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

摘要 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(LONG)有关 QT综合征、Brugada综合征、先天性心脏传导阻滞)和扩张型心肌病。而函数的增益 破坏NaV1.5失活的突变解释了动作电位时程(APD)和QTC延长， 功能丧失的NaV1.5突变导致其他不同的致病结果的机制是 悬而未决。在心脏中表达的其他Na+通道基因的生理意义也不确定。 带有基因靶向SCN5A突变的啮齿动物模型可以概括疾病的一些临床特征，但他们的 可能发生在发育早期的补偿机制使使用变得复杂。此外，可用的 NaV1.5的药物阻滞剂可阻断脑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相关生物学的了解， 我们开发了一个化学遗传模型来实现NaV1.5在原位的急性可逆沉默。我们 设计了一个NaV1.5通道，该通道包含与酰基磺胺(GX)高亲和力的异构体特异性结合位点 药物，使化学策略能够在药理学上驱动非传导通道构象。这个 NaV1.5-Gx通道具有WT电压依赖的门控，与WT NaV1.5和大多数其他假定的心脏 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 在小鼠体内用Gx结合位点替换内源性SCN5A基因，创造了一种新的NaV1.5GX株。 纯合子NaV1.5GX/GX小鼠心脏表型正常，但急性应用纳米分子GX 化合物对NaV1.5GX/GX分离的心肌细胞具有消融Na+电流(INa)的作用。全身用药在体内的应用 结果NaV1.5GX/WT小鼠出现传导减慢，NaV1.5GX/GX小鼠出现传导阻滞和猝死。 从而为研究NaV1.5的功能和SCN5A介导的疾病提供了一种简便的手段。我们首先提议 GX化合物急性阻断NaV1.5对基因表达、钙离子转运、ROS的影响 将使用NaV1.5GX/WT和NaV1.5GX/GX心脏研究生产、纤维化、心功能和心律失常 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 然后将确定钠离子通道阻断对结构和电生理重塑的影响，以及 横断性主动脉缩窄(TAC)后心律失常易感性。最后，我们将在体内和EX中开发 体内平台研究患者中发现的SCN5A突变。Scn5aGX鼠标提供了一种独特的 在心脏环境中检查人类SCN5A突变表型的机会。总的来说，我们 预计这些努力将揭示起源于 SCN5A在心脏兴奋性控制中的作用

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.