Wnt Signaling Plays a Key Role in the Regulation of the Immune Response and Cardiac Damage during Trypanosoma cruzi Infection.

Chagas cardiomyopathy is the consequence of a compromised electrical and mechanical cardiac function, with parasite persistence, unbalanced inflammation, and pathological tissue remodelling, being intricately related to myocardial aggression and impaired function. Recent studies have shown that Wnt signaling pathways play a critical role in the pathogenesis of cardiac and vascular diseases. In addition, we have reported that Trypanosoma cruzi infection activates Wnt signaling to promote intracellular replication of the parasites in macrophages, with the treatment of mice with IWP-L6 (an inhibitor of the O-acyl-transferase, PORCN, responsible for the post-translational modifications necessary for Wnt protein secretion) being able to diminish parasitemia and tissue parasitism. Here, we show that inhibition of Wnt signaling during the acute phase of T. cruzi infection controls the parasite replication, inhibits the development of parasite-prone and fibrosis-prone Th2-type immune response, and prevents the development of cardiac abnormalities characteristics of chronic Chagas disease. Our results suggest that the Wnt signaling pathway might be a potential target to prevent the development of T. cruzi-induced cardiomyopathy.

SCN5A gene variants as potential markers of the progression of chronic chagasic cardiac alterations.

Host genetic factors have been proposed as determinants of the variable progression of Chagas disease (ChD). Two polymorphisms, H558R and A572D, of the voltage-gated sodium channel α-subunit SCN5A gene were studied in chagasic patients in order to determine their contribution to the susceptibility to the development and/or to the progression of the cardiovascular disease. A total of 104 patients were classified as seronegative or seropositive for Trypanosoma cruzi antibodies. Clinical evaluation, electrocardiograms (ECG) and echocardiograms (Echo) were performed to detect any conduction and/or structural alteration. Patients were classified into: G1: without ECG and/or Echo alterations, G2: with ECG alterations and G3: with ECG and Echo alterations. H558R and A572D polymorphisms were detected by PCR. Cardiac alterations were more frequent in G2 + G3 seropositive patients. For H558R polymorphism, the C allele was significantly increased in seropositive G2 + G3 patients (P = 0.049. OR = 2.08; 95% CI = 1.12-4.33). When comparing the disease cardiac progression (G2 vs G3), the genotypes from the H558R polymorphism were associated to more intense cardiac alterations (P = 0.018). For A572D polymorphism, no associations were found. The results suggest a possible involvement of SCN5A polymorphisms in the susceptibility to chronic ChD and the disease progression, contributing to the elucidation of the molecular mechanism underlying this complex myocardiopathy. In this regard, this is the first work that studies this gene in the context of chagasic cardiomyopathy.

In vitro and in vivo drug combination for the treatment of Trypanosoma cruzi infection: A multivariate approach.

Combination therapies based on the available drugs have been proposed as promising therapeutic alternatives for many diseases. Clomipramine (CLO) has been found to modify the evolution of the experimental infection. The objective of this study was to evaluate the combined effect of benznidazole (BZ) and clomipramine (CLO) against different life-stages of Trypanosoma cruzi in vitro and their efficacy in a murine model. Life-stages of T. cruzi, BZ-partially-resistant (Y) strain, were incubated with BZ and CLO and isobolograms and combination index (CI) were obtained. Swiss mice were infected with trypomastigotes and different treatment schedules were performed, each of which consisted of 30 consecutive daily doses. Treatment efficacy was evaluated by comparing parasitemia, qPCR, survival and histological analysis. These results were analyzed using multivariate analysis to determine the combined effect of the drugs in vivo. CLO + BZ showed synergistic activity in vitro against the clinically relevant life-stages of T. cruzi. The most susceptible forms were the intracellular amastigotes (CI: 0.20), followed by trypomastigotes (CI: 0.60), with no toxicity upon mammalian cells. The combination of both drugs CLO (1.25 mg/kg) and BZ (6.25 mg/kg), in vivo, significantly diminished the parasitic load in blood and the mortality rate. CLO + BZ presented a similar inflammatory response in cardiac and skeletal muscle (amount of inflammatory cells) to BZ (6.25 mg/kg). Finally, the results from the principal component analysis reaffirmed that both drugs administered in combination presented higher activity compared with the individual administration in the acute experimental model.

[Analysis of the mitochondrial function and ultrastructure in healthy albino mice treated with heart failure medications] / Análisis de la función y ultraestructura mitocondrial en ratones albinos sanos tratados con medicamentos para insuficiencia cardíaca.

Background: Mitochondrial activity is essential for cardiac and skeletal muscle. The relationship between mitochondrial dysfunction and different cardiovascular conditions has been well described. Pharmacological treatment for heart failure involves different drugs as: angiotensin-converting enzyme inhibitors, B-adrenergic blockers, digitalis glycosides and diuretics. The clinical benefit from medication is clear, however, the role of this drugs in mitochondrial metabolisms is not well understood. Aim of the study: The objective of our study was to analyze structural and functional characteristics of cardiac and skeletal muscle mitochondria in mice treated with drugs normally used for heart failure and compare it to a control group. Methods: Twenty-five Albino Mice divided in five groups were treated with heart failure medication during 30 days (group I to IV). 30 days after treatment they were sacrificed, heart and skeletal muscle were analyzed and compared with a control group (V). Results: Enzymatic activity was slightly increased in groups treated with heart failure medication compared to control group (p>0.05). Mitochondrial morphology was significantly altered in groups treated compared to control group, in addition, mitochondrial area was significantly increased in the treated groups, in both cardiac and skeletal muscle. Conclusions: We concluded that heart failure medication could produce modifications in mitochondrial function; we believe that mitochondria maintains the enzymatic activity by increasing size and modifying morphology. Methods: Twenty-five Albino Mice divided in five groups were treated with heart failure medication during 30 days (group I to IV). 30 days after treatment they were sacrificed, heart and skeletal muscle were analyzed and compared with a control group (V). Results: Enzymatic activity was slightly increased in groups treated with heart failure medication compared to control group (p>0.05). Mitochondrial morphology was significantly altered in groups treated compared to control group, in addition, mitochondrial area was significantly increased in the treated groups, in both cardiac and skeletal muscle. Conclusions: We concluded that heart failure medication could produce modifications in mitochondrial function; we believe that mitochondria maintains the enzymatic activity by increasing size and modifying morphology. Results: Enzymatic activity was slightly increased in groups treated with heart failure medication compared to control group (p>0.05). Mitochondrial morphology was significantly altered in groups treated compared to control group, in addition, mitochondrial area was significantly increased in the treated groups, in both cardiac and skeletal muscle. Conclusions: We concluded that heart failure medication could produce modifications in mitochondrial function; we believe that mitochondria maintains the enzymatic activity by increasing size and modifying morphology. Conclusions: We concluded that heart failure medication could produce modifications in mitochondrial function; we believe that mitochondria maintains the enzymatic activity by increasing size and modifying morphology.

Introducción: la actividad mitocondrial es esencial para el músculo cardíaco y esquelético. La relación entre la disfunción mitocondrial y diferentes condiciones cardiovasculares ha sido bien descrita. El tratamiento farmacológico de la insuficiencia cardíaca implica diferentes medicamentos como: inhibidores de la enzima convertidora de la angiotensina, bloqueadores B-adrenérgicos, glucósidos digitálicos y diuréticos. Los beneficios clínicos del tratamiento son claros, sin embargo, el papel de estos fármacos en el metabolismo mitocondrial no esta bien establecido.Objetivo del estudio: El objetivo de nuestro estudio fue analizar las características estructurales y funcionales de las mitocondrias del músculo cardíaco y esquelético en ratones tratados con fármacos habitualmente utilizados para la insuficiencia cardíaca y compararlo con un grupo control.Métodos: Veinticinco ratones albinos divididos en cinco grupos fueron tratados con la medicación para insuficiencia cardíaca durante 30 días (grupo I a IV). 30 días después del tratamiento se sacrificaron, el corazón y el músculo esquelético se analizaron y se compararon con un grupo control (V).Resultados: La actividad enzimática se incrementó ligeramente en los grupos tratados con medicamentos insuficiencia cardiaca en comparación con el grupo control (p> 0,05). morfología mitocondrial se modificó significativamente en los grupos tratados en comparación con el grupo control, además, el área mitocondrial fue significativamente mayor en los grupos tratados, tanto en el músculo cardíaco y estriado.Conclusiones: Concluimos que la medicación insuficiencia cardíaca podría producir modificaciones en la función mitocondrial; creemos que las mitocondrias pueden mantener la actividad enzimática mediante el aumento de tamaño y modificación de la morfología. Objetivo: El objetivo de nuestro estudio fue analizar las características estructurales y funcionales de las mitocondrias del músculo cardíaco y esquelético en ratones tratados con fármacos habitualmente utilizados para la insuficiencia cardíaca y compararlo con un grupo control. Métodos: Veinticinco ratones albinos divididos en cinco grupos fueron tratados con la medicación para insuficiencia cardíaca durante 30 días (grupo I a IV). 30 días después del tratamiento se sacrificaron, el corazón y el músculo esquelético se analizaron y se compararon con un grupo control (V).Resultados: La actividad enzimática se incrementó ligeramente en los grupos tratados con medicamentos insuficiencia cardiaca en comparación con el grupo control (p> 0,05). morfología mitocondrial se modificó significativamente en los grupos tratados en comparación con el grupo control, además, el área mitocondrial fue significativamente mayor en los grupos tratados, tanto en el músculo cardíaco y estriado.Conclusiones: Concluimos que la medicación insuficiencia cardíaca podría producir modificaciones en la función mitocondrial; creemos que las mitocondrias pueden mantener la actividad enzimática mediante el aumento de tamaño y modificación de la morfología. Resultados: La actividad enzimática se incrementó ligeramente en los grupos tratados con medicamentos insuficiencia cardiaca en comparación con el grupo control (p> 0,05). morfología mitocondrial se modificó significativamente en los grupos tratados en comparación con el grupo control, además, el área mitocondrial fue significativamente mayor en los grupos tratados, tanto en el músculo cardíaco y estriado.Conclusiones: Concluimos que la medicación insuficiencia cardíaca podría producir modificaciones en la función mitocondrial; creemos que las mitocondrias pueden mantener la actividad enzimática mediante el aumento de tamaño y modificación de la morfología. Conclusiones: Concluimos que la medicación insuficiencia cardíaca podría producir modificaciones en la función mitocondrial; creemos que las mitocondrias pueden mantener la actividad enzimática mediante el aumento de tamaño y modificación de la morfología.

Quantitative PCR and unconventional serological methods to evaluate clomipramine treatment effectiveness in experimental Trypanosoma cruzi infection.

Clomipramine (CLO), a tricyclic antidepressant drug, has been used for the treatment of mice infected with Trypanosoma cruzi. In this work we evaluated the effectiveness of CLO treatment upon T. cruzi-infected mice in the chronic phase of the experimental infection using Quantitative polymerase chain reaction (qPCR) and recombinant ELISA. Sixty Swiss albino mice were inoculated with 50 trypomastigote forms of T. cruzi (Tulahuen strain). CLO treatment consisted of 5mg/kg/day during 60days by intraperitoneal injection, beginning on day 90 post infection (p.i) when the mice presented electrocardiographic (ECG) alterations compatible with the chronic phase of the disease. The evolution of experimental infection and the treatment efficacy were studied through survival, electrocardiography, serology using a mixture and individual (1, 2, 13, 30, 36 and SAPA) recombinant proteins from epimastigotes and trypomastigotes of T. cruzi; and qPCR on days 180 and 270 p.i. CLO treatment in the chronic phase decreased the parasite load, reduced the levels of antibodies against antigen 13 throughout 270days p.i and reversed the ECG abnormalities in the treated animals, from 100% of the mice with alterations at the beginning of the treatment to only 20% of the mice with alterations by day 270 p.i. This study shows that qPCR and the use of recombinant antigens are more sensitive to evaluate the effectiveness of the treatment and proves that clomipramine may be considered as a new chemotherapy for the chronic phase of the disease.

CD73 Inhibition Shifts Cardiac Macrophage Polarization toward a Microbicidal Phenotype and Ameliorates the Outcome of Experimental Chagas Cardiomyopathy.

Increasing evidence demonstrates that generation of extracellular adenosine from ATP, which is hydrolyzed by the CD39/CD73 enzyme pair, attenuates the inflammatory response and deactivates macrophage antimicrobial mechanisms. Although CD73 is emerging as a critical pathway and therapeutic target in cardiovascular disorders, the involvement of this ectonucleotidase during myocardial infection has not been explored. Using a murine model of infection with Trypanosoma cruzi, the causal agent of Chagas cardiomyopathy, we observed a sudden switch from the classical M1 macrophage (microbicidal) phenotype toward an alternative M2 (repairing/anti-inflammatory) phenotype that occurred within the myocardium very shortly after BALB/c mice infection. The observed shift in M1/M2 rate correlated with the cardiac cytokine milieu. Considering that parasite persistence within myocardium is a necessary and sufficient condition for the development of the chronic myocarditis, we hypothesized that CD73 activity may counteract cardiac macrophage microbicidal polarization, rendering the local immune response less effective. In fact, a transient treatment with a specific CD73 inhibitor (adenosine 5'-α,ß-methylene-diphosphate) enhanced the microbicidal M1 subset predominance, diminished IL-4- and IL-10-producing CD4(+) T cells, promoted a proinflammatory cytokine milieu, and reduced parasite load within the myocardium during the acute phase. As a direct consequence of these events, there was a reduction in serum levels of creatine kinase muscle-brain isoenzyme, a myocardial-specific injury marker, and an improvement in the electrocardiographic characteristics during the chronic phase. Our results demonstrate that this purinergic system drives the myocardial immune response postinfection and harbors a promising potential as a therapeutic target.

Clomipramine kills Trypanosoma brucei by apoptosis.

Drug repositioning, i.e. use of existing medicals to treat a different illness, is especially rewarding for neglected tropical diseases (NTD), since in this field the pharmaceutical industry is rather reluctant to spend vast investments for drug development. NTDs afflict primarily poor populations in under-developed countries, which minimizes financial profit. Here we investigated the trypanocidal effect of clomipramine, a commercial antipsychotic drug, on Trypanosoma brucei. The data showed that this drug killed the parasite with an IC50 of about 5µM. Analysis of the involved cell death mechanism revealed furthermore an initial autophagic stress response and finally the induction of apoptosis. The latter was substantiated by a set of respective markers such as phosphatidylserine exposition, DNA degradation, loss of the inner mitochondrial membrane potential and characteristic morphological changes. Clomipramine was described as a trypanothione inhibitor, but as judged from our results it also showed DNA binding capacities and induced substantial morphological changes. We thus consider it likely that the drug induces a multifold adverse interaction with the parasite's physiology and induces stress in a way that trypanosomes cannot cope with.

Use of clomipramine as chemotherapy of the chronic phase of Chagas disease.

Chagas infection is a major endemic disease affecting Latin American countries. The persistence of Trypanosoma cruzi generates a chronic inflammatory reactivity that induces an immune response directed to the host's tissues. The effectiveness of the treatment in the chronic phase is still unsatisfactory due, amongst other reasons, to the collateral effects of the drugs used. We investigated the effect of clomipramine, a tricyclic antidepressant that, when used as a treatment of T. cruzi-chronically infected mice, inhibits trypanothione reductase, an exclusive and vital enzyme of T. cruzi. Clomipramine improved survival (P<0.05) by diminishing the parasite intensity as demonstrated by PCR studies in the heart and skeletal muscle, and significantly prevented the evolution to fibrosis of the inflammatory infiltrates. Clomipramine could be a good candidate for the treatment of chronic Chagas disease.

Chronic indeterminate phase of Chagas' disease: mitochondrial involvement in infection with two strains.

Chagasic cardiopathy has become one of the most frequent causes of heart failure and sudden death, as well as one of the most common causes of cardio-embolic stroke in Latin America. The myocyte response to oxidative stress involves the progression of cellular changes, primarily targeting the mitochondria and modifying therefore the energy supply. In this paper we analysed the effect of the infection of mice with 2 different strains of Trypanosoma cruzi (Tulahuen and SGO Z12) in the chronic indeterminate stage (75 days post-infection), upon the structure and function of cardiac mitochondria. The structural results showed that 83% of the mitochondria from the Tulahuen-infected mice presented an increase in their matrix and 91% of the mitochondria from the SGO Z12-infected group showed a reduction in their diameter (P < 0.05). When the Krebs cycle and mitochondrial respiratory chain functionality was analysed through the measurement of the citrate synthase and complexes I to IV activity, it showed that their activity was altered in all cases in a similar manner in both infected groups. In this paper we have demonstrated that the chronic indeterminate phase is not 'silent' and that cardiac mitochondria are clearly involved in the genesis and progression to the chronic chagasic cardiopathy when different factors alter the host-parasite equilibrium.

Mitochondrial involvement in chronic chagasic cardiomyopathy.

The pathogenesis of chronic chagasic cardiopathy is still under discussion; there is considerable evidence that inflammatory infiltrates and their mediators have a direct effect on cardiac cells. Here we studied the structure and function of cardiac mitochondria in chronic chagasic myocardiopathy. Cardiac mitochondrial structure and enzyme activity of citrate synthase and complexes I to IV of the respiratory chain were studied in albino Swiss mice infected with Trypanosoma cruzi (Tulahuen strain or SGO Z12 isolate) on 365 days post-infection (dpi). The presence of parasites in cardiac and skeletal muscle was also investigated. The activity of complexes I to IV was altered in different ways, according to the strain employed (P<0.0001), in relation to the cristae disorganisation and the parasite persistence found in the Tulahuen group, and the chronic inflammatory process described in both groups; citrate synthase activity also increased in both infected groups. Changes in mitochondrial structure were detected in 89% of Tulahuen- and 58% of SGO Z12-infected mice. In this paper we demonstrate that parasite persistence and inflammation are likely to be involved in the structural and functional alterations in cardiac mitochondria from chronically T. cruzi-infected mice, demonstrating that the parasite strain determines different mitochondrial changes in chagasic cardiopathy.

Association of clomipramine and allopurinol for the treatment of the experimental infection with Trypanosoma cruzi.

We have previously shown that clomipramine and allopurinol used separately are effective in preventing chronic chagasic cardiomyopathy. The aim of the present study was to evaluate the effect of the association of clomipramine (Clo--5 mg/kg/day/90 days) and allopurinol (Allo--5, 10, or 15 mg/kg/day/90 days) for the treatment of experimental Chagas disease in the acute stage. Treatment effectiveness was evaluated through parasitemia, survival, electrocardiography, serology, and cardiac histopathology. Groups treated showed no electrocardiographic abnormalities, in contrast to those untreated which presented 25% of mice with conduction alterations. The myocardium of treated mice (Clo, Allo10+Clo, and Allo15+Clo) presented no structural alterations. Cardiac b-receptor affinity was preserved in mice treated with Clo or Clo+Allo at the different doses; receptor density of the Clo and Allo15+Clo groups did not differ from the non-infected group. Anti-cruzipain antibody levels were similar in treated and untreated groups. Survival was significantly increased in the treated groups (p < 0.05), with Clo and all the Clo+Allo groups presenting the highest rates. These results show that the association of clomipramine + allopurinol is effective for Chagas disease treatment and has the same effect as clomipramine alone.

Chemotherapy of chronic indeterminate Chagas disease: a novel approach to treatment.

Treatment of Chagas disease is a controversial issue because the available drugs are highly toxic. Clomipramine is a tricyclic antidepressant drug that inhibits Trypanosoma cruzi's trypanothione reductase, provoking the death of the parasite and preventing the cardiac damage when used for the treatment of acutely infected mice. Here, we studied the effectiveness of clomipramine (5 mg/kg/day for one month) as chemotherapy for T. cruzi-infected mice in the chronic indeterminate stage of the infection. The animals were analyzed in the cardiac chronic phase. Survival of treated animals was 84% while for the untreated ones was 40%; most of the animals presented electrocardiographic abnormalities. Affinity and density of cardiac beta receptors from infected and treated mice were similar to those in the indeterminate phase, showing that clomipramine treatment stopped the increment of functional alterations provoked by the infection, while untreated mice presented affinity and density significantly diminished. Hearts from infected and untreated mice in the chronic stage presented mononuclear cells, necrosis and fiber dissolution while hearts from treated animals showed only isolated inflammatory infiltrates. Present results demonstrate that clomipramine used in the chronic indeterminate phase of the T. cruzi infection modified the natural evolution of the chagasic cardiopathy.