified its antifungal and antiprotozoal activities, which take place through Ca2 homeostasis disruption and ergosterol biosynthesis blockage. We tested different concentrations of amiodarone (2.5 to 10 M) on infected primary cultures of heart muscle cells and observed a dose- and time-dependent effect on growth of the clinically relevant intracellular amastigote form of T. cruzi. Ultrastructural analyses revealed that amiodarone had a profound effect on intracellular amastigotes, including mitochondrial swelling and disorganization of reservosomes and the kinetoplast and a blockade of amastigote-trypomastigote differentiation. Amiodarone showed no toxic effects on host cells, which recovered their F-actin fibrillar organization, connexin43 distri- bution, and spontaneous contractility concomitant with the drug-induced eradication of the intracellular parasites. Amiodarone is, therefore, a promising compound for the development of new drugs against T. cruzi. Chagas’ disease is the largest parasitic disease burden and a major cause of heart disease and heart-related deaths in Latin America, where it affects approximately 16 to 18 million people (25, 38). The disease is caused by the protozoan parasite Trypanosoma cruzi, which possesses a life cycle involving a mammalian host and an insect vector (31). Chemotherapy against T. cruzi is limited to two compounds, namely, benznidazole (a 2-nitroimidazole) and nifurtimox (a 5-nitrofuran), which are mostly active in acute- and early chronic-phase patients but are of limited efficacy in the prev- alent chronic stage (8, 34). Moreover, T. cruzi exhibits consid- erable biological variability, indicating possible variations in virulence, pathogenicity, oxidative stress, and drug resistance (23, 27, 36), which may pose an important challenge in the search of safer and more effective chemotherapeutic agents for the specific treatment of Chagas’ disease. Studies in the last 2 decades have permitted the identifica- tion of several new drug targets for this parasite. Among the most promising are (i) the essential cathepsin L-like protease cruzipain, (ii) the unique Kinetoplastida enzymes trypano- thione reductase and trypanothione synthase, and (iii) the in- hibitors of de novo sterol biosynthesis pathways, such as imi- dazole and triazole derivatives (5, 34, 35). There is also strong evidence that bisphosphonates can accumulate in the parasite’s acidocalcisomes and interfere with the activity of enzymes in- volved in isoprenoid biosynthesis, such as farnesyl diphosphate synthase (13). Using T. cruzi-infected cardiomyocytes, we pre- viously demonstrated that the bisphosphonate risedronate has a potent and selective in vitro effect against this parasite, re- sulting in recovery of the cardiac cells after the treatment (16). This compound also exhibited marked in vivo antiparasitic activity in a murine model of acute Chagas’ disease (18). An- other promising approach is the recent discovery of the anti-T. cruzi activity of the antiarrhythmic drug amiodarone, which is frequently prescribed for the symptomatic treatment of Cha- gas’ disease patients (4). In the heart, the effects of this com- pound include inhibition of Na channels, L-type Ca2 chan- nels, K channels, and the Na/Ca2 exchanger, leading to its characteristic antiarrhythmic action. It was found that the in vitro and in vivo activity against T. cruzi was mediated by dis- ruption of the parasite’s Ca2 homeostasis and a blockade of ergosterol biosynthesis at the level of oxidosqualene cy- clase (4). Although the antiparasitic activity of amiodarone has been demonstrated previously, there is a lack of data regarding the effect of this compound on the ultrastructure of T. cruzi and its host cells and the recovery of these cells after the antiparasitic treatment. Primary cultures of murine cardiac myocytes have been the method of choice to demonstrate alterations in the host cell induced by this parasite. In these studies, many as- * Corresponding author. Mailing address for Luciana R. Garzoni: Laborato´rio de Ultraestrutura Celular, Instituto Oswaldo Cruz, FIOCRUZ, Avenida Brasil 4365, Pavilha˜o Carlos Chagas, 2° andar, 21040-900 Rio de Janeiro, Brazil. Phone: 55 (21) 2598-4535. Fax: 55 (21) 2260-4434. E-mail: largarz@ioc.fiocruz.br. Present address for Julio A. Urbina: 734 Springhill Lane, Cincinnati, OH 45226. Phone: (513) 321-2981. Fax: (●●●) ●●●-●●●●. E-mail: jurbina@mac.com. † D.A. and E.M.A. equally contributed to this work and should be considered first coauthors.  Published ahead of print on ●●●●●●●●. 1 AUTHOR: Publication of this article cannot proceed without the signature of the person who read and corrected the proof on behalf of all the authors: signature date zac00111/zac9559d11z xppws S1 11/16/10 4/C Fig: 5 Art: 1129-10 zjs / DOCHEADARTICLE zjss / DOCTOPICMECHANISMS OF ACTION: PHYSIOLOGICAL EFFECTS FROM COVER SHEET: Editor: Edlind Article No.: T Section: Mech of Action AQ: A Fn2 AQ: B AQ: E pects of this relationship were clarified, such as alterations in intracellular calcium dynamics (2, 17), changes in the cell cy- toskeleton (24, 30), and cell-cell junction (1, 12). Gap junction channels are critical to maintaining cardiac homeostasis by allowing the free flow of ions and metabolites between cardiac myocytes, which contributes to the synchronized contraction of and signal exchange throughout the tissue. Gap junctions are composed of the connexin family of transmembrane proteins that assemble as end-to-end alignments of hexameric con- nexon subunits, thereby forming intercellular conduits for mol- ecules of up to 1 kDa. Connexin43 (Cx43) is the most abundant gap junction protein in ventricular myocytes, being localized at intercalated disks in normal myocardium (15). In the present study, we demonstrate the effects of amioda- rone on the proliferation and ultrastructure of intracellular amastigote forms of T. cruzi growing in cardiomyocytes and the recovery of the host cells. We evaluated the recovery of spon- taneous contractility of cardiac myocytes and the distribution of F-actin and Cx43 after treatment. MATERIALS AND METHODS Parasites. The Y (MHOM/BR/1950/Y) strain of T. cruzi was used in this work. Trypomastigote forms of T. cruzi were obtained from the supernatant of infected heart muscle cells grown in Dulbecco’s modified Eagle medium (DMEM; Sigma Aldrich, St. Louis, MO) supplemented with 5% fetal bovine serum (FBS; Cul- tilab, Sa˜o Paulo, Brazil), 1 mM CaCl2, 1 mM L-glutamine, 2% chicken embryo extract, 1,000 U/ml penicillin, and 50 g/ml streptomycin. After 96 h of infection, the parasites were collected, centrifuged, and resuspended in DMEM. Cardiac cell cultures. Hearts of 18-day-old Swiss Webster mouse embryos were submitted to mechanical and enzymatic dissociation as previously described (22). Briefly, cells were harvested using 0.05% trypsin and 0.01% collagenase in phosphate-buffered saline (PBS) at 37°C. Ventricular heart muscle cells (HMCs) were plated on 0.02% gelatin-coated plastic flasks, on glass coverslips, in 24-well plates, or in petri dishes. Cells were maintained at 37°C in a 5% CO2 atmosphere in DMEM for 72 h before the experiments. Use and handling of the animals were approved by the Ethics Committee for the Use of Laboratory Animals, FIOCRUZ (CEUA), protocol P70/09.2. Infection of cultures and treatments. Heart muscle cells were plated in 24-well plates at a density of 1.5  105 cells/well in glass coverslips and infected with culture-derived trypomastigotes (20:1, parasites/host cells) in a final volume of 300 l DMEM. After 2 h, the cultures were washed with PBS to remove non- adherent parasites and maintained in DMEM. Treatment with 2.5 to 10 M amiodarone was performed by using the following two protocols: (i) addition immediately after the interaction step and (ii) addition at 48 h after infection. The total volume in each well was 500 l. At specific times, coverslips (in triplicate) were collected, fixed with Bouin’s fixative solution (Electron Micros- copy Sciences, Hatfield, PA), and stained in Giemsa solution (Merck, Darmstadt, Germany). The percentage of infection was quantified by randomly counting at least 300 cells. In addition, supernatants were collected, and released parasites were counted in a hemocytometer. Ultrastructural studies. Cells were plated in 35-mm plastic petri dishes and, at chosen times, were fixed (60 min/4°C) with 2.5% glutaraldehyde (Sigma-Al- drich), 2.5 mM CaCl2, and 0.1 M Na-cacodylate buffer (pH 7.2), followed by postfixation for 1 h in cacodylate buffer solution containing 1% OsO4, 0.8% potassium ferricyanide, and 2.5 mM CaCl2. Samples were dehydrated in acetone and then embedded in Poly/Bed 812 resin (Electron Microscopy Sciences). Thin sections (Ultracut UCT; Leica, Vienna, Austria) were stained with uranyl acetate and lead citrate and were examined by transmission electron microscopy using a Zeiss EM10C microscope. Immunofluorescence. Cells were washed with PBS and fixed with 4% parafor- maldehyde for 5 min at 20°C at the desired time points. After being washed in PBS, cells were permeabilized with 0.5% Triton X-100, and nonspecific staining was blocked with 4% bovine serum albumin (BSA). Primary anti-Cx43 antibody (Sigma-Aldrich, St. Louis, MO) was incubated overnight at 4°C, after which cells were washed and incubated with secondary polyclonal goat anti-rabbit Alexa Fluor 488 antibody (Invitrogen, Eugene, OR) for 1 h at 37°C. F-actin filaments were stained with Alexa Fluor 594 phalloidin (Invitrogen) for 30 min at 37°C, and DNA was stained with DAPI (4,6-diamidino-2-phenylindole). Images were ac- quired with an Olympus laser scanning confocal microscope. Statistical analysis. Mean value comparisons were performed by using analysis of variance (ANOVA). P values below 0.05 were considered significant. IC50s (50% inhibitory concentrations) were calculated from dose-response curves by using nonlinear regression analysis with the GraFit software package. Materials. Amiodarone was purchased from Sigma-Aldrich. Stock solutions were prepared in phosphate-buffered saline (PBS) (pH adjusted to 7.4) and sterilized by using a 0.2-M filter (Millipore, Billerica, MA). Trypsin was pur- chased from Sigma Chemical Co. (St. Louis, MO), and collagenase was pur- chased from Worthington Laboratories (Lakewood, NJ). All other reagents were analytical grade. RESULTS Effect of amiodarone on intracellular T. cruzi amastigotes and host cells. Using two different treatment protocols, amio- darone caused a strong inhibition of the infection of HMCs (Fig. 1 to 3). When the drug was added immediately after infection (protocol i) (Fig. 1), dose- and time-dependent re- FIG. 1. Time and concentration dependence of the effects of ami- odarone on infection of HMCs by T. cruzi (protocol i). Amiodarone (2.5 to 10 M) was added to the cultures after 2 h of infection. (A, B) We observed significant effects of the drug on the percentage of in- fected cells (A) and the number of intracellular parasites (B), which were followed as a function of time. (C) The concentrations of the drug used in these studies were not toxic to the cardiac myocytes. Asterisks indicate statistical differences in relation to control cultures. The graphs show the means and standard deviations from triplicates of one representative experiment of four independent experiments. Cont, control. 2 ADESSE ET AL. ANTIMICROB. AGENTS CHEMOTHER. zac00111/zac9559d11z xppws S1 11/16/10 4/C Fig: 5 Art: 1129-10 F1-3 ductions in the percentages of infected cells were observed. The inhibition was statistically significant after 24 h of treat- ment with 10 M amiodarone (P  0.05), 48 h with 5 M (P  0.01), and 96 h with 2.5 M (P  0.01) (Fig. 1A). IC50s with protocol i were 5.85  1.4 M at 96 h and 3.14  1.2 M at 120 h. Amiodarone had also an inhibitory effect on the number of intracellular amastigotes (Fig. 1B), as revealed by light mi- croscopy (Fig. 3), significant for 10 M amiodarone at 24 h of treatment (P  0.05). Addition of amiodarone to cultures at 48 h after infection (protocol ii) also led to a highly significant reduction in the percentage of infected cells (Fig. 2). After 48 h of treatment (96 h of infection), 5 M amiodarone already reduced significantly (P  0.05) the parasitism of cultures, which was also observed at 96 h treatment with 2.5 M ami- odarone (Fig. 2A). In this protocol, IC50s were 4.47  0.3 M and 2.24  0.24 M at 96 and 120 h, respectively. This inhib- itory effect was also observed for the number of intracellular amastigotes in the HMCs (Fig. 2B). Moreover, amiodarone inhibited the release of trypomastigotes from infected cells in this model after completion of the intracellular cycle of the parasite. In control (untreated)-infected HMC cultures, the first two peaks of trypomastigote release to the supernatant occurred at 96 h and 192 h postinfection (Fig. 2C). Treatment with 5 and 10 M amiodarone, starting 48 h postinfection, drastically inhibited such release after 48 h of treatment (96 h postinfection), while at 96 and 144 h, no trypomastigotes were detected in the supernatant (Fig. 2C). Amiodarone at the con- centrations used in the experiments (up to 10 M) had no apparent cytotoxic effects on the myocytes, as shown in Fig. 1C and 2D. Light microscopy observations showed that treatment with amiodarone induced drastic morphological alterations on in- tracellular amastigotes (Fig. 3B, C, and E, insets). We con- firmed this observation through transmission electron micros- copy of infected HMCs and released parasites after 144 h of treatment of infected HMCs with 5 M amiodarone. Infected untreated HMCs displayed abundant intracellular amastigotes in the cells’ cytoplasm, with the expected kinetoplast morphol- ogy (bar shaped). A drastic loss of cytoplasmatic content and the formation of membrane inclusions inside the amastigotes were visualized in treated cultures; these cells also exhibited mitochondrial swelling, disorganization of reservosomes and the kinetoplast, probably associated with the disruption of Ca2 homeostasis (4), and a blockade of amastigote-trypomas- tigote differentiation. Untreated amastigotes spontaneously re- leased from their host cells also displayed their characteristic morphology (Fig. 4C). There was a marked damage in amas- tigotes released to the medium from amiodarone-treated (5 M) cultures, such as kinetoplast alteration, and in the Golgi apparatus (Fig. 4D). Cell physiology recovery after treatment with amiodarone. In order to assess the recovery of host cell ultrastructure and physiology after the treatment of infected cultures with amio- darone, we evaluated gap junction protein Cx43 (detected by immunofluorescence) and actin filaments (stained with phal- loidin). T. cruzi infection is known to disrupt gap junctional FIG. 2. Time and concentration dependence of the effects of amiodarone on infection of HMCs by T. cruzi (protocol ii). (A to C) Amiodarone (5 to 10 M) was added to the cultures after 48 h of infection, and the percentage of infected cells (A), number of intracellular parasites per host cell (B), and number of released trypomastigotes in the supernatant (C) were followed as a function of time. (D) We did not observe significant cytotoxic effects of amiodarone with the concentrations used throughout the experiments. Asterisks indicate statistical differences in relation to control cultures. The graphs show the means and standard deviations from triplicates of one representative experiment of four independent experiments. VOL. 55, 2011 AMIODARONE EFFECTS ON T. CRUZI-INFECTED CARDIOMYOCYTES 3 zac00111/zac9559d11z xppws S1 11/16/10 4/C Fig: 5 Art: 1129-10 F4 AQ: C communication through Cx43 protein reduction (1). Figure 5 shows that infected untreated HMCs (Fig. 5D to F) totally lost Cx43 immunoreactivity at 192 h postinfection compared to that of control uninfected cells (Fig. 5A to C), which displayed well-formed gap junction plaques (Fig. 5C) and abundant myo- fibrils, as revealed by phalloidin staining. After treatment with 5 M amiodarone for 144 h, infection was nearly abolished, as revealed by DAPI staining. In these cultures, Cx43 levels were comparable to those of age-matched uninfected cultures (Fig. 5I), and phalloidin staining revealed the presence of both po- FIG. 3. Effect of amiodarone on the intracellular cycle of T. cruzi. Primary cardiac myocytes were obtained and infected with the Y strain of T. cruzi. After 2 h of infection, cultures were treated with 5 or 10 M amiodarone. At 96 h postinfection (hpi), untreated cultures (A) displayed cells with trypomastigote forms of the parasite already evading host cells. Cultures treated with 5 M (B) and 10 M (C) of amiodarone displayed intracellular parasites with severe morphological alterations (arrows). (C) Interestingly, an amiodarone concentration of 10 M induced a drastic reduction of parasitism. After 144 h of infection (corresponding to 142 h of treatment), we observed that in nontreated cultures (D), 70 to 80% of cardiomyocytes were infected. Treatment with 5 M amiodarone (E) drastically reduced the number of parasites, whereas the use of 10 M amiodarone (F) almost eliminated the parasitism of the cultures. Insets show details of intracellular parasites in higher magnification. P, parasites; N, nucleus. 4 ADESSE ET AL. ANTIMICROB. AGENTS CHEMOTHER. zac00111/zac9559d11z xppws S1 11/16/10 4/C Fig: 5 Art: 1129-10 F5 lygonal and filamentous structures, indicative of cytoskeleton reassembly (Fig. 5H). We also assessed the impact of T. cruzi infection on cardiac myocyte spontaneous electrocontractility, and the results are shown in Fig. 5J. The parasites’ proliferation was associated with a progressive reduction in the cardiomyocytes’ contrac- tion, which followed the disorganization of the contractile ap- paratus as well as the disappearance of the gap junctions of these cells. After 48, 96, and 144 hours of infection (protocol i), untreated controls displayed a 28, 31, and 45% reduction in spontaneous contractility (P  0.05), respectively, whereas treatment with amiodarone (5 M) restored the number of spontaneous beatings to levels indistinguishable from those of uninfected cultures at 96 (P  0.05) and 144 (P  0.01) hours FIG. 4. (A) Effects of amiodarone on the ultrastructure of intracellular T. cruzi (Y strain) amastigotes. Untreated HMCs after 192 h of infection with T. cruzi, displaying severe intracellular damage and intact amastigote forms (P) and presenting a bar-shaped kinetoplast (K), nucleus (N), and flagellum (F). (B) Infected cultures treated with 5 M amiodarone for 144 h showed parasites with membrane blebs (arrows), loss of intracellular material (stars), and kinetoplast (K) alterations. (C) Released parasites from untreated cultures displayed acidocalcisomes (AC) and a bar-shaped kinetoplast (K), reservosome (R), and flagellum (F), as expected. (D) Parasites obtained from cultures treated with 5 M amiodarone (144 h) showed important alterations in the kinetoplast and Golgi apparatus (arrow). VOL. 55, 2011 AMIODARONE EFFECTS ON T. CRUZI-INFECTED CARDIOMYOCYTES 5 zac00111/zac9559d11z xppws S1 11/16/10 4/C Fig: 5 Art: 1129-10 FIG. 5. Amiodarone induced the recovery of host cell homeostasis. After eradicating the infection, we observed the recovery of morphological and functional aspects of cardiac myocytes cultures, as assessed by immunofluorescence and measuring of the spontaneous electrocontractility of the cells. (A to C) Uninfected cultured myocytes displayed abundant connexin43 immunoreactivity (C) as well as striated patterns of F-actin staining (B). (D to F) Highly infected cultures, at 144 h of infection, presented destruction of the F-actin cytoskeleton (E) and loss of Cx43 plaques (F). (G to I) Treatment with 5 M amiodarone for 142 h decreased parasitism and induced recovery of host cells, as evidenced by F-actin and Cx43 recovery, which included striations (H) and the presence of gap junction plaques (I). (J) The spontaneous electrocontractility of the myocytes culture was evaluated by counting the number of contractions per 10 s. We observed that T. cruzi-infected cultures had a progressive decrease in electrocontractility, whereas treatment with 5 and 10 M amiodarone restored cultures to normal levels of electrocontractility. *, P  0.05; **, P  0.01 (ANOVA). 6 ADESSE ET AL. ANTIMICROB. AGENTS CHEMOTHER. zac00111/zac9559d11z xppws S1 11/16/10 4/C Fig: 5 Art: 1129-10 4 C O L O R of treatment. We observed no significant effect on contractility when the same dose of amiodarone was added to uninfected cultures (not shown). DISCUSSION Human Chagas’ disease results from infection by T. cruzi, and tissue damage arises from both direct parasite action and the inflammatory process that ensues (7). Cytolysis and fibrosis are key components associated with Chagas’ disease’s patho- logical manifestations, along with a sustained and diffuse in- flammation of the affected organs (31). The growing percep- tion that chronic-phase manifestations are associated with the persistence of the parasite in the mammalian tissues (32) and that the two available drugs (benznidazole and nifurtimox) have important limitations, particularly in the chronic phase (6), has stimulated the search for new trypanocidal com- pounds. Amiodarone is frequently used as an antiarrhythmic in chronic-phase Chagas’ disease patients with cardiac compro- mise (21, 26), and it also has antifungal (9, 10) and antiproto- zoal (4, 28) activities, which were recently described. It was shown that the drug disrupts the parasite’s Ca2 homeostasis and also blocks ergosterol biosynthesis, resulting in excellent parasiticidal activity with low cytotoxicity (4). The results ob- tained in this work clearly confirm that amiodarone has potent and selective activity against T. cruzi, with no significant effects on their preferred host cells, cardiomyocytes, and at doses that do not induce the well-known antiarrhythmic action of the drug on these cells. This selective action was confirmed by ultrastructural analysis of amiodarone-treated cultures (Fig. 4), which revealed massive alterations of the parasites, allowing at the same time the full structural and functional recovery of the cardiomyocytes (see below). The ultrastructural effects of amiodarone on intracellular amastigotes were similar to those previously described for other sterol biosynthesis in- hibitors, such as risedronate (16), ketoconazole, and terbi- nafine (19, 37). We also investigated whether amiodarone treatment would induce host-cell recovery, concomitant with the elimination of the intracellular parasites’ burden. We have previously shown that infection by T. cruzi induces a cytoskeletal disruption in HMCs due to myofibrillar breakdown (24) and that treatment with risedronate and posaconazole allowed reassembly of cy- toskeleton elements (16, 29). The present results show that amiodarone allowed the reorganization of the actin-containing myofibrils to their normal state, with the occurrence of char- acteristic polygonal arrangement. Lin and coworkers (20) have described this polygonal configuration in dissociated cardiac myocytes as an indicator of myofibrillar reassembly. Moreover, it is known that the infection of cultured cardiac myocytes with T. cruzi decreases gap junction communication and Cx43 ex- pression (1, 11). These observations were extended to acutely infected mice, which displayed reduced cardiac Cx43 expres- sion (1). Our results demonstrate that Cx43 distribution among cardiac myocytes was fully restored after treatment of infected HMCs with amiodarone, and treated cultures displayed gap junction plaques comparable to those of uninfected controls. Cx43 has a turnover of approximately 1.5 h in vitro (3), and in T. cruzi-infected myocytes, Cx43 mRNA levels were largely unaffected (D. Adesse, unpublished data). This may provide evidence that the infection affects Cx43 translation and/or its subsequent trafficking to the plasma membrane, possibly due to microtubular damage (24). After the amiodarone-induced disappearance of the intracellular parasites, the host cells were capable of synthesizing new Cx43 molecules and made their successful delivery to the plasma membrane. More impor- tantly, HMCs restored their spontaneous contractility after treatment with 5 M amiodarone to levels comparable to those in control cultures. This observation is of interest since it permits us to speculate that during in vivo infection, T. cruzi disturbs synchronous contractility, which can be reverted with the use of amiodarone alone or in combination with another inhibitor of the parasite’s sterol biosynthesis, such as posacon- azole, which results in synergistic effects (4). To summarize, our results showed that amiodarone has a selective antiproliferative effect on T. cruzi in an in vitro model of infection of cardiac cells. Treatment induced ultrastructural damage to intracellular amastigotes but promoted full struc- tural and functional recovery of the host cells. This compound should be considered, beyond its known antiarrhythmic activ- ity, as an important antiparasitic agent and a lead for the development of new specific treatments of this neglected dis- ease. ACKNOWLEDGMENTS This work was supported with grants from FIOCRUZ, CNPq, FAPERJ, and the Howard Hughes Medical Institute (grant 55000620 to J.A.U.), in part by the U.S. Public Health Service (NIH grant GM 65307 to E.O. and R.D. and NIH NRSA grant GM 65782 to G.L.M.), and by the American Heart Association, Midwest Affiliate (supporting E.O.) and National Center (supporting R.D.). D.A. was in part sup- ported by grant NIH D43 W007129 from the Fogarty International Center. REFERENCES 1. Adesse, D., L. R. Garzoni, H. Huang, H. B. Tanowitz, M. de Nazareth Meirelles, and D. C. Spray. 2008. Trypanosoma cruzi induces changes in cardiac connexin43 expression. Microbes Infect. 10:21–28. 2. Barr, S. C., W. Han, N. W. Andrews, J. W. Lopez, B. A. Ball, T. L. Panna- becker, and R. F. Gilmour, Jr. 1996. A factor from Trypanosoma cruzi induces repetitive cytosolic free Ca2 transients in isolated primary canine cardiac myocytes. Infect. Immun. 64:1770–1777. 3. Beardslee, M. A., J. G. Laing, E. C. Beyer, and J. E. Saffitz. 1998. Rapid turnover of connexin43 in the adult rat heart. Circ. Res. 21:629–635. 4. Benaim, G., J. M. Sanders, Y. Garcia-Marcha´n, C. Colina, R. Lira, A. R. Caldera, G. Payares, C. Sanoja, J. M. Burgos, A. Leon-Rossell, J. L. Con- cepcion, A. G. Schijman, M. Levin, E. Oldfield, and J. A. Urbina. 2006. Amiodarone has intrinsic anti-Trypanosoma cruzi activity and acts synergis- tically with posaconazole. J. Med. Chem. 9:892–899. 5. Buckner, F., K. Yokoyama, J. Lockman, K. Aikenhead, J. Ohkanda, M. Sadilek, S. Sebti, W. Van Voorhis, A. Hamilton, and M. H. Gelb. 2003. A class of sterol 14-demethylase inhibitors as anti-Trypanosoma cruzi agents. Proc. Natl. Acad. Sci. U. S. A. 9:15149–15153. 6. Canc¸ado, J. R. 2002. Long term evaluationtermevaluation of etiological treatment of Chagas disease with disease with benznidazole. Rev. Inst. Med. Trop. Sao Paulo 44:29–37. 7. Chagas, C. 1909. Nova tripanozomiaze humana. Estudos sobre a morfolojia e o ciclo evolutivo do Schizotrypanum cruzi n. gen, n. sp., ajente etiolojico de nova entidade morbida do homem. Mem. Inst. Oswaldo Cruz 1:11–80. 8. Coura, J. R., and S. L. de Castro. 2002. A critical review on Chagas disease chemotherapy. Mem. Inst. Oswaldo Cruz 97:3–24. 9. Courchesne, W. E. 2002. Characterization of a novel, broad-based fungicidal activity for the antiarrhythmic drug amiodarone. J. Pharmacol. Exp. Ther. 300:195–199. 10. Courchesne, W. E., M. Tunc, and S. Liao. 2009. Amiodarone induces stress responses and calcium flux mediated by the cell wall in Saccharomyces cerevisiae. Can. J. Microbiol. 55:288–303. 11. de Carvalho, A. C., H. B. Tanowitz, M. Wittner, R. Dermietzel, C. Roy, E. L. Hertzberg, and D. C. Spray. 1992. Gap junction distribution is altered be- tween cardiac myocytes infected with Trypanosoma cruzi. Circ. Res. 70:733– 742. VOL. 55, 2011 AMIODARONE EFFECTS ON T. CRUZI-INFECTED CARDIOMYOCYTES 7 zac00111/zac9559d11z xppws S1 11/16/10 4/C Fig: 5 Art: 1129-10 AQ: D 12. de Melo, T. G., M. N. Meirelles, and M. C. Pereira. 2008. Trypanosoma cruzi alters adherens junctions in cardiomyocytes. Microbes Infect. 10:1405–1410. 13. Docampo, R., and S. N. Moreno. 2008. The acidocalcisome as a target for chemotherapeutic agents in protozoan parasites. Curr. Pharm. Des. 14:882– 888. 14. Docampo, R. 2001. Recent developments in the chemotherapy of Chagas disease. Curr. Pharm. Des. 7:1157–1164. 15. Duffy, H. S., A. G. Fort, and D. C. Spray. 2006. Cardiac connexins: genes to nexus. Adv. Cardiol. 42:1–17. 16. Garzoni, L. R., A. Caldera, M. de N. Meirelles, S. L. de Castro, R. Docampo, G. A. Meints, E. Oldfield, and J. A. Urbina. 2004. Selective in vitro effects of the farnesyl pyrophosphate synthase inhibitor risedronate on Trypanosoma cruzi. Int. J. Antimicrob. Agents 23:273–285. 17. Garzoni, L. R., M. O. Masuda, M. M. Capella, A. G. Lopes, and M. de N. Meirelles. 2003. Characterization of [Ca2]i responses in primary cultures of mouse cardiomyocytes induced by Trypanosoma cruzi trypomastigotes. Mem. Inst. Oswaldo Cruz 98:487–493. 18. Garzoni, L. R., M. C. Waghabi, M. M. Baptista, S. L. de Castro, M. de N. Meirelles, C. C. Britto, R. Docampo, E. Oldfield, and J. A. Urbina. 2004. Antiparasitic activity of risedronate in a murine model of acute Chagas’ disease. Int. J. Antimicrob. Agents 23:286–290. 19. Lazardi, K., J. A. Urbina, and W. de Souza. 1990. Ultrastructural alterations induced by two ergosterol biosynthesis inhibitors, ketoconazole and terbin- afine, on epimastigotes and amastigotes of Trypanosoma (Schizotrypanum) cruzi. Antimicrob. Agents Chemother. 34:2097–2105. 20. Lin, Z., S. Holtzer, T. Schultheiss, J. Murray, T. Masaki, D. A. Fischman, and H. Holtzer. 1989. Polygons and adhesion plaques and disassembly and assembly of myofibrils in cardiac myocytes. J. Cell Biol. 108:2355–2367. 21. Mason, J. W. 1987. Amiodarone. N. Engl. J. Med. 316:455–466. 22. Meirelles, M. N. L., T. C. Arau´jo-Jorge, C. F. Miranda, W. DeSouza, and H. S. Barbosa. 1986. Interaction of Trypanosoma cruzi with heart muscle cells: ultrastructural and cytochemical analysis of endocytic vacuole forma- tion and effect upon myogenesis in vitro. Eur. J. Cell Biol. 41:198–206. 23. Mielniczki-Pereira, A. A., C. M. Chiavegatto, J. A. Lo´pez, W. Colli, M. J. Alves, and F. R. Gadelha. 2007. Trypanosoma cruzi strains, Tulahuen 2 and Y, besides the difference in resistance to oxidative stress, display differential glucose-6-phosphate and 6-phosphogluconate dehydrogenases activities. Acta Trop. 101:54–60. 24. Pereira, M. C. S., M. Costa, C. Chagas Filho, and M. N. L. Meirelles. 1993. Myofibrillar breakdown and cytoskeletal alterations in heart muscle cells during invasion by Trypanosoma cruzi: immunological and ultrastructural study. J. Submicrosc. Cytol. Pathol. 25:559–569. 25. Rassi, A., Jr., J. C. Dias, J. A. Marin-Neto, and A. Rassi. 2009. Challenges and opportunities for primary, secondary, and tertiary prevention of Chagas’ disease. Heart 95:524–534. 26. Rassi, A., Jr., A. Rassi, and W. C. Little. 2000. Chagas’ heart disease. Clin. Cardiol. 23:883–889. 27. Sanchez, G., A. Wallace, M. Olivares, N. Diaz, X. Aguilera, W. Apt, and A. Solari. 1990. Biological characterization of Trypanosoma cruzi zymodemes: in vitro differentiation of epimastigotes and infectivity of culture metacyclic trypomastigotes to mice. Exp. Parasitol. 71:125–133. 28. Serrano-Martín, X., Y. García-Marchan, A. Fernandez, N. Rodriguez, H. Rojas, G. Visbal, and G. Benaim. 2009. Amiodarone destabilizes intracellu- lar Ca2 homeostasis and biosynthesis of sterols in Leishmania mexicana. Antimicrob. Agents Chemother. 53:1403–1410. 29. Silva, D. T., M. de Nazareth S. L. de Meirelles, D. Almeida, J. A. Urbina, and M. C. Pereira. 2006. Cytoskeleton reassembly in cardiomyocytes infected by Trypanosoma cruzi is triggered by treatment with ergosterol biosynthesis inhibitors. Int. J. Antimicrob. Agents 27:530–537. 30. Taniwaki, N. N., F. S. Machado, A. R. Massensini, and R. A. Mortara. 2006. Trypanosoma cruzi disrupts myofibrillar organization and intracellular cal- cium levels in mouse neonatal cardiomyocytes. Cell Tissue Res. 324:489–496. 31. Tanowitz, H. B., L. V. Kirchhoff, D. Simon, S. A. Morris, L. M. Weiss, and M. Wittner. 1992. Chagas’ disease. Clin. Microbiol. Rev. 5:400–419. 32. Tarleton, R. L., L. Zhang, and M. O. Downs. 1997. “Autoimmune rejection” of neonatal heart transplants in experimental Chagas disease is a parasite- specific response to infected host tissue. Proc. Natl. Acad. Sci. U. S. A. 15:3932–3937. 33. Urbina, J. A. 2009. Ergosterol biosynthesis and drug development for Chagas disease. Mem. Inst. Oswaldo Cruz 104(Suppl. 1):311–318. 34. Urbina, J. A. 2010. Specific chemotherapy of Chagas disease: relevance, current limitations and new approaches. Acta Trop. 115:55–68. 35. Urbina, J. A., and R. Docampo. 2003. Specific chemotherapy of Chagas disease: controversies and advances. Trends Parasitol. 19:495–501. 36. Veloso, V. M., C. M. Carneiro, M. J. Toledo, M. Lana, E. Chiari, W. L. Tafuri, and M. T. Bahia. 2001. Variation in susceptibility to benznidazole in isolates derived from Trypanosoma cruzi parental strains. Mem. Inst. Oswaldo Cruz 96:1005–1011. 37. Vivas, J., J. A. Urbina, and W. de Souza. 1996. Ultrastructural alterations in Trypanosoma (Schizotrypanum) cruzi induced by delta(24(25)) sterol methyl transferase inhibitors and their combinations with ketoconazole. Int. J. An- timicrob. Agents 7:235–240. 38. WHO. 2002. Control of Chagas disease. Technical reports series. 905:1–109. 8 ADESSE ET AL. ANTIMICROB. AGENTS CHEMOTHER. zac00111/zac9559d11z xppws S1 11/16/10 4/C Fig: 5 Art: 1129-10 Discussão 79 DISCUSSÃO Discussão 80 Um dos mais importantes desafios para a compreensão da fisiopatologia da doença de Chagas está no estudo dos mecanismos envolvidos na interação entre parasito e hospedeiro. Nesta tese, utilizamos modelos in vitro e in vivo, incluindo culturas primárias de cardiomiócito e a infecção experimental de camundongos por T. cruzi visando elucidar alguns destes aspectos. Um importante fator consiste na diversidade de populações de T. cruzi que diferem em propriedades bioquímicas, que influenciam sua infectividade (Yoshida, 2006), virulência (Espinoza et al., 2010) e patogênese (Revollo et al., 1998). Diversas abordagens foram utilizadas para caracterizar a estrutura das populações de T. cruzi, com objetivo de definir subgrupos de relevância biológica. Estes subgrupos receberam diferentes designações, que incluem zimodema, esquizodema, biodema, linhagens e, mais recentemente, unidades de tipificação distintas (DTUs) (revisto em Zingales et al., 2009). As diferentes combinações entre cepas de T. cruzi e hospedeiros com backgrounds diversos, gera um amplo espectro de manifestações da doença humana e experimental (Miles et al., 1981). Durante a fase aguda da doença, estas diferenças podem gerar um amplo espectro de sintomas e manifestações no hospedeiro, que podem determinar o curso da fase crônica, e o possível desenvolvimento da doença cardíaca crônica (Higuchi et al., 2003). Por esta razão os estudos in vitro da interação parasito-célula hospedeira são de extrema relevância, e já geraram uma vasta quantidade de informações a respeito do reconhecimento e invasão da célula hospedeira (revisto em Alves & Mortara, 2009); da instalação do ciclo intracelular do parasito e fatores relacionados à replicação das formas amastigotas (Schettino et al., 1995) e das alterações sofridas pela célula. No entanto verificamos que alguns estudos apresentaram resultados conflitantes a depender do modelo experimental utilizado. Em nosso modelo de interação, cardiomiócitos de camundongos infectados pela cepa Y de T. cruzi apresentaram aumento na contratilidade espontânea, resposta a neurotransmissores diminuída e não sofreram alterações na miogênese (Meirelles et al., 1986; Aprigliano et al., 1993). Células musculares de ratos infectados pela cepa Tulahuen de T. cruzi tiveram resposta Discussão 81 ànorepinefrina aumentada (Bergdolt et al., 1996) e quando aqueles infectados pela cepa Brazil apresentaram inibição de miogênese (Rowin et al., 1983). Estas diferenças nos motivaram a avaliar até que ponto as alterações na expressão gênica global seriam similares após a infecção da linhagem L6E9 com quatro diferentes cepas de T. cruzi. Para isso utilizamos a técnica de microarranjos (microarrays) de oligonucleotídeos, que permite o estudo rápido e imparcial de alterações na expressão de um grande número de genes, no nível do RNAm. Alguns trabalhos utilizaram a tecnologia de microarrays para investigar o impacto da infecção por T. cruzi no hospedeiro, tanto in vitro (Vaena-de-Avalos et al., 2002; Shigihara et al., 2008; Imai et al., 2005) como in vivo (Mukherjee et al., 2003; Mukherjee et al., 2008; Garg et al., 2003; Soares et al., 2010). No entanto, não podemos afirmar que os resultados demonstrados por estes autores são aplicáveis a outras combinações parasito-hospedeiro. Um estudo mais recente comparou, por microarrays, a resposta inicial à infecção intradérmica por três diferentes cepas deT. cruzi (Y, Brazil e G), verificando que o parasita induz uma resposta dependente de IFN- tipo I (Chessler et al., 2009). Nós utilizamos a linhagem de mioblastos de rato L6E9, já que o músculo esquelético é também um alvo primário da infecção aguda. Esta linhagem apresenta crescimento rápido em cultura e foi muito utilizada em estudos de infecção por T. cruzi (Rowin et al., 1983; Morris et al., 1987; Rodriguez et al., 1996). Além disso, o uso de cultura de células evita a interferência de variáveis externas presentes na infecção in vivo como migração de células inflamatórias, fatores solúveis presentes no soro e presença de outros tipos celulares como fibroblastos e células endoteliais. As análises de microarray descritas no Trabalho #1 foram feitas utilizando células L6E9 após 72 horas de infecção com quatro cepas referência de T. cruzi, todas bem caracterizadas quanto à infectividadein vitro e in vivo, resistência à quimioterapia e patogênese. Nós realizamos a classificação filogenética segundo Fernandes e colaboradores (2001) e verificamos que as cepas Y, CL e Tulahuen são representativas do grupo T. cruzi II (TCII), encontrado em regiões do Centro e no Leste do Brasil e estão em geral associadas com as “mega” síndromes (cardiomegalia, megacólone megaesôfago). A cepa Brazil foi selecionada por ser utilizada há muitos anos em modelos in vivo, gerando cardiomiopatia dilatada severa, associada com redução na Discussão 82 fração de ejeção do ventrículo esquerdo e afinamento da parede do miocárdio em camundongos (Mukherjee et al., 2008). A genotipagem revelou que esta cepa pertence ao grupo TCI, predominante na Venezuela e no centro do Brasil e está geralmente associada a importantes alterações eletrocardiográficas (Miles et al., 1981). Nós analisamos os resultados obtidos destes microarrays de duas maneiras diferentes, porém complementares com relação ao impacto da infecção por T. cruzi nas células hospedeiras in vitro. Quando identificamos os genes dos mioblastos que sofreram alterações em sua expressão após a infecção, verificamos que uma pequena quantidade destes foi significativamente alterada pelas quatro cepas (>50%, p<0,05). No entanto, apenas um pequeno número de genes (21) foi comum às quatro cepas utilizadas, permitindo que concluíssemos que cada cepa de T. cruzi possui uma assinatura distinta na patologia. Um gene comumente alterado foi o cardiotrophin-like cytokine factor 1, que pertence à família da interleucina-6 (IL-6), que inclui IL-11, LIF, oncostatina M, fator neurotrófico ciliar e cardiotrofina-1 (CT-1). Estas citocinas estão envolvidas em processos inflamatórios e imunológicos, assim como hematopoiese, regeneração hepática e neuronal, desenvolvimento embrionário e fisiologia cardiovascular. Através da ativação de genes-alvo envolvidos nos processos de diferenciação, sobrevivência, apoptose e proliferação, as citocinas tipo IL-6 tem um papel na homeostase celular e tissular. A proteína STAT3 (transdutor de sinal e ativador de fator de transcrição-3) é um mediador crítico para a sobrevivência dos cardiomiócitos (Sano et al., 2000) e parece ser essencial na indução de hipertrofia cardíaca através da glicoproteína gp130 (Kunisada et al., 1998, 2000). Todas as citocinas tipo IL-6 potencialmente ativam STAT3 e, em menor escala, STAT1, através de uma subunidade de gp130 em comum. A infecção de ratos Lewis por T. cruzi (cepa Sylvio X10/7) induziu um aumento transitório da expressão de cardiotrofina-1 e gp130 durante a fase aguda da infecção experimental (Chandrasekar et al., 1998). Estes resultados sugerem que a ativação desta via de sinalização possa ter um efeito cardioprotetor, uma vez que a superexpressão destas proteínas em culturas de cardiomiócitos promoveu aumento na sobrevida e hipertrofia destas células (Sheng et al., 1997). Apesar de haver aumento da expressão de gp130 no coração dos animais infectados (Chandrasekar et al.,1998) e da infecção de cardiomiócitos de ratos induzir hipertrofia destas células (Petersen & Discussão 83 Burleigh, 2003), ainda não foi elucidada a participação da via ativada por cardiotrofina durante a infecção por T. cruzi. No entanto, a redução na expressão deste transcrito observada em nossos experimentos sugere que a ativação de cardiotrofina-1 descrita por Chandrasekar e colaboradores (1998) pode ser proveniente de macrófagos ativados (Harris et al., 2006), presentes em infiltrados inflamatórios. Outra observação interessante foi a de que diversos genes que codificam proteínas juncionais foram afetados pela infecção por T. cruzi, incluindo plakoglobin, junctional adhesion molecule 3, e adipocyte-specific adhesion molecule, bem como cadherin 15 (pela cepa Y). Nosso grupo já demonstrou que a infecção de cardiomiócitos por T. cruzi altera a expressão de moléculas de adesão como Cx43 (Adesse et al., 2008) e caderina/catenina (de Melo et al., 2008). Como demonstrado num modelo murino de sepse (Celes et al., 2007), alguns tipos de cardiomiopatias apresentam alterações ou mutações em genes relacionados a proteínas do disco intercalar, sugerindo que estas doenças, possivelmente incluindo a CCC, são “doenças juncionais” (revisto por Spray & Tanowitz, 2007). O segundo tipo de análise que realizamos em nossos estudos de microarray foi uma comparação global entre os transcriptomas dos mioblastos infectados com cada cepa, considerando inclusive as pequenas alterações (ou seja, menores que 1,5 vezes). Nestas análises, comparamos os dados obtidos com cada cepa com aqueles obtidos com as outras três cepas, gerando seis combinações altamente correlacionadas (p<0,001).Esta abordagem enfatiza o conceito de que apesar da doença de Chagas apresentar um amplo espectro de manifestações in vivo, na realidade representa uma síndrome de alterações fenotípicas em comum, como revelado por nossos resultados. Os genes comumente alterados pelas quatro cepas estudadas podem servir como biomarcadores de doença aguda em áreas endêmicas, uma vez que tais cepas são usadas como referência para diversos estudos (Gruson & Borodovitz, 2010). Para compreender melhor o impacto da infecção por T. cruzi na fisiologia da célula hospedeira, estudamos a expressão de Cav-3 em cardiomiócitos infectados in vitro e no coração de camundongos durante infecção in vivo. Este estudo foi motivado por observações prévias de nosso laboratório, que mostraram alterações na homeostase de cálcio de cardiomiócitos durante infecção in vitro (Garzoni et al., 2003), que incluía a redução de sítios de cálcio de superfície e cavéolas das células Discussão 84 hospedeiras. Nossos resultados (Trabalho #2) mostraram que a infecção reduziu os níveis de Cav-3 nos cardiomiócitos após 48 h de infecção pela cepa Y e no coração dos camundongos após 66 dias de infecção pela cepa Brazil. Estes resultados corroboram observações prévias que demonstraram redução de caveolinas durante a infecção in vitro de adipócitos e no coração de camundongos infectados (Nagajyothi et al., 2006, 2008). A redução nos níveis de Cav-3 cardíaca pode estar relacionada a alterações na homeostase de cálcio intracelular e, consequentemente, àgeração de arritmias no tecido cardíaco durante a infecção (Sargiacomo et al., 1993). Diversos patógenos, incluindo vírus, bactérias, fungos e protozoários utilizam caveolina para invasão da célula hospedeira ou causam profundos danos à composição das cavéolas, como discutido no Trabalho #3. O tratamento de macrófagos peritoneais (Barrias et a., 2007) ou células HeLa (Fernandes et al., 2007) com metil--ciclodextrina, que elimina o colesterol da membrana plasmática, inibiu a invasão de formas tripomastigotas, sugerindo que as cavéolas participem do processo de invasão da célula hospedeira (Barrias et al., 2007). Além disso, a correta expressão de caveolinas inibe a ativação da via de MAP quinases incluindo a ativação (fosforilação) de ERK 1/2, como demonstrado em modelos KO de Cav-3 e Cav-1/-3 (Engelman et al., 1998, Park et al., 2002; Woodman et al., 2002). Verificamos no Trabalho#2 que a redução na expressão de Cav-3 induzida pela infecção foi concomitante com a ativação de ERK nas culturas de cardiomiócito a partir da segunda hora de infecção in vitro e também na infecção in vivo. A ativação de ERK pode estar relacionada com o remodelamento cardíaco observado durante a doença de Chagas e à hipertrofia compensatória dos cardiomiócitos (Lorenz et al., 2009), através da redução de Cav-3. Para avaliar a recuperação da célula hospedeira após a infecção por T. cruzi, testamos diferentes concentrações de amiodarona em culturas primárias de cardiomiócito (Trabalho #4). Utilizando dois protocolos de tratamento, observamos que este composto teve um efeito anti-proliferativo dose-dependente nas formas amastigotas intracelulares (cepa Y), sem efeitos citotóxicos significativos. Este composto já é utilizado como antiarrítmico em pacientes com CCC e recentemente foi demonstrado seu efeito na homeostase de cálcio intracelular de tripanossomatídeos e Discussão 85 na inibição da biossíntese de ergosterol de fungos e protozoários (Benaim et al., 2006; Serrano-Martin et al., 2009a, 2009b). Utilizando microscopia eletrônica de transmissão observamos que o tratamento com amiodarona induziu importantes alterações morfológicas em amastigotas intracelulares e também nos parasitos liberados pelos cardiomiócitos após 192 h de infecção. Estas alterações incluíram perda de material citoplasmático, danos ao cinetoplasto e a formação de estruturas membranares intracelulares, induzidas possivelmente pela inibição da síntese de esteróis do parasito. Estas alterações foram similares a observações prévias que utilizaram outras classes de inibidores desta via tais como risedronato (Garzoni et al., 2004a), cetoconazol e terbinafina (Lazardi et al., 1990; Vivas et al., 1996). Nós investigamos se após o tratamento as culturas infectadas seriam capazes de recuperar sua fisiologia, uma vez que a infecção de cardiomiócitos por T. cruzi causa a quebra do citoesqueleto da célula hospedeira, incluindo actina, tubulina, desmina, miosina e -actinina (Pereira et al., 1993; Taniwaki et al., 2006; Melo et al, 2006), alterando a capacidade contrátil das células. Através de microscopia confocal de varredura a laser observamos que nas culturas não infectadas os miócitos exibiam abundantes miofibrilas e que, após 192 h de infecção, as células infectadas e não tratadas apresentaram total ausência de marcação para F-actina. O tratamento com amiodarona por 144 h permitiu o restabelecimento do citoesqueleto de actina nas células cardíacas. A marcação por faloidina demonstrou que as células apresentaram estruturas poligonais típicas de recuperação das miofibrilas (Lin et al., 1989), corroborando resultados prévios que demonstraram que o tratamento com os compostos risedronato (Garzoni et al., 2004a) e pozaconazol (Silva et al., 2006) também induziram recuperação do citoesqueleto de actina e tubulina em cardiomiócitos. Em seguida avaliamos o efeito do tratamento na imunolocalização de Cx43 nestas culturas. Nós descrevemos previamente que a infecção de cardiomiócitos pela cepa Y de T. cruzi induz uma redução de 61% na expressão de Cx43 (Adesse et al., 2008), que pode estar relacionada àredução no acoplamento célula-célula causada pela infecção (de Carvalho et al., 1992). Após o tratamento com amiodarona verificamos a total recuperação da expressão desta proteína, em regiões de contato Discussão 86 célula-célula, como revelado por microscopia confocal. A meia-vida da Cx43 in vitro é de aproximadamente 1,5 horas, desde sua síntese no retículo sarcoplasmático, processamento e oligomerização, transporte até o sarcolema e degradação (Beardslee et al., 1998). Seu transporte até a membrana plasmática e estabilização depende de uma interação direta com tubulina (Giepmans et al., 2001), através de uma proteína associada chamada EB1 (Shaw et al., 2007). Esta interação é fundamental para a correta expressão da Cx43 no coração e, em casos de dano ao miocárdio, o transporte desta proteína para o sarcolema é reduzida (Smyth et al., 2010), afetando o acoplamento celular. Apesar de sua ação anti-arrítmica, através da inibição de canais de K+, a adição de amiodarona por 10 min não alterou a resistência juncional de cardiomócitos, mesmo na dose de 100 M (Daleau et al., 1998). No entanto, o tratamento de camundongos com amiodarona por seis meses induziu uma importante redução na expressão de RNAm para Cx43 (Le Bouter et al., 2004). Estas observações indicam que a recuperação de Cx43 após o tratamento das culturas infectadas não está relacionada a uma ação direta de amiodarona sobre esta proteína e sim ao seu efeito tripanocida. É provável que os danos ao citoesqueleto causados por T. cruzi sejam responsáveis pela redução nos níveis de Cx43 e que, após o tratamento com amiodarona, haja reconstituição do citoesqueleto e, conseqüentemente, das junções comunicantes. A expressão de Cx43 cardíaca também está diretamente relacionada à capacidade contrátil dos cardiomiócitos, como demonstrado por diferentes modelos de dano cardíaco (Haefliger & Meda, 2000, Lin et al., 2005). Nós verificamos uma redução significativa na contratilidade espontânea das culturas de cardiomiócito após 96 h de infecção, que foi revertida com o tratamento com amiodarona. Estes resultados estão de acordo com observações prévias demonstrando que a infecção in vivo por T. cruzi induz diminuição na contração do miocárdio já na fase aguda, consequência também da ação de citocinas pró-inflamatórias (Roman-Campos et al., 2009) ou ainda dos receptores -adrenérgicos (Enders et al., 2004) no remodelamento mecânico do coração. Em resumo, nossos resultados demonstraram que a amiodarona é um composto promissor para o tratamento da doença de Chagas, devido ao seu efeito Discussão 87 seletivo tripanocida e por promovera recuperação da fisiologia das células cardíacas, além de sua já conhecida ação antiarrítmica. Conclusões 88 CONCLUSÕES 1) A infecção por diferentes cepas de T. cruzi gera assinaturas transcriptômicas únicas na célula hospedeira; 2) Os genes alterados da mesma maneira pelas quatro cepas (ex: DNA-damage inducible transcript 3 e serologically defined colon cancer antigen 3) são possíveis candidatos a biomarcadores para fase aguda da doença de Chagas; 3) A infecção por T. cruzi altera diversas vias envolvidas em adesão/comunicação celular (incluindo junction plakoglobin e junctional adhesion molecule 3), que no tecido cardíaco podem contribuir para geração de arritmias; 4) Tanto no modelo de infecção in vitro quando no tecido cardíaco de camundongos infectados, há redução na expressão de Cav-3 induzida por T. cruzi, que pode contribuir com as alterações na homeostase de cálcio intracelular em cardiomiócitos; 5) A redução de caveolina-3 pela infecção foi acompanhada da ativação da via da MAP quinase ERK, tanto in vitro quanto in vivo e pode estar envolvida em eventos de hipertrofia cardíaca; 6) O composto antiarrítmico amiodarona apresenta atividade seletiva anti-T. cruzi, sem causar danos citotóxicos aos cardiomiócitos; 7) Após erradicar o parasitismo, amiodarona promove o restabelecimento da fisiologia de cardiomiócitos, com recuperação de citoesqueleto de actina, Cx43 e da contratilidade espontânea. Referências Bibliográficas 89 REFERÊNCIASBIBLIOGRÁFICAS Referências Bibliográficas 90 1. Adesse D, Garzoni LR, Huang H, Tanowitz HB, de Nazareth Meirelles M, Spray DC. 2008. Trypanosoma cruzi induces changes in cardiac connexin43 expression. Microbes Infect. 10(1):21-8. 2. Almeida DR, Carvalho AC, Branco JN, Pereira AP, Correa L, Vianna PV, Buffolo E, Martinez EE. 1996. Chagas' disease reactivation after heart transplantation: efficacy of allopurinol treatment. J Heart Lung Transplant. 15(10):988-92. 3. Alves MJ, Mortara RA. 2009. A century of research: what have we learned about the interaction of Trypanosoma cruzi with host cells? Mem Inst Oswaldo Cruz. 104 Suppl 1:76-88. 4. Anderson, R. G. W., B. A. Kamen, K. G. Rothberg, and S. W. Lacey. 1992. Potocytosis: sequestration and transport of small molecules by caveolae. Science 255:410-411. 5. Andrade S. 1974. Caracterização de cepas do Trypanosoma cruzi isoladas no Reconcavo Baiano. Revista de Patologia Tropical 3:65-121. 6. Andrade ZA. 1999. Immunopathology of Chagas disease. Mem Inst Oswaldo Cruz. 94 Suppl 1:71-80. 7. Añez N, Crisante G, da Silva FM, Rojas A, Carrasco H, Umezawa ES, Stolf AM, Ramírez JL, Teixeira MM. 2004. Predominance of lineage I among Trypanosoma cruzi isolates from Venezuelan patients with different clinical profiles of acute Chagas' disease. Trop Med Int Health. 9(12):1319-26. 8. Aprigliano O, Masuda MO, Meirelles MN, Pereira MC, Barbosa HS, Barbosa JC. 1993. Heart muscle cells acutely infected with Trypanosoma cruzi: characterization of electrophysiology and neurotransmitter responses. J Mol Cell Cardiol. 25(10):1265-74. 9. Araújo-Jorge TC, Waghabi MC, Hasslocher-Moreno AM, Xavier SS, Higuchi Mde L, Keramidas M, Bailly S, Feige JJ. 2002. Implication of transforming growth factor-beta1 in Chagas disease myocardiopathy. J Infect Dis. 15;186(12):1823-8. 10.Araújo-Jorge TC, Waghabi MC, Soeiro Mde N, Keramidas M, Bailly S, Feige JJ. 2008. Pivotal role for TGF-beta in infectious heart disease: The case of Trypanosoma cruzi infection and consequent Chagasic myocardiopathy. Cytokine Growth Factor Rev. 19(5-6):405-13. Referências Bibliográficas 91 11.Balijepalli RC, Foell JD, Hall DD, Hell JW, Kamp TJ. 2006. Localization of cardiac L-type Ca(2+) channels to a caveolar macromolecular signaling complex is required for beta(2)-adrenergic regulation. Proc Natl Acad Sci U S A. 9;103(19):7500-5. 12.Balijepalli RC, Kamp TJ. 2008. Caveolae, ion channels and cardiac arrhythmias Prog Biophys Mol Biol. 98(2-3):149-60. 13.Barbosa HS, Carvalho EL, Ferreira CF, Meirelles MN. 1993. Trypanosoma cruzi- heart muscle cell interaction: the presence of two or more trypomastigotes within a single endocytic vacuole. J Submicrosc Cytol Pathol. 25(4):613-5. 14.Barbosa HS, de Meirelles Mde N. 1992. Ultrastructural detection in vitro of WGA-, RCA I-, and Con A-binding sites involved in the invasion of heart muscle cells by Trypanosoma cruzi. Parasitol Res. 78(5):404-9. 15.Barbosa HS, Meirelles MN. 1995. Evidence of participation of cytoskeleton of heart muscle cells during the invasion of Trypanosoma cruzi. Cell Struct Funct. 20(4):275-84. 16.Barr SC, Han W, Andrews NW, Lopez JW, Ball BA, Pannabecker TL, Gilmour RF. 1996. A factor from Trypanosoma cruzi induces repetitive cytosolic free Ca2+ transients in isolated primary canine cardiac myocytes. Infect Immun. 64(5):1770- 7. 17.Barrias ES, Dutra JM, De Souza W, Carvalho TM. 2007. Participation of macrophage membrane rafts in Trypanosoma cruzi invasion process. Biochem Biophys Res Commun. 23;363(3):828-34. 18.Beardslee MA, Laing JG, Beyer EC, Saffitz JE. 1998. Rapid turnover of connexin43 in the adult rat heart. Circ Res. 21;83(6):629-35. 19.Benaim G, Sanders JM, Garcia-Marchán Y, Colina C, Lira R, Caldera AR, Payares G, Sanoja C, Burgos JM, Leon-Rossell A, Concepcion JL, Schijman AG, Levin M, Oldfield E, Urbina JA. 2006. Amiodarone has intrinsic anti-Trypanosoma cruzi activity and acts synergistically with posaconazole. J Med Chem. 9;49(3):892-9. 20.Bénard J, Riou G, Saucier JM. 1979. Characterization by sedimentation analysis of kinetoplast DNA from Trypanosoma cruzi at different stages of culture. Nucleic Acids Res. 6(5):1941-52. Referências Bibliográficas 92 21.Benchimol Barbosa PR. 2006. The oral transmission of Chagas' disease: an acute form of infection responsible for regional outbreaks. Int J Cardiol. 10;112(1):132-3. 22.Bergdolt BA, Tanowitz HB, Wittner M, Morris SA, Bilezikian JP, Moreno AP, Spray DC. 1994. Trypanosoma cruzi: effects of infection on receptor-mediated chronotropy and Ca2+ mobilization in rat cardiac myocytes. Exp Parasitol. 78(2):149-60. 23.Berra HH, Piaggio E, Revelli SS, Luquita A. 2005. Blood viscosity changes in experimentally Trypanosoma cruzi-infected rats. Clin Hemorheol Microcirc. 32(3):175-82. 24.Borges CR, Rodrigues Junior V, dos Reis MA, Castellano LR, Chica JE, Pereira SA, Santos ES, Rodrigues DB. 2009. Role of nitric oxide in the development of cardiac lesions during the acute phase of experimental infection by Trypanosoma cruzi. Rev Soc Bras Med Trop. 42(2):170-4. 25.Bouzahzah B, Jelicks LA, Morris SA, Weiss LM, Tanowitz HB. 2005. Risedronate in the treatment of Murine Chagas' disease. Parasitol Res. 96(3):184-7. 26.Britto C, Cardoso MA, Vanni CM, Hasslocher-Moreno A, Xavier SS, Oelemann W, Santoro A, Pirmez C, Morel CM, Wincker P. 1995. Polymerase chain reaction detection of Trypanosoma cruzi in human blood samples as a tool for diagnosis and treatment evaluation. Parasitology. 110 ( Pt 3):241-7. 27.Caler EV, Vaena de Avalos S, Haynes PA, Andrews NW, Burleigh BA. 1998. Oligopeptidase B-dependent signaling mediates host cell invasion by Trypanosoma cruzi. EMBO J. 1;17(17):4975-86. 28.Campos de Carvalho AC, Goldenberg RC, Jelicks LA, Soares MB, Dos Santos RR, Spray DC, Tanowitz HB. 2009. Cell Therapy in Chagas Disease Interdiscip Perspect Infect Dis. 2009:484358. 29.Campos de Carvalho AC, Roy C, Hertzberg EL, Tanowitz HB, Kessler JA, Weiss LM, Wittner M, Dermietzel R, Gao Y, Spray DC. 1998. Gap junction disappearance in astrocytes and leptomeningeal cells as a consequence of protozoan infection. Brain Res. 20;790(1-2):304-14. Referências Bibliográficas 93 30.Carvalho TM, De Souza W, Coimbra ES. 1999. Internalization of components of the host cell plasma membrane during infection by Trypanosoma cruzi. Mem Inst Oswaldo Cruz. 94 Suppl 1:143-7. 31.Celes MR, Torres-Dueñas D, Alves-Filho JC, Duarte DB, Cunha FQ, Rossi MA. 2007. Reduction of gap and adherens junction proteins and intercalated disc structural remodeling in the hearts of mice submitted to severe cecal ligation and puncture sepsis. Crit Care Med. 2007 35(9):2176-85. 32.Chagas, C. 1909. Nova tripanozomiase humana. Estudos sobre a morfolojia e o ciclo evolutivo do Schizotrypanum cruzi n.g., n.s.p., ajente etiolójico de nova entidade mórbida no homem. Mem. Inst. Oswaldo Cruz 1: 159-218, 1909. 33.Chandrasekar B, Melby PC, Pennica D, Freeman GL. 1998. Overexpression of cardiotrophin-1 and gp130 during experimental acute Chagasic cardiomyopathy. Immunol Lett. 61(2-3):89-95. 34.Chang WJ, Rothberg KG, Kamen BA, Anderson RG. 1992. Lowering the cholesterol content of MA104 cells inhibits receptor-mediated transport of folate. J Cell Biol. 118(1):63-9. 35.Chang WJ, Ying YS, Rothberg KG, Hooper NM, Turner AJ, Gambliel HA, De Gunzburg J, Mumby SM, Gilman AG, Anderson RG. 1994. Purification and characterization of smooth muscle cell caveolae. J Cell Biol. 126(1):127-38. 36.Chessler AD, Unnikrishnan M, Bei AK, Daily JP, Burleigh BA. 2009. Trypanosoma cruzi triggers an early type I IFN response in vivo at the site of intradermal infection. J Immunol. 182(4):2288-96. 37.Combs TP, Nagajyothi, Mukherjee S, de Almeida CJ, Jelicks LA, Schubert W, Lin Y, Jayabalan DS, Zhao D, Braunstein VL, Landskroner-Eiger S, Cordero A, Factor SM, Weiss LM, Lisanti MP, Tanowitz HB, Scherer PE. 2005. The adipocyte as an important target cell for Trypanosoma cruzi infection. J Biol Chem. 280(25):24085-94. 38.Corrêa AF, Andrade LR, Soares MJ. 2002. Elemental composition of acidocalcisomes of Trypanosoma cruzi bloodstream trypomastigote forms. Parasitol Res. 88(10):875-80. 39.Costa PC, Fortes FS, Machado AB, Almeida NA, Olivares EL, Cabral PR, Pedrosa RC, Goldenberg RC, Campos-De-Carvalho AC, Masuda MO. 2000. Referências Bibliográficas 94 Sera from chronic chagasic patients depress cardiac electrogenesis and conduction. Braz J Med Biol Res. 33(4):439-46. 40.Costales JA, Daily JP, Burleigh BA. 2009. Cytokine-dependent and-independent gene expression changes and cell cycle block revealed in Trypanosoma cruzi- infected host cells by comparative mRNA profiling. BMC Genomics. 29;10:252. 41.Cottrell GT, Burt JM. 2005. Functional consequences of heterogeneous gap junction channel formation and its influence in health and disease. Biochim Biophys Acta. 10;1711(2):126-41. 42.Coura JR, de Castro SL. 2002. A critical review on Chagas disease chemotherapy. Mem Inst Oswaldo Cruz. 97(1):3-24. 43.Cronk LB, Ye B, Kaku T, Tester DJ, Vatta M, Makielski JC, Ackerman MJ. 2007. Novel mechanism for sudden infant death syndrome: persistent late sodium current secondary to mutations in caveolin-3. Heart Rhythm. 4(2):161-6. 44.Cunha-e-Silva NL, Atella GC, Porto-Carreiro IA, Morgado-Diaz JA, Pereira MG, De Souza W. 2002. Isolation and characterization of a reservosome fraction from Trypanosoma cruzi. FEMS Microbiol Lett. 27;214(1):7-12. 45.Daleau P. 1998. Effects of antiarrhythmic agents on junctional resistance of guinea pig ventricular cell pairs. J Pharmacol Exp Ther. 284(3):1174-9. 46.Das K, Lewis RY, Scherer PE, Lisanti MP. 1999. The membrane-spanning domains of caveolins-1 and -2 mediate the formation of caveolin hetero- oligomers. Implications for the assembly of caveolae membranes in vivo. J Biol Chem. 25;274(26):18721-8. 47.De Araujo-Jorge TC, Barbosa HS, Meirelles MN. 1992. Trypanosoma cruzi recognition by macrophages and muscle cells: perspectives after a 15-year study. Mem Inst Oswaldo Cruz. 87 Suppl 5:43-56. 48.de Carvalho AC, Tanowitz HB, Wittner M, Dermietzel R, Roy C, Hertzberg EL, Spray DC. 1992. Gap junction distribution is altered between cardiac myocytes infected with Trypanosoma cruzi. Circ Res. 70(4):733-42. 49.de Castro SL, de Meirelles Mde N. 1987. Effect of drugs on Trypanosoma cruzi and on its interaction with heart muscle cell "in vitro". Mem Inst Oswaldo Cruz. 82(2):209-18. Referências Bibliográficas 95 50.De Castro SL, Soeiro MN, De Meirelles Mde N. 1992. Trypanosoma cruzi: effect of phenothiazines on the parasite and its interaction with host cells. Mem Inst Oswaldo Cruz. 87(2):209-15. 51.de Meirelles MN, de Araujo Jorge TC, de Souza W. 1980. Interaction of epimastigote and trypomastigote forms of Trypanosoma cruzi with chicken macrophages in vitro.Parasitology. 81(2):373-81. 52.de Melo TG, Meirelles Mde N, Pereira MC. 2008. Trypanosoma cruzi alters adherens junctions in cardiomyocytes. Microbes Infect. 10(12-13):1405-10. 53.de Souza W, de Carvalho TU, Benchimol M, Chiari E. 1978b. Trypanosoma cruzi: ultrastructural, cytochemical and freeze-fracture studies of protein uptake. Exp Parasitol. 45(1):101-15. 54.De Souza W, Martínez-Palomo A, González-Robles A. 1978a. The cell surface of Trypanosoma cruzi: cytochemistry and freeze-fracture. J Cell Sci. 33:285-99. 55.de Souza W, Rodrigues JC. 2009. Sterol Biosynthesis Pathway as Target for Anti-trypanosomatid Drugs. Interdiscip Perspect Infect Dis. 2009:642502. 56.de Souza W. 2002. Special organelles of some pathogenic protozoa. Parasitol Res. 88(12):1013-25. 57.de Souza W. 2007. Chagas' disease: facts and reality. Microbes Infect. 9(4):544- 5. 58.de Souza W. 2008. Electron microscopy of trypanosomes – A historical review. Mem Inst Oswaldo Cruz 103 (4): 313-325. 59.Deane MP, Lenzi HL, Jansen A. 1984. Trypanosoma cruzi: vertebrate and invertebrate cycles in the same mammal host, the opossum Didelphis marsupialis. Mem Inst Oswaldo Cruz. 79(4):513-5. 60.Del Castillo M, Mendoza G, Oviedo J, Perez Bianco RP, Anselmo AE, Silva M. 1990. AIDS and Chagas' disease with central nervous system tumor-like lesion. Am J Med. 88(6):693-4. 61.Dias JCP. 2000. Epidemiologia. In Z Brener, Z Andrade, M Barral-Netto (eds), Trypanosoma cruzi e Doença de Chagas, 2a ed., Guanabara Koogan, Rio de Janeiro, p. 48-74. 62.Docampo R, Scott DA, Vercesi AE, Moreno SN. 1995. Intracellular Ca2+ storage in acidocalcisomes of Trypanosoma cruzi. Biochem J. 15;310 ( Pt 3):1005-12 Referências Bibliográficas 96 63.Dorn GW 2nd, Force T. 2005. Protein kinase cascades in the regulation of cardiac hypertrophy. J Clin Invest. 115(3):527-37. 64.Enders JE, Fernández AR, Rivarola HW, Paglini PA, Palma JA. 2004. Studies of membrane fluidity and heart contractile force in Trypanosoma cruzi infected mice. Mem Inst Oswaldo Cruz. 99(7):691-6. 65.Engel JC, Doyle PS, Hsieh I, McKerrow JH. 1988. Cysteine protease inhibitors cure an experimental Trypanosoma cruzi infection. J Exp Med. 17;188(4):725-34. 66.Engelman JA, Chu C, Lin A, Jo H, Ikezu T, Okamoto T, Kohtz DS, Lisanti MP. 1998. Caveolin-mediated regulation of signaling along the p42/44 MAP kinase cascade in vivo. A role for the caveolin-scaffolding domain. FEBS Lett. 29;428(3):205-11. 67.Espinoza B, Rico T, Sosa S, Oaxaca E, Vizcaino-Castillo A, Caballero ML, Martínez I. 2010. Mexican Trypanosoma cruzi T. cruzi I strains with different degrees of virulence induce diverse humoral and cellular immune responses in a murine experimental infection model. J Biomed Biotechnol. 2010:890672. 68.Fernandes MC, Cortez M, Geraldo Yoneyama KA, Straus AH, Yoshida N, Mortara RA. 2007. Novel strategy in Trypanosoma cruzi cell invasion: implication of cholesterol and host cell microdomains. Int J Parasitol. 37(13):1431-41. 69.Fernandes O, Santos SS, Cupolillo E, Mendonça B, Derre R, Junqueira AC, Santos LC, Sturm NR, Naiff RD, Barret TV, Campbell DA, Coura JR. 2001. A mini-exon multiplex polymerase chain reaction to distinguish the major groups of Trypanosoma cruzi and T. rangeli in the Brazilian Amazon. Trans R Soc Trop Med Hyg. 95(1):97-9. 70.Filardi LS, Brener Z. 1987. Susceptibility and natural resistance of Trypanosoma cruzi strains to drugs used clinically in Chagas disease. Trans R Soc Trop Med Hyg. 81(5):755-9. 71.Fonseca SG, Moins-Teisserenc H, Clave E, Ianni B, Nunes VL, Mady C, Iwai LK, Sette A, Sidney J, Marin ML, Goldberg AC, Guilherme L, Charron D, Toubert A, Kalil J, Cunha-Neto E. 2005. Identification of multiple HLA-A*0201-restricted cruzipain and FL-160 CD8+ epitopes recognized by T cells from chronically Trypanosoma cruzi-infected patients. Microbes Infect. 7(4):688-97. Referências Bibliográficas 97 72.Frank JS, Langer GA, Nudd LM, Seraydarian K. 19n77. The myocardial cell surface, its histochemistry, and the effect of sialic acid and calcium removal on its stucture and cellular ionic exchange. Circ Res. 41(5):702-14. 73.Fürstenau M, Löhn M, Ried C, Luft FC, Haller H, Gollasch M. 2000. Calcium sparks in human coronary artery smooth muscle cells resolved by confocal imaging. J Hypertens. 18(9):1215-22. 74.Galbiati F, Engelman JA, Volonte D, Zhang XL, Minetti C, Li M, Hou H Jr, Kneitz B, Edelmann W, Lisanti MP. 2001. Caveolin-3 null mice show a loss of caveolae, changes in the microdomain distribution of the dystrophin-glycoprotein complex, and t-tubule abnormalities. J Biol Chem. 15;276(24):21425-33. 75.Galvão LM, Chiari E, Macedo AM, Luquetti AO, Silva SA, Andrade AL. 2003. PCR assay for monitoring Trypanosoma cruzi parasitemia in childhood after specific chemotherapy. J Clin Microbiol. 41(11):5066-70. 76.Garcia S, Ramos CO, Senra JF, Vilas-Boas F, Rodrigues MM, Campos-de- Carvalho AC, Ribeiro-Dos-Santos R, Soares MB. 2005. Treatment with benznidazole during the chronic phase of experimental Chagas' disease decreases cardiac alterations. Antimicrob Agents Chemother. 49(4):1521-8. 77.Garg N, Popov VL, Papaconstantinou J. 2003. Profiling gene transcription reveals a deficiency of mitochondrial oxidative phosphorylation in Trypanosoma cruzi- infected murine hearts: implications in chagasic myocarditis development. Biochim Biophys Acta. 14;1638(2):106-20. 78.Garzoni LR, Adesse D, Soares MJ, Rossi MI, Borojevic R, de Meirelles Mde N. 2008. Fibrosis and hypertrophy induced by Trypanosoma cruzi in a three- dimensional cardiomyocyte-culture system. J Infect Dis. 15;197(6):906-15. 79.Garzoni LR, Caldera A, Meirelles Mde N, de Castro SL, Docampo R, Meints GA, Oldfield E, Urbina JA. 2004a. Selective in vitro effects of the farnesyl pyrophosphate synthase inhibitor risedronate on Trypanosoma cruzi. Int J Antimicrob Agents 23(3):273-85. 80.Garzoni LR, Masuda MO, Capella MM, Lopes AG, de Meirelles Mde N. 2003. Characterization of [Ca2+]i responses in primary cultures of mouse cardiomyocytes induced by Trypanosoma cruzi trypomastigotes. Mem Inst Oswaldo Cruz. 98(4):487-93. Referências Bibliográficas 98 81.Garzoni LR, Rossi MI, de Barros AP, Guarani V, Keramidas M, Balottin LB, Adesse D, Takiya CM, Manso PP, Otazú IB, Meirelles Mde N, Borojevic R. 2009. Dissecting coronary angiogenesis: 3D co-culture of cardiomyocytes with endothelial or mesenchymal cells. Exp Cell Res. 15;315(19):3406-18. 82.Garzoni LR, Waghabi MC, Baptista MM, de Castro SL, Meirelles Mde N, Britto CC, Docampo R, Oldfield E, Urbina JA. 2004b. Antiparasitic activity of risedronate in a murine model of acute Chagas' disease. Int J Antimicrob Agents 23(3):286-90. 83.Giepmans BN, Verlaan I, Hengeveld T, Janssen H, Calafat J, Falk MM, Moolenaar WH. 2001. Gap junction protein connexin-43 interacts directly with microtubules. Curr Biol. 4;11(17):1364-8. 84.Gillespie-Brown J, Fuller SJ, Bogoyevitch MA, Cowley S, Sugden PH. 1995. The mitogen-activated protein kinase kinase MEK1 stimulates a pattern of gene expression typical of the hypertrophic phenotype in rat ventricular cardiomyocytes. J Biol Chem. 24;270(47):28092-6. 85.Goldenberg RC, Jelicks LA, Fortes FS, Weiss LM, Rocha LL, Zhao D, Carvalho AC, Spray DC, Tanowitz HB. 2008. Bone marrow cell therapy ameliorates and reverses chagasic cardiomyopathy in a mouse mode J Infect Dis. 15;197(4):544- 7. 86.Gourdie RG, Ghatnekar GS, O'Quinn M, Rhett MJ, Barker RJ, Zhu C, Jourdan J, Hunter AW. 2006. The unstoppable connexin43 carboxyl-terminus: new roles in gap junction organization and wound healing. Ann N Y Acad Sci. 1080:49-62. 87.Grant IH, Gold JW, Wittner M, Tanowitz HB, Nathan C, Mayer K, Reich L, Wollner N, Steinherz L, Ghavimi F, et al. 1989. Transfusion-associated acute Chagas disease acquired in the United States. Ann Intern Med. 15;111(10):849-51. 88.Gruson D, Bodovitz S. 2010. Rapid emergence of multimarker strategies in laboratory medicine. Biomarkers. 15(4):289-96. 89.Haefliger JA, Meda P. 2000. Chronic hypertension alters the expression of Cx43 in cardiovascular muscle cells. Braz J Med Biol Res. 33(4):431-8. 90.Hardin CD, Vallejo J. 2009. Dissecting the functions of protein-protein interactions: caveolin as a promiscuous partner. Focus on "Caveolin-1 scaffold Referências Bibliográficas 99 domain interacts with TRPC1 and IP3R3 to regulate Ca2+ store release-induced Ca2+ entry in endothelial cells". Am J Physiol Cell Physiol. 296(3):C387-9. 91.Harris TH, Cooney NM, Mansfield JM, Paulnock DM. 2006. Signal transduction, gene transcription, and cytokine production triggered in macrophages by exposure to trypanosome DNA. Infect Immun. 74(8):4530-7. 92.Hassan GS, Mukherjee S, Nagajyothi F, Weiss LM, Petkova SB, de Almeida CJ, Huang H, Desruisseaux MS, Bouzahzah B, Pestell RG, Albanese C, Christ GJ, Lisanti MP, Tanowitz HB. 2006. Trypanosoma cruzi infection induces proliferation of vascular smooth muscle cells. Infect Immun. 74(1):152-9. 93.Higuchi Mde L, Benvenuti LA, Martins Reis M, Metzger M. 2003. Pathophysiology of the heart in Chagas' disease: current status and new developments. Cardiovasc Res. 15;60(1):96-107. 94.Higuchi Mde L, Gutierrez PS, Aiello VD, Palomino S, Bocchi E, Kalil J, Bellotti G, Pileggi F. 1993. Immunohistochemical characterization of infiltrating cells in human chronic chagasic myocarditis: comparison with myocardial rejection process. Virchows Arch A Pathol Anat Histopathol. 423(3):157-60. 95.Higuchi ML, De Morais CF, Pereira Barreto AC, Lopes EA, Stolf N, Bellotti G, Pileggi F. 1987. The role of active myocarditis in the development of heart failure in chronic Chagas' disease: a study based on endomyocardial biopsies. Clin Cardiol. 10(11):665-70. 96.Higuchi ML, Fukasawa S, De Brito T, Parzianello LC, Bellotti G, Ramires JA. 1999. Different microcirculatory and interstitial matrix patterns in idiopathic dilated cardiomyopathy and Chagas' disease: a three dimensional confocal microscopy study. Heart. 82(3):279-85. 97.Hiss FC, Lascala TF, Maciel BC, Marin-Neto JA, Simões MV. 2009. Changes in myocardial perfusion correlate with deterioration of left ventricular systolic function in chronic Chagas' cardiomyopathy. JACC Cardiovasc Imaging. 2(2):164-72. 98.Hoare CA. 1966. The classification of mammalian trypanosomes. Ergeb Mikrobiol Immunitatsforsch Exp Ther. 39:43-57. Referências Bibliográficas 100 99. Imai K, Mimori T, Kawai M, Koga H. 2005. Microarray analysis of host gene- expression during intracellular nests formation of Trypanosoma cruzi amastigotes. Microbiol Immunol. 49(7):623-31. 100. Jeong K, Kwon H, Min C, Pak Y. 2009. Modulation of the caveolin-3 localization to caveolae and STAT3 to mitochondria by catecholamine-induced cardiac hypertrophy in H9c2 cardiomyoblasts. Exp Mol Med. 30;41(4):226-35. 101. Jones EM, Colley DG, Tostes S, Lopes ER, Vnencak-Jones CL, McCurley TL. 1993. Amplification of a Trypanosoma cruzi DNA sequence from inflammatory lesions in human chagasic cardiomyopathy. Am J Trop Med Hyg. 48(3):348-57. 102. Jongsma HJ, Wilders R. 2000. Gap junctions in cardiovascular disease. Circ Res. 23;86(12):1193-7. 103. Kanno S, Saffitz JE. 2001. The role of myocardial gap junctions in electrical conduction and arrhythmogenesis. Cardiovasc Pathol. 10(4):169-77. 104. Kierszenbaum F. 2005. Where do we stand on the autoimmunity hypothesis of Chagas disease? Trends Parasitol. 21(11):513-6. 105. Krajewska WM, Masłowska I. 2004. Caveolins: structure and function in signal transduction. Cell Mol Biol Lett. 9(2):195-220. 106. Kreuzberg MM, Schrickel JW, Ghanem A, Kim JS, Degen J, Janssen- Bienhold U, Lewalter T, Tiemann K, Willecke K. 2006. Connexin30.2 containing gap junction channels decelerate impulse propagation through the atrioventricular node. Proc Natl Acad Sci U S A. 11;103(15):5959-64. 107. Kunisada K, Negoro S, Tone E, Funamoto M, Osugi T, Yamada S, Okabe M, Kishimoto T, Yamauchi-Takihara K. 2000. Signal transducer and activator of transcription 3 in the heart transduces not only a hypertrophic signal but a protective signal against doxorubicin-induced cardiomyopathy. Proc Natl Acad Sci U S A. 4;97(1):315-9. 108. Kunisada K, Tone E, Fujio Y, Matsui H, Yamauchi-Takihara K, Kishimoto T. 1998. Activation of gp130 transduces hypertrophic signals via STAT3 in cardiac myocytes. Circulation. 28;98(4):346-52. 109. Langlois S, Cowan KN, Shao Q, Cowan BJ, Laird DW. 2008. Caveolin-1 and -2 interact with connexin43 and regulate gap junctional intercellular communication in keratinocytes. Mol Biol Cell. 19(3):912-28. Referências Bibliográficas 101 110. Lazardi K, Urbina JA, de Souza W. 1990. Ultrastructural alterations induced by two ergosterol biosynthesis inhibitors, ketoconazole and terbinafine, on epimastigotes and amastigotes of Trypanosoma (Schizotrypanum) cruzi. Antimicrob Agents Chemother. 34(11):2097-105. 111. Le Bouter S, El Harchi A, Marionneau C, Bellocq C, Chambellan A, van Veen T, Boixel C, Gavillet B, Abriel H, Le Quang K, Chevalier JC, Lande G, Léger JJ, Charpentier F, Escande D, Demolombe S. 2004. Long-term amiodarone administration remodels expression of ion channel transcripts in the mouse heart. Circulation. 9;110(19):3028-35. 112. Leite LR, Fenelon G, Paes AT, de Paola AA. 2001. The impact of syncope during clinical presentation of sustained ventricular tachycardia on total and cardiac mortality in patients with chronic Chagasic heart disease. Arq Bras Cardiol. 77(5):439-52. 113. Lin LC, Wu CC, Yeh HI, Lu LS, Liu YB, Lin SF, Lee YT. 2005. Downregulated myocardial connexin 43 and suppressed contractility in rabbits subjected to a cholesterol-enriched diet. Lab Invest. 85(10):1224-37. 114. Lin ZX, Holtzer S, Schultheiss T, Murray J, Masaki T, Fischman DA, Holtzer H. 1989. Polygons and adhesion plaques and the disassembly and assembly of myofibrils in cardiac myocytes. J Cell Biol. 108(6):2355-67. 115. Lisboa CV, Mangia RH, De Lima NR, Martins A, Dietz J, Baker AJ, Ramon-Miranda CR, Ferreira LF, Fernandes O, Jansen AM. 2004. Distinct patterns of Trypanosoma cruzi infection in Leontopithecus rosalia in distinct Atlantic coastal rainforest fragments in Rio de Janeiro--Brazil. Parasitology. 129(Pt 6):703-11. 116. Liu L, Li Y, Lin J, Liang Q, Sheng X, Wu J, Huang R, Liu S, Li Y. 2010. Connexin43 interacts with Caveolin-3 in the heart. Mol Biol Rep. 37(4):1685-91. 117. Löhn M, Fürstenau M, Sagach V, Elger M, Schulze W, Luft FC, Haller H, Gollasch M. 2000. Ignition of calcium sparks in arterial and cardiac muscle through caveolae. Circ Res. 24;87(11):1034-9. 118. Lorenz K, Schmitt JP, Vidal M, Lohse MJ. 2009. Cardiac hypertrophy: targeting Raf/MEK/ERK1/2-signaling. Int J Biochem Cell Biol. 41(12):2351-5. Referências Bibliográficas 102 119. Machado FS, Martins GA, Aliberti JC, Mestriner FL, Cunha FQ, Silva JS. 2000. Trypanosoma cruzi-infected cardiomyocytes produce chemokines and cytokines that trigger potent nitric oxide-dependent trypanocidal activity. Circulation. 12;102(24):3003-8. 120. Marin-Neto JA, Cunha-Neto E, Maciel BC, Simões MV. 2007. Pathogenesis of chronic Chagas heart disease. Circulation. 6;115(9):1109-23. 121. Marin-Neto JA, Rassi A Jr, Avezum A Jr, Mattos AC, Rassi A, Morillo CA, Sosa- Estani S, Yusuf S; BENEFIT Investigators. 2009. The BENEFIT trial: testing the hypothesis that trypanocidal therapy is beneficial for patients with chronic Chagas heart disease. Mem Inst Oswaldo Cruz. 104 Suppl 1:319-24. 122. Marino AP, da Silva A, dos Santos P, Pinto LM, Gazzinelli RT, Teixeira MM, Lannes-Vieira J. 2004. Regulated on activation, normal T cell expressed and secreted (RANTES) antagonist (Met-RANTES) controls the early phase of Trypanosoma cruzi-elicited myocarditis. Circulation. 14;110(11):1443-9. 123. Martin D, Tarleton R. 2004. Generation, specificity, and function of CD8+ T cells in Trypanosoma cruzi infection. Immunol Rev. 201:304-17. 124. Mazza S, Freire R, Urcelay G, Miyara S, Basso G, Basso R, Conte D 1940. Chagomas. MEPRA 46. p. 48-74. 125. Meirelles MN, de Araujo-Jorge TC, Miranda CF, de Souza W, Barbosa HS. 1986. Interaction of Trypanosoma cruzi with heart muscle cells: ultrastructural and cytochemical analysis of endocytic vacuole formation and effect upon myogenesis in vitro. Eur J Cell Biol. 41(2):198-206. 126. Melo TG, Almeida DS, de Meirelles Mde N, Pereira MC. 2004. Trypanosoma cruzi infection disrupts vinculin costameres in cardiomyocytes. Eur J Cell Biol. 83(10):531-40. 127. Melo TG, Almeida DS, Meirelles MN, Pereira MC. 2006. Disarray of sarcomeric alpha-actinin in cardiomyocytes infected by Trypanosoma cruzi. Parasitology. 133(Pt 2):171-8. 128. Meyer H, Oliveira MX. 1948. Cultivation of Trypanosoma cruzi in tissue culture, a four year study. Parasitology 39:91-94. 129. Mielniczki-Pereira A.A., Chiavegatto C.M., López J.A., Colli W., Alves M.J., Gadelha F.R. 2007. Trypanosoma cruzi strains, Tulahuen 2 and Y, besides the Referências Bibliográficas 103 difference in resistance to oxidative stress, display differential glucose-6- phosphate and 6-phosphogluconate dehydrogenases activities. Acta Trop. 101 (1):54-60. 130. Miles MA, Cedillos RA, Póvoa MM, de Souza AA, Prata A, Macedo V. 1981. Do radically dissimilar Trypanosoma cruzi strains (zymodemes) cause Venezuelan and Brazilian forms of Chagas' disease? Lancet. 20;1(8234):1338-40. 131. Momen H. 1999. Taxonomy of Trypanosoma cruzi: a commentary on characterization and nomenclature. Mem Inst Oswaldo Cruz. 94 Suppl 1:181-4. 132. Morris SA, Bilezikian JP, Tanowitz H, Wittner M. 1987. Infection of L6E9 myoblasts with Trypanosoma cruzi alters adenylate cyclase activity and guanine nucleotide binding proteins. J Cell Physiol. 133(1):64-71. 133. Morris SA, Weiss LM, Factor S, Bilezikian JP, Tanowitz H, Wittner M. 1989. Verapamil ameliorates clinical, pathologic and biochemical manifestations of experimental chagasic cardiomyopathy in mice. J Am Coll Cardiol. 14(3):782-9. 134. Mukherjee S, Belbin TJ, Spray DC, Iacobas DA, Weiss LM, Kitsis RN, Wittner M, Jelicks LA, Scherer PE, Ding A, Tanowitz HB. 2003. Microarray analysis of changes in gene expression in a murine model of chronic chagasic cardiomyopathy. Parasitol Res. 91(3):187-96. 135. Mukherjee S, Nagajyothi F, Mukhopadhyay A, Machado FS, Belbin TJ, Campos de Carvalho A, Guan F, Albanese C, Jelicks LA, Lisanti MP, Silva JS, Spray DC, Weiss LM, Tanowitz HB. 2008. Alterations in myocardial gene expression associated with experimental Trypanosoma cruzi infection. Genomics. 91(5):423-32. 136. Nagajyothi F, Desruisseaux M, Bouzahzah B, Weiss LM, Andrade Ddos S, Factor SM, Scherer PE, Albanese C, Lisanti MP, Tanowitz HB. 2006. Cyclin and caveolin expression in an acute model of murine Chagasic myocarditis. Cell Cycle. 5(1):107-12. 137. Nagajyothi F, Desruisseaux MS, Thiruvur N, Weiss LM, Braunstein VL, Albanese C, Teixeira MM, de Almeida CJ, Lisanti MP, Scherer PE, Tanowitz HB. 2008. Trypanosoma cruzi infection of cultured adipocytes results in an inflammatory phenotype. Obesity (Silver Spring). 16(9):1992-7 Referências Bibliográficas 104 138. Nunes Mdo C, Barbosa Mde M, Rocha ES, Rocha MO. 2005. Function of the left atrium in Chagas' cardiomyopathy. Arq Bras Cardiol. 84(6):452-6. 139. Olivieri BP, Molina JT, de Castro SL, Pereira MC, Calvet CM, Urbina JA, Araújo-Jorge TC. 2010. A comparative study of posaconazole and benznidazole in the prevention of heart damage and promotion of trypanocidal immune response in a murine model of Chagas disease. Int J Antimicrob Agents. 36(1):79-83. 140. Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B, Pickel J, McKay R, Nadal-Ginard B, Bodine DM, Leri A, Anversa P. 2001. Bone marrow cells regenerate infarcted myocardium. Nature. 5;410(6829):701-5. 141. Osuna A, Castanys S, Rodriguez-Cabezas MN, Gamarro F. 1990. Trypanosoma cruzi: calcium ion movement during internalization in host HeLa cells. Int J Parasitol. 20(5):673-6. 142. Paiva CN, Figueiredo RT, Kroll-Palhares K, Silva AA, Silvério JC, Gibaldi D, Pyrrho Ados S, Benjamim CF, Lannes-Vieira J, Bozza MT. 2009. CCL2/MCP-1 controls parasite burden, cell infiltration, and mononuclear activation during acute Trypanosoma cruzi infection. J Leukoc Biol. 86(5):1239-46. 143. Palade GE. 1953. An electron microscope study of the mitochondrial structure. J Histochem Cytochem. 1(4):188-211. 144. Park DS, Woodman SE, Schubert W, Cohen AW, Frank PG, Chandra M, Shirani J, Razani B, Tang B, Jelicks LA, Factor SM, Weiss LM, Tanowitz HB, Lisanti MP. 2002. Caveolin-1/3 double-knockout mice are viable, but lack both muscle and non-muscle caveolae, and develop a severe cardiomyopathic phenotype. Am J Pathol. 160(6):2207-17. 145. Parton RG, Way M, Zorzi N, Stang E. 1997. Caveolin-3 associates with developing T-tubules during muscle differentiation. J Cell Biol. 136: 137–154. 146. Pellegrino J. 1949. Transmissão da doença de Chagas pela transfusão de sangue. Primeiras comprovações sorológicas em doadores e em candidatos a doadores de sangue. Rev Bras Med 6: 297-301. 147. Pereira MC, Costa M, Chagas Filho C, de Meirelles MN. 1993. Myofibrillar breakdown and cytoskeletal alterations in heart muscle cells during invasion by Referências Bibliográficas 105 Trypanosoma cruzi: immunological and ultrastructural study. J Submicrosc Cytol Pathol. 25(4):559-69. 148. Pereira MC, Singer RH, de Meirelles MN. 2000. Trypanosoma cruzi infection affects actin mRNA regulation in heart muscle cells. J Eukaryot Microbiol. 47(3):271-9. 149. Petersen CA, Burleigh BA. 2003. Role for interleukin-1 beta in Trypanosoma cruzi-induced cardiomyocyte hypertrophy. Infect Immun. 71(8):4441-7. 150. Pinho AP, Cupolillo E, Mangia RH, Fernandes O, Jansen AM. 2000. Trypanosoma cruzi in the sylvatic environment: distinct transmission cycles involving two sympatric marsupials. Trans R Soc Trop Med Hyg. 94(5):509-14. 151. Punukollu G, Gowda RM, Khan IA, Navarro VS, Vasavada BC. 2007. Clinical aspects of the Chagas' heart disease. Int J Cardiol. 14;115(3):279-83. 152. Razani B, Wang XB, Engelman JA, Battista M, Lagaud G, Zhang XL, Kneitz B, Hou H Jr, Christ GJ, Edelmann W, Lisanti MP. 2002. Caveolin-2-deficient mice show evidence of severe pulmonary dysfunction without disruption of caveolae. Mol Cell Biol. 22(7):2329-44. 153. Reis MM, Higuchi Mde L, Benvenuti LA, Aiello VD, Gutierrez PS, Bellotti G, Pileggi F. 1997. An in situ quantitative immunohistochemical study of cytokines and IL-2R+ in chronic human chagasic myocarditis: correlation with the presence of myocardial Trypanosoma cruzi antigens. Clin Immunol Immunopathol. 83(2):165-72. 154. Revollo S, Oury B, Laurent JP, Barnabé C, Quesney V, Carrière V, Noël S, Tibayrenc M. 1998. Trypanosoma cruzi: impact of clonal evolution of the parasite on its biological and medical properties. Exp Parasitol. 89(1):30-9. 155. Rey, L. 2008. Parasitologia: parasitos e doenças parasitárias do homem nos trópicos ocidentais. Rio de Janeiro: Guanabara Koogan. 156. Ribeiro dos Santos R, Marquez JO, Von Gal Furtado CC, Ramos de Oliveira JC, Martins AR, Köberle F. 1979. Antibodies against neurons in chronic Chagas' disease. Tropenmed Parasitol. 30(1):19-23. 157. Rivera J, Hillis LD, Levine BD. 2004. Reactivation of cardiac Chagas' disease in acquired immune deficiency syndrome. Am J Cardiol. 15;94(8):1102-3. Referências Bibliográficas 106 158. Rocha GM, Brandao BA, Mortara RA, Attias M, de Souza W, Carvalho TM. 2006. The flagellar attachment zone of Trypanosoma cruzi epimastigote forms. J Struct Biol. 154(1):89-99. 159. Rodríguez A, Rioult MG, Ora A, Andrews NW. 1995. A trypanosome- soluble factor induces IP3 formation, intracellular Ca2+ mobilization and microfilament rearrangement in host cells. J Cell Biol. 129(5):1263-73. 160. Rodríguez A, Samoff E, Rioult MG, Chung A, Andrews NW. 1996. Host cell invasion by trypanosomes requires lysosomes and microtubule/kinesin-mediated transport. J Cell Biol. 134(2):349-62. 161. Roffê E, Oliveira F, Souza AL, Pinho V, Souza DG, Souza PR, Russo RC, Santiago HC, Romanha AJ, Tanowitz HB, Valenzuela JG, Teixeira MM. 2010. Role of CCL3/MIP-1alpha and CCL5/RANTES during acute Trypanosoma cruzi infection in rats. Microbes Infect. 12(8-9):669-76. 162. Roman-Campos D, Duarte HL, Sales PA Jr, Natali AJ, Ropert C, Gazzinelli RT, Cruz JS. 2009. Changes in cellular contractility and cytokines profile during Trypanosoma cruzi infection in mice. Basic Res Cardiol. 104(3):238-46. 163. Romanha AJ, Castro SL, Soeiro Mde N, Lannes-Vieira J, Ribeiro I, Talvani A, Bourdin B, Blum B, Olivieri B, Zani C, Spadafora C, Chiari E, Chatelain E, Chaves G, Calzada JE, Bustamante JM, Freitas-Junior LH, Romero LI, Bahia MT, Lotrowska M, Soares M, Andrade SG, Armstrong T, Degrave W, Andrade Zde A. 2010. In vitro and in vivo experimental models for drug screening and development for Chagas disease. Mem Inst Oswaldo Cruz. 105(2):233-8. 164. Rothberg KG, Heuser JE, Donzell WC, Ying YS, Glenney JR, Anderson RG. 1992. Caveolin, a protein component of caveolae membrane coats. Cell. 21;68(4):673-82. 165. Rowin KS, Tanowitz HB, Wittner M, Nguyen HT, Nadal-Ginard B. Inhibition of muscle differentiation by trypanosoma cruzi. 1983. Proc Natl Acad Sci U S A. 80(20):6390-4. 166. Sanchez G., Wallace A., Olivares M., Diaz N., Aguilera X., Apt W., Solari A. 1990. Biological characterization of Trypanosoma cruzi zymodemes: in vitro differentiation of epimastigotes and infectivity of culture metacyclic trypomastigotes to mice. Exp Parasitol. 71 (1):125-133. Referências Bibliográficas 107 167. Sano M, Fukuda K, Kodama H, Pan J, Saito M, Matsuzaki J, Takahashi T, Makino S, Kato T, Ogawa S. 2000. Interleukin-6 family of cytokines mediate angiotensin II-induced cardiac hypertrophy in rodent cardiomyocytes. J Biol Chem. 22;275(38):29717-23. 168. Sargiacomo M, Sudol M, Tang Z, Lisanti MP. 1993. Signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells. J Cell Biol. 122(4):789-807. 169. Schenkman S, Eichinger D, Pereira ME, Nussenzweig V. 1994. Structural and functional properties of Trypanosoma trans-sialidase. Annu Rev Microbiol. 48:499-523. 170. Scherer PE, Lewis RY, Volonte D, Engelman JA, Galbiati F, Couet J, Kohtz DS, van Donselaar E, Peters P, Lisanti MP. 1997. Cell-type and tissue-specific expression of caveolin-2. J Biol Chem. 14;272(46):29337-46. 171. Schettino PM, Majumder S, Kierszenbaum F. 1995. Regulatory effect of the level of free Ca2+ of the host cell on the capacity of Trypanosoma cruzi to invade and multiply intracellularly. J Parasitol. 81(4):597-602. 172. Schofield CJ, Jannin J, Salvatella R. 2006. The future of Chagas disease control. Trends Parasitol. 22(12):583-8. 173. Schwab W, Galbiati F, Volonte D, Hempel U, Wenzel KW, Funk RH, Lisanti MP, Kasper M. 1999. Characterisation of caveolins from cartilage: expression of caveolin-1, -2 and -3 in chondrocytes and in alginate cell culture of the rat tibia. Histochem Cell Biol. 112(1):41-9. 174. Serrano-Martín X, García-Marchan Y, Fernandez A, Rodriguez N, Rojas H, Visbal G, Benaim G. 2009b. Amiodarone destabilizes intracellular Ca2+ homeostasis and biosynthesis of sterols in Leishmania mexicana. Antimicrob Agents Chemother 53(4):1403-10. 175. Serrano-Martín X, Payares G, De Lucca M, Martinez JC, Mendoza-León A, Benaim G. 2009a. Amiodarone and miltefosine act synergistically against Leishmania mexicana and can induce parasitological cure in a murine model of cutaneous leishmaniasis. Antimicrob Agents Chemother. 53(12):5108-13. Referências Bibliográficas 108 176. Severs NJ, Dupont E, Coppen SR, Halliday D, Inett E, Baylis D, Rothery S. 2004. Remodelling of gap junctions and connexin expression in heart disease. Biochim Biophys Acta. 23;1662(1-2):138-48. 177. Shaw RM, Fay AJ, Puthenveedu MA, von Zastrow M, Jan YN, Jan LY. 2007. Microtubule plus-end-tracking proteins target gap junctions directly from the cell interior to adherens junctions. Cell. 9;128(3):547-60. 178. Sheng Z, Knowlton K, Chen J, Hoshijima M, Brown JH, Chien KR. 1997. Cardiotrophin 1 (CT-1) inhibition of cardiac myocyte apoptosis via a mitogen- activated protein kinase-dependent pathway. Divergence from downstream CT-1 signals for myocardial cell hypertrophy. J Biol Chem. 28;272(9):5783-91. 179. Shigihara T, Hashimoto M, Shindo N, Aoki T. Transcriptome profile of 2008. Trypanosoma cruzi-infected cells: simultaneous up- and down-regulation of proliferation inhibitors and promoters. Parasitol Res. 102(4):715-22. 180. Silva DT, de Nazareth S L de Meirelles M, Almeida D, Urbina JA, Pereira MC. 2006. Cytoskeleton reassembly in cardiomyocytes infected by Trypanosoma cruzi is triggered by treatment with ergosterol biosynthesis inhibitors. Int J Antimicrob Agents. 27(6):530-7. 181. Silva NN, Kuhn G, Santos JFC, Von Eye G, Chaer JAB. 1974. Eficácia e tolerância do nitrofurfurilidene na fase crônica da moléstia de Chagas. Rev. Soc. Bras. Med. Trop. 8: 325-334. 182. Simões MV, Soares FA, Marin-Neto JA. 1995. Severe myocarditis and esophagitis during reversible long standing Chagas' disease recrudescence in immunocompromised host. Int J Cardiol. 49(3):271-3. 183. Smart EJ, Graf GA, McNiven MA, Sessa WC, Engelman JA, Scherer PE, Okamoto T, Lisanti MP. 1999. Caveolins, liquid-ordered domains, and signal transduction. Mol Cell Biol. 19(11):7289-304. 184. Smyth JW, Hong TT, Gao D, Vogan JM, Jensen BC, Fong TS, Simpson PC, Stainier DY, Chi NC, Shaw RM. 2010. Limited forward trafficking of connexin 43 reduces cell-cell coupling in stressed human and mouse myocardium. J Clin Invest. 4;120(1):266-79. Referências Bibliográficas 109 185. Soares MB, de Lima RS, Rocha LL, Vasconcelos JF, Rogatto SR, dos Santos RR, Iacobas S, Goldenberg RC, Iacobas DA, Tanowitz HB, de Carvalho AC, Spray DC. 2010. Gene expression changes associated with myocarditis and fibrosis in hearts of mice with chronic chagasic cardiomyopathy. J Infect Dis. 15;202(3):416-26. 186. Soares MB, Lima RS, Rocha LL, Takyia CM, Pontes-de-Carvalho L, de Carvalho AC, Ribeiro-dos-Santos R. 2004. Transplanted bone marrow cells repair heart tissue and reduce myocarditis in chronic chagasic mice. Am J Pathol. 164(2):441-7. 187. Soares MB, Santos RR. Immunopathology of cardiomyopathy in the experimental Chagas disease. 1999. Mem Inst Oswaldo Cruz. 94 Suppl 1:257-62. 188. Soares MJ, de Souza W. 1991. Endocytosis of gold-labeled proteins and LDL by Trypanosoma cruzi. Parasitol Res. 77(6):461-8. 189. Soares MJ. 1999. The reservosome of Trypanosoma cruzi epimastigotes: an organelle of the endocytic pathway with a role on metacyclogenesis. Mem Inst Oswaldo Cruz. 94 Suppl 1:139-41. 190. Soeiro Mde N, Costa e Silva Filho F, Leal de Meirelles Mde N. 1995. Alterations in the surface charge of heart muscle cells during interaction with Trypanosoma cruzi. Cell Biophys. 26(1):21-44. 191. Soeiro Mde N, Mota RA, Batista Dda G, Meirelles Mde N. 2002. Endocytic pathway in mouse cardiac cells. Cell Struct Funct. 27(6):469-78. 192. Solan JL, Lampe PD. 2005. Connexin phosphorylation as a regulatory event linked to gap junction channel assembly. Biochim Biophys Acta. 10;1711(2):154-63. 193. Song KS, Scherer PE, Tang Z, Okamoto T, Li S, Chafel M, Chu C, Kohtz DS, Lisanti MP. 1996. Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells. Caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins. J Biol Chem. 21;271(25):15160-5. 194. Spray DC, Burt JM. 1990. Structure-activity relations of the cardiac gap junction channel. Am J Physiol. 258(2 Pt 1):C195-205. Referências Bibliográficas 110 195. Spray DC, Tanowitz HB. 2007. Pathology of mechanical and gap junctional co- coupling at the intercalated disc: Is sepsis a junctionopathy? Crit Care Med. 35(9):2231-2. 196. Suzuki S, Sugi H. 1989. Evidence for extracellular localization of activator calcium in dog coronary artery smooth muscle as studied by the pyroantimonate method. Cell Tissue Res. 257(2):237-46. 197. Takeo S, Elmoselhi AB, Goel R, Sentex E, Wang J, Dhalla NS. 2000. Attenuation of changes in sarcoplasmic reticular gene expression in cardiac hypertrophy by propranolol and verapamil. Mol Cell Biochem. 213(1-2):111-8. 198. Tang Z, Scherer PE, Okamoto T, Song K, Chu C, Kohtz DS, Nishimoto I, Lodish HF, Lisanti MP. 1996. Molecular cloning of caveolin-3, a novel member of the caveolin gene family expressed predominantly in muscle. J Biol Chem. 26;271(4):2255-61. 199. Taniwaki NN, Andreoli WK, Calabrese KS, da Silva S, Mortara RA. 2005. Disruption of myofibrillar proteins in cardiac muscle of Calomys callosus chronically infected with Trypanosoma cruzi and treated with immunosuppressive agent. Parasitol Res. 97(4):323-31. 200. Taniwaki NN, Machado FS, Massensini AR, Mortara RA. 2006. Trypanosoma cruzi disrupts myofibrillar organization and intracellular calcium levels in mouse neonatal cardiomyocytes. Cell Tissue Res. 324(3):489-96. 201. Tanowitz HB, Burns ER, Sinha AK, Kahn NN, Morris SA, Factor SM, Hatcher VB, Bilezikian JP, Baum SG, Wittner M. 1990. Enhanced platelet adherence and aggregation in Chagas' disease: a potential pathogenic mechanism for cardiomyopathy. Am J Trop Med Hyg. 43(3):274-81. 202. Tanowitz HB, Kaul DK, Chen B, Morris SA, Factor SM, Weiss LM, Wittner M. 1996. Compromised microcirculation in acute murine Trypanosoma cruzi infection. J Parasitol. 82(1):124-30. 203. Tanowitz HB, Kirchhoff LV, Simon D, Morris SA, Weiss LM, Wittner M. 1992. Chagas' disease. Clin Microbiol Rev. 5(4):400-19. 204. Tardieux I, Webster P, Ravesloot J, Boron W, Lunn JA, Heuser JE, Andrews NW. 1992. Lysosome recruitment and fusion are early events required for trypanosome invasion of mammalian cells. Cell. 24;71(7):1117-30. Referências Bibliográficas 111 205. Tarleton RL. 2001. Parasite persistence in the aetiology of Chagas disease. Int J Parasitol. 1;31(5-6):550-4. 206. Teunissen BE, Jansen AT, Mutsaers NA, Vuerhard MJ, Vos MA, Bierhuizen MF. 2007. Primary structure, organization, and expression of the rat connexin45 gene. DNA Cell Biol. 26(2):108-15. 207. Tourkina E, Richard M, Gööz P, Bonner M, Pannu J, Harley R, Bernatchez PN, Sessa WC, Silver RM, Hoffman S. 2008. Antifibrotic properties of caveolin-1 scaffolding domain in vitro and in vivo. Am J Physiol Lung Cell Mol Physiol. 294(5):L843-61. 208. Unger VM, Kumar NM, Gilula NB, Yeager M. 1999. Three-dimensional structure of a recombinant gap junction membrane channel. Science. 19;283(5405):1176-80. 209. Urban M, Rozental R, Spray DC. 1999. A simple RT-PCR-based strategy for screening connexin identity. Braz J Med Biol Res. 32(8):1029-37. 210. Urbina JA, Docampo R. 2003. Specific chemotherapy of Chagas disease: controversies and advances. Trends Parasitol. 19(11):495-501. 211. Urbina JA. 2002. Chemotherapy of Chagas disease. Curr Pharm Des. 2002;8(4):287-95. 212. Urbina JA. 2009. Ergosterol biosynthesis and drug development for Chagas disease. Mem Inst Oswaldo Cruz. 104 Suppl 1:311-8. 213. Urbina JA. 2010. Specific chemotherapy of Chagas disease: relevance, current limitations and new approaches. Acta Trop. 115(1-2):55-68. 214. Vaena de Avalos S, Blader IJ, Fisher M, Boothroyd JC, Burleigh BA. 2002. Immediate/early response to Trypanosoma cruzi infection involves minimal modulation of host cell transcription. J Biol Chem. 4;277(1):639-44. 215. Vaidian AK, Weiss LM, Tanowitz HB. 2004. Chagas' disease and AIDS. Kinetoplastid Biol Dis. 13;3(1):2. 216. Valente N, Pimenta J, Paola AA. 2006. Serial electrophysiological studies of the heart's excito conductor system in patients with chronic chagasic cardiopathy. Arq Bras Cardiol. 86(1):19-25. 217. Vatta M, Ackerman MJ, Ye B, Makielski JC, Ughanze EE, Taylor EW, Tester DJ, Balijepalli RC, Foell JD, Li Z, Kamp TJ, Towbin JA. 2006. Mutant caveolin-3 Referências Bibliográficas 112 induces persistent late sodium current and is associated with long-QT syndrome. Circulation. 14;114(20):2104-12. 218. Veloso V.M., Carneiro C.M., Toledo M.J., Lana M., Chiari E., Tafuri W.L., Bahia M.T. 2001. Variation in susceptibility to benznidazole in isolates derived from Trypanosoma cruzi parental strains. Mem Inst Oswaldo Cruz. 96 (7):1005-1011. 219. Viotti R, Vigliano C, Lococo B, Bertocchi G, Petti M, Alvarez MG, Postan M, Armenti A. 2006. Long-term cardiac outcomes of treating chronic Chagas disease with benznidazole versus no treatment: a nonrandomized trial. Ann Intern Med. 16;144(10):724-34. 220. Vivas J, Urbina JA, de Souza W. 1996. Ultrastructural alterations in Trypanosoma (Schizotrypanum) cruzi induced by Delta(24(25)) sterol methyl transferase inhibitors and their combinations with ketoconazole. Int J Antimicrob Agents. 7(4):235-40. 221. Volonte D, Peoples AJ, Galbiati F. 2003. Modulation of myoblast fusion by caveolin-3 in dystrophic skeletal muscle cells: Implications for Duchenne muscular dystrophy and limb-girdle muscular dystrophy-1C. Mol Biol Cell. 14: 4075–4088. 222. Waghabi MC, Coutinho-Silva R, Feige JJ, Higuchi Mde L, Becker D, Burnstock G, Araújo-Jorge TC. 2009. Gap junction reduction in cardiomyocytes following transforming growth factor-beta treatment and Trypanosoma cruzi infection. Mem Inst Oswaldo Cruz. 104(8):1083-90. 223. Waghabi MC, Keramidas M, Feige JJ, Araujo-Jorge TC, Bailly S. 2005. Activation of transforming growth factor beta by Trypanosoma cruzi. Cell Microbiol. 7(4):511-7. 224. Wilkinson SR, Kelly JM. 2009. Trypanocidal drugs: mechanisms, resistance and new targets. Expert Rev Mol Med. 29;11:e31. 225. Williams TM, Lisanti MP. 2005. Caveolin-1 in oncogenic transformation, cancer, and metastasis. Am J Physiol Cell Physiol. 288(3):C494-506. 226. Woodman SE, Park DS, Cohen AW, Cheung MW, Chandra M, Shirani J, Tang B, Jelicks LA, Kitsis RN, Christ GJ, Factor SM, Tanowitz HB, Lisanti MP. 2002. Caveolin-3 knock-out mice develop a progressive cardiomyopathy and Referências Bibliográficas 113 show hyperactivation of the p42/44 MAPK cascade. J Biol Chem. 11;277(41):38988-97. 227. Yee HF Jr, Kuwata JH, Langer GA. 1991. Effects of neuraminidase on cellular calcium and contraction in cultured cardiac myocytes. J Mol Cell Cardiol. 23(2):175-85. 228. Yoshida N. 2006. Molecular basis of mammalian cell invasion by Trypanosoma cruzi. An Acad Bras Cienc. 78(1):87-111. 229. Zingales B, Andrade SG, Briones MR, Campbell DA, Chiari E, Fernandes O, Guhl F, Lages-Silva E, Macedo AM, Machado CR, Miles MA, Romanha AJ, Sturm NR, Tibayrenc M, Schijman AG; Second Satellite Meeting. 2009. A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI. Mem Inst Oswaldo Cruz. 104(7):1051-4. 230. Zingales B, Andrade SG, Briones MR, Campbell DA, Chiari E, Fernandes O, Guhl F, Lages-Silva E, Macedo AM, Machado CR, Miles MA, Romanha AJ, Sturm NR, Tibayrenc M, Schijman AG; Second Satellite Meeting. 2009. A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI. Mem Inst Oswaldo Cruz. 104(7):1051-4.