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.

Metabolomic insights in advanced cardiomyopathy of chronic chagasic and idiopathic patients that underwent heart transplant.

Heart failure (HF) studies typically focus on ischemic and idiopathic heart diseases. Chronic chagasic cardiomyopathy (CCC) is a progressive degenerative inflammatory condition highly prevalent in Latin America that leads to a disturbance of cardiac conduction system. Despite its clinical and epidemiological importance, CCC molecular pathogenesis is poorly understood. Here we characterize and discriminate the plasma metabolomic profile of 15 patients with advanced HF referred for heart transplantation - 8 patients with CCC and 7 with idiopathic dilated cardiomyopathy (IDC) - using gas chromatography/quadrupole time-of-flight mass spectrometry. Compared to the 12 heart donor individuals, also included to represent the control (CTRL) scenario, patients with advanced HF exhibited a metabolic imbalance with 21 discriminating metabolites, mostly indicative of accumulation of fatty acids, amino acids and important components of the tricarboxylic acid (TCA) cycle. CCC vs. IDC analyses revealed a metabolic disparity between conditions, with 12 CCC distinctive metabolites vs. 11 IDC representative metabolites. Disturbances were mainly related to amino acid metabolism profile. Although mitochondrial dysfunction and loss of metabolic flexibility may be a central mechanistic event in advanced HF, metabolic imbalance differs between CCC and IDC populations, possibly explaining the dissimilar clinical course of Chagas' patients.

Unraveling the role of miRNAs as biomarkers in Chagas cardiomyopathy: Insights into molecular pathophysiology

BACKGROUND: Chagas cardiomyopathy (ChCM) is a severe form of Chagas disease and a major cause of cardiovascular morbidity and mortality. The dysregulation of the immune response leads to cardiac remodeling and functional disruptions, resulting in life-threatening complications. Conventional diagnostic methods have limitations, and therapeutic response evaluation is challenging. MicroRNAs (miRNAs), important regulators of gene expression, show potential as biomarkers for diagnosis and prognosis. AIM: This review aims to summarize experimental findings on miRNA expression in ChCM and explore the potential of these miRNAs as biomarkers of Chagas disease. METHODS: The search was conducted in the US National Library of Medicine MEDLINE/PubMed public database using the terms "Chagas cardiomyopathy" OR "Chagas disease" AND "microRNA" OR "miRNA" OR "miR." Additionally, bioinformatics analysis was performed to investigate miRNA-target interactions and explore enrichment pathways of gene ontology biological processes and molecular functions. RESULTS: The miR-21, miR-146b, miR-146a, and miR-155 consistently exhibited up-regulation, whereas miR-145 was down-regulated in ChCM. These specific miRNAs have been linked to fibrosis, immune response, and inflammatory processes in heart tissue. Moreover, the findings from various studies indicate that these miRNAs have the potential as biomarkers for the disease and could be targeted in therapeutic strategies for ChCM. CONCLUSION: In this review, we point out miR-21, miR-146b, miR-146a, miR-155, and miR-145-5p role in the complex mechanisms of ChCM. These miRNAs have been shown as potential biomarkers for precise diagnosis, reliable prognostic evaluation, and effective treatment strategies in the ChCM.

Unraveling the role of miRNAs as biomarkers in Chagas cardiomyopathy: Insights into molecular pathophysiology.

BACKGROUND: Chagas cardiomyopathy (ChCM) is a severe form of Chagas disease and a major cause of cardiovascular morbidity and mortality. The dysregulation of the immune response leads to cardiac remodeling and functional disruptions, resulting in life-threatening complications. Conventional diagnostic methods have limitations, and therapeutic response evaluation is challenging. MicroRNAs (miRNAs), important regulators of gene expression, show potential as biomarkers for diagnosis and prognosis. AIM: This review aims to summarize experimental findings on miRNA expression in ChCM and explore the potential of these miRNAs as biomarkers of Chagas disease. METHODS: The search was conducted in the US National Library of Medicine MEDLINE/PubMed public database using the terms "Chagas cardiomyopathy" OR "Chagas disease" AND "microRNA" OR "miRNA" OR "miR." Additionally, bioinformatics analysis was performed to investigate miRNA-target interactions and explore enrichment pathways of gene ontology biological processes and molecular functions. RESULTS: The miR-21, miR-146b, miR-146a, and miR-155 consistently exhibited up-regulation, whereas miR-145 was down-regulated in ChCM. These specific miRNAs have been linked to fibrosis, immune response, and inflammatory processes in heart tissue. Moreover, the findings from various studies indicate that these miRNAs have the potential as biomarkers for the disease and could be targeted in therapeutic strategies for ChCM. CONCLUSION: In this review, we point out miR-21, miR-146b, miR-146a, miR-155, and miR-145-5p role in the complex mechanisms of ChCM. These miRNAs have been shown as potential biomarkers for precise diagnosis, reliable prognostic evaluation, and effective treatment strategies in the ChCM.

Inflammation and mitochondria in the pathogenesis of chronic Chagas disease cardiomyopathy.

Chagas disease (CD), caused by the protozoan parasite Trypanosoma cruzi, is a neglected disease affecting around 6 million people. About 30% of CD patients develop chronic Chagas disease cardiomyopathy (CCC), an inflammatory cardiomyopathy that occurs decades after the initial infection, while most infected patients (60%) remain asymptomatic in the so-called indeterminate form (IF). Death results from heart failure or arrhythmia in a subset of CCC patients. Myocardial fibrosis, inflammation, and mitochondrial dysfunction are involved in the arrhythmia substrate and triggering events. Survival in CCC is worse than in other cardiomyopathies, which may be linked to a Th1-T cell rich myocarditis with abundant interferon (IFN)-γ and tumor necrosis factor (TNF)-α, selectively lower levels of mitochondrial energy metabolism enzymes in the heart, and reduced levels of high-energy phosphate, indicating poor adenosine triphosphate (ATP) production. IFN-γ and TNF-α signaling, which are constitutively upregulated in CD patients, negatively affect mitochondrial function in cardiomyocytes, recapitulating findings in CCC heart tissue. Genetic studies such as whole-exome sequencing (WES) in nuclear families with multiple CCC/IF cases has disclosed rare heterozygous pathogenic variants in mitochondrial and inflammatory genes segregating in CCC cases. In this minireview, we summarized studies showing how IFN-γ and TNF-α affect cell energy generation, mitochondrial health, and redox homeostasis in cardiomyocytes, in addition to human CD and mitochondria. We hypothesize that cytokine-induced mitochondrial dysfunction in genetically predisposed patients may be the underlying cause of CCC severity and we believe this mechanism may have a bearing on other inflammatory cardiomyopathies.

Metabolomic Profiling of End-Stage Heart Failure Secondary to Chronic Chagas Cardiomyopathy.

Chronic Chagas cardiomyopathy (CCC) is the most frequent and severe clinical form of chronic Chagas disease, representing one of the leading causes of morbidity and mortality in Latin America, and a growing global public health problem. There is currently no approved treatment for CCC; however, omics technologies have enabled significant progress to be made in the search for new therapeutic targets. The metabolic alterations associated with pathogenic mechanisms of CCC and their relationship to cellular and immunopathogenic processes in cardiac tissue remain largely unknown. This exploratory study aimed to evaluate the potential underlying pathogenic mechanisms in the failing myocardium of patients with end-stage heart failure (ESHF) secondary to CCC by applying an untargeted metabolomic profiling approach. Cardiac tissue samples from the left ventricle of patients with ESHF of CCC etiology (n = 7) and healthy donors (n = 7) were analyzed using liquid chromatography-mass spectrometry. Metabolite profiles showed altered branched-chain amino acid and acylcarnitine levels, decreased fatty acid uptake and oxidation, increased activity of the pentose phosphate pathway, dysregulation of the TCA cycle, and alterations in critical cellular antioxidant systems. These findings suggest processes of energy deficit, alterations in substrate availability, and enhanced production of reactive oxygen species in the affected myocardium. This profile potentially contributes to the development and maintenance of a chronic inflammatory state that leads to progression and severity of CCC. Further studies involving larger sample sizes and comparisons with heart failure patients without CCC are needed to validate these results, opening an avenue to investigate new therapeutic approaches for the treatment and prevention of progression of this unique and severe cardiomyopathy.

Galectin-3 and fibrosis intensity in Chronic Chagas Cardiomyopathy: a systematic review.

Chronic Chagas Cardiomyopathy (CCC) is the most prevalent type of myocarditis and the main clinical form of the Chagas disease, which has peculiarities such as focal inflammation, structural derangement, hypertrophy, dilation, and intense reparative fibrosis. Many cellular compounds contribute to CCC development. Galectin-3 is a partaker in inflammation and contributes to myocardial fibrosis formation. Some studies showed the connection between Galectin-3 and fibrosis in Chagas disease but are still inconclusive on the guidance for the early implementation of pharmacological therapy. This systematic review evaluated Galectin-3 as a biomarker for fibrosis intensity in CCC. Two independent reviewers have searched five databases (PubMed, EMBASE, Cochrane Library, Scopus, and Lilacs), using the following search terms: galectin-3, biomarkers, fibrosis, Chagas cardiomyopathy, and Chagas disease. Overall, seven studies met the inclusion criteria and made up this review. There were four trials conducted through animal model experiments and three trials with humans. Experimental data in mice indicate an association between Galectin-3 expression and fibrosis in CCC (75% of studies). Data from human studies showed no direct connection between myocardial fibrosis and Galectin-3 expression (80% of studies). Thus, human findings do not provide significant evidence indicating that Galectin-3 is related to fibrosis formation in Chagas disease. Based on the analyzed studies, it is suggested that Galectin-3 might not be a good fibrosis marker in CCC.

Transforming growth factor-ß as a therapeutic target for the cardiac damage of Chagas disease.

Transforming growth factor beta (TGF-ß) is deeply involved on the pathogenesis of Chagas disease. Our group has been investigating the participation of this pleiotropic cytokine in different aspects of Chagas disease over the last 20 years. Important observations have been made, such as: (i) the ability of Trypanosoma cruzi in activating latent TGF-ß; (ii) the potential involvement of TGF-ß pathway on T. cruzi invasion of host cells; (iii) association of TGF-ß with parasite intracellular replication; (iv) cardiac fibrosis development and maintenance; (v) disruption of Connexin-43 plaque structures and (vi) inflammation and immune response. In this perspective article we intend to discuss the advances of the potential use of new therapies targeting TGF-ß to treat the cardiac alterations of Chagas disease-affected patients.

Impairment of Multiple Mitochondrial Energy Metabolism Pathways in the Heart of Chagas Disease Cardiomyopathy Patients.

Chagas disease cardiomyopathy (CCC) is an inflammatory dilated cardiomyopathy occurring in 30% of the 6 million infected with the protozoan Trypanosoma cruzi in Latin America. Survival is significantly lower in CCC than ischemic (IC) and idiopathic dilated cardiomyopathy (DCM). Previous studies disclosed a selective decrease in mitochondrial ATP synthase alpha expression and creatine kinase activity in CCC myocardium as compared to IDC and IC, as well as decreased in vivo myocardial ATP production. Aiming to identify additional constraints in energy metabolism specific to CCC, we performed a proteomic study in myocardial tissue samples from CCC, IC and DCM obtained at transplantation, in comparison with control myocardial tissue samples from organ donors. Left ventricle free wall myocardial samples were subject to two-dimensional electrophoresis with fluorescent labeling (2D-DIGE) and protein identification by mass spectrometry. We found altered expression of proteins related to mitochondrial energy metabolism, cardiac remodeling, and oxidative stress in the 3 patient groups. Pathways analysis of proteins differentially expressed in CCC disclosed mitochondrial dysfunction, fatty acid metabolism and transmembrane potential of mitochondria. CCC patients' myocardium displayed reduced expression of 22 mitochondrial proteins belonging to energy metabolism pathways, as compared to 17 in DCM and 3 in IC. Significantly, 6 beta-oxidation enzymes were reduced in CCC, while only 2 of them were down-regulated in DCM and 1 in IC. We also observed that the cytokine IFN-gamma, previously described with increased levels in CCC, reduces mitochondrial membrane potential in cardiomyocytes. Results suggest a major reduction of mitochondrial energy metabolism and mitochondrial dysfunction in CCC myocardium which may be in part linked to IFN-gamma. This may partially explain the worse prognosis of CCC as compared to DCM or IC.

Co-Exposure of Cardiomyocytes to IFN-γ and TNF-α Induces Mitochondrial Dysfunction and Nitro-Oxidative Stress: Implications for the Pathogenesis of Chronic Chagas Disease Cardiomyopathy.

Infection by the protozoan Trypanosoma cruzi causes Chagas disease cardiomyopathy (CCC) and can lead to arrhythmia, heart failure and death. Chagas disease affects 8 million people worldwide, and chronic production of the cytokines IFN-γ and TNF-α by T cells together with mitochondrial dysfunction are important players for the poor prognosis of the disease. Mitochondria occupy 40% of the cardiomyocytes volume and produce 95% of cellular ATP that sustain the life-long cycles of heart contraction. As IFN-γ and TNF-α have been described to affect mitochondrial function, we hypothesized that IFN-γ and TNF-α are involved in the myocardial mitochondrial dysfunction observed in CCC patients. In this study, we quantified markers of mitochondrial dysfunction and nitro-oxidative stress in CCC heart tissue and in IFN-γ/TNF-α-stimulated AC-16 human cardiomyocytes. We found that CCC myocardium displayed increased levels of nitro-oxidative stress and reduced mitochondrial DNA as compared with myocardial tissue from patients with dilated cardiomyopathy (DCM). IFN-γ/TNF-α treatment of AC-16 cardiomyocytes induced increased nitro-oxidative stress and decreased the mitochondrial membrane potential (ΔΨm). We found that the STAT1/NF-κB/NOS2 axis is involved in the IFN-γ/TNF-α-induced decrease of ΔΨm in AC-16 cardiomyocytes. Furthermore, treatment with mitochondria-sparing agonists of AMPK, NRF2 and SIRT1 rescues ΔΨm in IFN-γ/TNF-α-stimulated cells. Proteomic and gene expression analyses revealed that IFN-γ/TNF-α-treated cells corroborate mitochondrial dysfunction, transmembrane potential of mitochondria, altered fatty acid metabolism and cardiac necrosis/cell death. Functional assays conducted on Seahorse respirometer showed that cytokine-stimulated cells display decreased glycolytic and mitochondrial ATP production, dependency of fatty acid oxidation as well as increased proton leak and non-mitochondrial oxygen consumption. Together, our results suggest that IFN-γ and TNF-α cause direct damage to cardiomyocytes' mitochondria by promoting oxidative and nitrosative stress and impairing energy production pathways. We hypothesize that treatment with agonists of AMPK, NRF2 and SIRT1 might be an approach to ameliorate the progression of Chagas disease cardiomyopathy.

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.

GWAS loci associated with Chagas cardiomyopathy influences DNA methylation levels.

A recent genome-wide association study (GWAS) identified a locus in chromosome 11 associated with the chronic cardiac form of Chagas disease. Here we aimed to elucidate the potential functional mechanism underlying this genetic association by analyzing the correlation among single nucleotide polymorphisms (SNPs) and DNA methylation (DNAm) levels as cis methylation quantitative trait loci (cis-mQTL) within this region. A total of 2,611 SNPs were tested against 2,647 DNAm sites, in a subset of 37 chronic Chagas cardiomyopathy patients and 20 asymptomatic individuals from the GWAS. We identified 6,958 significant cis-mQTLs (False Discovery Rate [FDR]<0.05) at 1 Mb each side of the GWAS leading variant, where six of them potentially modulate the expression of the SAC3D1 gene, the reported gene in the previous GWAS. In addition, a total of 268 cis-mQTLs showed differential methylation between chronic Chagas cardiomyopathy patients and asymptomatic individuals. The most significant cis-mQTLs mapped in the gene bodies of POLA2 (FDR = 1.04x10-11), PLAAT3 (FDR = 7.22x10-03), and CCDC88B (FDR = 1.89x10-02) that have been associated with cardiovascular and hematological traits in previous studies. One of the most relevant interactions correlated with hypermethylation of CCDC88B. This gene is involved in the inflammatory response, and its methylation and expression levels have been previously reported in Chagas cardiomyopathy. Our findings support the functional relevance of the previously associated genomic region, highlighting the regulation of novel genes that could play a role in the chronic cardiac form of the disease.

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.

MICA and KIR: Immunogenetic Factors Influencing Left Ventricular Systolic Dysfunction and Digestive Clinical Form of Chronic Chagas Disease.

Tissue damage observed in the clinical forms of chronic symptomatic Chagas disease seems to have a close relationship with the intensity of the inflammatory process. The objective of this study was to investigate whether the MICA (MHC class I-related chain A) and KIR (killer cell immunoglobulin-like receptors) polymorphisms are associated with the cardiac and digestive clinical forms of chronic Chagas disease. Possible influence of these genes polymorphisms on the left ventricular systolic dysfunction (LVSD) in patients with chronic Chagas heart disease was also evaluated. This study enrolled 185 patients with positive serology for Trypanosoma cruzi classified according to the clinical form of the disease: cardiac (n=107) and digestive (n=78). Subsequently, patients with the cardiac form of the disease were sub-classified as with LVSD (n=52) and without LVSD (n=55). A control group was formed of 110 healthy individuals. Genotyping was performed by polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSOP). Statistical analyzes were carried out using the Chi-square test and odds ratio with 95% confidence interval was also calculated to evaluate the risk association. MICA-129 allele with high affinity for the NKG2D receptor was associated to the LVSD in patients with CCHD. The haplotype MICA*008~HLA-C*06 and the KIR2DS2-/KIR2DL2-/KIR2DL3+/C1+ combination were associated to the digestive clinical form of the disease. Our data showed that the MICA and KIR polymorphisms may exert a role in the LVSD of cardiac patients, and in digestive form of Chagas disease.

Tissue Inhibitor of Metalloproteinase-1 Is Increased in Chagasic Cardiomyopathy.

Chagas disease (CD) mainly conveys stroke risk through structural cardiac disease. However, stroke and cognitive impairment are seen in CD independently of cardiac disease severity. Chronic inflammation may be an explanation for this association, because inflammation plays an important role in the pathogenesis of acute ischemic stroke and dementia. In the present study, we selected five candidate biomarkers for Chagas disease: interleukin-6, membrane metalloproteinase-9, tissue inhibitor of metalloproteinase-1 (TIMP1), orosomucoid, and neprilysin. We sought to determine if mean levels of proinflammatory biomarkers are higher in patients with heart failure (HF) associated with Chagas disease when compared with other etiologies of HF. Patients were consecutively enrolled from subspecialty HF outpatient clinics at two university-based hospitals. Serum biomarker levels from blood samples were analyzed by ELISA. Severity of HF on echocardiography was worse in non-CD when compared with CD patients. No significant difference was observed in the levels of candidate biomarkers between the CD and non-CD groups. We found a significantly 2.2 ng/mL higher level of TIMP1 in CD when compared with non-CD patients with HF after adjustment for age and gender (95% confidence interval = 0.1 to 4.5, P = 0.037). In patients with heart failure, serum TIMP1 is increased in Chagas patients despite a lower myocardial disease severity on echocardiography when compared with non-Chagas patients. TIMP1 is probably one of multiple mediators of inflammatory injury.

Therapeutic miR-21 Silencing Reduces Cardiac Fibrosis and Modulates Inflammatory Response in Chronic Chagas Disease.

Chagas disease, caused by the parasite Trypanosoma cruzi (T. cruzi), remains a serious public health problem for which there is no effective treatment in the chronic stage. Intense cardiac fibrosis and inflammation are hallmarks of chronic Chagas disease cardiomyopathy (CCC). Previously, we identified upregulation of circulating and cardiac miR-21, a pro-fibrotic microRNA (miRNA), in subjects with CCC. Here, we explored the potential role of miR-21 as a therapeutic target in a model of chronic Chagas disease. PCR array-based 88 microRNA screening was performed in heart samples obtained from C57Bl/6 mice chronically infected with T. cruzi and serum samples collected from CCC patients. MiR-21 was found upregulated in both human and mouse samples, which was corroborated by an in silico analysis of miRNA-mRNA target prediction. In vitro miR-21 functional assays (gain-and loss-of-function) were performed in cardiac fibroblasts, showing upregulation of miR-21 and collagen expression upon transforming growth factor beta 1 (TGFß1) and T. cruzi stimulation, while miR-21 blockage reduced collagen expression. Finally, treatment of T. cruzi-infected mice with locked nucleic acid (LNA)-anti-miR-21 inhibitor promoted a significant reduction in cardiac fibrosis. Our data suggest that miR-21 is a mediator involved in the pathogenesis of cardiac fibrosis and indicates the pharmacological silencing of miR-21 as a potential therapeutic approach for CCC.

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.

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.