Cardiomyocyte infection by Trypanosoma cruzi promotes innate immune response and glycolysis activation.

Introduction: Chagas cardiomyopathy, a disease caused by Trypanosoma cruzi (T. cruzi) infection, is a major contributor to heart failure in Latin America. There are significant gaps in our understanding of the mechanism for infection of human cardiomyocytes, the pathways activated during the acute phase of the disease, and the molecular changes that lead to the progression of cardiomyopathy. Methods: To investigate the effects of T. cruzi on human cardiomyocytes during infection, we infected induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) with the parasite and analyzed cellular, molecular, and metabolic responses at 3 hours, 24 hours, and 48 hours post infection (hpi) using transcriptomics (RNAseq), proteomics (LC-MS), and metabolomics (GC-MS and Seahorse) analyses. Results: Analyses of multiomic data revealed that cardiomyocyte infection caused a rapid increase in genes and proteins related to activation innate and adaptive immune systems and pathways, including alpha and gamma interferons, HIF-1α signaling, and glycolysis. These responses resemble prototypic responses observed in pathogen-activated immune cells. Infection also caused an activation of glycolysis that was dependent on HIF-1α signaling. Using gene editing and pharmacological inhibitors, we found that T. cruzi uptake was mediated in part by the glucose-facilitated transporter GLUT4 and that the attenuation of glycolysis, HIF-1α activation, or GLUT4 expression decreased T. cruzi infection. In contrast, pre-activation of pro-inflammatory immune responses with LPS resulted in increased infection rates. Conclusion: These findings suggest that T. cruzi exploits a HIF-1α-dependent, cardiomyocyte-intrinsic stress-response activation of glycolysis to promote intracellular infection and replication. These chronic immuno-metabolic responses by cardiomyocytes promote dysfunction, cell death, and the emergence of cardiomyopathy.

mTOR signaling inhibition decreases lysosome migration and impairs the success of Trypanosoma cruzi infection and replication in cardiomyocytes.

Chagas disease is caused by the parasite Trypanosoma cruzi (T. cruzi) and, among all the chronic manifestations of the disease, Chronic Chagas Cardiomyopathy (CCC) is the most severe outcome. Despite high burden and public health importance in Latin America, there is a gap in understanding the molecular mechanisms that results in CCC development. Previous studies showed that T. cruzi uses the host machinery for infection and replication, including the repurposing of the responses to intracellular infection such as mitochondrial activity, vacuolar membrane, and lysosomal activation in benefit of parasite infection and replication. One common signaling upstream to many responses to parasite infection is mTOR pathway, previous associated to several downstream cellular mechanisms including autophagy, mitophagy and lysosomal activation. Here, using human iPSC derived cardiomyocytes (hiPSCCM), we show the mTOR pathway is activated in hiPSCCM after T. cruzi infection, and the inhibition of mTOR with rapamycin reduced number of T. cruzi 48 h post infection (hpi). Rapamycin treatment also reduced lysosome migration from nuclei region to cell periphery resulting in less T. cruzi inside the parasitophorous vacuole (PV) in the first hour of infection. In addition, the number of parasites leaving the PV to the cytoplasm to replicate in later times of infection was also lower after rapamycin treatment. Altogether, our data suggest that host's mTOR activation concomitant with parasite infection modulates lysosome migration and that T. cruzi uses this mechanism to achieve infection and replication. Modulating this mechanism with rapamycin impaired the success of T. cruzi life cycle independent of mitophagy.

Different Transcriptomic Response to T. cruzi Infection in hiPSC-Derived Cardiomyocytes From Chagas Disease Patients With and Without Chronic Cardiomyopathy.

Chagas disease is a tropical zoonosis caused by Trypanosoma cruzi. After infection, the host present an acute phase, usually asymptomatic, in which an extensive parasite proliferation and intense innate immune activity occurs, followed by a chronic phase, characterized by low parasitemia and development of specific immunity. Most individuals in the chronic phase remain without symptoms or organ damage, a state called indeterminate IND form. However, 20 to 40% of individuals develop cardiac or gastrointestinal complications at any time in life. Cardiomyocytes have an important role in the development of Chronic Chagas Cardiomyopathy (CCC) due to transcriptional and metabolic alterations that are crucial for the parasite survival and replication. However, it still not clear why some infected individuals progress to a cardiomyopathy phase, while others remain asymptomatic. In this work, we used hiPSCs-derived cardiomyocytes (hiPSC-CM) to investigate patterns of infection, proliferation and transcriptional response in IND and CCC patients. Our data show that T. cruzi infection and proliferation efficiency do not differ significantly in PBMCs and hiPSC-CM from both groups. However, RNA-seq analysis in hiPSC-CM infected for 24 hours showed a significantly different transcriptional response to the parasite in cells from IND or CCC patients. Cardiomyocytes from IND showed significant differences in the expression of genes related to antigen processing and presentation, as well as, immune co-stimulatory molecules. Furthermore, the downregulation of collagen production genes and extracellular matrix components was significantly different in these cells. Cardiomyocytes from CCC, in turn, showed increased expression of mTORC1 pathway and unfolded protein response genes, both associated to increased intracellular ROS production. These data point to a differential pattern of response, determined by baseline genetic differences between groups, which may have an impact on the development of a chronic outcome with or without the presentation of cardiac symptoms.

Renal ischemia/reperfusion-induced cardiac hypertrophy in mice: Cardiac morphological and morphometric characterization.

BACKGROUND: Tissue remodeling is usually dependent on the deposition of extracellular matrix that may result in tissue stiffness and impaired myocardium contraction. OBJECTIVES: We had previously demonstrated that renal ischemia/reperfusion (I/R) is able to induce development of cardiac hypertrophy in mice. Therefore, we aimed to characterize renal I/R-induced cardiac hypertrophy. DESIGN: C57BL/6 J mice were subjected to 60 minutes' unilateral renal pedicle occlusion, followed by reperfusion (I/R) for 5, 8, 12 or 15 days. Gene expression, protein abundance and morphometric analyses were performed in all time points. RESULTS: Left ventricle wall thickening was increased after eight days of reperfusion (p < 0.05). An increase in the number of heart ventricle capillaries and diameter after 12 days of reperfusion (p < 0.05) was observed; an increase in the density of capillaries starting at 5 days of reperfusion (p < 0.05) was also observed. Analyses of MMP2 protein levels showed an increase at 15 days compared to sham (p < 0.05). Moreover, TGF-ß gene expression was downregulated at 12 days as well TIMP 1 and 2 (p < 0.05). The Fourier-transform infrared spectroscopy analysis showed that collagen content was altered only in the internal section of the heart (p < 0.05); such data were supported by collagen mRNA levels. CONCLUSIONS: Renal I/R leads to impactful changes in heart morphology, accompanied by an increase in microvasculature. Although it is clear that I/R is able to induce cardiac remodeling, such morphological changes is present in only a section of the heart tissue.