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SCN 5A基因突变与遗传性心律失常综合征（长时间心律失常）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。当函数增益 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NaV1.5突变导致其他不同致病结果的机制是 悬而未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Scn 5a突变的啮齿动物模型可以重现疾病的一些临床特征，但其 由于发育早期可能出现的补偿机制，使用变得复杂。此外，可用的 NaV1.5的药理学阻断剂阻断脑Na+通道和其他潜在的心脏Na+通道， 相等或更大的效力，限制其效用。为了促进我们对NaV1.5相关生物学的理解， 我们已经开发了一种化学-遗传模型，以实现NaV1.5的急性和可逆的原位沉默。我们 设计了一个NaV1.5通道，该通道含有一个高亲和力的酰基磺酰胺（GX）亚型特异性结合位点 药物，使化学策略能够驱动非导电通道构象。的 NaV1.5-GX通道具有WT电压依赖性门控，与WT NaV1.5和大多数其他推定的心脏 Na+通道被纳摩尔浓度的GX化合物阻断。我们使用CRISPR基因编辑技术 在小鼠中用GX结合位点取代内源性Scn 5a位点，产生新的NaV1.5GX菌株。 纯合子NaV1.5GX/GX小鼠具有正常的心脏表型，但急性应用纳摩尔GX 化合物对NaV1.5GX/GX分离的心肌细胞的Na+电流（INa）的抑制作用。体内全身给药 导致NaV1.5GX/WT小鼠的传导减慢，NaV1.5GX/GX小鼠的传导阻滞和猝死， 从而为研究NaV1.5功能和SCN 5A介导的疾病提供了一种简便的方法。我们建议首先 检查GX化合物急性Nav1.5阻断对基因表达、Ca 2+处理、ROS 将使用NaV1.5GX/WT和NaV1.5GX/GX心脏研究产率、纤维化、心功能和心律失常 肌细胞和小鼠，并与使用Scn 5a +/-杂合敲除小鼠的慢性Nav1.5阻断进行比较。我们 然后将确定Na+通道阻滞对结构和电生理重塑的影响，以及对 横向主动脉缩窄（TAC）后的心律失常易感性。最后，我们将在体内和体外开发 体内平台来研究患者中发现的SCN 5A突变。Scn 5aGX小鼠呈现出独特的 有机会检查心脏环境中人类SCN 5A突变的表型。我们总共 预计这些努力将揭示基因型-表型偶联的新分子机制， SCN 5A在控制心脏兴奋性中的作用

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.