Biomarkers and Echocardiographic Predictors of Cardiovascular Outcome in Patients With Chronic Chagas Disease.

Background Chagas disease (CD) presents an ominous prognosis. The predictive value of biomarkers and new echocardiogram parameters in adjusted models have not been well studied. Methods and Results There were 361 patients with chronic CD (57.6% men, 61±11 years of age, clinical forms: indeterminate 27.1%, cardiac 56.6%, digestive 3.6%, cardiodigestive 12.7%) included in this single-center, observational, prospective longitudinal study. Echocardiographic evaluation included strain analyses of left atrial, left ventricular (LV), and right ventricular and 3-dimensional analyses of left atrial and LV volumes. Biomarkers included cardiac troponin I, brain natriuretic peptide, transforming growth factor ß1, tumor necrosis factor, matrix metalloproteinases, and Trypanosoma cruzi polymerase chain reaction. The studied end point was a composite of CD-related mortality, heart transplant, hospital admission due to worsening heart failure, or new cardiac device insertion. Event-free survival was analyzed by multivariable regression analyses adjusted for competing risks. P values <0.05 were considered significant. The composite event occurred in 79 patients after 4.9±2.0 years follow-up. LV end-diastolic volume (hazard ratio [HR], 1.01 [95% CI, 1.00-1.02]; P=0.02), peak negative global atrial strain (HR, 1.08 [95% CI, 1.00-1.17]; P=0.04), LV global circumferential strain (HR, 1.12 [95% CI, 1.04-1.21]; P=0.003), LV torsion (HR, 0.55 [95% CI, 0.35-0.81]; P=0.003), brain natriuretic peptide (HR, 2.03 [95% CI, 1.23-3.34]; P=0.005), and positive T cruzi polymerase chain reaction (HR, 1.80 [95% CI, 1.12-2.91]; P=0.01) were end point predictors independent from age, sex, 2-dimensional echocardiographic indexes, hypertension, previous cardiac device, and CD cardiac form. Conclusions Two-dimensional strain- and 3-dimensional-derived parameters, brain natriuretic peptide, and positive T cruzi polymerase chain reaction can be useful for prediction of CD cardiovascular events.

Selenium, TGF-Beta and Infectious Endemic Cardiopathy: Lessons from Benchwork to Clinical Application in Chagas Disease.

For over 60 years, selenium (Se) has been known as an essential microelement to many biological functions, including cardiovascular homeostasis. This review presents a compilation of studies conducted in the past 20 years related to chronic Chagas disease cardiomyopathy (CCC), caused by Trypanosoma cruzi infection, a neglected disease that represents a global burden, especially in Latin America. Experimental and clinical data indicate that Se may be used as a complementary therapy to prevent heart failure and improve heart function. Starting from the main questions "Is Se deficiency related to heart inflammation and arrhythmogenesis in CCC?" and "Could Se be recommended as a therapeutic strategy for CCC?", we show evidence implicating the complex and multidetermined CCC physiopathology, discussing its possible interplays with the multifunctional cytokine TGF-ß as regulators of immune response and fibrosis. We present two new proposals to face this global public health challenge in vulnerable populations affected by this parasitic disease: fibrosis modulation mediated by TGF-ß pathways and the possible use of selenoproteins as antioxidants regulating the increased reactive oxygen stress present in CCC inflammatory environments. We assess the opportunity to consider the beneficial effects of Se in preventing heart failure as a concept to be applied for CCC patients.

Effects of Selenium treatment on cardiac function in Chagas heart disease: Results from the STCC randomized Trial.

BACKGROUND: Chagas disease (caused by Trypanosoma cruzi infection) evolves to chronic chagasic cardiomyopathy (CCC) affecting 1.8 million people worldwide. This is the first randomized, placebo-controlled, double-blinded, clinical trial designed to estimate efficacy and safety of selenium (Se) treatment in CCC. METHODS: 66 patients with CCC stages B1 (left ventricular ejection fraction [LVEF] > 45% and no heart failure; n = 54) or B2 (LVEF < 45% and no heart failure; n = 12) were randomly assigned to receive 100 mcg/day sodium selenite (Se, n = 32) or placebo (Pla, n = 34) for one year (study period: May 2014-September 2018). LVEF changes over time and adverse effects were investigated. Trial registration number: NCT00875173 (clinicaltrials.gov). FINDINGS: No significant differences between the two groups were observed for the primary outcome: mean LVEF after 6 (ß= +1.1 p = 0.51 for Se vs Pla) and 12 months (ß= +2.1; p = 0.23). In a subgroup analysis, statistically significant longitudinal changes were observed for mean LVEF in the stage B2 subgroup (ß= +10.1; p = 0.02 for Se [n = 4] vs Pla [n = 8]). Se treatment was safe for CCC patients, and the few adverse effects observed were similarly distributed across the two groups. INTERPRETATION: Se treatment did not improve cardiac function (evaluated from LVEF) in CCC. However, in the subgroup of patients at B2 stage, a potential beneficial influence of Se was observed. Complementary studies are necessary to explore diverse Se dose and/or associations in different CCC stages (B2 and C), as well as in A and B1 stages with longer follow-up. FUNDING: Brazilian Ministry of Health, Fiocruz, CNPq, FAPERJ.

Experimental Social Stress: Dopaminergic Receptors, Oxidative Stress, and c-Fos Protein Are Involved in Highly Aggressive Behavior.

Aggression is defined as hostile behavior that results in psychological damage, injury and even death among individuals. When aggression presents itself in an exacerbated and constant way, it can be considered escalating or pathological. The association between social stress and the emergence of exacerbated aggressiveness is common and is suggested to be interconnected through very complex neurobiological factors. For example, alterations in the expression of the dopaminergic receptors D1 and D2, reactive oxygen species (ROS) and the c-Fos protein in the cortex have been observed. Our objective was to analyze which factors are involved at the neurobiological level in the highly aggressive response of Swiss Webster adult male mice in a vivarium. In this work, we investigated the relationship among dopaminergic receptors, the production of ROS and the expression of c-Fos. Mice with exacerbated aggression were identified by the model of spontaneous aggression (MSA) based on the grouping of young mice and the regrouping of the same animals in adulthood. During the regrouping, we observed different categories of behavior resulting from social stress, such as (i) highly aggressive animals, (ii) defeated animals, and (iii) harmonic groups. To evaluate the dopaminergic system and the c-Fos protein, we quantified the expression of D1 and D2 dopaminergic receptors by Western blotting and fluorescence immunohistochemistry and that of the c-Fos protein by fluorescence immunohistochemistry. The possible production of ROS was also evaluated through the dihydroethidium (DHE) assay. The results showed that aggressive and subordinate mice showed a reduction in the expression of the D1 receptor, and no significant difference in the expression of the D2 receptor was observed between the groups. In addition, aggressive mice exhibited increased production of ROS and c-Fos protein. Based on our results, we suggest that exacerbated aggression is associated with social stress, dysregulation of the dopaminergic system and exacerbated ROS production, which leads to a state of cellular oxidative stress. The overexpression of c-Fos due to social stress suggests an attempt by the cell to produce antioxidant agents to reduce the toxic cellular concentration of ROS.

"Chagas Express XXI": A new ArtScience social technology for health and science education-A case study in Brazilian endemic areas of Chagas disease with an active search of chronic cases.

BACKGROUND: Chagas Disease (CD) affects 6-7 million people worldwide and is related to poverty-promoting conditions. Chronic asymptomatic cases are mostly invisible to health systems. Aiming (1) to translate CD discoveries into education/information practices to raise alertness and empowerment of affected people; and (2) to perform an active search of CD cases, articulating intersectoral actions to improve the access of infected people to the local health service for the treatment of CD; our research group developed and tested under field conditions as innovative social technology: an itinerant education interdisciplinary setting named "Chagas Express XXI" (CE21). METHODOLOGY: CE21 was created as an "imaginary train" with ~40 ArtScience workshops, games, laboratory activities and conversation circles. An entry/exit plus six activity modules combined associations of affected people, microscopic observations, One Health education, and wellness activities. CE21 was conceived as a social technology, since all the processes were co-created with CD patients and inter-sector local partners. Descriptive statistics showed quantitative data collected throughout the expeditions (CD knowledge, serological results). Qualitative data accessed the public perceptions about the education activities. PRINCIPAL FINDINGS: CE21 was exhibited in local educational institutions (schools, universities) in four cities, engaging 2,117 people that evaluated the 41 activities carried out. Citizens and health professionals enjoyed acquisition of information related to blood, parasites, vectors, reservoirs, environmental changes, and social determinants of CD. Further, local legacies of 600 participants volunteer for health promotion groups and CD associations, local empowerment groups to fight for better health conditions, and 05 mural paintings. We observed that 81% of the participants ignored the possibility of treating CD while 52% of the participants requested a blood test for CD showing seropositivity in 20% of them. CONCLUSIONS: CE21 is a social technology potentially useful for health and science education and active search of asymptomatic CD chronic cases. Moreover, this technology may be adapted to understand and to cooperate in other potentially epidemic situations, especially NTDs related.

Trypanosoma cruzi Promotes Transcriptomic Remodeling of the JAK/STAT Signaling and Cell Cycle Pathways in Myoblasts.

Chagas disease is responsible for more than 10,000 deaths per year and about 6 to 7 million infected people worldwide. In its chronic stage, patients can develop mega-colon, mega-esophagus, and cardiomyopathy. Differences in clinical outcomes may be determined, in part, by the genetic background of the parasite that causes Chagas disease. Trypanosoma cruzi has a high genetic diversity, and each group of strains may elicit specific pathological responses in the host. Conflicting results have been reported in studies using various combinations of mammalian host-T. cruzi strains. We previously profiled the transcriptomic signatures resulting from infection of L6E9 rat myoblasts with four reference strains of T. cruzi (Brazil, CL, Y, and Tulahuen). The four strains induced similar overall gene expression alterations in the myoblasts, although only 21 genes were equally affected by all strains. Cardiotrophin-like cytokine factor 1 (Clcf1) was one of the genes found to be consistently upregulated by the infection with all four strains of T. cruzi. This cytokine is a member of the interleukin-6 family that binds to glycoprotein 130 receptor and activates the JAK/STAT signaling pathway, which may lead to muscle cell hypertrophy. Another commonly upregulated gene was tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein theta (Ywhaq, 14-3-3 protein Θ), present in the Cell Cycle Pathway. In the present work, we reanalyzed our previous microarray dataset, aiming at understanding in more details the transcriptomic impact that each strain has on JAK/STAT signaling and Cell Cycle pathways. Using Pearson correlation analysis between the expression levels of gene pairs in biological replicas from each pathway, we determined the coordination between such pairs in each experimental condition and the predicted protein interactions between the significantly altered genes by each strain. We found that although these highlighted genes were similarly affected by all four strains, the downstream genes or their interaction partners were not necessarily equally affected, thus reinforcing the idea of the role of parasite background on host cell transcriptome. These new analyses provide further evidence to the mechanistic understanding of how distinct T. cruzi strains lead to diverse remodeling of host cell transcriptome.

Current understanding of the Trypanosoma cruzi-cardiomyocyte interaction.

Trypanosoma cruzi, the etiological agent of Chagas disease, exhibits multiple strategies to ensure its establishment and persistence in the host. Although this parasite has the ability to infect different organs, heart impairment is the most frequent clinical manifestation of the disease. Advances in knowledge of T. cruzi-cardiomyocyte interactions have contributed to a better understanding of the biological events involved in the pathogenesis of Chagas disease. This brief review focuses on the current understanding of molecules involved in T. cruzi-cardiomyocyte recognition, the mechanism of invasion, and on the effect of intracellular development of T. cruzi on the structural organization and molecular response of the target cell.

Recent developments in the interactions between caveolin and pathogens.

The role of caveolin and caveolae in the pathogenesis of infection has only recently been appreciated. In this chapter, we have highlighted some important new data on the role of caveolin in infections due to bacteria, viruses and fungi but with particular emphasis on the protozoan parasites Leishmania spp., Trypanosoma cruzi and Toxoplasma gondii. This is a continuing area of research and the final chapter has not been written on this topic.

Amiodarone inhibits Trypanosoma cruzi infection and promotes cardiac cell recovery with gap junction and cytoskeleton reassembly in vitro.

We present the results of the first detailed study of the antiproliferative and ultrastructural effects of amiodarone on Trypanosoma cruzi, the causative agent of Chagas' disease. Moreover, we report the effects of this compound on the recovery of F-actin fibrils, connexin43, and contractility in T. cruzi-infected cardiac myocytes. Amiodarone is the most prescribed class III antiarrhythmic agent and is frequently used for the symptomatic treatment of Chagas' disease patients with cardiac compromise. In addition, recent studies identified its antifungal and antiprotozoal activities, which take place through Ca(2+) homeostasis disruption and ergosterol biosynthesis blockade. We tested different concentrations of amiodarone (2.5 to 10 µM) on infected primary cultures of heart muscle cells and observed a dose- and time-dependent effect on growth of the clinically relevant intracellular amastigote form of T. cruzi. Ultrastructural analyses revealed that amiodarone had a profound effect on intracellular amastigotes, including mitochondrial swelling and disorganization of reservosomes and the kinetoplast and a blockade of amastigote-trypomastigote differentiation. Amiodarone showed no toxic effects on host cells, which recovered their F-actin fibrillar organization, connexin43 distribution, and spontaneous contractility concomitant with the drug-induced eradication of the intracellular parasites. Amiodarone is, therefore, a promising compound for the development of new drugs against T. cruzi.

Transcriptomic signatures of alterations in a myoblast cell line infected with four distinct strains of Trypanosoma cruzi.

We examined the extent to which different Trypanosoma cruzi strains induce transcriptomic changes in cultured L(6)E(9) myoblasts 72 hours after infection with Brazil (TC I), Y (TC II), CL (TC II), and Tulahuen (TC II) strains. Expression of 6,289 distinct, fully annotated unigenes was quantified with 27,000 rat oligonucleotide arrays in each of the four replicas of all control and infected RNA samples. Considering changes greater than 1.5-fold and P values < 0.05, the Tulahuen strain was the most disruptive to host transcriptome (17% significantly altered genes), whereas the Y strain altered only 6% of the genes. The significantly altered genes in the infected cells were largely different among the strains, and only 21 genes were similarly changed by all four strains. However, myoblasts infected with different strains showed proportional overall gene-expression alterations. These results indicate that infection with different parasite strains modulates similar but not identical pathways in the host cells.

Osteoblasts and bone marrow mesenchymal stromal cells control hematopoietic stem cell migration and proliferation in 3D in vitro model.

BACKGROUND: Migration, proliferation, and differentiation of hematopoietic stem cells (HSCs) are dependent upon a complex three-dimensional (3D) bone marrow microenvironment. Although osteoblasts control the HSC pool, the subendosteal niche is complex and its cellular composition and the role of each cell population in HSC fate have not been established. In vivo models are complex and involve subtle species-specific differences, while bidimensional cultures do not reflect the 3D tissue organization. The aim of this study was to investigate in vitro the role of human bone marrow-derived mesenchymal stromal cells (BMSC) and active osteoblasts in control of migration, lodgment, and proliferation of HSCs. METHODOLOGY/PRINCIPAL FINDINGS: A complex mixed multicellular spheroid in vitro model was developed with human BMSC, undifferentiated or induced for one week into osteoblasts. A clear limit between the two stromal cells was established, and deposition of extracellular matrix proteins fibronectin, collagens I and IV, laminin, and osteopontin was similar to the observed in vivo. Noninduced BMSC cultured as spheroid expressed higher levels of mRNA for the chemokine CXCL12, and the growth factors Wnt5a and Kit ligand. Cord blood and bone marrow CD34(+) cells moved in and out the spheroids, and some lodged at the interface of the two stromal cells. Myeloid colony-forming cells were maintained after seven days of coculture with mixed spheroids, and the frequency of cycling CD34(+) cells was decreased. CONCLUSIONS/SIGNIFICANCE: Undifferentiated and one-week osteo-induced BMSC self-assembled in a 3D spheroid and formed a microenvironment that is informative for hematopoietic progenitor cells, allowing their lodgment and controlling their proliferation.

Dissecting coronary angiogenesis: 3D co-culture of cardiomyocytes with endothelial or mesenchymal cells.

In embryogenesis, coronary blood vessels are formed by vasculogenesis from epicardium-derived progenitors. Subsequently, growing or regenerating myocardium increases its vasculature by angiogenesis, forming new vessels from the pre-existing ones. Recently, cell therapies for myocardium ischemia that used different protocols have given promising results, using either extra-cardiac blood vessel cell progenitors or stimulating the cardiac angiogenesis. We have questioned whether cardiomyocytes could sustain both vasculogenesis and angiogenesis. We used a 3D culture model of tissue-like spheroids in co-cultures of cardiomyocytes supplemented either with endothelial cells or with bone marrow-derived mesenchymal stroma cells. Murine foetal cardiomyocytes introduced into non-adherent U-wells formed 3D contractile structures. They were coupled by gap junctions. Cardiomyocytes segregated inside the 3D structure into clumps separated by connective tissue septa, rich in fibronectin. Three vascular endothelial growth factor isoforms were produced (VEGF 120, 164 and 188). When co-cultured with human umbilical cord endothelial cells, vascular structures were produced in fibronectin-rich external layer and in radial septa, followed by angiogenic sprouting into the cardiomyocyte microtissue. Presence of vascular structures led to the maintenance of long-term survival and contractile capacity of cardiac microtissues. Conversely, bone marrow mesenchymal cells formed isolated cell aggregates, which progressively expressed the endothelial markers von Willebrand's antigen and CD31. They proceeded to typical vasculogenesis forming new blood vessels organised in radial pattern. Our results indicate that the in vitro 3D model of cardiomyocyte spheroids provides the two basic elements for formation of new blood vessels: fibronectin and VEGF. Within the myocardial environment, endothelial and mesenchymal cells can proceed to formation of new blood vessels either through angiogenesis or vasculogenesis, respectively.

Effects of antihypertensive drugs on capillary rarefaction in spontaneously hypertensive rats: intravital microscopy and histologic analysis.

We investigated the effects of chronic oral antihypertensive treatment on functional and structural capillary rarefaction in spontaneously hypertensive rats (SHR). Wistar Kyoto rats (WKY) were used as a normotensive control group. In untreated rats, intravital videomicroscopy showed that functional capillary density was lower in SHR skeletal muscle (WKY 395 +/- 17 and SHR 258 +/- 13 capillaries/mm, P < 0.01) and ear skin (WKY 391 +/- 18 and SHR 210 +/- 15 capillaries/mm, P < 0.01). A linear relationship was seen between skeletal muscle and skin capillary densities (r = 0.654, P < 0.0001). Histologic analysis showed that SHR had a lower capillary-to-fiber ratio in the skeletal muscle (WKY 1.74 +/- 0.08 and SHR 1.40 +/- 0.06, P < 0.01). Capillary volume density-to-fiber volume density ratio in the left ventricle of SHR was also reduced (WKY 0.55 +/- 0.09 and SHR 0.42 +/- 0.09, P < 0.01). The animals were treated with the angiotensin-converting enzyme (ACE) inhibitor enalapril, the angiotensin II type I receptor (AT1) receptor antagonist losartan, the beta-blocker atenolol, or the calcium channel blocker nifedipine, resulting in similar reductions in systolic blood pressure (19.8%, 19.1%, 17.4%, and 18.2%, respectively, P > 0.05). Atenolol did not induce any change in functional capillary density of SHR. Losartan and nifedipine completely reversed functional capillary rarefaction in both muscle and cutaneous tissues, whereas enalapril significantly increased functional capillary density only in the skin. The skeletal muscle capillary-to-fiber ratio was normalized by enalapril, losartan, and nifedipine. Treatments with enalapril or losartan normalized the cardiac structural capillary rarefaction of SHRs, whereas atenolol and nifedipine had no effect. Our results suggest that different pharmacologic classes of antihypertensive drugs with similar effect on blood pressure differ in terms of their effect on the microcirculation.

Trypanosoma cruzi induces changes in cardiac connexin43 expression.

Gap junction proteins (connexins) are required for myocardial function, since they allow intercellular transmission of current carrying ions and signaling molecules. Previous studies demonstrated that rat cardiac myocytes infected with Trypanosoma cruzi lost gap junctional communication and decreased automaticity. We infected mouse cardiac myocytes with trypomastigotes of the Y strain of T. cruzi and observed alterations in connexin43 (Cx43) distribution. One hour post infection Cx43 levels were significantly increased. However, at longer time points post infection there was a significant loss of Cx43 staining in membranes of infected cardiac myocytes. Interestingly, there was also a significant reduction in myocardial Cx43 protein levels during acute infection. These data indicate that T. cruzi infection alters Cx43 expression both in vitro and in vivo. Disruptions in Cx43 may contribute to the pathogenesis of cardiac electrical alterations observed in T. cruzi infection.

Selective in vitro effects of the farnesyl pyrophosphate synthase inhibitor risedronate on Trypanosoma cruzi.

We present the results of the first detailed study of the molecular and cellular basis of the antiproliferative effects of the bisphosphonate risedronate (Ris) on Trypanosoma cruzi, the causative agent of Chagas' disease. Ris and related compounds, which block poly-isoprenoid biosynthesis at the level of farnesyl pyrophosphate synthase, are currently used for the treatment of bone resorption disorders, but also display selective activity against trypanosomatid and apicomplexan parasites. Ris induced a dose-dependent effect on growth of the extracellular epimastigote form of T. cruzi; complete growth arrest and cell lysis ensued at 150 microM. Growth inhibition was associated with depletion of the parasite's endogenous sterols, but complete growth arrest and loss of cell viability took place before full depletion of these compounds, suggesting that disappearance of other essential poly-isoprenoids is involved in its anti-parasitic action. Ris had a variety of effects on cellular ultrastructure, including mitochondrial swelling, disorganisation of other organelles, such as reservosomes and the kinetoplast, together with the appearance of autophagic vesicles and progressive vacuolization of the cytoplasm. Ris had selective antiproliferative effects against the clinically relevant amastigote form of T. cruzi, and at 100 microM, was able to prevent completely the development of T. cruzi infection of murine muscle heart or Vero cells, and to cure cultures which were already infected. Ris induced drastic ultrastructural alterations in the intracellular parasites and blocked amastigote to trypomastigote differentiation, with no biochemical or ultrastructural effects on the host cells, which fully recovered their normal structure and activity after treatment. Ris is, therefore, a promising lead compound for the development of new drugs against T. cruzi.

Antiparasitic activity of risedronate in a murine model of acute Chagas' disease.

We report the results of a study on the activity of the farnesyl-pyrophosphate synthase inhibitor risedronate (Ris) in a murine model of acute Chagas' disease. This compound displays rapid, cytocidal activity in vitro against Trypanosoma cruzi, but its in vivo activity had not been investigated previously. A murine model of acute Chagas' disease was used, in which experimental animals were infected with 10(3) trypomastigotes and intravenous treatment was started 24 h post-infection. In this model, Ris, at doses as low as 1 mg/kg per day given for 7 days, induced > 90% reductions in parasitaemia and increased very significantly (P = 0.001) the survival of treated animals. Higher doses (up to 10 mg/kg per day) led to further reductions in parasitaemia and mortality, with no deleterious effects on weight gain and general physical condition of the treated animals. There was no relapse of parasitaemia after discontinuation of treatment, suggesting trypanocidal, rather than trypanostatic, activity. This interpretation was confirmed by the almost complete disappearance of amastigote nests in the hearts of treated animals. However, no parasitological cures were observed in infected animals that received the bisphosphonate, probably due to the short treatment period. Taken together, these results indicate that Ris could be a useful lead compound for the development of new drugs effective against Chagas' disease.

Characterization of [Ca2+]i responses in primary cultures of mouse cardiomyocytes induced by Trypanosoma cruzi trypomastigotes.

Trypanosoma cruzi, the protozoan responsible for Chagas disease, employs distinct strategies to invade mammalian host cells. In the present work we investigated the participation of calcium ions on the invasion process using primary cultures of embryonic mice cardiomyocytes which exhibit spontaneous contraction in vitro. Using Fura 2-AM we found that T. cruzi was able to induce a sustained increase in basal intracellular Ca2+ level in heart muscle cells (HMC), the response being associated or not with Ca2+ transient peaks. Assays performed with both Y and CL strains indicated that the changes in intracellular Ca2+ started after parasites contacted with the cardiomyocytes and the evoked response was higher than the Ca2+ signal associated to the spontaneous contractions. The possible role of the extracellular and intracellular Ca2+ levels on T. cruzi invasion process was evaluated using the extracellular Ca2+ chelator EGTA alone or in association with the calcium ionophore A23187. Significant dose dependent inhibition of the invasion levels were found when intracellular calcium release was prevented by the association of EGTA +A23187 in calcium free medium. Dose response experiments indicated that EGTA 2.5 mM to 5 mM decreased the invasion level by 15.2 to 35.1% while A23187 (0.5 M) alone did not induce significant effects (17%); treatment of the cultures with the protease inhibitor leupeptin did not affect the endocytic index, thus arguing against the involvement of leupeptin sensitive proteases in the invasion of HMC.

Characterization of [Ca2+]i responses in primary cultures of mouse cardiomyocytes induced by Trypanosoma cruzi trypomastigotes

Trypanosoma cruzi, the protozoan responsible for Chagas disease, employs distinct strategies to invade mammalian host cells. In the present work we investigated the participation of calcium ions on the invasion process using primary cultures of embryonic mice cardiomyocytes which exhibit spontaneous contraction in vitro. Using Fura 2-AM we found that T. cruzi was able to induce a sustained increase in basal intracellular Ca2+ level in heart muscle cells (HMC), the response being associated or not with Ca2+ transient peaks. Assays performed with both Y and CL strains indicated that the changes in intracellular Ca2+ started after parasites contacted with the cardiomyocytes and the evoked response was higher than the Ca2+ signal associated to the spontaneous contractions. The possible role of the extracellular and intracellular Ca2+ levels on T. cruzi invasion process was evaluated using the extracellular Ca2+ chelator EGTA alone or in association with the calcium ionophore A23187. Significant dose dependent inhibition of the invasion levels were found when intracellular calcium release was prevented by the association of EGTA +A23187 in calcium free medium. Dose response experiments indicated that EGTA 2.5 mM to 5 mM decreased the invasion level by 15.2 to 35.1 percent while A23187 (0.5 æM) alone did not induce significant effects (17 percent); treatment of the cultures with the protease inhibitor leupeptin did not affect the endocytic index, thus arguing against the involvement of leupeptin sensitive proteases in the invasion of HMC

Trypanosoma cruzi-cardiomyocytes: new contributions regarding a better understanding of this interaction

The present paper summarizes new approaches regarding the progress done to the understanding of the interaction of Trypanosoma cruzi-cardiomyocytes. Mannose receptors localized at the surface of heart muscle cell are involved in binding and uptake of the parasite. One of the most striking events in the parasite-heart muscle cells interaction is the disruption of the actin cytoskeleton. We have investigated the regulation of the actin mRNA during the cytopathology induced in myocardial cells by the parasite. T. cruzi invasion increases calcium resting levels in cardiomyocytes. We have previously shown that Ca2+ ATPase of the sarcoplasmic reticulum (SERCA) is involved in the invasion of T. cruzi in cardiomyocytes. Treating the cells with thapsigargin, a drug that binds to all SERCA ATPases and causes depletion of intracellular calcium stores, we found a 75 per cent inhibition in the T. cruzi-cardiomyocytes invasion.