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

以 Nav1.5 贩运为目标作为致命遗传性心律失常的治疗方法

• 批准号: 9041020
• 负责人: QING Kenneth WANG
• 金额: $ 39.62万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9041020
• 关键词: Action Potentials; Adverse effects; Animal Model; Area; Arrhythmia; Attenuated; Binding; Biochemical; Biogenesis; Biological; Biological Assay; Blood Circulation; Brugada syndrome; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Caveolae; Cell membrane; Cell surface; Cells; Computers; Coronary Arteriosclerosis; Coupled; Data; Defect; Dependovirus; Development; Disease; Dominant-Negative Mutation; Down-Regulation; Electrophysiology (science); Event; Fluorescence; Functional disorder; Gene Transfer; Generations; Genes; Genetic; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Heart; Heart failure; Human; Implantable Defibrillators; Inherited; Ion Channel; Knock-in Mouse; Knockout Mice; Lead; Life; Malignant - descriptor; Mediating; Membrane Microdomains; Modeling; Molecular; Mus; Mutagenesis; Mutation; Myocardial Infarction; Myocardial Ischemia; Nature; Pacemakers; Patients; Phenotype; Physiology; Process; Proteins; Reporting; Research; Role; Rough endoplasmic reticulum; Sarcolemma; Series; Sick Sinus Syndrome; Sinus; Sinus Bradycardia; Sodium; Sodium Channel; Structural Models; Sudden Death; System; Testing; Therapeutic; Translating; Ventricular Arrhythmia; Vesicle; Yeasts; associated symptom; base; caveolin-3; clinical phenotype; density; disease phenotype; effective therapy; gene therapy; genetic regulatory protein; implantation; interest; loss of function; loss of function mutation; mouse model; mutant; novel; overexpression; protein protein interaction; public health relevance; sudden cardiac death; tool; trafficking; yeast two hybrid system

 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.

 描述（由申请人提供）：在美国，心律失常每年导致超过 400,000 例猝死。心脏钠通道基因 SCN5A 的突变会导致多种遗传性心律失常，包括布鲁格达综合征 (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 中的 BrS 和 SSS 突变通过功能丧失机制（即 INa 丢失或减少）起作用。 INa 的减少与 Nav1.5 向质膜运输的缺陷有关。然而，Nav1.5 运输至质膜的分子机制大多未知。为了确定 Nav1.5 运输所需的关键分子决定因素，我们进行了酵母双杂交筛选，并鉴定了一种小蛋白 MOG1，它直接与 Nav1.5 相互作用，可以促进 Nav1.5 运输到质膜并增加 INa。据报道，BrS 中存在 MOG1 (E83D) 的一种显性失活突变，该突变也会导致 Nav1.5 的运输缺陷和 INa 减少。我们发现，在贩运过程中，MOG1 是 Nav1.5 的 ER 导出所必需的。基于计算机的蛋白质结构建模以及蛋白质-蛋白质相互作用研究表明 MOG1 与 Sar1-GTPase 相互作用，Sar1-GTPase 是调节 ER 输出的最重要的蛋白质之一。基于这些新发现，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SCN5A中BrS突变G1743R和SSS突变D1275N运输缺陷引起的INa减少。我们推测，MOG1 的过度表达可以挽救含有突变 G1743R 和 D1275N 的动物模型以及杂合子 Scn5a+/- 小鼠（BrS 的现有模型）中导致 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度表达 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 小鼠模型中的相关疾病表型（目标 1）。目前，除了侵入性植入 ICD（植入式心脏复律除颤器）或起搏器外，尚无针对 BrS 或 SSS 的有效疗法。由于 ICD 和起搏器具有侵入性和许多副作用，我们相信开发一种非侵入性疗法，即一种新型的基于 MOG1 的基因疗法，对人类患者非常有价值。然后，我们将利用一系列综合生化、分子生物学和细胞方法来确定MOG1控制Nav1.5向细胞表面运输的分子机制（目标2），这可能用于增强MOG1基因治疗BrS和SSS的疗效。