The effects of inflammation on connexin 43 in chronic Chagas disease cardiomyopathy.

Background: Cardiac arrhythmias are the main cause of sudden death due to Chronic Chagasic Cardiomyopathy (CCC). Here we investigated alterations in connexin 43 (Cx43) expression and phosphorylation in cardiomyocytes as well as associations with cardiac arrhythmias in CCC. Methods: C57Bl/6 mice infected with Trypanosoma cruzi underwent cardiac evaluations at 6 and 12 months after infection via treadmill testing and EKG. Histopathology, cytokine gene expression, and distribution of total Cx43 and its phosphorylated forms Cx43S368 and Cx43S325/328/330 were investigated. Human heart samples obtained from subjects with CCC were submitted to immunofluorescence analysis. In vitro simulation of a pro-inflammatory microenvironment (IL-1ß, TNF, and IFN-γ) was performed in H9c2 cells and iPSC-derived cardiomyocytes to evaluate Cx43 distribution, action potential duration, and Lucifer Yellow dye transfer. Results: Mice chronically infected with T. cruzi exhibited impaired cardiac function associated with increased inflammation, fibrosis and upregulated IL-1ß, TNF, and IFN-γ gene expression. Confocal microscopy revealed altered total Cx43, Cx43S368 and Cx43S325/328/330 localization and phosphorylation patterns in CCC, with dispersed staining outside the intercalated disc areas, i.e., in lateral membranes and the cytoplasm. Reduced co-localization of total Cx43 and N-cadherin was observed in the intercalated discs of CCC mouse hearts compared to controls. Similar results were obtained in human CCC heart samples, which showed Cx43 distribution outside the intercalated discs. Stimulation of human iPSC-derived cardiomyocytes or H9c2 cells with IL-1ß, TNF, and IFN-γ induced alterations in Cx43 localization, reduced action potential duration and dye transfer between adjacent cells. Conclusion: Heart inflammation in CCC affects the distribution and phosphorylation pattern of Cx43, which may contribute to the generation of conduction disturbances in Chagas disease.

Pirfenidone Prevents Heart Fibrosis during Chronic Chagas Disease Cardiomyopathy.

Cardiac fibrosis is a severe outcome of Chagas disease (CD), caused by the protozoan Trypanosoma cruzi. Clinical evidence revealed a correlation between fibrosis levels with impaired cardiac performance in CD patients. Therefore, we sought to analyze the effect of inhibitors of TGF-ß (pirfenidone), p38-MAPK (losmapimod) and c-Jun (SP600125) on the modulation of collagen deposition in cardiac fibroblasts (CF) and in vivo models of T. cruzi chronic infection. Sirius Red/Fast Green dye was used to quantify both collagen expression and total protein amount, assessing cytotoxicity. The compounds were also used to treat C57/Bl6 mice chronically infected with T. cruzi, Brazil strain. We identified an anti-fibrotic effect in vitro for pirfenidone (TGF-ß inhibitor, IC50 114.3 µM), losmapimod (p38 inhibitor, IC50 17.6 µM) and SP600125 (c-Jun inhibitor, IC50 3.9 µM). This effect was independent of CF proliferation since these compounds do not affect T. cruzi-induced host cell multiplication as measured by BrdU incorporation. Assays of chronic infection of mice with T. cruzi have shown a reduction in heart collagen by pirfenidone. These results propose a novel approach to fibrosis therapy in CD, with the prospect of repurposing pirfenidone to prevent the onset of ECM accumulation in the hearts of the patients.

Arrhythmogenic Manifestations of Chagas Disease: Perspectives From the Bench to Bedside.

Chagas cardiomyopathy caused by infection with the intracellular parasite Trypanosoma cruzi is the most common and severe expression of human Chagas disease. Heart failure, systemic and pulmonary thromboembolism, arrhythmia, and sudden cardiac death are the principal clinical manifestations of Chagas cardiomyopathy. Ventricular arrhythmias contribute significantly to morbidity and mortality and are the major cause of sudden cardiac death. Significant gaps still exist in the understanding of the pathogenesis mechanisms underlying the arrhythmogenic manifestations of Chagas cardiomyopathy. This article will review the data from experimental studies and translate those findings to draw hypotheses about clinical observations. Human- and animal-based studies at molecular, cellular, tissue, and organ levels suggest 5 main pillars of remodeling caused by the interaction of host and parasite: immunologic, electrical, autonomic, microvascular, and contractile. Integrating these 5 remodeling processes will bring insights into the current knowledge in the field, highlighting some key features for future management of this arrhythmogenic disease.

Identification of host antioxidant effectors as thioridazine targets: Impact on cardiomyocytes infection and Trypanosoma cruzi-induced acute myocarditis.

Phenothiazines inhibit antioxidant enzymes in trypanosomatids. However, potential interferences with host cell antioxidant defenses are central concerns in using these drugs to treat Trypanosoma cruzi-induced infectious myocarditis. Thus, the interaction of thioridazine (TDZ) with T. cruzi and cardiomyocytes antioxidant enzymes, and its impact on cardiomyocytes and cardiac infection was investigated in vitro and in vivo. Cardiomyocytes and trypomastigotes in culture, and mice treated with TDZ and benznidazole (Bz, reference antiparasitic drug) were submitted to microstructural, biochemical and molecular analyses. TDZ was more cytotoxic and less selective against T. cruzi than Bz in vitro. TDZ-pretreated cardiomyocytes developed increased infection rate, reactive oxygen species (ROS) production, lipid and protein oxidation; similar catalase (CAT) and superoxide dismutase (SOD) activity, and reduced glutathione's (peroxidase - GPx, S-transferase - GST, and reductase - GR) activity than infected untreated cells. TDZ attenuated trypanothione reductase activity in T. cruzi, and protein antioxidant capacity in cardiomyocytes, making these cells more susceptible to H2O2-based oxidative challenge. In vivo, TDZ potentiated heart parasitism, total ROS production, myocarditis, lipid and protein oxidation; as well as reduced GPx, GR, and GST activities compared to untreated mice. Benznidazole decreased heart parasitism, total ROS production, heart inflammation, lipid and protein oxidation in T. cruzi-infected mice. Our findings indicate that TDZ simultaneously interact with enzymatic antioxidant targets in cardiomyocytes and T. cruzi, potentiating the infection by inducing antioxidant fragility and increasing cardiomyocytes and heart susceptibility to parasitism, inflammation and oxidative damage.

Preclinical evaluation of combined therapy with amiodarone and low-dose benznidazole in a mouse model of chronic Trypanosoma cruzi infection.

Chagasic chronic cardiomyopathy (CCC) is the primary clinical manifestation of Chagas disease (CD), caused by Trypanosoma cruzi. Current therapeutic options for CD are limited to benznidazole (Bz) and nifurtimox. Amiodarone (AMD) has emerged as most effective drug for treating the arrhythmic form of CCC. To address the effects of Bz and AMD we used a preclinical model of CCC. Female C57BL/6 mice were infected with T. cruzi and subjected to oral treatment for 30 consecutive days, either as monotherapy or in combination. AMD in monotherapy decreased the prolonged QTc interval, the incidence of atrioventricular conduction disorders and cardiac hypertrophy. However, AMD monotherapy did not impact parasitemia, parasite load, TNF concentration and production of reactive oxygen species (ROS) in cardiac tissue. Alike Bz therapy, the combination of Bz and AMD (Bz/AMD), improved cardiac electric abnormalities detected T. cruzi-infected mice such as decrease in heart rates, enlargement of PR and QTc intervals and increased incidence of atrioventricular block and sinus arrhythmia. Further, Bz/AMD therapy ameliorated the ventricular function and reduced parasite burden in the cardiac tissue and parasitemia to a degree comparable to Bz monotherapy. Importantly, Bz/AMD treatment efficiently reduced TNF concentration in the cardiac tissue and plasma and had beneficial effects on immunological abnormalities. Moreover, in the cardiac tissue Bz/AMD therapy reduced fibronectin and collagen deposition, mitochondrial damage and production of ROS, and improved sarcomeric and gap junction integrity. Our study underlines the potential of the Bz/AMD therapy, as we have shown that combination increased efficacy in the treatment of CCC.

The mitochondrial uncoupler 2,4-dinitrophenol modulates inflammatory and oxidative responses in Trypanosoma cruzi-induced acute myocarditis in mice.

By uncoupling oxidative phosphorylation, 2,4-dinitrophenol (DNP) attenuates reactive oxygen species (ROS) biosynthesis, which are known to aggravate infectious myocarditis in Chagas disease. Thus, the impact of DNP-based chemotherapy on Trypanosoma cruzi-induced acute myocarditis was investigated. C56BL/6 mice uninfected and infected untreated and treated daily with 100 mg/kg benznidazole (Bz, reference drug), 5 and 10 mg/kg DNP by gavage for 11 days after confirmation of T. cruzi infection were investigated. Twenty-four hours ​after the last treatment, the animals were euthanized and the heart was collected for microstructural, immunological and biochemical analyses. T. cruzi inoculation induced systemic inflammation (e.g., cytokines and anti-T. cruzi IgG upregulation), cardiac infection (T. cruzi DNA), oxidative stress, inflammatory infiltrate and microstructural myocardial damage in untreated mice. DNP treatment aggravated heart infection and microstructural damage, which were markedly attenuated by Bz. DNP (10 mg/kg) was also effective in attenuating ROS (total ROS, H2O2, and O2-), nitric oxide (NO), lipid (malondialdehyde - MDA) and protein (protein carbonyl - PCn) oxidation, TNF, IFN-γ, IL-10, and MCP-1/CCL2, anti-T. cruzi IgG, cardiac troponin I levels, as well as inflammatory infiltrate and cardiac damage in T. cruzi-infected mice. Our findings indicate that DNP aggravated heart infection and microstructural cardiomyocytes damage in infected mice. These responses were related to the antioxidant and anti-inflammatory properties of DNP, which favors infection by weakening the pro-oxidant and pro-inflammatory protective mechanisms of the infected host. Conversely, Bz-induced cardioprotective effects combined effective anti-inflammatory and antiparasitic responses, which protect against heart infection, oxidative stress, and microstructural damage in Chagas disease.

Oxidative stress, cardiomyocytes premature senescence and contractile dysfunction in in vitro and in vivo experimental models of Chagas disease.

AIMS: The relationship between redox imbalance and cardiovascular senescence in infectious myocarditis is unknown. Thus, the aim of this study was to investigate whether cardiomyocytes parasitism, oxidative stress and contractile dysfunction can be correlated to senescence-associated ß-galactosidase (SA-ß-Gal) activity in Trypanosoma cruzi-infection in vitro and in vivo. METHODS: Uninfected, T. cruzi-infected untreated and benznidazole (BZN)-treated H9c2 cardiomyocytes and rats were investigated. Parasitological, prooxidant, antioxidant, microstructural, and senescence-associated markers were quantified in vitro and in vivo. RESULTS: T. cruzi infection triggered intense cardiomyocytes parasitism in vitro and in vivo, which was accompanied by reactive oxygen species (ROS) upregulation, lipids, proteins and DNA oxidation in cardiomyocytes and cardiac tissue. Oxidative stress was parallel to microstructural cell damage (e.g., increased cardiac toponin I levels) and contractile dysfunction in cardiomyocytes in vitro and in vivo, whose severity accompanied a premature cellular senescence-like phenotype revealed by increased senescence-associated ß-galactosidase (SA-ß-Gal) activity and DNA oxidation (8-OHdG). Cellular parasitism (e.g., infection rate and parasite load), myocarditis and T. cruzi-induced prooxidant responses were attenuated by early BZN administration to interrupt the progression of T. cruzi infection, protecting against SA-ß-gal-based premature cellular senescence, microstructural damage and contractile deterioration in cardiomyocytes from T. cruzi-infected animals. CONCLUSION: Our findings indicated that cell parasitism, redox imbalance and contractile dysfunction were correlated to SA-ß-Gal-based cardiomyocytes premature senescence in acute T. cruzi infection. Therefore, in addition to controlling parasitism, inflammation and oxidative stress; inhibiting cardiomyocytes premature senescence should be further investigated as an additional target of specific Chagas disease therapeutics.

Risk Factors for Progression to Chronic Chagas Cardiomyopathy: A Systematic Review and Meta-Analysis.

Approximately one-third of people with chronic Trypanosoma cruzi infection develop Chagas cardiomyopathy, which carries a poor prognosis. Accurate prediction of which individuals will go on to develop Chagas cardiomyopathy remains elusive. We performed a systematic review of literature comparing characteristics of individuals with chronic Chagas disease with or without evidence of cardiomyopathy. Studies were not excluded on the basis of language or publication date. Our review yielded a total of 311 relevant publications. We further examined the subset of 170 studies with data regarding individual age, sex, or parasite load. A meta-analysis of 106 eligible studies indicated that male sex was associated with having Chagas cardiomyopathy (Hedge's g: 1.56, 95% CI: 1.07-2.04), and a meta-analysis of 91 eligible studies indicated that older age was associated with having Chagas cardiomyopathy (Hedge's g: 0.66, 95% CI: 0.41-0.91). A meta-analysis of four eligible studies did not find an association between parasite load and disease state. This study provides the first systematic review to assess whether age, sex, and parasite load are associated with Chagas cardiomyopathy. Our findings suggest that older and male patients with Chagas disease are more likely to have cardiomyopathy, although we are unable to identify causal relationships due to the high heterogeneity and predominantly retrospective study designs in the current literature. Prospective, multidecade studies are needed to better characterize the clinical course of Chagas disease and identify risk factors for progression to Chagas cardiomyopathy.

Macrophages Mediate Healing Properties of Fenofibrate in Experimental Chagasic Cardiomyopathy.

Chronic cardiomyopathy is one of the most relevant outcomes of Chagas disease associated with parasite persistence and exacerbated inflammatory response. Fenofibrate, a third generation fibric acid derivative and peroxisome proliferator-activated receptor-α ligand, is involved in the regulation of inflammatory response. However, the participation of macrophages in this scenario has not been elucidated. Here we show, for the first time, that macrophages play a fundamental role in the fenofibrate-mediated modulation of heart pro-inflammatory response and fibrosis caused by the infection with Trypanosoma cruzi. Furthermore, macrophages are required for fenofibrate to improve the loss of ventricular function and this restoration correlates with an anti-inflammatory microenvironment. Understanding the contributions of macrophages to the healing properties of fenofibrate reinforces its potential use as a therapeutic drug, with the aim of helping to solve a public health problem, such as chronic Chagas disease.

Correlation of TcII discrete typing units with severe chronic Chagas cardiomyopathy in patients from various Brazilian geographic regions.

BACKGROUND: Chagas disease (ChD) is caused by Trypanosoma cruzi. The genetic structure of the species is divided into seven distinct genetic groups, TcI to TcVI, and Tcbat, which have shown differences in terms of geographic distribution, biological properties, and susceptibility to drugs. However, the association between genetic variability and clinical forms of ChD has not yet been fully elucidated. The predominance of TcII and TcVI discrete typing units (DTUs) (genetic groups) is known to occur in several Brazilian regions and is associated with both the domestic and the wild cycles of ChD. Thus, this study aimed to verify the genotypes of the parasites present in 330 patients with chronic Chagas cardiomyopathy (CCC) from different Brazilian states attended at the Clinical Hospital of the Ribeirão Preto Medical School and to assess the existence of a correlation between the clinical forms with the main cardiovascular risk factors and the genetics of the parasite. METHODOLOGY PRINCIPAL FINDINGS: All patients with CCC were clinically evaluated through anamnesis, physical examination, biochemical tests, 12-lead electrocardiogram, echocardiogram and chest X-ray. Peripheral blood (5 mL) was collected in guanidine/ethylenediaminetetraacetic acid from each patient for DNA extraction and real-time polymerase chain reaction (PCR) for Chagas disease and genotyping of the parasite in the 7 DTUs. Parasite genotyping was performed using conventional multilocus PCR. Samples of only 175 patients were positive after amplification of the specific genes contained in the T. cruzi genotyping criteria. TcII (64/175), TcVI (9/175), and TcI (3/175) DTUs were predominant, followed by TcII/TcV/TcVI (74/175), and TcII/TcVI (23/175). The TcIII and TcIV DTU´s was detected in only one sample of CCC patients. CONCLUSIONS/SIGNIFICANCE: Our data corroborate previous findings, indicating the predominance of the TcII genotype in patients with CCC of Brazilian origin. Moreover, this study pioneered disclosing a direct correlation between the TcII DTU and severe CCC.

Long-term cardiology outcomes in children after early treatment for Chagas disease, an observational study.

BACKGROUND: Parasite persistence after acute infection with Trypanosoma cruzi is an important factor in the development of Chagas disease (CD) cardiomyopathy. Few studies have investigated the clinical effectiveness of CD treatment through the evaluation of cardiological events by long term follow-up of treated children. Cardiological evaluation in children is challenging since features that would be diagnosed as abnormal in an adult's ECG may be normal, age-related findings in a pediatric ECG trace. The objective was to evaluate cardiac involvement in patients with Chagas disease with a minimum follow-up of 6 years post-treatment. METHODOLOGY: A descriptive study of a cohort of pediatric patients with CD treated with benznidazole (Bz) or nifurtimox (Nf) was conducted. Children (N = 234) with at least 6 years post CD treatment followed at the Parasitology and Chagas Service, Buenos Aires Children's Hospital (Argentina) were enrolled. By convenience sampling, children who attended a clinical visit between August 2015 and November 2019 were also invited to participate for additional cardiovascular studies like 24-hour Holter monitoring and speckle-tracking 2D echocardiogram (STE). Benznidazole was prescribed in 171 patients and nifurtimox in 63 patients. Baseline parasitemia data was available for 168/234 patients. During the follow-up period, alterations in routine ECG were observed in 11/234 (4.7%, 95% CI [2-7.4%]) patients. In only four patients, with complete right bundle branch block (cRBBB) and left anterior fascicular block (LAFB), ECG alterations were considered probably related to CD. During follow-up, 129/130 (99%) treated patients achieved persistent negative parasitemia by qPCR. Also decrease in T.cruzi antibodies titers was observed in all patients and negative seroconversion occurred in 123/234 (52%) patients. CONCLUSIONS: A low incidence of cardiological lesions related to CD was observed in patients treated early for pediatric CD. This suggests a protective effect of parasiticidal treatment on the development of cardiological lesions and highlights the importance of early treatment of infected children. TRIAL REGISTRATION: ClinicalTrials.gov NCT04090489.

Insights from the use of erythropoietin in experimental Chagas disease.

In addition to the long-established role in erythropoiesis, erythropoietin (Epo) has protective functions in a variety of tissues, including the heart. This is the most affected organ in chronic Chagas disease, caused by the protozoan Trypanosoma cruzi. Despite seven million people being infected with T. cruzi worldwide, there is no effective treatment preventing the disease progression to the chronic phase when the pathological involvement of the heart is often observed. Chronic chagasic cardiomyopathy has a wide variety of manifestations, like left ventricular systolic dysfunction, dilated cardiomyopathy, and heart failure. Since Epo may help maintain cardiac function by reducing myocardial necrosis, inflammation, and fibrosis, this study aimed to evaluate whether the Epo has positive effects on experimental Chagas disease. For that, we assessed the earlier (acute phase) and also the later (chronic phase) use of Epo in infected C57BL/6 mice. Blood cell count, biochemical parameters, parasitic load, and echocardiography data were evaluated. In addition, histopathological analysis was carried out. Our data showed that Epo had no trypanocide effect nor did it modify the production of anti-T. cruzi antibodies. Epo-treated groups exhibited parasitic burden much lower in the heart compared to blood. No pattern of hematological changes was observed combining infection with treatment with Epo. Chronic Epo administration reduced CK-MB serum activity from d0 to d180, irrespectively of T. cruzi infection. Likewise, echocardiography and histological results indicate that Epo treatment is more effective in the chronic phase of experimental Chagas disease. Since treatment is one of the greatest challenges of Chagas disease, alternative therapies should be investigated, including Epo combined with benznidazole.

Trypanosoma cruzi-specific CD8+ T cells and other immunological hallmarks in chronic Chagas cardiomyopathy: Two decades of research.

Trypanosoma cruzi, the causal agent of Chagas disease, has coexisted with humans for thousands of years. Therefore, the parasite has developed several mechanisms of antigenic variability that has allowed it to live inside the cells and evade the host immune response. Since T. cruzi displays an intracellular cycle-stage, our research team focused on providing insights into the CD8+ T cells immune response in chronic Chagas cardiomyopathy. We began our work in the 2000s studying parasite antigens that induce natural immune responses such as the KMP11 protein and TcTLE, its N-terminal derived peptide. Different approaches allowed us to reveal TcTLE peptide as a promiscuous CD8+ T cell epitope, able of inducing multifunctional cellular immune responses and eliciting a humoral response capable of decreasing parasite movement and infective capacity. Next, we demonstrated that as the disease progresses, total CD8+ T cells display a dysfunctional state characterized by a prolonged hyper-activation state along with an increase of inhibitory receptors (2B4, CD160, PD-1, TIM-3, CTLA-4) expression, an increase of specific terminal effector T cells (TTE), a decrease of proliferative capacity, a decrease of stem cell memory (TSCM) frequency, and a decrease of CD28 and CD3ζ expression. Thus, parasite-specific CD8+ T cells undergo clonal exhaustion, distinguished by an increase in late-differentiated cells, a mono-functional response, and enhanced expression of inhibitory receptors. Finally, it was found that anti-parasitic treatment induces an improved CD8+ T cell response in asymptomatic individuals, and a mouse animal model led us to establish a correlation between the quality of the CD8+ T cell responses and the outcome of chronic infection. In the future, using OMICs strategies, the identification of the specific cellular signals involved in disease progression will provide an invaluable resource for discovering new biomarkers of progression or new vaccine and immunotherapy strategies. Also, the inclusion of the TcTLE peptide in the rational design of epitope-based vaccines, the development of immunotherapy strategies using TSCM or the blocking of inhibitory receptors, and the use of the CD8+ T cell response quality to follow treatments, immunotherapies or vaccines, all are alternatives than could be explored in the fight against Chagas disease.

Distinct CD4-CD8- (Double-Negative) Memory T-Cell Subpopulations Are Associated With Indeterminate and Cardiac Clinical Forms of Chagas Disease.

CD4-CD8- (double-negative, DN) T cells are critical orchestrators of the cytokine network associated with the pathogenic inflammatory response in one of the deadliest cardiomyopathies known, Chagas heart disease, which is caused by Trypanosoma cruzi infection. Here, studying the distribution, activation status, and cytokine expression of memory DN T-cell subpopulations in Chagas disease patients without cardiac involvement (indeterminate form-IND) or with Chagas cardiomyopathy (CARD), we report that while IND patients displayed a higher frequency of central memory, CARD had a high frequency of effector memory DN T cells. In addition, central memory DN T cells from IND displayed a balanced cytokine profile, characterized by the concomitant expression of IFN-γ and IL-10, which was not observed in effector memory DN T cells from CARD. Supporting potential clinical relevance, we found that the frequency of central memory DN T cells was associated with indicators of better ventricular function, while the frequency of effector memory DN T cells was not. Importantly, decreasing CD1d-mediated activation of DN T cells led to an increase in IL-10 expression by effector memory DN T cells from CARD, restoring a balanced profile similar to that observed in the protective central memory DN T cells. Targeting the activation of effector memory DN T cells may emerge as a strategy to control inflammation in Chagas cardiomyopathy and potentially in other inflammatory diseases where these cells play a key role.

Aspirin-triggered resolvin D1 reduces parasitic cardiac load by decreasing inflammation in a murine model of early chronic Chagas disease.

BACKGROUND: Chagas disease, caused by the protozoan Trypanosoma cruzi, is endemic in Latin America and is widely distributed worldwide because of migration. In 30% of cases, after years of infection and in the absence of treatment, the disease progresses from an acute asymptomatic phase to a chronic inflammatory cardiomyopathy, leading to heart failure and death. An inadequate balance in the inflammatory response is involved in the progression of chronic Chagas cardiomyopathy. Current therapeutic strategies cannot prevent or reverse the heart damage caused by the parasite. Aspirin-triggered resolvin D1 (AT-RvD1) is a pro-resolving mediator of inflammation that acts through N-formyl peptide receptor 2 (FPR2). AT-RvD1 participates in the modification of cytokine production, inhibition of leukocyte recruitment and efferocytosis, macrophage switching to a nonphlogistic phenotype, and the promotion of healing, thus restoring organ function. In the present study, AT-RvD1 is proposed as a potential therapeutic agent to regulate the pro-inflammatory state during the early chronic phase of Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS: C57BL/6 wild-type and FPR2 knock-out mice chronically infected with T. cruzi were treated for 20 days with 5 µg/kg/day AT-RvD1, 30 mg/kg/day benznidazole, or the combination of 5 µg/kg/day AT-RvD1 and 5 mg/kg/day benznidazole. At the end of treatment, changes in immune response, cardiac tissue damage, and parasite load were evaluated. The administration of AT-RvD1 in the early chronic phase of T. cruzi infection regulated the inflammatory response both at the systemic level and in the cardiac tissue, and it reduced cellular infiltrates, cardiomyocyte hypertrophy, fibrosis, and the parasite load in the heart tissue. CONCLUSIONS/SIGNIFICANCE: AT-RvD1 was shown to be an attractive therapeutic due to its regulatory effect on the inflammatory response at the cardiac level and its ability to reduce the parasite load during early chronic T. cruzi infection, thereby preventing the chronic cardiac damage induced by the parasite.

Spatial metabolomics identifies localized chemical changes in heart tissue during chronic cardiac Chagas Disease.

Chagas disease (CD), caused by the parasite Trypanosoma cruzi, is one of nineteen neglected tropical diseases. CD is a vector-borne disease transmitted by triatomines, but CD can also be transmitted through blood transfusions, organ transplants, T. cruzi-contaminated food and drinks, and congenital transmission. While endemic to the Americas, T. cruzi infects 7-8 million people worldwide and can induce severe cardiac symptoms including apical aneurysms, thromboembolisms and arrhythmias during the chronic stage of CD. However, these cardiac clinical manifestations and CD pathogenesis are not fully understood. Using spatial metabolomics (chemical cartography), we sought to understand the localized impact of chronic CD on the cardiac metabolome of mice infected with two divergent T. cruzi strains. Our data showed chemical differences in localized cardiac regions upon chronic T. cruzi infection, indicating that parasite infection changes the host metabolome at specific sites in chronic CD. These sites were distinct from the sites of highest parasite burden. In addition, we identified acylcarnitines and glycerophosphocholines as discriminatory chemical families within each heart region, comparing infected and uninfected samples. Overall, our study indicated global and positional metabolic differences common to infection with different T. cruzi strains and identified select infection-modulated pathways. These results provide further insight into CD pathogenesis and demonstrate the advantage of a systematic spatial perspective to understand infectious disease tropism.

Inhibition of calcium/calmodulin (Ca2+ /CaM)-Calcium/calmodulin-dependent protein kinase II (CaMKII) axis reduces in vitro and ex vivo arrhythmias in experimental Chagas disease.

Chagasic cardiomyopathy (CCC) is one of the main causes of heart failure and sudden death in Latin America. To date, there is no available medication to prevent or reverse the onset of cardiac symptoms. CCC occurs in a scenario of disrupted calcium dynamics and enhanced oxidative stress, which combined, may favor the hyper activation of calcium/calmodulin (Ca2+ /CaM)-calcium/calmodulin-dependent protein kinase II (CaMKII) (Ca2+ /CaM-CaMKII) pathway, which is fundamental for heart physiology and it is implicated in other cardiac diseases. Here, we evaluated the association between Ca2+ /CaM-CaMKII in the electro-mechanical (dys)function of the heart in the early stage of chronic experimental Trypanosoma cruzi infection. We observed that in vitro and ex vivo inhibition of Ca2+ /CaM-CaMKII reversed the arrhythmic profile of isolated hearts and isolated left-ventricles cardiomyocytes. The benefits of the limited Ca2+ /CaM-CaMKII activation to cardiomyocytes' electrical properties are partially related to the restoration of Ca2+ dynamics in a damaged cellular environment created after T. cruzi infection. Moreover, Ca2+ /CaM-CaMKII inhibition prevented the onset of arrhythmic contractions on isolated heart preparations of chagasic mice and restored the responsiveness to the increase in the left-ventricle pre-load. Taken together, our data provide the first experimental evidence for the potential of targeting Ca2+ /CaM-CaMKII pathway as a novel therapeutic target to treat CCC.

α-Gal immunization positively impacts Trypanosoma cruzi colonization of heart tissue in a mouse model.

Chagas disease, caused by the parasite Trypanosoma cruzi, is considered endemic in more than 20 countries but lacks both an approved vaccine and limited treatment for its chronic stage. Chronic infection is most harmful to human health because of long-term parasitic infection of the heart. Here we show that immunization with a virus-like particle vaccine displaying a high density of the immunogenic α-Gal trisaccharide (Qß-αGal) induced several beneficial effects concerning acute and chronic T. cruzi infection in α1,3-galactosyltransferase knockout mice. Approximately 60% of these animals were protected from initial infection with high parasite loads. Vaccinated animals also produced high anti-αGal IgG antibody titers, improved IFN-γ and IL-12 cytokine production, and controlled parasitemia in the acute phase at 8 days post-infection (dpi) for the Y strain and 22 dpi for the Colombian strain. In the chronic stage of infection (36 and 190 dpi, respectively), all of the vaccinated group survived, showing significantly decreased heart inflammation and clearance of amastigote nests from the heart tissue.

Fat tissue regulates the pathogenesis and severity of cardiomyopathy in murine chagas disease.

Chronic Chagas cardiomyopathy (CCC) caused by a parasite Trypanosoma cruzi is a life-threatening disease in Latin America, for which there is no effective drug or vaccine. The pathogenesis of CCC is complex and multifactorial. Previously, we demonstrated T. cruzi infected mice lose a significant amount of fat tissue which correlates with progression of CCC. Based on this an investigation was undertaken during both acute and chronic T. cruzi infection utilizing the FAT-ATTAC murine model (that allows modulation of fat mass) to understand the consequences of the loss of adipocytes in the regulation of cardiac parasite load, parasite persistence, inflammation, mitochondrial stress, ER stress, survival, CCC progression and CCC severity. Mice were infected intraperitoneally with 5x104 and 103 trypomastigotes to generate acute and chronic Chagas models, respectively. Ablation of adipocytes was carried out in uninfected and infected mice by treatment with AP21087 for 10 days starting at 15DPI (acute infection) and at 65DPI (indeterminate infection). During acute infection, cardiac ultrasound imaging, histological, and biochemical analyses demonstrated that fat ablation increased cardiac parasite load, cardiac pathology and right ventricular dilation and decreased survival. During chronic indeterminate infection ablation of fat cells increased cardiac pathology and caused bi-ventricular dilation. These data demonstrate that dysfunctional adipose tissue not only affects cardiac metabolism but also the inflammatory status, morphology and physiology of the myocardium and increases the risk of progression and severity of CCC in murine Chagas disease.

L-arginine supplementation increases cardiac collagenogenesis in mice chronically infected with Berenice-78 Trypanosoma cruzi strain.

Chagas disease, caused by Trypanosoma cruzi, is a major neglected tropical disease that occurs mainly as chronic infection and systemic infection. Currently, there is no suitable and effective drug to treat this parasitic disease. Administration of nutrients with immunomodulatory properties, such as arginine and nitric oxide radicals, may be helpful as antiparasitic therapy. In this study, we evaluated the effects of arginine supplementation during the acute phase of infection under the development of chronic Chagas' heart disease in Swiss mice inoculated with the Berenice-78 strain of T. cruzi. The effectiveness of arginine was determined by daily detection of the parasite in the blood and long-term serum levels of nitric oxide and tumor necrosis factor-alpha, in addition to evaluation of heart tissue damage. Arginine could flatten parasitemia and prevent elevation of tumor necrosis factor-alpha in T. cruzi-infected mice. Regarding chronic inflammatory myocardial derangements, similar findings were verified among T. cruzi-infected groups. Arginine promoted collagenogenesis in the heart muscle tissue of T. cruzi-infected arginine-supplemented group. These data show the paradoxical benefits of arginine in improving the outcome of Chagas chronic cardiomyopathy.