In this study, we demonstrate that human cardiomyocytes (AC16) produce reactive oxygen species (ROS) and inflammatory cytokines in response to Trypanosoma cruzi. ROS were primarily produced by mitochondria, some of which diffused to cytosol of infected cardiomyocytes. These ROS resulted in an increase in 8

There is growing evidence to suggest that chagasic myocardia are exposed to sustained oxidative stress induced injuries that may contribute to disease progression. Trypanosoma cruzi invasion- and replication-mediated cellular injuries and immune-mediated cytotoxic reactions are the common source of reactive oxygen species (ROS) during acute infection. Mitochondria are proposed to be the major source of ROS in chronic chagasic hearts. However, it has not been established yet, whether mitochondrial dysfunction is a causative factor in chagasic cardiomyopathy or a consequence of other pathological events. A better understanding of oxidative stress in relation to cardiac tissue damage would be useful in the evaluation of its true role in the pathogenesis of Chagas disease and other heart diseases. In this review, we discuss the evidence for increased oxidative stress in chagasic disease, with emphasis on mitochondrial abnormalities, and its role in sustaining oxidative stress in myocardium.

Objectives Determine the pathological importance of oxidative stress-induced injurious processes in chagasic heart dysfunction. Background Trypanosoma cruzi-induced inflammatory pathology and a feedback cycle of mitochondrial dysfunction and oxidative stress may contribute to Chagas disease. Methods Sprague Dawley rats were infected with T. cruzi, and treated with phenyl-α-tert-butylnitrone (PBN/antioxidant) and/or benzonidazole (BZ/anti-parasite). We monitored myocardial parasite burden, oxidative adducts, mitochondrial complex activities, respiration and ATP synthesis rates, and inflammatory and cardiac remodeling responses during disease development. Cardiac hemodynamics was determined for all rats. Results BZ (not PBN) decreased the parasite persistence and immune adverse events (proinflammatory cytokine expression, NADPH (β-Nicotinamide Adenine Dinucleotide Phosphate, reduced) oxidase and myeloperoxidase activities, and inflammatory infiltrate) in chronic hearts. PBN±BZ (not BZ alone) decreased the mtROS level, oxidative adducts (malonyldialdehyde, 4-hydroxynonenal, carbonyls), hypertrophic gene expression (ANP, BNP, αsk-Actin), and collagen deposition, and preserved the respiratory chain efficiency and energy status in chronic hearts. Subsequently, left ventricular dysfunction was prevented in PBN±BZ-treated chagasic rats. Conclusions BZ treatment after acute stage decreased the parasite persistence and inflammatory pathology. Yet, oxidative adducts, mitochondrial dysfunction and remodeling responses persisted and contributed to declining cardiac function in chagasic rats. Combinatorial treatment (PBN+BZ) was beneficial in arresting the T. cruzi-induced inflammatory and oxidative pathology and chronic heart failure in chagasic rats.

In this study, we investigated the role of Trypanosoma cruzi invasion and inflammatory processes in reactive oxygen species (ROS) production in mouse atrial cardiomyocyte line (HL-1) and primary adult rat ventricular cardiomyocytes. Cardiomyocytes were incubated with T. cruzi (Tc) trypomastigotes, Tc lysate (TcTL) or Tc secreted proteins (TcSP) for 0-72 h, and ROS measured by amplex red assay. Cardiomyocytes infected by T. cruzi (but not those incubated with TcTL or TcSP) exhibited a linear increase in ROS production during 2-48 h post-infection (max.18-fold increase) which was further enhanced by recombinant cytokines (IL-1β, TNF-α and IFN-γ). We observed no increase in NADPH oxidase, xanthine oxidase, and myeloperoxidase activities, and specific inhibitor of these enzymes did not block the increased rate of ROS production in infected cardiomyocytes. Instead, the mitochondrial membrane potential was perturbed, and resulted in inefficient electron transport chain (ETC) activity, and enhanced electron leakage and ROS formation in infected cardiomyocytes. HL-1 rho (ρ) cardiomyocytes lacked a functional ETC, and exhibited no increase in ROS formation in response to T. cruzi. Together, these results demonstrate that invasion by T. cruzi and inflammatory milieu affect mitochondrial integrity and contribute to electron transport chain inefficiency and ROS production in cardiomyocytes.

BackgroundChagas disease is a major health problem in Latin America, and an emerging infectious disease in the US. Previously, we have screened the Trypanosoma cruzi sequence database by a computational/bioinformatics approach, and identified antigens that exhibited the characteristics of vaccine candidates.MethodologyWe investigated the protective efficacy of a multi-component DNA-prime/protein-boost vaccine (TcVac2) constituted of the selected candidates and cytokine (IL-12 and GM-CSF) expression plasmids in a murine model. C57BL/6 mice were immunized with antigen-encoding plasmids plus cytokine adjuvants, followed by recombinant proteins; and two-weeks later, challenged with T. cruzi trypomastigotes. ELISA and flow cytometry were employed to measure humoral (antibody isotypes) and cellular (lymphocyte proliferation, CD4+ and CD8+ T cell phenotype and cytokines) responses. Myocardial pathology was evaluated by H&E and Masson's trichrome staining.Principal FindingsTcVac2 induced a strong antigen-specific antibody response (IgG2b>IgG1) and a moderate level of lymphocyte proliferation in mice. Upon challenge infection, TcVac2-vaccinated mice expanded the IgG2b/IgG1 antibodies and elicited a substantial CD8+ T cell response associated with type 1 cytokines (IFN-γ and TNF-α) that resulted in control of acute parasite burden. During chronic phase, antibody response persisted, splenic activation of CD8+ T cells and IFN-γ/TNF-α cytokines subsided, and IL-4/IL-10 cytokines became dominant in vaccinated mice. The tissue parasitism, inflammation, and fibrosis in heart and skeletal muscle of TcVac2-vaccinated chronic mice were undetectable by histological techniques. In comparison, mice injected with vector or cytokines only responded to T. cruzi by elicitation of a mixed (type 1/type 2) antibody, T cell and cytokine response, and exhibited persistent parasite burden and immunopathology in the myocardium.ConclusionTcVac2-induced activation of type 1 antibody and lymphocyte responses provided resistance to acute T. cruzi infection, and consequently, prevented the evolution of chronic immunopathology associated with parasite persistence in chagasic hearts.