Role of the ATP-dependent chromatin-remodeling enzyme Brg1 in the regulation of cardiac Na+ channel

NaV1.5由SCN5A基因编码，是心肌Na+通道的主要α亚基。Na+通道活动 决定心脏的兴奋性和电导性。抑制引起的Na+通道活性降低 NaV1.5的表达与缺血性心脏病(IHD)的室性心动过速和室颤(VT/VF)有关， 但NaV1.5下调监管的机制在很大程度上尚不清楚。染色质重塑，由梵天- 相关基因-1(BRG1)是许多染色质修饰酶复合体的中心催化亚单位，发挥作用 通过重新编程基因表达在心脏发育和功能障碍中的重要作用 病理生理条件。BRG1通过相互作用重塑染色质活性并促进基因的子集 序列特异的转录因子。我们的初步结果表明：1.NaV1.5的表达是 IHD人心脏和心肌梗塞(MI)小鼠心脏降低，2.Na+通道活性 在小鼠心肌梗死周围区(PIZ)，3.BRG1和β-连环蛋白使BRG1增加 人IHD和小鼠MI心脏PIZ心肌细胞的核积聚--4.活性氧 IHD中(ROS)升高通过增强β-1而增加BRG1NaV1.5的表达 连环蛋白/TCF4信号转导，5.BRG1/β-连环蛋白/TCF4复合体聚集在TCF4结合部位抑制 在HL-细胞中存在SCN5A启动子，并抑制NaV1.5的表达和Na~+通道活性，6.检测到BRG1 然而，BRG1的心脏特异性敲除(KO)不影响NAV1.5 表达和Na+通道活性。有趣的是，通过心脏特异性缺失β-Catenin/Tcf4来增强β-Catenin/TCF4 连环蛋白外显子3(β-CAT∆E3)抑制NaV1.5的表达和Na+通道活性，导致易感性 用氟卡胺(IC抗心律失常药)激发小鼠的室性心动过速，BRG1KO对其有预防作用。 免疫沉淀结果显示，在成年心肌细胞中，BRG1与β-CAT∆E3相互作用，而不是与β-连环蛋白相互作用。 提示BRG1NaV1.5表达的抑制依赖于β-catenin/TCF4信号的增强。 这些初步发现支持我们的假设，即BRG1与β-连环蛋白相互作用促进β-连环蛋白/TCF4 信号介导的NaV1.5抑制，导致心脏去极化减速和发展 IHD中的VT/VF。我们将在两个特定的目标中检验这一假设，目标1：确定BRG1增加 是增强的β-连环蛋白/TCF4信号介导的抑制NaV1.5表达所必需的， 导致小鼠心肌Na+通道活性降低；目的2：确定BRG1 通过抑制SCN5A启动子活性抑制NaV1.5的表达和Na+通道活性 β-连环蛋白/TCF4.为了实现这两个特定目的，我们将使用心脏BRG1 KO转基因小鼠， β-CAT∆E3、BRG1 KO/β-CAT∆E3和TCF4 KO、BRG1 OE和BRG1 OE/TCF4 KO与MI的组合 确定BRG1是否促进β-连环蛋白/TCF4抑制NaV1.5的程序和体外研究 表达，导致心肌梗死小鼠Na+通道活性和VT降低。

NaV1.5, encoded by SCN5A gene, is a main α subunit of the cardiac Na+ channel. Na+ channel activity determines cardiac excitability and electrical conduction. Decreased Na+ channel activity induced by suppression of NaV1.5 expression is linked to ventricular tachycardia and fibrillation (VT/VF) in ischemic heart disease (IHD), but the mechanisms of the NaV1.5 downregulation are largely unknown. Chromatin remodeling by Brahma- related gene-1 (BRG1), a central catalytic subunit of numerous chromatin-modifying enzymatic complexes, play an essential role in cardiac development and dysfunction by reprogramming gene expression under pathophysiological conditions. BRG1 remodels chromatin activity and facilitates a subset of genes via interaction with sequence-specific transcription factors. Our preliminary results showed that 1. NaV1.5 expression is decreased in human hearts with IHD and mouse hearts with myocardial infarction (MI), 2. Na+ channel activity is decreased in the peri-infarct zone (PIZ) of mouse MI hearts, 3. BRG1 is increased with BRG1 and β-catenin nuclear accumulation of cardiomyocytes in human IHD and PIZ of mouse MI hearts, 4. Reactive oxygen species (ROS) elevated in IHD increases BRG1 expression and decreases NaV1.5 expression by enhancing β- catenin/TCF4 signaling, 5. A complex of BRG1/β-catenin/TCF4 complex recruited in TCF4 binding site inhibits SCN5A promoter in HL-cells and suppresses NaV1.5 expression and Na+ channel activity, 6. BRG1 was detected in the nuclei of adult cardiomyocytes; however, cardiac-specific knockout (KO) of BRG1 did not affect NaV1.5 expression and Na+ channel activity. Interestingly, enhanced β-catenin/TCF4 by cardiac-specific deletion of β- catenin exon 3 (β-cat∆E3) suppressed NaV1.5 expression and Na+ channel activity, leading to susceptibility to VT in mice challenging with flecainide (Ic antiarrhythmic drug) which was prevented by BRG1 KO. Immunoprecipitation showed BRG1 interacts with β-cat∆E3 rather than β-catenin in adult cardiomyocytes, suggesting BRG1 suppressing NaV1.5 expression is dependent on the enhanced β-catenin/TCF4 signaling. These preliminary findings support our hypothesis that BRG1 interacts with β-catenin to promote β-catenin/TCF4 signaling-mediated suppression of NaV1.5, leading to deceleration of cardiac depolarization and development of VT/VF in IHD. We will test this hypothesis in 2 specific aims, AIM 1: To determine whether increased BRG1 is necessary for enhanced β-catenin/TCF4 signaling-mediated suppression of NaV1.5 expression, leading to decreased Na+ channel activity in moue MI hearts; AIM 2: To determine whether BRG1 suppressing NaV1.5 expression and Na+ channel activity by inhibiting SCN5A promoter activity through β-catenin/TCF4. In order to achieve these 2 specific AIMs, we will use transgenic mice with cardiac BRG1 KO, β-cat∆E3, BRG1 KO/β-cat∆E3 and TCF4 KO, BRG1 OE, and BRG1 OE/TCF4 KO in combination of MI procedure and in vitro studies to determine whether BRG1 facilitates β-catenin/TCF4 suppression of NaV1.5 expression, leading to decreased Na+ channel activity and VT in mice with MI.