Targeting Nav1.5 trafficking as a therapy for lethal genetic cardiac arrhythmias

 描述(申请人提供)：美国每年有40多万人因心律失常而猝死。心脏钠通道基因SCN5A的突变导致几种遗传性心律失常，包括Brugada综合征(BRS)和病态窦房结综合征(SSS)。SCN5A编码心脏钠通道NaV1.5，它产生心脏钠电流(INa)，负责心脏动作电位的产生和传播。SCN5A的BRS和SSS突变通过功能机制的丧失(即INA的丢失或减少)起作用。为了确定NaV1.5转运所需的关键分子决定因素，我们进行了酵母双杂交筛选，并鉴定了一个与NaV1.5直接相互作用的小蛋白MOG1，它可以促进NaV1.5转运到质膜并增加INA。在BRS中发现了一个MOG1(E83D)的显性负突变，该突变还导致NaV1.5的转运缺陷和INA的减少。基于计算机的蛋白质结构模拟和蛋白质-蛋白质相互作用研究表明，MOG1与调控内质网输出的最重要的蛋白质之一Sar1-GTP酶相互作用。基于这些新的发现，我们假设MOG1通过调节Sar1-GTP循环来调节NaV1.5的ER输出。有趣的是，我们发现在HEK293/tsA201细胞中过表达MOG1可以完全挽救因SCN5A中BRS突变G1743R和SSS突变D1275N的运输缺陷而导致的INA减少。我们推测，在含有G1743R和D1275N突变的动物模型以及杂合子SCN5A+/-小鼠(BRS的现有模型)中，过表达MOG1可以挽救导致BRS和SSS的NaV1.5突变的运输缺陷。因此，在本项目中，我们将首先确定通过腺相关病毒介导的基因转移过表达MOG1是否可以挽救NAV1.5突变G1743R和D1275N的运输缺陷，并减轻BRS和SSS小鼠模型的相关疾病表型(目标1)。目前，除了分别植入ICD(植入式心脏复律除颤器)或起搏器外，还没有有效的治疗BRS或SSS的方法。由于ICD和起搏器的侵袭性和许多副作用，我们认为开发一种非侵入性治疗方法，即新型的基于MOG1的基因治疗，对人类患者具有很高的价值。然后，我们将利用一系列综合的生化、分子生物学和细胞学方法来确定MOG1控制NaV1.5转运到细胞表面的分子机制(AIM 2)，这可能用于提高MOG1基因治疗BRS和SSS的疗效。

 DESCRIPTION (provided by applicant): Cardiac arrhythmias cause more than 400,000 sudden deaths each year in the U.S. Mutations in the cardiac sodium channel gene SCN5A cause several inherited arrhythmias, including Brugada syndrome (BrS) and sick sinus syndrome (SSS). SCN5A encodes the cardiac sodium channel Nav1.5, which produces the cardiac sodium current (INa) responsible for generation and propagation of the cardiac action potential. BrS and SSS mutations in SCN5A act by a loss of function mechanism (i.e. loss or reduction of INa). Reduction of INa is associated with defective trafficking of Nav1.5 to the plasma membrane. However, the molecular mechanisms underlying trafficking of Nav1.5 to the plasma membrane are mostly unknown. To identify critical molecular determinants required for Nav1.5 trafficking, we performed a yeast two-hybrid screen and identified a small protein MOG1 that interacts directly with Nav1.5 and can facilitate trafficking of Nav1.5 to the plasma membrane and increase INa. One dominant negative mutation of MOG1 (E83D) was reported in BrS and also causes a trafficking defect of Nav1.5 and reduced INa. We have found that MOG1 is required for ER export of Nav1.5 during trafficking. Computer-based protein structural modeling followed by protein-protein interaction studies indicate that MOG1 interacts with Sar1-GTPase, one of the most important proteins regulating ER export. Based on these novel findings, we hypothesize that MOG1 regulates ER export of Nav1.5 by regulating the Sar1-GTP cycle. Interestingly, we have found that overexpression of MOG1 in HEK293/tsA201 cells can fully rescue the reduced INa caused by trafficking defects of BrS mutation G1743R and SSS mutation D1275N in SCN5A. We surmise that overexpression of MOG1 can rescue trafficking defects of Nav1.5 mutations causing BrS and SSS in animal models containing mutations G1743R and D1275N as well as heterozygous Scn5a+/- mice (an existing model for BrS). Thus, in this project we will first determine whether overexpression of MOG1 by adeno- associated virus-mediated gene transfer can rescue the trafficking defects of Nav1.5 mutations G1743R and D1275N and attenuate related disease phenotypes in mouse models for BrS and SSS (Aim 1). Currently, no effective therapies exist for BrS or SSS except for invasive implantation of ICDs (Implantable Cardioverter Defibrillators) or pacemakers, respectively. Due to the invasiveness and many side effects associated with ICDs and pacemakers, we believe that the development of a non-invasive therapy, i.e. a novel MOG1- based gene therapy, is highly valuable for human patients. Then, we will utilize a series of integrative biochemical, molecular biological and cellular approaches to identify the molecular mechanisms by which MOG1 controls trafficking of Nav1.5 to cell surface (Aim 2), which may be used to enhance the efficacy of MOG1 gene therapy for BrS and SSS.