Whole-Genome Cardiac DNA Methylation Fingerprint and Gene Expression Analysis Provide New Insights in the Pathogenesis of Chronic Chagas Disease Cardiomyopathy

Abstract

Background: Chagas disease, caused by the protozoan Trypanosoma cruzi, is endemic in Latin America and affects 10 million people worldwide. Approximately 12000 deaths attributable to Chagas disease occur annually due to chronic Chagas disease cardiomyopathy (CCC), an inflammatory cardiomyopathy presenting with heart failure and arrythmia; 30% of infected subjects develop CCC years after infection. Genetic mechanisms play a role in differential progression to CCC, but little is known about the role of epigenetic modifications in pathological gene expression patterns in CCC patients' myocardium. DNA methylation is the most common modification in the mammalian genome.

Methods: We investigated the impact of genome-wide cardiac DNA methylation on global gene expression in myocardial samples from end-stage CCC patients, compared to control samples from organ donors.

Results: In total, 4720 genes were differentially methylated between CCC patients and controls, of which 399 were also differentially expressed. Several of them were related to heart function or to the immune response and had methylation sites in their promoter region. Reporter gene and in silico transcription factor binding analyses indicated promoter methylation modified expression of key genes. Among those, we found potassium channel genes KCNA4 and KCNIP4, involved in electrical conduction and arrythmia, SMOC2, involved in matrix remodeling, as well as enkephalin and RUNX3, potentially involved in the increased T-helper 1 cytokine-mediated inflammatory damage in heart.

Conclusions: Results support that DNA methylation plays a role in the regulation of expression of pathogenically relevant genes in CCC myocardium, and identify novel potential disease pathways and therapeutic targets in CCC.

Figure 1.

Percentage of methylation in heart biopsy samples. Mean DNA methylation levels in all tested sites in samples from 14 patients with chronic Chagas disease cardiomyopathy (CCC) and 7 controls. Upper and lower hinges of the box, 75th and 25th percentiles, respectively; whiskers represent highest and lowest values.

Figure 2.

Unsupervised hierarchical clustering of methylation in 14 patients with severe chronic Chagas disease cardiomyopathy (CCC) and 7 controls. Unsupervised hierarchical clustering of DNA methylation based on the top 500 CpG probes. Each row is a CpG site and each column is a sample. The DNA-methylation delta β-values are represented using a color scale from green (low DNA methylation) to red (high DNA methylation).

Figure 3.

Distribution of CpG sites in differentially methylated genes. A total of 4720 genes were differentially methylated between patients with chronic Chagas disease cardiomyopathy and controls; each has between 1 and 24 CpG sites (P < 10^-7).

Figure 4.

Distribution of CpG sites in differentially expressed genes and differentially methylated genes. Three hundred ninety-nine genes are differentially expressed and methylated in the myocardial samples of 10 patients with chronic Chagas disease cardiomyopathy and 7 controls (1 to 12 CpG sites, P < 10^-7). Thirty-four of these genes are differentially expressed and contain at least 5 differentially methylated sites irrespective of their position (arbitrary cutoff). Among them, 23 play a role in immune response or heart functions. The remaining genes that contain >5 differentially methylated sites and not further investigated are C16orf54, PITX1, PRLHR, PTPRVP, SLFN12L, STAG3, SYTL1, TMC8, WDFY4, XAF1, and ZNRD1-AS1.

Figure 5.

A–W, Relationship between gene expression levels and methylation. Correlation between messenger RNA expression and the mean methylation percentage of all CpG sites in 23 selected genes in patients with chronic Chagas disease cardiomyopathy (CCC) and controls, according to linear regression analysis. P < .05 was considered statistically significant. DNA methylation in promoters is closely linked to downstream gene repression. DNA methylation may affect the affinity of transcription factors for their binding sites. An average promoter methylation is usually used in studies, whereas recent results suggested that methylation of individual cytosines can also be important. However, it is absolutely not obvious if this dogma may be applied to CpG sites located in the gene body or 3ʹ region. This may explain why we detected direct or inverse correlations between our expression level and methylation level.

Figure 6.

Effect of vector backbone CpG methylation on a CpG-free promoter by reporter gene analysis in transient transfections of the HEK 293 cell line. A and B, Gray bar represents reporter gene expression in the absence of promoter insert. White bars represent vector with unmethylated insert. Black bars represent vector with methylated insert (±2 standard deviations). Experimental points were done in quadruplicate and experiments were repeated 4 times. For statistical analysis, a cutoff value of .004 was used. *The difference is statistically significant (the P value is under the cutoff value).

Figure 7.

Effect of vector backbone CpG methylation on a CpG-free promoter by reporter gene analysis in transient transfections of the AC16 cell line. A and B, Gray bar represents reporter gene expression in the absence of promoter insert. White bars represent vector with unmethylated insert. Black bars represent vector with methylated insert (± 2 standard deviations). Experimental points were done in quadruplicate and experiments were repeated 4 times. For statistical analysis, a cutoff value of .004 was used. *The difference is statistically significant (the P value is under the cutoff value).