Cardiac gene expression profiling provides evidence for cytokinopathy as a molecular mechanism in Chagas' disease cardiomyopathy

Abstract

Chronic Chagas' disease cardiomyopathy is a leading cause of congestive heart failure in Latin America, affecting more than 3 million people. Chagas' cardiomyopathy is more aggressive than other cardiomyopathies, but little is known of the molecular mechanisms responsible for its severity. We characterized gene expression profiles of human Chagas' cardiomyopathy and dilated cardiomyopathy to identify selective disease pathways and potential therapeutic targets. Both our customized cDNA microarray (Cardiochip) and real-time reverse transcriptase-polymerase chain reaction analysis showed that immune response, lipid metabolism, and mitochondrial oxidative phosphorylation genes were selectively up-regulated in myocardial tissue of the tested Chagas' cardiomyopathy patients. Interferon (IFN)-gamma-inducible genes represented 15% of genes specifically up-regulated in Chagas' cardiomyopathy myocardial tissue, indicating the importance of IFN-gamma signaling. To assess whether IFN-gamma can directly modulate cardio-myocyte gene expression, we exposed fetal murine cardiomyocytes to IFN-gamma and the IFN-gamma-inducible chemokine monocyte chemoattractant protein-1. Atrial natriuretic factor expression increased 15-fold in response to IFN-gamma whereas combined IFN-gamma and monocyte chemoattractant protein-1 increased atrial natriuretic factor expression 400-fold. Our results suggest IFN-gamma and chemokine signaling may directly up-regulate cardiomyocyte expression of genes involved in pathological hypertrophy, which may lead to heart failure. IFN-gamma and other cytokine pathways may thus be novel therapeutic targets in Chagas' cardiomyopathy.

Figure 1

Cluster analysis of gene expression (Cardiochip version 6.0 microarray data) in the CCC, DCM, and N left ventricular-free wall heart tissue mRNA samples. Data reveals CCC- and DCM-specific global gene expression profiles, with disease-specific and shared clusters of up- or down-regulated genes. Each individual column stands for the gene expression pattern of an individual heart mRNA sample belonging to CCC, DCM, or N groups; green dendrograms on the top of the figure represent degrees of similarity of gene expression among samples. Each horizontal line (y-axis) corresponds to the expression of a single gene across all samples. Gene expression clusters (green dendrograms to the left of the figure) represent relatedness of expression patterns among the 10,368 genes across several samples and patient groups. Expression values for each gene in each sample in relation to average control values normal tissue samples are expressed in a color scale (right of figure): yellow, unchanged; orange-red, up-regulated; blue, down-regulated. Nonsupervised hierarchical clustering performed with Genespring version.6.0, using correlation by up-regulated expression with default parameters, of microarray results for five CCC, seven DCM, and four normal heart tissue samples. Samples from each disease group are marked to facilitate their identification, with red, green, and white, standing for samples for CCC, DCM, and normal donor heart, respectively.

Figure 2

Venn diagram, CCC-specific, DCM-specific, and shared classified, nonredundant genes of known function in end-stage heart failure samples, as assessed by cDNA microarrays. Selected genes and gene groups (in boldface) are listed in boxes below each area. A: Genes up-regulated ≥1.5-fold, P < 0.05. B: Genes down-regulated ≥1.5-fold, P < 0.05.

Figure 3

A: The percentage of known genes in each functional category that are up-regulated exclusively in myocardial samples from CCC, DCM, or shared by both diseases. B: Percentage of down-regulated known genes in each functional category in CCC. Representation of genes in each category among up-regulated genes in a patient group was calculated as follows: number of up-regulated genes in the category/total number of known up-regulated genes in that patient group. The same was performed for each patient sample, for the shared gene list, and for the down-regulated CCC gene list.

Figure 4

Real-time RT-PCR analysis of selected genes in CCC or DCM myocardium. A: Chemokine/chemokine receptor genes. B: Genes identified by microarray analysis. C: Analysis of ANF expression in fetal cardiomyocytes after 8 hours of stimulus with MCP-1, IFN-γ and their combination. M, culture medium. Cardiomyocyte culture performed as described. In A and B, results are expressed as average fold change from normal heart (column) and SD (error bar). Asterisks, P < 0.05 versus normal. Human (A, B) or murine (C) GAPDH was used as the reference mRNA species, and GAPDH Ct values did not change significantly between disease categories or in vitro stimulus.