Discovery of circulating miRNAs as biomarkers of chronic Chagas heart disease via a small RNA-Seq approach.

Chagas disease affects approximately 7 million people worldwide in Latin America and is a neglected tropical disease. Twenty to thirty percent of chronically infected patients develop chronic Chagas cardiomyopathy decades after acute infection. Identifying biomarkers of Chagas disease progression is necessary to develop better therapeutic and preventive strategies. Circulating microRNAs are increasingly reliable biomarkers of disease and therapeutic targets. To identify new circulating microRNAs for Chagas disease, we performed exploratory small RNA sequencing from the plasma of patients and performed de novo miRNA prediction, identifying potential new microRNAs. The levels of the new microRNAs temporarily named miR-Contig-1519 and miR-Contig-3244 and microRNAs that are biomarkers for nonchagasic cardiomyopathies, such as miR-148a-3p and miR-224-5p, were validated by quantitative reverse transcription. We found a specific circulating microRNA signature defined by low miR-Contig-3244, miR-Contig-1519, and miR-148a-3 levels but high miR-224-5p levels for patients with chronic Chagas disease. Finally, we predicted in silico that these altered circulating microRNAs could affect the expression of target genes involved in different cellular pathways and biological processes, which we will explore in the future.

Molecular Remodeling of Cardiac Sinus Node Associated with Acute Chagas Disease Myocarditis.

Chagas disease principally affects Latin-American people, but it currently has worldwide distribution due to migration. Death among those with Chagas disease can occur suddenly and without warning, even in those who may not have evidence of clinical or structural cardiac disease and who are younger than 60 years old. HCN4 channels, one of the principal elements responsible for pacemaker currents, are associated with cardiac fetal reprogramming and supraventricular and ventricular arrhythmias, but their role in chagasic arrhythmias is not clear. We found that a single-dose administration of ivabradine, which blocks HCN4, caused QTc and QRS enlargement and an increase in P-wave amplitude and was associated with ventricular and supraventricular arrhythmias in mice challenged with isoproterenol, a chronotropic/ionotropic positive agent. Continuous treatment with ivabradine did not alter the QTc interval, but P-wave morphology was deeply modified, generating supraventricular arrhythmias. In addition, we found that repolarization parameters improved with ivabradine treatment. These effects could have been caused by the high HCN4 expression observed in auricular and ventricular tissue in infected mice. Thus, we suggest, for the first time, that molecular remodeling by overexpression of HCN4 channels may be related to supraventricular arrhythmias in acute Chagas disease, causing ivabradine over-response. Thus, ivabradine treatment should be administered with caution, while HCN4 overexpression may be an indicator of heart failure and/or sudden death risk.

Autoantibodies against the immunodominant sCha epitope discriminate the risk of sudden death in chronic Chagas cardiomyopathy.

In Chagas disease (ChD) caused by Trypanosoma cruzi, new biomarkers to predict chronic cardiac pathology are urgently needed. Previous studies in chagasic patients with mild symptomatology showed that antibodies against the immunodominant R3 epitope of sCha, a fragment of the human basic helix-loop-helix transcription factor like 5, correlated with cardiac pathology. To validate sCha as a biomarker and to understand the origin of anti-sCha antibodies, we conducted a multicenter study with several cohorts of chagasic patients with severe cardiac symptomatology. We found that levels of antibodies against sCha discriminated the high risk of sudden death, indicating they could be useful for ChD prognosis. We investigated the origin of the antibodies and performed an alanine scan of the R3 epitope. We identified a minimal epitope MRQLD, and a BLAST search retrieved several T. cruzi antigens. Five of the hits had known or putative functions, of which phosphonopyruvate decarboxylase showed the highest cross-reactivity with sCha, confirming the role of molecular mimicry in the development of anti-sCha antibodies. Altogether, we demonstrate that the development of antibodies against sCha, which originated by molecular mimicry with T. cruzi antigens, could discriminate electrocardiographic alterations associated with a high risk of sudden death.

L-arginine supplementation reduces mortality and improves disease outcome in mice infected with Trypanosoma cruzi.

Chagas disease caused by Trypanosoma cruzi is a neglected disease that affects about 7 million people in Latin America, recently emerging on other continents due to migration. As infection in mice is characterized by depletion of plasma L-arginine, the effect on infection outcome was tested in mice with or without L-arginine supplementation and treatment with 1400W, a specific inhibitor of inducible nitric oxide synthase (iNOS). We found that levels of L-arginine and citrulline were reduced in the heart and plasma of infected mice, whereas levels of asymmetric dimethylarginine, an endogenous iNOS inhibitor, were higher. Moreover, L-arginine supplementation decreased parasitemia and heart parasite burden, improving clinical score and survival. Nitric oxide production in heart tissue and plasma was increased by L-arginine supplementation, while pharmacological inhibition of iNOS yielded an increase in parasitemia and worse clinical score. Interestingly, electrocardiograms improved in mice supplemented with L-arginine, suggesting that it modulates infection and heart function and is thus a potential biomarker of pathology. More importantly, L-arginine may be useful for treating T. cruzi infection, either alone or in combination with other antiparasitic drugs.

Differential cytokine profiling in Chagasic patients according to their arrhythmogenic-status.

BACKGROUND: Chagas disease is caused by the protozoan Trypanosoma cruzi and is characterized by heart failure and sudden death. Identifying which factors are involved in evolution and treatment response is actually challenging. Thus, the aim of this work was to determine the Th1/Th17 (IL-6, IL-2, TNF, IL-17 and IFN-γ) and Th2 (IL-4 and IL-10) serum profile in Venezuelan Chagasic patients stratified according amiodarone treatment, hypertension and arrhythmias. METHODS: Sera from 38 chagasic patients were analyzed to determine the level of cytokines by Multiplexed Bead-Based Immunoassays. ANOVA test was applied to determine differences for each group. Additionally, a Linear Discriminant Analysis (LDA) was applied to observe the accuracy of different cytokines to discriminate between the groups. RESULTS: The levels of several cytokines were significantly higher in the high-risk of sudden death and untreated group. LDA showed that IL-2, IFN-γ and IL-10 were the best cytokines for discriminating between high-risk of sudden death and untreated patients versus low-risk of sudden death, treated and control groups. CONCLUSIONS: High IL-2 levels seem to identify patients with high-risk of sudden death and seems adequate as treatment efficacy marker. To our knowledge, this is the first report about the anti-inflammatory role of the amiodarone in Chagas disease, suggesting an inmunomodulatory effect that may be exploited as coadjutant therapy in chronic Chagas disease.

Evidence of reversible bradycardia and arrhythmias caused by immunogenic proteins secreted by T. cruzi in isolated rat hearts.

RATIONALE: Chagas cardiomyopathy, caused by the protozoan Trypanosoma cruzi, is characterized by alterations in intracellular ion, heart failure and arrhythmias. Arrhythmias have been related to sudden death, even in asymptomatic patients, and their molecular mechanisms have not been fully elucidated. OBJECTIVE: The aim of this study is to demonstrate the effect of proteins secreted by T. cruzi on healthy, isolated beating rat heart model under a non-damage-inducing protocol. METHODS AND RESULTS: We established a non-damage-inducing recirculation-reoxygenation model where ultrafiltrate fractions of conditioned medium control or conditioned infected medium were perfused at a standard flow rate and under partial oxygenation. Western blotting with chagasic patient serum was performed to determine the antigenicity of the conditioned infected medium fractions. We observed bradycardia, ventricular fibrillation and complete atrioventricular block in hearts during perfusion with >50 kDa conditioned infected culture medium. The preincubation of conditioned infected medium with chagasic serum abolished the bradycardia and arrhythmias. The proteins present in the conditioned infected culture medium of >50 kDa fractions were recognized by the chagasic patient sera associated with arrhythmias. CONCLUSIONS: These results suggest that proteins secreted by T. cruzi are involved in Chagas disease arrhythmias and may be a potential biomarker in chagasic patients.

O etanercepte induz QRS de baixa tensão e disfunção autonômica em camundongos com doença de Chagas experimental / Etanercept induces low QRS voltage and autonomic dysfunction in mice with experimental Chagas disease

FUNDAMENTO: A doença de Chagas é uma doença parasitária tropical causada pelo protozoário flagelado Trypanosoma cruzi. A cardiomiopatia chagásica é caracterizada por distúrbios na regulação autonômica e na condução do potencial de ação nas fases aguda e crônica da infecção. Embora o fator de necrose tumoral alfa (TNF-α) tenha sido associadoà cardiomiopatia em modelos experimentais e em pacientes com doença de Chagas, outros relatos sugerem que o TNF-α pode exercer ações antiparasitárias durante a fase aguda da infecção. OBJETIVOS: Este estudo teve como objetivo determinar os efeitos de um blocker TNF-α solúvel, o etanercepte, em parâmetros eletrocardiográficos na fase aguda da infecção experimental com Trypanosoma cruzi. MÉTODOS: Foram feitos eletrocardiogramas em camundongos infectados não tratados e camundongos infectados que foram tratados com etanercepte 7 dias após a infecção. Os parâmetros de variabilidade onda do eletrocardiograma e frequência cardíaca foram determinados utilizando o Chart para Windows. RESULTADOS: O tratamento com etanercepte resultou em uma baixa tensão do complexo QRS e uma redução da variabilidade da frequência cardíaca em comparação com a ausência de tratamento. No entanto, os camundongos tratados apresentaram um atraso na queda da curva de sobrevivência durante a fase aguda. CONCLUSÃO: Os resultados deste estudo sugerem que, embora o tratamento com etanercepte promova a sobrevivência em camundongos infectados com uma linhagem virulenta de T. cruzi, o bloqueio do TNF-α gera um complexo de baixa tensão e disfunção autonômica durante a fase aguda da infecção. Esses resultados indicam que a mortalidade durante a fase aguda pode ser atribuída a uma resposta inflamatória sistêmica, em vez da disfunção cardíaca.

BACKGROUND: Chagas disease is a tropical parasitic disease caused by the flagellate protozoan Trypanosoma cruzi. Chagasic cardiomyopathy is characterized by disorders of autonomic regulation and action potential conduction in the acute and chronic phases of infection. Although tumor necrosis factor alpha (TNF-α) has been linked to cardiomyopathy in experimental models and in patients with Chagas disease, other reports suggest that TNF-α may exert anti-parasitic actions during the acute phase of infection. OBJECTIVES: This study aimed to determine the effects of a soluble TNF-α agonist, etanercept, on electrocardiographic parameters in the acute phase of experimental infection with Trypanosoma cruzi. METHODS: Electrocardiograms were obtained from untreated infected mice and infected mice who were treated with etanercept 7 days after infection. ECG wave and heart rate variability parameters were determined using Chart for Windows. RESULTS: Etanercept treatment resulted in a low QRS voltage and decreased heart rate variability compared with no treatment. However, the treated mice exhibited a delay in the fall of the survival curve during the acute phase. CONCLUSION: The results of this study suggest that although etanercept treatment promotes survival in mice infected with a virulent T. cruzi strain, TNF-α blockade generates a low voltage complex and autonomic dysfunction during the acute phase of infection. These findings indicate that mortality during the acute phase can be attributed to a systemic inflammatory response rather than cardiac dysfunction.).

Etanercept induces low QRS voltage and autonomic dysfunction in mice with experimental Chagas disease.

BACKGROUND: Chagas disease is a tropical parasitic disease caused by the flagellate protozoan Trypanosoma cruzi. Chagasic cardiomyopathy is characterized by disorders of autonomic regulation and action potential conduction in the acute and chronic phases of infection. Although tumor necrosis factor alpha (TNF-α) has been linked to cardiomyopathy in experimental models and in patients with Chagas disease, other reports suggest that TNF-α may exert anti-parasitic actions during the acute phase of infection. OBJECTIVES: This study aimed to determine the effects of a soluble TNF-α agonist, etanercept, on electrocardiographic parameters in the acute phase of experimental infection with Trypanosoma cruzi. METHODS: Electrocardiograms were obtained from untreated infected mice and infected mice who were treated with etanercept 7 days after infection. ECG wave and heart rate variability parameters were determined using Chart for Windows. RESULTS: Etanercept treatment resulted in a low QRS voltage and decreased heart rate variability compared with no treatment. However, the treated mice exhibited a delay in the fall of the survival curve during the acute phase. CONCLUSION: The results of this study suggest that although etanercept treatment promotes survival in mice infected with a virulent T. cruzi strain, TNF-α blockade generates a low voltage complex and autonomic dysfunction during the acute phase of infection. These findings indicate that mortality during the acute phase can be attributed to a systemic inflammatory response rather than cardiac dysfunction.