Elevated EZH2 in ischemic heart disease epigenetically mediates suppression of Na_V1.5 expression

Highlights

• EZH2 and H3K27me3 are increased while Na_V1.5 expression is reduced in ischemic hearts.

• Silencing expression or suppressing activity of EZH2 leads to a decrease of H3K27me3 and an elevation of Na_V1.5.

• Suppressing activity of EZH2 enhances Scn5a promoter activity by decreasing H3K27me3.

• Suppressing activity of EZH2 increases Na⁺ channel activity.

• EZH2-mediated suppression of Na_V1.5 is one of the mechanisms underlying arrhythmias in patients with IHD.

Abstract

Suppression of the cardiac sodium channel Na_V1.5 leads to fatal arrhythmias in ischemic heart disease (IHD). However, the transcriptional regulation of Na_V1.5 in cardiac ischemia is still unclear. Our studies are aimed to investigate the expression of enhancer of zeste homolog 2 (EZH2) in IHD and regulation of cardiac Na_V1.5 expression by EZH2. Human heart tissue was obtained from IHD and non-failing heart (NFH) patients; mouse heart tissue was obtained from the peri-infarct zone of hearts with myocardial infarction (MI) and hearts with a sham procedure. Protein and mRNA expression were measured by immunoblotting, immunostaining, and qRT-PCR. Protein-DNA binding and promoter activity were analyzed by ChIP-qPCR and luciferase assays, respectively. Na⁺ channel activity was assessed by whole-cell patch clamp recordings. EZH2 and H3K27me3 were increased while Na_V1.5 expression was reduced in IHD hearts and in mouse MI hearts compared to the controls. Reduced Na_V1.5 and increased EZH2 mRNA levels were observed in mouse MI hearts. A selective EZH2 inhibitor, GSK126 decreased H3K27me3 and elevated Na_V1.5 in HL-1 cells. Silencing of EZH2 expression decreased H3K27me3 and increased Na_V1.5 in these cells. EZH2 and H3K27me3 were enriched in the promoter regions of Scn5a and were decreased by treatment with EZH2 siRNA. GSK126 inhibited the enrichment of H3K27me3 in the Scn5a promoter and enhanced Scn5a transcriptional activity. GSK126 significantly increased Na⁺ channel activity. Taken together, EZH2 is increased in ischemic hearts and epigenetically suppresses Scn5a transcription by H3K27me3, leading to decreased Na_V1.5 expression and Na⁺ channel activity underlying the pathogenesis of arrhythmias.

Introduction

Ischemic heart disease (IHD) is a leading cause of fatal cardiac arrhythmias including ventricular tachycardia (VT) and ventricular fibrillation (VF) [1]. Patients with severely depressed left ventricular ejection fraction are at a high risk of developing VT/VF and sudden cardiac death (SCD) [2]. In the ischemic myocardium, altered expression and function of ion channels affect cardiac electrical excitability, conduction, and automaticity [3]. In infarcted canine hearts, Na⁺ current density is significantly decreased in the epicardial border zone, leading to slow conduction velocity [4]. Encoded by SCN5A, the pore-forming Na_V1.5 α-subunit of the cardiac Na⁺ channel governs cardiac depolarization [5]. Loss-of-function of Na_V1.5 is associated with arrhythmogenicity in patients with cardiac ischemia and end-stage heart failure [6,7]. The decrease in Na⁺ current density is caused by reduced Na_V1.5 protein levels on the cell membrane and/or altered Na⁺ channel kinetics [4]. The decrease of Na_V1.5 expression in ischemic myocardium from mice with myocardial infarction (MI) suggests that the repression of Scn5a gene transcription contributes to the downregulation of Na_V1.5 expression, suppression of Na⁺ channel activity, and cardiac arrhythmias [8].

Transcription factors, including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), T-box transcription factor 5 (TBX5), and forkhead box protein O1 (FOXO1), are crucial regulators of Na_V1.5 expression [[8], [9], [10], [11]]. We previously showed that enhanced β-catenin/T-cell factor 4 (TCF-4) signaling transcriptionally suppresses Na_V1.5 expression [[12], [13], [14]] and increases arrhythmogenicity in mice [13]. High-throughput studies have revealed an essential role of epigenetic modulation, including DNA methylation and histone post-translational modification (PTM), underlying cardiovascular homeostasis [15]. Enhancer of zeste homolog 2 (EZH2), the functional enzymatic component of polycomb repressive complex 2 (PRC2), promotes heterochromatin formation via catalyzing trimethylation of histone 3 lysine 27 (H3K27me3), which leads to transcriptional suppression, serving as a potential barrier for cardiac regeneration [16,17]. In ischemic cardiomyopathy, EZH2 is elevated and regulates cardiac gene expression [18]. EZH2 functions as a methyltransferase to mediate Lamin A/C mutation-induced suppression of SCN5A transcription in human cardiac stem cells [19]. Interestingly, EZH2 interacts with β-catenin/TCF-4 to suppress target gene expressions, thereby regulating endothelial cell function [20]. Whether EZH2 epigenetically regulates Na⁺ channel expression in IHD warrants further investigation.

In this study, we report that EZH2 expression is increased in human IHD and mouse MI hearts. This upregulation correlates with increased H3K27me3 and decreased Na_V1.5 expression. Inhibition of EZH2 methyltransferase activity or knockdown of EZH2 increases Na_V1.5 expression at mRNA and protein levels in HL-1 cardiomyocytes. Also, we found that EZH2 is enriched in the promoter of Scn5a and is associated with a high level of H3K27me3. Both EZH2 expression and function are required to suppress the promoter activity of Scn5a. Moreover, GSK126, a selective EZH2 inhibitor, significantly increases Na⁺ channel activity in HL-1 cardiomyocytes. These findings demonstrate the indispensable role of EZH2 in the epigenetic downregulation of Na_V1.5, highlighting EZH2 inhibition as a potential antiarrhythmic therapy in IHD.