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

NaV1.5由SCN 5A基因编码，是心肌Na+通道的主要α亚基。Na+通道活性 决定心脏的兴奋性和电传导。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下调的机制在很大程度上是未知的。染色质重塑由梵天- 相关基因-1（BRG 1）是许多染色质修饰酶复合物的中心催化亚基， 在心脏发育和功能障碍中的重要作用，通过重编程基因表达， 病理生理条件。BRG 1重塑染色质活性并通过相互作用促进基因子集 与序列特异性转录因子结合。我们的初步结果表明，1。NaV1.5表达是 在患有IHD的人心脏和患有心肌梗死（MI）的小鼠心脏中降低，2. Na+通道活性 在小鼠MI心脏的梗塞周围区（PIZ）中降低，3. BRG 1与BRG 1和β-连环蛋白一起增加 人IHD和小鼠MI心脏PIZ中心肌细胞的核积聚，4.活性氧 (ROS)IHD升高可通过增强β-MG表达增加BRG 1表达，并降低NaV 1.5表达。 连环蛋白/TCF 4信号传导，5.在TCF 4结合位点募集的BRG 1/β-catenin/TCF 4复合物的复合物抑制 SCN 5A启动子在HL-细胞中的表达并抑制NaV1.5表达和Na+通道活性，6.检测到BRG 1 然而，心脏特异性敲除（KO）BRG 1并不影响NaV 1.5 表达和Na+通道活性。有趣的是，通过心脏特异性缺失β-catenin/TCF 4， 连环蛋白外显子3（β-cat β E3）抑制NaV1.5表达和Na+通道活性，导致对 用氟卡尼（Ic类抗心律失常药物）激发的小鼠中的VT可通过BRG 1 KO预防。 免疫沉淀显示BRG 1在成年心肌细胞中与β-cat β-catenin E3而不是β-catenin相互作用， 提示BRG 1抑制NaV 1.5表达依赖于增强的β-catenin/TCF 4信号传导。 这些初步发现支持了我们的假设，即BRG 1与β-catenin相互作用，促进β-catenin/TCF 4 信号介导的NaV1.5抑制，导致心脏去极化减速和 IHD中的VT/VF。我们将在2个具体目标中检验这一假设，目的1：确定BRG 1增加是否 是增强β-catenin/TCF 4信号介导的NaV1.5表达抑制所必需的， 导致小鼠心肌梗死后Na+通道活性降低;目的2：确定BRG 1是否 通过抑制SCN 5A启动子活性抑制NaV1.5表达和Na+通道活性， β-连环蛋白/TCF 4。为了实现这2种特异性AIM，我们将使用具有心脏BRG 1 KO的转基因小鼠， β-cat BGE 3、BRG 1 KO/β-cat BGE 3和TCF 4 KO、BRG 1 OE和BRG 1 OE/TCF 4 KO联合MI 程序和体外研究，以确定BRG 1是否有助于β-连环蛋白/TCF 4抑制NaV 1.5 表达，导致MI小鼠中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.