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

Abstract

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.

Keywords: chronic Chagas disease cardiomyopathy; energy metabolism; idiopathic dilated cardiomyopathy; interferon-gamma; ischemic cardiomyopathy; mitochondria; proteomics; two-dimensional electrophoresis with fluorescent labeling.

Figure 1

Venn diagrams representing the occurrence of proteins differentially expressed in common or unique relationships between groups of patients with cardiomyopathy group compared with individuals without cardiomyopathies. Number of proteins with increased (A) or decreased (B) expression of at least one spot in samples from patients when compared with samples from subjects without cardiomyopathy. Over 67% of proteins differentially expressed in CCC were contained in a single spot.

Figure 2

Toxicity function pathways analysis of differentially expressed proteins in CCC, DCM and IC myocardium. Proteins differentially expressed in heart tissue were analyzed using Ingenuity Pathways Analysis^® (Qiagen) using the tox-list function, which classifies gene or protein sets into pathological/toxicological pathways. Bars indicate the –log p value for a given pathway or process.

Figure 3

Cartoon depicting fatty acid β-oxidation enzymes differentially expressed in CCC, DCM and IC myocardium. Arrows indicate up-regulated (red) or down-regulated (green) expression as compared to control myocardial samples in the proteomic analysis. *Indicates the right place for in the metabolic pathway for Acyl coA dehydrogenases ACADVL and ACADM.

Figure 4

Protein and mRNA levels of ACADVL (Very long-chain specific acyl-CoA dehydrogenase) in myocardial tissue of CCC, DCM, IC and controls. (A) Densitometry measurement of ACADVL protein levels using immunoblot (One-Way ANOVA p = 0.0011). (B). ACADVL mRNA levels assessed using real time RT-qPCR (One-Way ANOVA p = 0.0154). The horizontal lines show statistically significant changes between groups by the Tukey-Kramer test: *p < 0.05; ***p < 0.001.

Figure 5

Analysis of antioxidant enzyme Catalase and lipid peroxidation status. (A) Catalase protein levels measured by immunoblotting; the densitometric values were normalized by the total protein for each sample (One-Way ANOVA p = 0.0008). (B) Malondialdehyde (MDA) production, measured by the thiobarbituric acid reactive substances (TBARS) assay (One-Way ANOVA p = 0.0113). The horizontal lines show statistically significant changes between groups by the Tukey-Kramer test: **p < 0.01; ***p < 0.001.

Figure 6

Effect of IFN-gamma on cardiomyocyte mitochondrial membrane potential. (A) Human cardiomyocytes AC16 were stimulated with 5, 10 or 25ng/ml of IFN-gamma for 48 hours. Then, cells were stained using 1µM TMRM, 400nM of mitotracker DeepRed, 500ng/ml of PI and 1µM of Hoechst 33342 and micrographs were captured in ImageXpress Micro XLS Widefield High-Content Analysis system at 100x magnification. Fluorescence colocalization of TMRE and mitotracker deepred in live cells (PI negative) was used to calculate mitochondrial ΔΨ. Data are reported as the ratio to not-treated cells. Cell viability is the ratio of the number of live cells (PI-negative) and total cells (PI-negative plus PI-positive cells) x 100. n = 3. *p < 0.05. (B) Representative fluorescence microscopy (100x) showing decrease in TMRE fluorescence after incubation with IFN-gamma for 48h.

Figure 7

Hypothetical chain of events subsequent to reduced beta-oxidation and oxidative stress related proteins in CCC myocardium leading to cardiac damage.