Arq Bras Cardiol abc Arquivos Brasileiros de Cardiologia Arq. Bras. Cardiol. 0066-782X 1678-4170 Sociedade Brasileira de Cardiologia - SBC 4ZCFKWtHGK87XqVTpXWShGp S0066-782X2021000900132 abc.20200330 10.36660/abc.20200330 Artigo de Revisão Treinamento com Exercício Físico e Doença de Chagas: Função Potencial dos MicroRNAs 0000-0002-8779-1188 Improta-Caria Alex Cleber 1 2 Aras Roque Júnior 1 Programa de Pós-Graduação em Medicina e Saúde Faculdade de Medicina Universidade Federal da Bahia Salvador BA Brasil Programa de Pós-Graduação em Medicina e Saúde, Faculdade de Medicina, Universidade Federal da Bahia, Salvador, BA - Brasil Departamento de Educação Física em Cardiologia do Estado da Bahia Sociedade Brasileira de Cardiologia Salvador BA Brasil Departamento de Educação Física em Cardiologia do Estado da Bahia, Sociedade Brasileira de Cardiologia,Salvador, BA - Brasil Correspondência: Alex Cleber Improta-Caria • Universidade Federal da Bahia - Programa de Pós-Graduação em Medicina e Saúde - Rua Dr. Augusto Viana, s/n. CEP 40110-060, Canela, Salvador, BA - Brasil E-mail: alexcaria.personal@hotmail.com

Contribuição dos autores

Concepção e desenho da pesquisa, Obtenção de dados e Redação do manuscrito: Improta-Caria AC; Análise e interpretação dos dados e Revisão crítica do manuscrito quanto ao conteúdo intelectual importante: Improta-Caria AC, Aras Júnior R.

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 26 07 2021 07 2021 117 1 132 141 06 05 2020 02 07 2020 16 08 2020 This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Resumo

A doença de Chagas (DC) é causada pelo Trypanosoma Cruzi. Esse parasita pode infectar vários órgãos do corpo humano, especialmente o coração, causando inflamação, fibrose, arritmias e remodelação cardíaca, e promovendo a cardiomiopatia chagásica crônica (CCC) no longo prazo. Entretanto, poucas evidências científicas elucidaram os mecanismos moleculares que regulam os processos fisiopatológicos nessa doença. Os microRNAs (miRNAs) são reguladores de expressão gênica pós-transcricional que modulam a sinalização celular, participando de mecanismos fisiopatológicos da DC, mas o entendimento dos miRNAs nessa doença é limitado. Por outro lado, há muitas evidências científicas demonstrando que o treinamento com exercício físico (TEF) modula a expressão de miRNAs, modificando a sinalização celular em indivíduos saudáveis. Alguns estudos também demonstram que o TEF traz benefícios para indivíduos com DC, porém esses não avaliaram as expressões de miRNA. Dessa forma, não há evidências demonstrando o papel do TEF na expressão dos miRNAs na DC. Portanto, essa revisão teve o objetivo de identificar os miRNAs expressos na DC que poderiam ser modificados pelo TEF.

 Exercício Físico Doença de Chagas MicroRNAs Coordenação de Aperfeiçoamento de Pessoal de Nível Superior 001 Fontes de financiamento: O presente estudo foi parcialmente financiado pela CAPES. Introdução

A doença de Chagas (DC) é uma doença complexa causada pela infecção por Trypanosoma Cruzi (T. Cruzi), um parasita protozoário flagelado, no nível intracelular.1 Na fase aguda, a infecção por T. Cruzi gera grande inflamação dos tecidos e há uma resposta inicial do sistema imune inato na tentativa de combater a parasitemia.2

Entretanto, a infecção persiste e o sistema imune adaptativo ativa tanto os linfócitos T, como também as células T citotóxicas e auxiliares, que produzem citocinas, tais como o interferon gama (IFN-γ), que podem, por sua vez, levar à morte de parasitas intracelulares ao induzir um aumento nas espécies reativas do oxigênio e nitrogênio, que são microbicidas. Essa infecção também aumenta a expressão do fator de necrose tumoral (TNF-α), bem como anticorpos específicos para combater o T. Cruzi, que controlam o parasitismo, com o estabelecimento de uma infecção de baixo grau.3

Ainda na fase aguda da doença, há um aumento na expressão do peptídeo vasoativo endotelina-1 (ET-1) e da cardiotrofina-1 (CT-1), ambos induzindo a hipertrofia cardíaca, bem como um aumento na expressão da interleucina-1 beta (IL-1β), induzindo uma resposta inflamatória e pró-hipertrófica do miocárdio, o que pode iniciar a hipertrofia até mesmo nesse estágio.4,5

Com o passar dos anos, a parasitemia diminui; entretanto, os antígenos parasíticos persistem, gerando um infiltrado inflamatório difuso e miocardite, com a presença de linfócitos T CD4+ e CD8+ e macrófagos que continuam a expressar TNF-α e IFN-γ.3 O IFN-γ tem a função essencial de controlar e combater parasitas, mas também contribui para a patogênese cardíaca, uma vez que lesiona o miocárdio por vários mecanismos moleculares que geram a disfunção miocárdica.6

Portanto, a doença evolui e passa para a fase crônica, que pode ser subdividida em duas formas: indeterminada e sintomática. Na forma indeterminada, os indivíduos podem passar anos sem manifestar nenhum tipo de sintoma mais sério, já que existe um equilíbrio entre a parasitemia e o sistema imune do hospedeiro. Entretanto, cerca de 30% desses pacientes desenvolvem uma forma sintomática ou determinada, que pode desencadear disfunções em vários órgãos, incluindo o coração, desenvolvendo a cardiomiopatia chagásica crônica (CCC) associada à miocardite e a fibrose miofibrilar cardíaca, e, dessa forma, reduzindo a condutividade elétrica cardíaca, levando à remodelação miocárdica.7

A CCC gera inflamação do tecido cardíaco, causando miocardite focal ou difusa, hipertrofia ou dilatação do ventrículo esquerdo, e morte progressiva de alguns cardiomiócitos, necrose e depósito de colágeno,8 aumentando, assim, o tecido fibrótico, levando à redução de sua capacidade de contração. Esse resultado é geralmente associada a arritmias e insuficiência cardíaca,9 mas é possível que os microRNAs (miRNAs) participem desses mecanismos. Em geral, os mecanismos moleculares que regulam esses processos não são bem entendidos.

MiRNAs são pequenos RNAs com comprimento de apenas 18 a 25 nucleotídeos,10 proteínas não codificantes, e reguladores da expressão gênica pós-transcricional com a função de inibir ou degradar seus genes alvo.11,12 Já se demonstrou que vários tipos de treinamento com exercício físico (TEF) modulam a expressão dos miRNAs.13 Entretanto, há poucos artigos na literatura que tenham analisado os efeitos do TEF na expressão dos miRNAs na DC. Portanto, o objetivo desta revisão de literatura foi analisar os miRNAs expressos na DC e comparar esse resultado aos miRNAs expressos durante ou depois do TEF.

Doença de Chagas e miRNAs

Poucos estudos na literatura analisaram o perfil de expressão de miRNAs na DC, seja na fase aguda ou na crônica, bem como a sinalização celular que é regulada pelos miRNAs nessa doença negligenciada. Portanto, este trabalho incluiu todos os estudos que avaliaram o padrão de expressão dos miRNAs na DC (Tabela 1).

– MicroRNAs na doença de Chagas
MicroRNAs Fonte Achados Referência
↓ miR-1, miR-133a, miR-133b, miR-208a, miR-208b Amostras cardíacas Associação a distúrbios do tecido conjuntivo e fibrose 16
↑ miR-208b Amostras plasmáticas Associação à disfunção cardiovascular e hipertrofia miocárdica 17
↑ miR-20, miR-20b, miR-21, miR-142, miR-146a, miR-146b, miR-155, miR-182, miR-203, miR-222 ↓ miR-139, miR-145, miR-149, miR-322, miR-503, Amostras cardíacas Associação com o intervalo QT (QTc) corrigido pela frequência cardíaca. Despolarização e repolarização ventricular. 14
↑ miR-19a, miR-21, miR-29b, miR-30a, miR-199b Amostras cardíacas e modelo celular Associação à fibrose e remodelação cardíaca 18
↑ miR-16, miR-26b, miR-190b, miR-3586, let-7f-2 ↓ miR-190b Células H9c2, infectadas com T. Cruzi Associação com crescimento celular, hipertrofia e sobrevivência celular 19

Doença de Chagas (fase aguda) e miRNAs

Durante a fase aguda da DC, os pesquisadores avaliaram a expressão de miRNAs aos 15, 30 e 45 dias após a infecção, e identificaram que os miRNAs se expressaram diferentemente durante a parasitemia e que mudanças no intervalo QT sofreram regulação ascendente: miR-20, miR-20b, miR-21, miR-142, miR-146a, miR-146b, miR-155, miR-182, miR-203, miR-222, e descendente: miR-139, miR-145, miR-149, miR-322, miR-503.14

Outro estudo realizou uma análise in silico para identificar a expressão diferencial de miRNAs e seus genes alvo em vários processos biológicos durante a fase aguda da infecção pelo T. Cruzi, demonstrando que alguns podem estar associados ao processo patológico, tais como os miRNAs miR-238-3p, miR-149-5p, miR-143-3p, miR-145-5p e miR-486-5p. Outros miRNAs podem estar associados à imunidade e função cardiovascular, por exemplo: miR-10a-5p, miR-16-5p, miR-30c-5p, miR-34a-5p, miR-138-5p, miR-146a-5p, miR-149, miR-191-5p, miR-204-5p, miR-320b e miR-653-3p, bem como miRNAs relacionados ao processo de fibrose de tecidos: miR-34a-5p, miR-142-3p, miR-200b-3p e 203a-3p.15

 Doença de Chagas (fase crônica) e miRNAs

A expressão dos miRNAs do tecido cardíaco dos pacientes com CCC após o transplante cardíaco foi analisada e comparada à expressão de miRNAs do tecido cardíaco de doadores saudáveis. De todos os miRNAs analisados, cinco tiveram sua expressão reduzida (miR-1, miR-133a, miR-133b, miR-208a e miR-208b) em pacientes com CCC em comparação ao grupo de controle.16 Em contraste, o miR-208a circulante em uma amostra de plasma foi superexpresso em pacientes com DC. Entretanto, eles foram indeterminados na fase crônica.17

A superexpressão de miR-19a, miR-21 e miR-29b já foi descrita em pacientes com CCC em comparação a indivíduos saudáveis. Inclusive, na análise histológica do tecido cardíaco de pacientes no estágio final da CCC, identificou-se que, além desses miRNAs mencionados acima, o miR-30a e o miR-199b também são superexpressos na DC.18

Esses estudos demonstram que muitos miRNAs participam de vários processos na DC, tanto na fase aguda quanto na crônica. Entretanto, são necessários mais estudos para elucidar o papel desses miRNAs e a sinalização celular que estão regulando na DC, incluindo a importância de terapias e tratamentos que podem modular o padrão de expressão apresentado na doença.

 Doença de Chagas e treinamento com exercício físico: miRNAs como possíveis moduladores

Vários tipos de TEF foram descritos como moduladores da expressão de miRNAs,13 em estudos experimentais e clínicos, tais como TEF de natação,20 maratona,21 corrida em esteira22 e treinamento de resistência (TR)23 (Tabela 2).

– MicroRNAs em Treinamento com exercício físico (estudos pré-clínicos e clínicos)
MicroRNAs Alvo Fonte Tipos de exercícios Referência
Modelos experimentais in vivo
↑ miR-27a, miR-155 ↓ miR-143 ACE, AT1R Amostras cardíacas Ratos Wistar-Kyoto Treinamento físico na esteira 39
↑ miR-17-3p TIMP-3 PTEN Amostras cardíacas Ratos C57Bl/6 Modelo de treinamento de nado em rampa Treinamento em roda voluntário 40
↑ miR-222 HIPK1 Amostras cardíacas Modelo de nado em rampa Treinamento em roda voluntário 41
↑ miR-19b, miR-30e, miR-133b, miR-208a ↓ miR-99b, miR-100, miR-191a, miR-22, miR-181a IGF-1 PI3K/AKT/mTOR MAPK Amostras cardíacas Plasma Ratos Wistar albinos Treinamento de natação 42
↑ miR-29a, miR-101a TG-β fos COL1A1 Amostras cardíacas Exercício de corrida intermitente 43
↑ miR-27a, miR-27b ↓ miR-143 ACE ACE2 Amostras cardíacas Ratos Wistar Treinamento de natação 44
↑ miR-126 PI3KR2 Amostras cardíacas Plasma Ratos Zucker Treinamento de natação 26
↓ miR-214 SERCA2A Amostras cardíacas Ratos Wistar Treinamento de resistência 23
↑ miR-1 ↓ miR-214 NCX SERCA2A Amostras cardíacas Ratos Wistar Treinamento de natação 27
↑ miR-29c ↓ miR-1, miR-133a, miR133b COL1A1 COL3A1 Amostras cardíacas Ratos Wistar Treinamento de natação 45
↑ miR-126 SPRED1 PI3KR2 Amostras cardíacas Ratos Wistar Treinamento de natação 46
↑ miR-21, miR-144, miR-145 ↓ miR-124 PTEN PIK3A TSC2 Amostras cardíacas Ratos Wistar Treinamento de natação 20
↑ miR-336-5p, miR-130b-5p, let7d-3p, miR-466c-5p, miR-324-3p, miR-146b-5p, miR-132-3p, miR-21-5p, miR-187-3p, miR-29b-5p, miR-324-5p, miR-214-5p, miR-140-5p, miR-152-5p, miR-99b-5p, miR-130a-5p, miR-455-5p, miR-27b-3p, miR-23b-3p, miR-652-5p, miR-199a-3p, miR-223-5p, miR-421-3p, miR-27a-5p, miR-24-5p, miR-34a-3p, miR-140-3p, miR-125b-5p, miR-145a-5p, miR-192-5p, miR-139-5p, miR-199a-5p, miR-674-3p, miR-191-5p, miR-28-3p, miR-195-5p, miR-598, miR-429, miR-224, miR-425, miR-221 ↓ miR-701-5p, miR-220, miR-144-3p, miR-694, miR-485-3p, miR-136-5p, miR-384-3p, miR-376c-3p, miR-208b-3p, miR-411-3p, miR-141-5p, miR-1894-3p, miR-9a, miR-687, miR-451-5p TNF-α COL1A1 MMP9 PTEN AKT1 AMPK BCL2 Amostras cardíacas Ratos Wistar Treinamento aeróbico de corrida 22
↑ miR-503, miR-465b-5p, miR-542-3p ↓ miR-652 Amostras cardíacas Ratos C57Bl6 Treinamento de natação 47
↓ miR-26b, miR-143 IGF1R GATA-4 NFAT1C GSK3B Amostras cardíacas Ratos Balb/c Treinamento aeróbico em rodas de metal 48
↑ miR-21, miR-30b ↓ miR-1 BCL-2 p53 PDCD4 Amostras cardíacas Treinamento de natação 49
↑ miR-23a, miR-27a PTEN Casp7 FoxO1 Amostras musculoesqueléticas Exercício de resistência 50
↑ miR-29c ↓ miR-1 COL1A1 COL3A1 Amostras cardíacas Treinamento de natação 51
↑ miR-382 Amostras de soro, tecido e células Exercício aeróbico em ratos IR 25
MicroRNAs Alvos Fonte Tipos de exercícios Referência
Estudos clínicos
↑ miR-126, miR-133 CPK Plasma Espiroergometria limitada a um único sintoma Corrida de maratona Exercício excêntrico de resistência 52
↓ miR-486 PTEN Soro Ciclismo sistemático a 70% VO2max 53
↑ miR-1, miR-126, miR-133a, miR-134, miR-146a, miR-208a, miR-499-5p CPK NT-proBNP hsCRP Plasma Corrida de maratona Imediatamente após a corrida 21
↑ miR-1, miR-133a, miR-206, miR-208b, miR-499 Plasma Corrida de maratona Imediatamente após a corrida 54
↑ miR-1, miR-133a, miR-206 Plasma Corrida de maratona Imediatamente após a corrida 55
↑ let-7d-3p, let-7f-3p miR-29a-3p, miR-34a-5p, miR-125b-5pmiR-132-3p, miR-143-3p, miR-148a-3p, miR-223-3p, miR-223-5p miR-424-3p, miR-424-5p Soro Corrida de maratona Imediatamente após a corrida 56
↑ miR-1, miR-30a, miR-133a ↓ miR-26a, -29b Plasma Corrida de maratona Imediatamente após a corrida 57
↑ miR-1, miR-133a, miR-206 Plasma Corrida de maratona Imediatamente após a corrida 58
↑ miR-1, miR-133a, miR-133b, miR-139-5p, miR-143, miR-145, miR-223, miR-330-3p, miR-338-3p, miR-424 ↓ miR-30b, miR-106a, miR-146, miR-151-3p, miR-151-5p, miR-221, miR-652, let-7i ↑ miR-103, miR-107 ↓ miR-21, miR-25, miR-29b, miR-92a, miR-133a, miR-148a, miR-148b, miR-185, miR-342-3p, miR-766, let-7d Plasma Teste cicloergômetro 1-3 h após o exercício Ciclo de resistência sistemática treinamento ergométrico 59
↑ miR-1, miR-133a, miR-133b, miR-206 miR-485-5p, miR-509-5p, miR-517a miR-518f, miR-520f, miR-522, miR-553, miR-888 Plasma Treinamento intervalado de alta intensidade Imediatamente após 60
↑ miR-181b, miR-214 ↑ miR-1, miR-133a, miR-133b, miR-208b Plasma Teste de esteira em aclive (concêntrico) Imediatamente após Teste de esteira em declive (excêntrico) 2-6 h após o exercício 61
↑ miR-149 ↓ miR-146a, miR-221 Soro Exercício de resistência 3 dias após o exercício 62
↑ miR-1, miR-133a, miR-133b, miR-206, miR-208b, miR-499 Plasma Treinamento de resistência sistemático 36-72 h após o exercício 63
↑ miR-1, miR-133a, miR-133b, miR-181a ↓ miR-9, miR-23a, miR-23b, miR-31 ↑ miR-1, miR-29b HDAC4 NRF1 Amostras musculoesqueléticas Teste cicloergômetro, ciclismo 64
↑ miR-136, miR-200c, miR-376a, miR-377, miR-499b, miR-558 ↓ miR-28, miR-30d, miR-204, miR-330, miR-345, miR-375, miR-449c, miR-483, miR-509, miR-520a, miR-548a, miR-628, miR-653, miR-670, miR-889, miR-1245a, miR-1270, miR-1280, miR-1322, miR-3180 Amostras musculoesqueléticas Treinamento de resistência 65
↑ miR-451 ↓ miR-26a, miR-29a, miR-378 Amostras musculoesqueléticas Exercício de resistência 66
↑ miR-125a, miR-145, miR-181b, miR-193a, miR-197, miR-212, miR-223, miR-340, miR-365, miR-485, miR-505, miR-520d, miR-629, miR-638, miR-939, miR-940, miR-1225, miR-1238 ↓ let-7i, miR-16, miR-17, miR-18a, miR-18b, miR-20a, miR-20b, miR-22, miR-93, miR- 96, miR-106a, miR-107, miR-126, miR-130a, miR-130b, miR-151, miR-185, miR-194, miR-363, miR-660 Soro Exercício em cicloergômetro, ciclismo 67
↑ miR-7, miR-15a, miR-21, miR-26b, miR-132, miR-140, miR-181a, miR-181b, miR-181c, miR-338, miR-363, miR-939, miR-940, miR-1225 ↓ let-7e, miR-23b, miR-31, miR-99a, miR-125a, miR-125b, miR-126, miR-130a, miR-145, miR-151, miR-199a, miR-199b, miR-221, miR-320, miR-451, miR-486, miR-584, miR-652 PBMC Exercício em cicloergômetro, ciclismo 68
↑ let-7f, miR-21, miR-29c, miR-223 ↓ let-7f, miR-21, miR-29c, miR-223 PBMC Exercício de corrida 69
↑ miR-7, miR-29a, miR-29b, miR-29c, miR-30e, miR-142, miR-192, miR-338, miR-363, miR-590 ↓ let-7e, miR-126, miR-130a, miR-151, miR-199a, miR-221, miR-223, miR-326, miR-328, miR-652 PBMC Exercício em cicloergômetro, ciclismo 70
↑ miR-15a, miR-29b, miR-29c, miR-30e, miR-140, miR-324, miR-338, miR-362, miR-532, miR-660 ↓ miR-23b, miR-130a, miR-151, miR-199a, miR-221 Soro Exercício em cicloergômetro, ciclismo 71
↑ miR-1, miR-486, miR-494 Soro (Atletas de resistência, corredores, ciclistas e triatletas) Teste de exercício cardiopulmonar 72
↑ miR-21, miR-146a, miR-221, miR-222 ↑ miR-20a, miR-21, miR-146a, miR-221, miR-222 Soro Treinamento por remada, 5 km, 1-3 h por sessão, 20-24 remadas/min) 73
↑ miR-376a ↓ miR-16, miR-27a, miR-28 Plasma Treinamento de exercício aeróbico - corrida (4 dias/semana) 74
↑ miR-19a, miR-19b, miR-20a, miR-26b, miR-143, miR-195 p-AKT p-S6K1 Soro Exercício de resistência 75
↑ miR-222 HIPK1 Plasma Teste ergométrico em bicicleta 41
↑ miR-221 ↓ miR-208b, miR-221, miR-21, miR-146a, miR-210 Soro Exercício de basquete 76

Alguns estudos também relataram a importância do TEF na modulação da expressão dos miRNAs em situações patológicas, bem como em diabéticos,24,25 na obesidade,26 após o infarto do miocárdio27 e com insuficiência cardíaca;22 entretanto, o papel do TEF na modulação dos miRNAs na DC ainda não foi evidenciado. A literatura apresenta apenas estudos que demonstraram os efeitos benéficos do TEF na DC; porém eles não analisaram o perfil do miRNA.

Realizando apenas TEF aeróbico com intensidade moderada (50 a 70% de frequência cardíaca máxima), três vezes por semana, por 30 minutos, em 12 semanas, obteve-se um aumento significativo na capacidade cardiorrespiratória metabólica máxima (VO2), aumento de tempo de exercício, distância percorrida e melhoria de aspectos emocionais,28 e, além disso, em associação com um programa de TR, foram obtidos resultados benéficos.29

Outro estudo, com um protocolo de TEF semelhante, também evidenciou uma melhoria da capacidade funcional, com melhoria da fração de ejeção e resistência respiratória, melhoria da pressão diastólica no ventrículo esquerdo, e da qualidade de vida de pacientes chagásicos após 8 meses de treinamento.30

Um programa de reabilitação cardíaca composto do mesmo protocolo de TEF mencionado acima, com TR e alongamentos, orientação nutricional adicional e aconselhamento farmacológico para pacientes com DC, demonstrou aumento da capacidade funcional e física, melhorando a qualidade de vida de pacientes chagásicos.31

Em outro estudo importante, os pesquisadores realizaram TEF três vezes por semana em pacientes chagásicos. Eles demonstraram que o grupo que realizou exercícios aumentou o consumo de oxigênio de pico durante o exercício e a ventilação máxima por minuto, melhorando a capacidade funcional desses pacientes.32

Entretanto, apesar de demonstrar que o TEF tem efeitos benéficos para pacientes com DC, é difícil analisar os efeitos desse tipo de treinamento no nível do tecido, celular e molecular, considerando que esses estudos foram realizados em seres humanos, para quem seriam necessárias biópsias. Portanto, para investigar os possíveis mecanismos associados a esses efeitos benéficos do TEF na DC, alguns estudos foram realizados em modelos experimentais de DC in vivo.

Camundongos Balb/c realizaram TEF em uma esteira antes de serem infectados com T. Cruzi. Observou-se que o TEF reduziu o pico da parasitemia, concluindo que o TEF pode promover alterações benéficas no sistema imune e obter melhores respostas a infecções.33

Em outros estudos, foram relatados os mesmos achados que os de estudos anteriores; entretanto, eles também observaram que ratos que fizeram o treinamento obtiveram maior proteção da atividade metabólica de NADH em neurônios mioentéricos e maior síntese de TNF-α e TGF-β.34 Isso contribuiu para a sobrevivência e/ou proteção de 10,3% dos neurônios mioentéricos e sua produção imunorreativa de sintase neuronal de óxido nítrico. O grupo em treinamento, inclusive, obteve maior expressão de TNF-α durante a fase aguda da infecção por T. Cruzi, oferecendo benefícios ao sistema imune para preservar os neurônios nitrérgicos.35

Nesse contexto, em outro estudo, pesquisadores observaram que o grupo de TEF obteve maior expressão de TNF-α, IFNγ, IL-6 e as quimiocinas MCP-1 e CX3CL1 durante a infecção aguda, além de alcançarem melhor capacidade física, aumento do limiar anaeróbio, aumento da atividade da catalase e do superóxido dismutase, e redução da oxidação lipídica e proteica no tecido cardíaco, demonstrando que o TEF pode ser uma estratégia interessante para aumentar a eficiência de mecanismos antioxidantes endógenos, reduzindo os danos oxidativos nesses animais.36

Outro estudo demonstrou que o TEF antes da infecção em ratos Wistar aumentou o tempo até que se atingisse a fadiga e o limiar anaeróbio, reduziu a expressão de TNF-α, CCL-2, MCP-1 e CX3CL1, bem como a oxidação lipídica e proteica, e aumentou a expressão de IL-10, catalase e superóxido dismutase, indicando que o TEF induz um fenótipo protetor, aumentando as defesas do hospedeiro contra o agente parasítico, inclusive atenuando o processo de remodelação patológica associado à miosite musculoesquelética.37

Finalmente, em outro estudo, ratos suíços foram infectados pelo T. Cruzi após TEF de intensidade moderada em uma esteira, realizado durante 9 semanas. Os pesquisadores identificaram que o TEF conseguiu reduzir a parasitemia latente dos animais infectados submetidos a treinamento, corroborando os achados de estudos anteriores. Eles chegaram a obter menor produção de citocinas pró-inflamatórias (TNF-α, INFγ, IL-12) e proteína quimiotática de monócitos 1 (MCP-1) durante os primeiros dias de infecção.38

Portanto, sugere-se que o TEF tenha um potencial terapêutico para a prevenção e o tratamento complementar de DC e CCC pela modulação do sistema imune. Entretanto, estudos clínicos carecem de análises morfométricas, celulares e moleculares especialmente pela análise de miRNAs para melhor entendimento dos efeitos benéficos do TEF na sinalização celular em seres humanos com DC, enquanto estudos pré-clínicos, in vivo, demandam estudos que avaliem os efeitos de TEF com DC e CCC já instaladas e não apenas no estágio de pré-infecção.

 Sobreposições entre miRNAs em DC e TEF

Além disso, realizamos uma análise utilizando o diagrama de Venn, para identificar os miRNAs que foram modulados por TEF, em estudos clínicos e pré-clínicos que podem possivelmente modular miRNAs na DC.

Houve apenas 7 miRNAs expressos em DC, 95 miRNAs expressos em estudos clínicos com TEF, e 36 miRNAs expressos em estudos pré-clínicos com TEF. É interessante notar que foram identificados 7 miRNAs que tinham modulação tanto na DC quanto em estudos clínicos com TEF, 3 miRNAs comuns modulados na DC e em estudos pré-clínicos com TEF, e, principalmente, 12 miRNAs comuns modulados na CD, estudos clínicos com TEF, e estudos pré-clínicos com TEF (Figura 1). Esses 12 miRNAs são: miR-1, miR-21, miR-26b, miR-29b, miR-133a, miR-133b, miR-139, miR-145, miR-146a, miR-208a, miR-208b, miR-222.

– O diagrama de Venn mostra sobreposições entre miRNAs: miRNAs (miRs) na doença de Chagas (azul), miRs TEF clin: estudos clínicos (rosa), e miRs TEF pre-clin: estudos pré-clínicos (verde).

Entretanto, desses 12 miRNAs comuns, apenas miR-133b, miR-139 e miR-208a foram identificados com um padrão de expressão diferente na DC e TEF; todos os 3 miRNAs passam por regulação descendente na DC, e ascendente, em TEF (Figura 2).

– miRNAs expressos na DC que podem provavelmente ser modulados por TEF

O miR-133b controla o fator de crescimento de tecido conjuntivo (CTGF)77 e pode suprimir a remodelação cardíaca;78 portanto, o TEF pode ser uma alternativa excelente para controlar a remodelação cardíaca, possivelmente pela modulação do miR-133b e a modificação da sinalização celular.

O miR-139 está associado à cardiomiopatia hipertrófica, regulando a expressão do c-Jun, um fator transcricional que liga a região promotora de alguns genes para induzir a hipertrofia cardíaca, e, portanto, a superexpressão desse miRNA reduz a expressão do c-Jun e consequentemente atenua a hipertrofia cardíaca patológica,79 que pode ser uma sinalização celular pela qual o TEF suprime a hipertrofia patológica na DC, porque o TEF também aumenta a expressão desse miRNA.22,59

Nesse contexto, o miR-208a regula a expressão de alguns fatores transcricionais, tais como GATA-4, que está associado à ativação de genes cardíacos pró-hipertróficos.80 Na DC, esse miRNA sofre regulação descendente,16 enquanto a TEF pode aumentar sua expressão,21,42 demonstrando, portanto, que possivelmente pode ser um mecanismo molecular pelo qual o TEF atenua a hipertrofia cardíaca nessa doença.

 Conclusões

Os miRNAs participam de vários processos na patogênese da DC. Muitas evidências mostram os efeitos benéficos do TEF na DC; entretanto, ainda não há artigos na literatura que demonstrem as alterações nos mecanismos moleculares dos miRNAs que o TEF induz na DC. Dessa forma, são necessários estudos posteriores para elucidar esses mecanismos.

 Agradecimentos

Este estudo foi financiado parcialmente pela Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Código Financeiro 001. Agradecemos ao programa de pós-graduação em Medicina e Saúde, Faculdade de Medicina, Universidade Federal da Bahia. Gostaríamos de agradecer a Mirela Correia Improta Caria pela ajuda na criação da Figura 2.

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Swimming training in rats increases cardiac MicroRNA-126 expression and angiogenesis. Med Sci Sports Exerc. 2012;44(8):1453-62. doi: 10.1249/MSS.0b013e31824e8a36. Silva ND Jr Fernandes T Soci UP Monteiro AW Phillips MI Oliveira EM Swimming training in rats increases cardiac MicroRNA-126 expression and angiogenesis Med Sci Sports Exerc 2012 44 8 1453 1462 10.1249/MSS.0b013e31824e8a36 47. McPherson NO, Owens JA, Fullston T, Lane M. Preconception diet or exercise intervention in obese fathers normalizes sperm microRNA profile and metabolic syndrome in female offspring. Am J Physiol Endocrinol Metab. 2015;308(9):E805-21. doi: 10.1152/ajpendo.00013.2015. McPherson NO Owens JA Fullston T Lane M Preconception diet or exercise intervention in obese fathers normalizes sperm microRNA profile and metabolic syndrome in female offspring Am J Physiol Endocrinol Metab 2015 308 9 E805 E821 10.1152/ajpendo.00013.2015 48. Martinelli NC, Cohen CR, Santos KG, Castro MA, Biolo A, Frick L, et al. 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Cui SF Wang C Yin X Tian D Lu QJ Zhang CY et al Similar Responses of Circulating MicroRNAs to Acute High-Intensity Interval Exercise and Vigorous-Intensity Continuous Exercise Front Physiol 2016 7 102 10.3389/fphys.2016.00102 61. Banzet S, Chennaoui M, Girard O, Racinais S, Drogou C, Chalabi H, et al. Changes in circulating microRNAs levels with exercise modality. J Appl Physiol. 2013;115(9):1237-44. doi: 10.1152/japplphysiol.00075.2013. Banzet S Chennaoui M Girard O Racinais S Drogou C Chalabi H et al Changes in circulating microRNAs levels with exercise modality J Appl Physiol 2013 115 9 1237 1244 10.1152/japplphysiol.00075.2013 62. Sawada S, Kon M, Wada S, Ushida T, Suzuki K, Akimoto T. Profiling of circulating microRNAs after a bout of acute resistance exercise in humans. PLoS One. 2013;8(7):e70823. doi: 10.1371/journal.pone.0070823. Sawada S Kon M Wada S Ushida T Suzuki K Akimoto T Profiling of circulating microRNAs after a bout of acute resistance exercise in humans PLoS One 2013 8 7 e70823 10.1371/journal.pone.0070823 63. Zhang T, Birbrair A, Wang ZM, Messi ML, Marsh AP, Leng I, et al. Improved knee extensor strength with resistance training associates with muscle specific miRNAs in older adults. Exp Gerontol. 2015;62:7-13. doi: 10.1016/j.exger.2014.12.014. Zhang T Birbrair A Wang ZM Messi ML Marsh AP Leng I et al Improved knee extensor strength with resistance training associates with muscle specific miRNAs in older adults Exp Gerontol 2015 62 7 13 10.1016/j.exger.2014.12.014 64. Russell AP, Lamon S, Boon H, Wada S, Güller I, Brown EL, et al. Regulation of miRNAs in human skeletal muscle following acute endurance exercise and short-term endurance training. J Physiol. 2013;591(18):4637-53. doi: 10.1113/jphysiol.2013.255695. Russell AP Lamon S Boon H Wada S Güller I Brown EL et al Regulation of miRNAs in human skeletal muscle following acute endurance exercise and short-term endurance training J Physiol 2013 591 18 4637 4653 10.1113/jphysiol.2013.255695 65. Ogasawara R, Akimoto T, Umeno T, Sawada S, Hamaoka T, Fujita S. MicroRNA expression profiling in skeletal muscle reveals different regulatory patterns in high and low responders to resistance training. Physiol Genomics. 2016;48(4):320-4. doi: 10.1152/physiolgenomics.00124.2015. Ogasawara R Akimoto T Umeno T Sawada S Hamaoka T Fujita S MicroRNA expression profiling in skeletal muscle reveals different regulatory patterns in high and low responders to resistance training Physiol Genomics 2016 48 4 320 324 10.1152/physiolgenomics.00124.2015 66. Davidsen PK, Gallagher IJ, Hartman JW, Tarnopolsky MA, Dela F, Helge JW, et al. High responders to resistance exercise training demonstrate differential regulation of skeletal muscle microRNA expression. 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Denham J Prestes PR Muscle-Enriched MicroRNAs Isolated from Whole Blood Are Regulated by Exercise and Are Potential Biomarkers of Cardiorespiratory Fitness Front Genet 2016 7 196 10.3389/fgene.2016.00196 73. Baggish AL, Hale A, Weiner RB, Lewis GD, Systrom D, Wang F, et al. Dynamic regulation of circulating microRNA during acute exhaustive exercise and sustained aerobic exercise training. J Physiol. 2011;589(16):3983-94. doi: 10.1113/jphysiol.2011.213363. Baggish AL Hale A Weiner RB Lewis GD Systrom D Wang F et al Dynamic regulation of circulating microRNA during acute exhaustive exercise and sustained aerobic exercise training J Physiol 2011 589 16 3983 3994 10.1113/jphysiol.2011.213363 74. Zhang T, Brinkley TE, Liu K, Feng X, Marsh AP, Kritchevsky S, et al. Circulating MiRNAs as biomarkers of gait speed responses to aerobic exercise training in obese older adults. Aging. 2017;9(3):900-913. doi: 10.18632/aging.101199. Zhang T Brinkley TE Liu K Feng X Marsh AP Kritchevsky S et al Circulating MiRNAs as biomarkers of gait speed responses to aerobic exercise training in obese older adults Aging 2017 9 3 900 913 10.18632/aging.101199 75. Margolis LM, Rivas DA, Berrone M, Ezzyat Y, Young AJ, McClung JP, et al. Prolonged Calorie Restriction Downregulates Skeletal Muscle mTORC1 Signaling Independent of Dietary Protein Intake and Associated microRNA Expression. Front Physiol. 2016;7:445. doi: 10.3389/fphys.2016.00445. Margolis LM Rivas DA Berrone M Ezzyat Y Young AJ McClung JP et al Prolonged Calorie Restriction Downregulates Skeletal Muscle mTORC1 Signaling Independent of Dietary Protein Intake and Associated microRNA Expression Front Physiol 2016 7 445 10.3389/fphys.2016.00445 76. Li Y, Yao M, Zhou Q, Cheng Y, Che L, Xu J, et al. Dynamic Regulation of Circulating microRNAs During Acute Exercise and Long-Term Exercise Training in Basketball Athletes. Front Physiol. 2018;9:282. doi: 10.3389/fphys.2018.00282. Li Y Yao M Zhou Q Cheng Y Che L Xu J et al Dynamic Regulation of Circulating microRNAs During Acute Exercise and Long-Term Exercise Training in Basketball Athletes Front Physiol 2018 9 282 10.3389/fphys.2018.00282 77. Gjymishka A, Pi L, Oh SH, Jorgensen M, Liu C, Protopapadakis Y, et al. miR-133b Regulation of Connective Tissue Growth Factor: A Novel Mechanism in Liver Pathology. Am J Pathol. 2016;186(5):1092-102. doi: 10.1016/j.ajpath.2015.12.022. Gjymishka A Pi L Oh SH Jorgensen M Liu C Protopapadakis Y et al miR-133b Regulation of Connective Tissue Growth Factor: A Novel Mechanism in Liver Pathology Am J Pathol 2016 186 5 1092 1102 10.1016/j.ajpath.2015.12.022 78. Li N, Zhou H, Tang Q. miR-133: A Suppressor of Cardiac Remodeling? Front Pharmacol. 2018;9:903. doi: 10.3389/fphar.2018.00903. Li N Zhou H Tang Q miR-133: A Suppressor of Cardiac Remodeling? Front Pharmacol 2018 9 903 10.3389/fphar.2018.00903 79. 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Fontes de financiamento: O presente estudo foi parcialmente financiado pela CAPES.

 Review Article Physical Exercise Training and Chagas Disease: Potential Role of MicroRNAs 0000-0002-8779-1188 Improta-Caria Alex Cleber 1 2 0000-0002-2161-3696 Aras Roque Júnior 1 Brasil Programa de Pós-Graduação em Medicina e Saúde, Faculdade de Medicina, Universidade Federal da Bahia, Salvador, BA - Brasil Brasil Departamento de Educação Física em Cardiologia do Estado da Bahia, Sociedade Brasileira de Cardiologia,Salvador, BA - Brasil Mailing Address: Alex Cleber Improta-Caria • Universidade Federal da Bahia - Programa de Pós-Graduação em Medicina e Saúde - Rua Dr. Augusto Viana, s/n. Postal Code 40110-060, Canela, Salvador, BA - Brasil E-mail: alexcaria.personal@hotmail.com

Author Contributions

Conception and design of the research, Acquisition of data and Writing of the manuscript: Improta-Caria AC; Analysis and interpretation of the data and Critical revision of the manuscript for intellectual content: Improta-Caria AC, Aras Júnior R.

Potential Conflict of Interest

No potential conflict of interest relevant to this article was reported.

 Abstract

Chagas disease (CD) is caused by Trypanosoma Cruzi. This parasite can infect several organs of the human body, mainly the heart, causing inflammation, fibrosis, arrhythmias, and cardiac remodeling, promoting long-term Chronic Chagas Cardiomyopathy (CCC). However, little scientific evidence has elucidated the molecular mechanisms that govern the pathophysiological processes in this disease. MicroRNAs (miRNAs) are regulators of post-transcriptional gene expression that modulate signaling pathways, participating in pathophysiological mechanisms in CD, but the understanding of miRNAs in this disease is limited. On the other hand, a wide range of scientific evidence shows that physical exercise training (PET) modulates the expression of miRNAs by modifying different signaling pathways in healthy individuals. Some studies also show that PET is beneficial for individuals with CD; however, these did not evaluate the miRNA expressions. Thus, there is no evidence showing the role of PET in the expression of miRNAs in CD. Therefore, this review aimed to identify miRNAs expressed in CD that could potentially be modified by PET.

 Exercise Chagas Disease MicroRNAs Introduction

Chagas Disease (CD) is a complex disease caused by Trypanosoma Cruzi (T. cruzi), a flagellated protozoan parasite, infection at the intracellular level.1 In the acute phase, the T. cruzi infection generates great tissue inflammation, and there is an initial response of the innate immune system in an attempt to fight parasitemia.2

However, the infection persists and the adaptive immune system activates the T lymphocytes, as well as the auxiliary and cytotoxic T cells, which produce cytokines, such as gamma interferon (IFN-γ), which can in turn lead to intracellular parasitic death by inducing an increase in the reactive oxygen species and nitrogen, which are microbicides. This infection also increases the expression of the tumor necrosis factor (TNF-α) and specific antibodies to combat T. cruzi, which control parasitism, with a low-grade infection being established.3

Still in the acute phase of the disease, there is an increase in the expression of the vasoactive peptide endothelin-1 (ET-1) and cardiotrophin-1 (CT-1), both inducing cardiac hypertrophy, as well as an increase in the expression of interleukin-1 beta. (IL-1β), inducing an inflammatory and pro-hypertrophic response of the myocardium, which may initiate cardiac hypertrophy even at this stage.4,5

Over the years, parasitemia is reduced; however, parasitic antigens persist, generating a diffuse inflammatory infiltrate and myocarditis, with the presence of CD4 + and CD8 + T lymphocytes and macrophages that continue to express TNF-α and IFN-γ.3 IFN-γ has an essential function to control and fight against parasites, but it also contributes to cardiac pathogenesis, as it damages the myocardium through several molecular mechanisms generating myocardial dysfunction.6

Thus, the disease evolves and passes to the chronic phase, which can be subdivided into two forms: indeterminate and symptomatic. In the indeterminate form, individuals can go for years without manifesting any type of more serious symptom, where there is a balance between parasitemia and the host’s immune system. However, about 30% of these patients develop a symptomatic or determined form, which can trigger dysfunctions in different organs, including the heart, developing Chronic Chagas Cardiomyopathy (CCC) associated with myocarditis and cardiac myofibrillary fibrosis, thereby reducing cardiac electrical conductivity and generating myocardial remodeling.7

CCC generates inflammation of the cardiac tissue, causing focal or diffuse myocarditis, hypertrophy, or dilation of the left ventricle and progressive death of some cardiomyocytes, necrosis, and collagen deposit,8 thereby increasing the fibrotic tissue, leading to a reduction in its contractile capacity. This outcome is mostly associated with arrhythmias and heart failure,9 but microRNAs (miRNAs) may also participate in these mechanisms. In general, the molecular mechanisms that govern these processes are poorly understood.

MiRNAs are small RNAs, with only 18 to 25 nucleotides in length;10 non-coding proteins; and regulators of post-transcriptional gene expression with the function of inhibiting or degrading its target genes.11,12 It has been shown that several types of physical exercise training (PET) modulate the expression of miRNAs.13 Nevertheless, articles that analyze the effects of PET on the expression of miRNAs in CD are still scarce in the literature. Thus, our literature review sought to analyze the miRNAs expressed in CD and to compare this finding with the miRNAs expressed during or after PET.

Chagas Disease and miRNAs

Few studies in the literature have analyzed the expression profile of miRNAs in CD, either in the acute or in the chronic phase, as well as the signaling pathways that are regulated by miRNAs in this neglected disease. Therefore, this study included all of the studies that evaluated the expression pattern of miRNAs in CD (Table 1).

– MicroRNAs in Chagas Disease
MicroRNAs Source Findings Reference
↓ miR-1, miR-133a, miR-133b, miR-208a, miR-208b Heart samples Association with connective tissue disorders and fibrosis 16
↑ miR-208b Plasma samples Association with cardiovascular dysfunction and myocardial hypertrophy 17
↑ miR-20, miR-20b, miR-21, miR-142, miR-146a, miR-146b, miR-155, miR-182, miR-203, miR-222 ↓ miR-139, miR-145, miR-149, miR-322, miR-503, Heart samples Association with heart rate-corrected QT (QTc) interval. Ventricular depolarization and repolarization. 14
↑ miR-19a, miR-21, miR-29b, miR-30a, miR-199b Heart samples and cell model Association with fibrosis and cardiac remodeling 18
↑ miR-16, miR-26b, miR-190b, miR-3586, let-7f-2 ↓ miR-190b H9c2 cells, infected with T. Cruzi Association with cell growth, hypertrophy, and cell survival 19

Chagas Disease (acute phase) and miRNAs

During the acute phase of CD, the researchers evaluated the expression of miRNAs at 15, 30, and 45 days post-infection, and identified that miRNAs were differentially expressed during parasitemia and that changes in the QT interval were upregulated: miR-20, miR-20b, miR-21, miR-142, miR-146a, miR-146b, miR-155, miR-182, miR-203, and miR-222, and downregulated: miR-139, miR-145, miR-149, miR-322, and miR-503.14

Another study performed an in silico analysis to identify the differential expression of miRNAs and their target genes in several biological processes during the acute phase of T. Cruzi infection, demonstrating that some miRNAs may be associated with the pathological process, such as miR-238-3p, miR-149-5p, miR-143-3p, miR-145-5p, and miR-486-5p. Other miRNAs may be associated with cardiovascular immunity and function, for example: miR-10a-5p, miR-16-5p, miR-30c-5p, miR-34a-5p, miR-138-5p, miR-146a-5p, miR-149, miR-191-5p, miR-204-5p, miR-320b and miR-653-3p, as well as miRNAs related to the tissue fibrosis process: miR-34a-5p, miR-142-3p, miR-200b-3p, and 203a-3p.15

 Chagas Disease (chronic phase) and miRNAs

The expression of miRNAs from the cardiac tissue of patients with CCC after heart transplantation was analyzed and compared with the expression of miRNAs from the cardiac tissue of healthy donor individuals. Of all miRNAs analyzed, five miRNAs had their expression reduced (miR-1, miR-133a, miR-133b, miR-208a, and miR-208b) in patients with CCC when compared to the control group.16 By contrast, the circulating miR-208a in a plasma sample was overexpressed in patients with CD; however, these were in the undetermined chronic phase.17

The overexpression of MiR-19a, miR-21, and miR-29b has been described in patients with CCC when compared to healthy individuals. In fact, in the histological analysis of the cardiac tissue of patients in the final stage of CCC, it was identified that, in addition to the miRNAs mentioned above, the miR-30a and miR-199b are also overexpressed in the CD.18

These studies demonstrate that many miRNAs participate in several processes in the CD both in the acute and chronic phase; however, further studies are needed to elucidate the role of these miRNAs and the signaling pathways they are regulating in the CD, including the importance of therapies or treatments that can modulate the pattern of expression shown in the disease.

 Chagas Disease and Physical Exercise Training: miRNAs as potential modulators

Several types of PET have been described as modulators of the expression of many miRNAs,13 in experimental and clinical studies, such as swimming PET,20 marathon,21 running on a treadmill,22 and resistance training (RT)23 (Table 2).

– MicroRNAs in Physical Exercise Training (pre-clinical and clinical studies)
MicroRNAs Target Source Types of Exercises Reference
In vivo experimental models
↑ miR-27a, miR-155 ↓ miR-143 ACE, AT1R Heart samples Wistar-Kyoto rats Exercise training on treadmill 39
↑ miR-17-3p TIMP-3 PTEN Heart samples C57Bl/6 mice Ramp swimming training model Voluntary wheel training 40
↑ miR-222 HIPK1 Heart samples Ramp swimming model Voluntary wheel training 41
↑ miR-19b, miR-30e, miR-133b, miR-208a ↓ miR-99b, miR-100, miR-191a, miR-22, miR-181a IGF-1 PI3K/AKT/mTOR MAPK Heart samples Plasma Wistar albino rats Swimming training 42
↑ miR-29a, miR-101a TG-β fos COL1A1 Heart samples Intermittent run exercise 43
↑ miR-27a, miR-27b ↓ miR-143 ACE ACE2 Heart samples Wistar rats Swimming training 44
↑ miR-126 PI3KR2 Heart samples Plasma Zucker rats Swimming training 26
↓ miR-214 SERCA2A Heart samples Wistar rats Resistance training 23
↑ miR-1 ↓ miR-214 NCX SERCA2A Heart samples Wistar rats Swimming training 27
↑ miR-29c ↓ miR-1, miR-133a, miR133b COL1A1 COL3A1 Heart samples Wistar rats Swimming training 45
↑ miR-126 SPRED1 PI3KR2 Heart samples Wistar rats Swimming training 46
↑ miR-21, miR-144, miR-145 ↓ miR-124 PTEN PIK3A TSC2 Heart samples Wistar rats Swimming training 20
↑ miR-336-5p, miR-130b-5p, let7d-3p, miR-466c-5p, miR-324-3p, miR-146b-5p, miR-132-3p, miR-21-5p, miR-187-3p, miR-29b-5p, miR-324-5p, miR-214-5p, miR-140-5p, miR-152-5p, miR-99b-5p, miR-130a-5p, miR-455-5p, miR-27b-3p, miR-23b-3p, miR-652-5p, miR-199a-3p, miR-223-5p, miR-421-3p, miR-27a-5p, miR-24-5p, miR-34a-3p, miR-140-3p, miR-125b-5p, miR-145a-5p, miR-192-5p, miR-139-5p, miR-199a-5p, miR-674-3p, miR-191-5p, miR-28-3p, miR-195-5p, miR-598, miR-429, miR-224, miR-425, miR-221 ↓ miR-701-5p, miR-220, miR-144-3p, miR-694, miR-485-3p, miR-136-5p, miR-384-3p, miR-376c-3p, miR-208b-3p, miR-411-3p, miR-141-5p, miR-1894-3p, miR-9a, miR-687, miR-451-5p TNF-α COL1A1 MMP9 PTEN AKT1 AMPK BCL2 Heart samples Wistar rats Aerobic run training 22
↑ miR-503, miR-465b-5p, miR-542-3p ↓ miR-652 Heart samples C57Bl6 mice Swimming training 47
↓ miR-26b, miR-143 IGF1R GATA-4 NFAT1C GSK3B Heart samples Balb/c mice Aerobic metal wheels training 48
↑ miR-21, miR-30b ↓ miR-1 BCL-2 p53 PDCD4 Heart samples Swimming training 49
↑ miR-23a, miR-27a PTEN Casp7 FoxO1 Skeletal muscle samples Resistance exercise 50
↑ miR-29c ↓ miR-1 COL1A1 COL3A1 Heart samples Swimming training 51
↑ miR-382 Serum, tissues, and cell samples IR mice Aerobic exercise 25
MicroRNAs Targets Source Types of Exercises Reference
Clinical studies
↑ miR-126, miR-133 CPK Plasma Single symptom-limited spiroergometry test Marathon run Eccentric resistance exercise 52
↓ miR-486 PTEN Serum Systematic-cycling at 70% VO2max 53
↑ miR-1, miR-126, miR-133a, miR-134, miR-146a, miR-208a, miR-499-5p CPK NT-proBNP hsCRP Plasma Marathon run Immediately after run 21
↑ miR-1, miR-133a, miR-206, miR-208b, miR-499 Plasma Marathon run Immediately after run 54
↑ miR-1, miR-133a, miR-206 Plasma Marathon run Immediately after run 55
↑ let-7d-3p, let-7f-3p miR-29a-3p, miR-34a-5p, miR-125b-5pmiR-132-3p, miR-143-3p, miR-148a-3p, miR-223-3p, miR-223-5p miR-424-3p, miR-424-5p Serum Marathon run Immediately after run 56
↑ miR-1, miR-30a, miR-133a ↓ miR-26a, -29b Plasma Marathon run Immediately after run 57
↑ miR-1, miR-133a, miR-206 Plasma Marathon run Immediately after run 58
↑ miR-1, miR-133a, miR-133b, miR-139-5p, miR-143, miR-145, miR-223, miR-330-3p, miR-338-3p, miR-424 ↓ miR-30b, miR-106a, miR-146, miR-151-3p, miR-151-5p, miR-221, miR-652, let-7i ↑ miR-103, miR-107 ↓ miR-21, miR-25, miR-29b, miR-92a, miR-133a, miR-148a, miR-148b, miR-185, miR-342-3p, miR-766, let-7d Plasma Cycle ergometry test 1-3 hs after exercise Systematic endurance cycle ergometry training 59
↑ miR-1, miR-133a, miR-133b, miR-206 miR-485-5p, miR-509-5p, miR-517a miR-518f, miR-520f, miR-522, miR-553, miR-888 Plasma High intensity interval exercise Immediately after 60
↑ miR-181b, miR-214 ↑ miR-1, miR-133a, miR-133b, miR-208b Plasma Uphill treadmill test (concentric) Immediately after Downhill treadmill test (eccentric) 2-6 hs after exercise 61
↑ miR-149 ↓ miR-146a, miR-221 Serum Resistance exercise 3 days after exercise 62
↑ miR-1, miR-133a, miR-133b, miR-206, miR-208b, miR-499 Plasma Systematic resistance training 36-72 hs after training 63
↑ miR-1, miR-133a, miR-133b, miR-181a ↓ miR-9, miR-23a, miR-23b, miR-31 ↑ miR-1, miR-29b HDAC4 NRF1 Skeletal muscle samples Cycle ergometer, Cycling 64
↑ miR-136, miR-200c, miR-376a, miR-377, miR-499b, miR-558 ↓ miR-28, miR-30d, miR-204, miR-330, miR-345, miR-375, miR-449c, miR-483, miR-509, miR-520a, miR-548a, miR-628, miR-653, miR-670, miR-889, miR-1245a, miR-1270, miR-1280, miR-1322, miR-3180 Skeletal muscle samples Resistance training 65
↑ miR-451 ↓ miR-26a, miR-29a, miR-378 Skeletal muscle samples Resistance exercise 66
↑ miR-125a, miR-145, miR-181b, miR-193a, miR-197, miR-212, miR-223, miR-340, miR-365, miR-485, miR-505, miR-520d, miR-629, miR-638, miR-939, miR-940, miR-1225, miR-1238 ↓ let-7i, miR-16, miR-17, miR-18a, miR-18b, miR-20a, miR-20b, miR-22, miR-93, miR- 96, miR-106a, miR-107, miR-126, miR-130a, miR-130b, miR-151, miR-185, miR-194, miR-363, miR-660 Serum Cycle ergometer exercise 67
↑ miR-7, miR-15a, miR-21, miR-26b, miR-132, miR-140, miR-181a, miR-181b, miR-181c, miR-338, miR-363, miR-939, miR-940, miR-1225 ↓ let-7e, miR-23b, miR-31, miR-99a, miR-125a, miR-125b, miR-126, miR-130a, miR-145, miR-151, miR-199a, miR-199b, miR-221, miR-320, miR-451, miR-486, miR-584, miR-652 PBMC Cycle ergometer exercise 68
↑ let-7f, miR-21, miR-29c, miR-223 ↓ let-7f, miR-21, miR-29c, miR-223 PBMC Running exercise 69
↑ miR-7, miR-29a, miR-29b, miR-29c, miR-30e, miR-142, miR-192, miR-338, miR-363, miR-590 ↓ let-7e, miR-126, miR-130a, miR-151, miR-199a, miR-221, miR-223, miR-326, miR-328, miR-652 PBMC Cycle ergometer exercise 70
↑ miR-15a, miR-29b, miR-29c, miR-30e, miR-140, miR-324, miR-338, miR-362, miR-532, miR-660 ↓ miR-23b, miR-130a, miR-151, miR-199a, miR-221 Serum Cycle ergometer exercise 71
↑ miR-1, miR-486, miR-494 Serum (Endurance athletes, runners, cyclists, and triathletes) Cardiopulmonary exercise test 72
↑ miR-21, miR-146a, miR-221, miR-222 ↑ miR-20a, miR-21, miR-146a, miR-221, miR-222 Serum Rowing training, 5Km, 1-3 h per session, 20-24 strokes/min) 73
↑ miR-376a ↓ miR-16, miR-27a, miR-28 Plasma Aerobic run exercise training (4 days/week) 74
↑ miR-19a, miR-19b, miR-20a, miR-26b, miR-143, miR-195 p-AKT p-S6K1 Serum Resistance exercise 75
↑ miR-222 HIPK1 Plasma Bicycle Ergometry Test 41
↑ miR-221 ↓ miR-208b, miR-221, miR-21, miR-146a, miR-210 Serum Basketball Exercise 76

Some studies have also reported the importance of PET modulating the expression of miRNAs in pathological situations, as well as in diabetics,24,25 in obesity,26 after myocardial infarction,27 and with heart failure;22 however, the role of PET-modulating miRNAs in CD has not yet been illustrated. The literature presents only studies that have shown beneficial effects of PET on CD, but they did not analyze the miRNA profile.

Performing only aerobic PET with moderate intensity (50% to 70% of maximum heart rate), three days a week, for 30 minutes, in 12 weeks, obtained a significant increase in maximum cardiorespiratory and metabolic capacity (VO2), increased time in exercise, distance covered, and improvement in emotional aspects,28 as well as association with an RT program, obtained similar beneficial results.29

In another study, with a similar PET protocol, an improvement in functional capacity was also evidenced, with an improvement in ejection fraction and respiratory strength, improvement in diastolic pressure in the left ventricle and in the quality of life of Chagas patients after 8 months of training.30

A cardiac rehabilitation program consisting of the same PET protocol mentioned above, with RT and stretches, adding nutritional guidance and pharmacological counseling for patients with CD, demonstrated an increase in the physical and functional capacity, improving the quality of life of Chagas patients.31

In another important study, researchers performed PET three times a week for six months on Chagas patients. They demonstrated that the exercise group increased peak exercise oxygen consumption and maximum minute ventilation, improving the functional capacity of these patients.32

However, even demonstrating that PET has beneficial effects for patients with CD, it is difficult to analyze the effects of this type of training at the tissue, cell, and molecular levels, given that these studies were performed in humans, where biopsies would be necessary. Therefore, to investigate the possible mechanisms associated with these beneficial effects of PET on CD, some studies have been carried out on experimental models of CD in vivo.

Balb/c mice performed PET on a treadmill before being infected by T. Cruzi. It was observed that PET reduced the peak of parasitemia, concluding that PET can promote beneficial changes in the immune system and obtain better responses to infections.33

In other studies, the same finding as in the previous study was reported; however, they also observed that trained mice obtained greater protection from the metabolic activity of NADH in myenteric neurons and greater synthesis of TNF-α and TGF-β.34 This contributed to the survival and/or protection of 10.3% of myenteric neurons and their immunoreactive production of nitric oxide neuronal synthase, in fact, the trained group obtained a greater expression of TNF-α during the acute phase of T. Cruzi infection, providing benefits to the host and improving the immune system to preserve nitrergic neurons.35

In this context, in another study, researchers observed that the PET group obtained a greater expression of TNF-α, IFNγ, IL-6, and chemokines MCP-1 and CX3CL1 during acute infection, and also obtained better physical capacity, increased anaerobic threshold, increased activity of catalase and superoxide dismutase and reduced lipid and protein oxidation in cardiac tissue, demonstrating that PET can be an interesting strategy to increase the efficiency of endogenous antioxidant mechanisms, reducing oxidative damage in these animals.36

Another study showed that PET before infection in Wistar rats, increased the time to reach fatigue and anaerobic threshold, reduced the expression of TNF-α, CCL-2, MCP-1, and CX3CL1, as well as lipid and protein oxidation, and increased the expression of IL-10, catalase, and superoxide dismutase, indicating that PET induces a protective phenotype, increasing the host’s defenses against the parasitic agent, including the attenuation of the pathological remodeling process associated with musculoskeletal myositis.37

Finally, in another study, Swiss mice were infected by T. Cruzi after PET with moderate intensity on a treadmill, being carried out for 9 weeks. Researchers identified that PET was able to reduce the latent parasitemia of the infected animals they trained, corroborating with previous studies, and even obtained less production of pro-inflammatory cytokines (TNF-α, INFγ, IL-12) and type-1 monocyte chemotactic protein (MCP-1) during the first days of infection.38

Thus, it is suggested that PET has a therapeutic potential for the prevention and complementary treatment of CD and CCC through the modulation of the immune system. However, clinical studies lack morphometric, cellular, and molecular analyzes, mainly through the analysis of miRNAs for a better understanding of the beneficial effects of PET on signaling pathways in humans with CD, while preclinical studies, in vivo, need studies that evaluate the effects of PET with CD and CCC already installed and not only in the pre-infection stage.

 Overlaps between miRNAs in CD and PET

Additionally, this study also performed an analysis using the Venn diagram to identify miRNAs that were modulated by PET in both clinical and pre-clinical studies that can possibly modulate miRNAs in CD.

There were only 7 miRNAs expressed in CD, 95 miRNAs expressed in PET clinical studies, and 36 miRNAs expressed in PET pre-clinical studies. Interestingly, the present study identified 7 miRNAs that had modulations in both CD and PET clinical studies, 3 common miRNAs modulated in CD and PET pre-clinical studies and, mainly, 12 common miRNAs modulated in CD, PET clinical studies, and PET pre-clinical studies (Figure 1). These 12 miRNAs are: miR-1, miR-21, miR-26b, miR-29b, miR-133a, miR-133b, miR-139, miR-145, miR-146a, miR-208a, miR-208b, and miR-222.

– Venn diagram shows overlaps between miRNAs: miRNAs (miRs) in Chagas Disease (blue), miRs PET clin: clinical studies (pink) and miRs PET pre-clin: pre-clinical studies (green).

Nevertheless, of these 12 common miRNAs, only miR-133b, miR-139, and miR-208a were identified with a different expression pattern in CD and PET; all 3 miRNAs are downregulated in CD and upregulated in PET (Figure 2).

– miRNAs expressed in CD that can likely be modulated by PET.

MiR-133b controls the connective tissue growth factor (CTGF)77 and can suppress cardiac remodeling;78 therefore, PET can be an excellent alternative to control cardiac remodeling, possibly through the modulation of miR-133b and the modification of some signaling pathways.

MiR-139 is associated with hypertrophic cardiomyopathy, regulating the expression of c-Jun, a transcriptional factor that binds in the promoter region of some genes to induce cardiac hypertrophy; thus, the overexpression of this miRNA reduces the expression of c-Jun, and consequently attenuates the pathological cardiac hypertrophy,79 which may be a signaling pathway by which PET suppresses the pathological hypertrophy in CD, since PET also increases the expression of this miRNA.22,59

In this context, miR-208a regulates the expression of some transcriptional factors, such as GATA-4, which is associated with the activation of pro-hypertrophic cardiac genes.80 In CD, this miRNA is downregulated,16 while PET can increase its expression,21,42 thus demonstrating that it may possibly be a molecular mechanism by which PET attenuates cardiac hypertrophy in this disease.

 Conclusions

miRNAs participate in several processes in the pathogenesis of CD. Much evidence shows the beneficial effects of PET on CD; however, there still are no articles in the literature that demonstrate the changes in the molecular mechanisms of miRNAs that PET induces in CD. Therefore, further studies are necessary to elucidate these mechanisms.

 Acknowledgments

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001. We would also like to acknowledge the Post-graduate Program in Medicine and Health, School of Medicine of the Federal University of Bahia. We would like to thank Mirela Correia Improta Caria for her help in creating Figure 2.

Study Association

This study is not associated with any thesis or dissertation work.

Sources of Funding: This study was partially funded by CAPES.