A Versatile Chemical-Genetic Approach to Determine Bases for Arrhythmogenesis and Sodium Channelopathies

确定心律失常发生和钠离子通道病基础的多功能化学遗传学方法

基本信息

批准号：
10608370
负责人：
Christopher A Ahern
金额：
$ 66.31万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-12-22 至 2026-11-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10608370
关键词：
Ablation Action Potentials Acute Affinity Animals Arrhythmia Binding Sites Biochemical Biology Brain Brugada syndrome Cardiac Cardiac Electrophysiologic Techniques Cardiac Myocytes Cardiovascular Diseases Cells Chemicals Chronic Clinical Clustered Regularly Interspaced Short Palindromic Repeats Congenital Heart Block Coupling Cytoskeleton Development Dilated Cardiomyopathy Disease Electrophysiology (science)Elements Engineering Environment Fibrosis Gene Expression Gene Targeting Generations Genes Genetic Models Genotype Goals Heart Heterozygote Human Human Pathology In Situ In Vitro Inherited Knockout Mice Long QT Syndrome Mediating Methodology Molecular Molecular Conformation Mouse Strains Mus Muscle Cells Mutation Oral Outcome Pathogenicity Patients Peripheral Pharmaceutical Preparations Pharmacology Phenotype Physiological Physiology Play Predisposition Production Protein Isoforms Proteins RNA Research Rodent Model Role Route Sodium Sodium Channel Sudden Death Syndrome System Variant aorta constriction base chemical genetics experimental study gain of function mutation genetic approach heart function imaging approach in vivo indium arsenide induced pluripotent stem cell intraperitoneal lentivirally transduced loss of function mouse model nanomolar novel pharmacologic response stem therapeutic target voltage

项目摘要

Abstract The voltage-gated sodium channel NaV1.5 controls cardiac excitability and is an established therapeutic target. Mutations in the SCN5A gene, which encodes NaV1.5, are associated with inherited arrhythmia syndromes (long QT syndrome, Brugada syndrome, congenital heart block) and dilated cardiomyopathy. While gain of function mutations that disrupt NaV1.5 inactivation explain action potential duration (APD) and QTc prolongation, the mechanisms by which loss of function NaV1.5 mutations cause the other diverse pathogenic outcomes are unresolved. The physiological significance of other Na+ channel genes expressed in the heart are also uncertain. Rodent models with gene-targeted Scn5a mutations can recapitulate some clinical features of disease, but their use is complicated by compensatory mechanisms that may occur early in development. In addition, the available pharmacological blockers of NaV1.5 block brain Na+ channels and other potential cardiac Na+ channels with equal or greater potency, limiting their utility. In order to advance our understanding of NaV1.5-related biology, we have developed a chemical-genetic model to achieve acute and reversible silencing of NaV1.5 in situ. We engineered a NaV1.5 channel that contains a high-affinity, isoform-specific binding site for acylsulfonamide (GX) drugs, enabling chemical strategies to pharmacologically drive nonconducting channel conformations. The NaV1.5-GX channel has WT voltage-dependent gating and, unlike WT NaV1.5 and most other putative cardiac Na+ channels, is blocked by nanomolar concentrations of GX compounds. We have used CRISPR gene-editing to replace the endogenous Scn5a locus with the GX binding site in mice, creating a novel NaV1.5GX strain. Homozygous NaV1.5GX/GX mice have normal cardiac phenotypes, yet the acute application of nanomolar GX compounds to NaV1.5GX/GX isolated cardiac myocytes ablates Na+ current (INa). Systemic drug application in vivo results in conduction slowing in NaV1.5GX/WT mice, and conduction block and sudden death in NaV1.5GX/GX mice, thus providing a facile means to study NaV1.5 function and SCN5A-mediated disease. We propose first to examine the effects of acute Nav1.5 blockade by GX compounds on gene expression, Ca2+ handling, ROS production, fibrosis, cardiac function and arrythmias will be studied using NaV1.5GX/WT and NaV1.5GX/GX cardiac myocytes and mice, and compared to chronic Nav1.5 blockade using Scn5a+/- heterozygous knockout mice. We will then identify the effects of Na+ channel blockade on structural and electrophysiological remodeling, and on arrhythmia susceptibility following Transverse Aortic Constriction (TAC). Lastly, we will develop in vivo and ex vivo platforms to study SCN5A mutations identified in patients. The Scn5aGX mouse presents a unique opportunity to examine the phenotypes of human SCN5A mutations in a cardiac environment. In total, we anticipate these efforts will reveal novel molecular mechanisms of genotype-phenotype coupling stemming from SCN5A's role in controlling cardiac excitability.

抽象的电压门控钠通道 NaV1.5 控制心脏兴奋性，是一个既定的治疗靶点。编码 NaV1.5 的 SCN5A 基因突变与遗传性心律失常综合征（长 QT 综合征、Brugada 综合征、先天性心脏传导阻滞）和扩张型心肌病。在功能获得的同时破坏 NaV1.5 失活的突变解释了动作电位持续时间 (APD) 和 QTc 延长， NaV1.5 功能丧失突变导致其他不同致病结果的机制是未解决。心脏中表达的其他Na+通道基因的生理意义也不确定。具有基因靶向 Scn5a 突变的啮齿动物模型可以重现疾病的一些临床特征，但它们的开发早期可能出现的补偿机制使使用变得复杂。此外，可用的 NaV1.5 的药理学阻断剂可阻断脑 Na+ 通道和其他潜在的心脏 Na+ 通道相同或更大的效力，限制了它们的效用。为了增进我们对 NaV1.5 相关生物学的理解，我们开发了一种化学遗传模型来实现 NaV1.5 原位急性和可逆沉默。我们设计了一个 NaV1.5 通道，其中包含酰基磺酰胺 (GX) 的高亲和力、亚型特异性结合位点药物，使化学策略能够在药理学上驱动非传导通道构象。这 NaV1.5-GX 通道具有 WT 电压依赖性门控，与 WT NaV1.5 和大多数其他假定的心脏不同 Na+ 通道被纳摩尔浓度的 GX 化合物阻断。我们已经使用了 CRISPR 基因编辑在小鼠中用 GX 结合位点替换内源性 Scn5a 位点，创建新的 NaV1.5GX 菌株。纯合 NaV1.5GX/GX 小鼠具有正常的心脏表型，但纳摩尔 GX 的急性应用 NaV1.5GX/GX 分离心肌细胞的化合物可消除 Na+ 电流 (INa)。体内全身药物应用导致 NaV1.5GX/WT 小鼠传导减慢，并导致 NaV1.5GX/GX 小鼠传导阻滞和猝死，从而为研究 NaV1.5 功能和 SCN5A 介导的疾病提供了一种简便的方法。我们首先建议检查 GX 化合物急性 Nav1.5 阻断对基因表达、Ca2+ 处理、ROS 的影响将使用 NaV1.5GX/WT 和 NaV1.5GX/GX 心脏研究生产、纤维化、心脏功能和心律失常肌细胞和小鼠，并与使用 Scn5a+/- 杂合基因敲除小鼠的慢性 Nav1.5 阻断进行比较。我们然后将确定 Na+ 通道阻断对结构和电生理重塑的影响，以及横主动脉缩窄（TAC）后心律失常的易感性。最后，我们将开发体内和体外用于研究患者中发现的 SCN5A 突变的体内平台。 Scn5aGX 鼠标呈现出独特的有机会在心脏环境中检查人类 SCN5A 突变的表型。总共，我们预计这些努力将揭示基因型-表型耦合的新分子机制 SCN5A 在控制心脏兴奋性中的作用。