Differential DNA methylation correlates with differential expression of angiogenic factors in human heart failure

Abstract

Epigenetic mechanisms such as microRNA and histone modification are crucially responsible for dysregulated gene expression in heart failure. In contrast, the role of DNA methylation, another well-characterized epigenetic mark, is unknown. In order to examine whether human cardiomyopathy of different etiologies are connected by a unifying pattern of DNA methylation pattern, we undertook profiling with ischaemic and idiopathic end-stage cardiomyopathic left ventricular (LV) explants from patients who had undergone cardiac transplantation compared to normal control. We performed a preliminary analysis using methylated-DNA immunoprecipitation-chip (MeDIP-chip), validated differential methylation loci by bisulfite-(BS) PCR and high throughput sequencing, and identified 3 angiogenesis-related genetic loci that were differentially methylated. Using quantitative RT-PCR, we found that the expression of these genes differed significantly between CM hearts and normal control (p<0.01). Moreover, for each individual LV tissue, differential methylation showed a predicted correlation to differential expression of the corresponding gene. Thus, differential DNA methylation exists in human cardiomyopathy. In this series of heterogeneous cardiomyopathic LV explants, differential DNA methylation was found in at least 3 angiogenesis-related genes. While in other systems, changes in DNA methylation at specific genomic loci usually precede changes in the expression of corresponding genes, our current findings in cardiomyopathy merit further investigation to determine whether DNA methylation changes play a causative role in the progression of heart failure.

Figure 1. Differential DNA methylation profile for 3 candidate CM-DMR.

(A) DMR24, (B) DMR36 and (C) DMR11. DNA methylation (%) was determined for a set of 8 LV samples (4 controls: B–E, 4 diseased (CM): IV–VII) by BS-PCR-sequencing as detailed in methods. Lower panel, DMR24 and DMR36 lie within the body of the genes: AMOTL2 and ARHGAP24; whereas DMR11 lies in the 5′ regulatory region of PECAM1.

Figure 2. Differential DNA methylation for 3 CM-DMR correlates with differential gene expression.

(A–C) Gene expression profiles for the gene corresponding to each DMR: AMOTL2, ARHGAP24, and PECAM1. Quantitative PCR was performed in a set of 11 LV samples (4 controls: C–F, 7 CM: II–VIII). QPCR experiments were performed in triplicate for each sample. ** p<0.05. (D–F) Correlation between gene expression and DNA methylation using Spearmans rank order correlation coefficient.