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

基本信息

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
项目类别：
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

项目摘要

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（由适用提供）：心律不齐每年在美国心脏钠通道基因SCN5A中每年造成400,000多次突然死亡，引起了几种遗传性心律不齐，包括Brugada综合征（BRS）和病态的Sinus综合征（SSS）。 SCN5A编码心脏钠通道NAV1.5，该通道导致心脏钠电流（INA）负责发电和传播心脏动作电位。 SCN5A中的BRS和SSS突变通过功能机制的丧失（即损失或减少INA）。 INA的减少与NAV1.5的贩运有缺陷有关。但是，NAV1.5向质膜运输的分子机制大多未知。为了确定NAV1.5运输所需的关键分子确定剂，我们进行了酵母两杂交筛选，并鉴定出一种与NAV1.5直接相互作用的小蛋白MOG1，并可以促进将NAV1.5运输到质膜上并增加INA。在BRS中报道了MOG1（E83D）的一个显性负突变，也导致NAV1.5的贩运缺陷并减少INA。我们发现MOG1在贩运过程中需要ER出口NAV1.5。基于计算机的蛋白质结构建模，然后进行蛋白质 - 蛋白质相互作用研究表明，MOG1与控制ER导出的最重要的蛋白质之一SAR1-GTPase相互作用。基于这些新颖的发现，我们假设MOG1通过控制SAR1-GTP周期来调节NAV1.5的ER导出。有趣的是，我们发现在HEK293/TSA201细胞中MOG1的过表达可以完全挽救BRS突变G1743R和SSS突变D1275N在SCN5A中引起的INA减少。我们表现出MOG1的过表达可以挽救NAV1.5突变的运输缺陷，从而在含有G1743R和D1275N的动物模型中引起BR和SSS，以及杂合SCN5A +/-小鼠（BRS的现有模型）。在这个项目中，我们将首先确定通过腺相关病毒介导的基因转移对MOG1的过表达是否可以挽救NAV1.5突变G1743R和D1275N的运输缺陷，并减弱BRS和SSS小鼠模型中相关的疾病表型（AIM 1）。目前，除了分别侵入性ICD（植入式心脏扭曲器除颤器）或起搏器外，BRS或SSS尚无有效的疗法。由于与ICD和起搏器相关的侵入性和许多副作用，我们认为非侵入性疗法的发展，即一种基于MOG1的新型基因疗法，对人类患者非常有价值。然后，我们将利用一系列集成的生化，分子生物学和细胞方法来识别MOG1控制NAV1.5对细胞表面的运输的分子机制（AIM 2），可用于提高BRS和SSS的MOG1基因治疗的效率。