Evaluating NAD Supplementation as a Novel Treatment for Arrhythmias

通过心肌钠通道NaV1.5的内向去极化钠电流(INa)，编码如下 SCN5A在心肌细胞动作电位的调节中起着关键作用。NaV1.5发布后- 翻译修饰(PTM)和结合蛋白可以改变通道丰度和/或 电生理特性。SCN5A功能突变缺失导致遗传性心律失常 症状包括Brugada综合征(BRS)和传导系统疾病 转录、翻译、通道特性和膜运输。NaV1.5中的更改也可以 在常见的获得性疾病中，如心力衰竭，会加剧心律失常。 NAD和NADH是心肌生物能量学和氧化还原状态的关键调节因子，NAD是一种 调节乙酰化的sirtuins所需的底物。我们的实验室报告说，基因突变 NAD/NADH依赖酶甘油-3磷酸脱氢酶-1样(GPD1-L)通过以下途径引起BRS 改变NaV1.5膜的运输。我们和其他人随后发现，NAD增加了细胞系中的INA 和心肌细胞，至少部分通过活性氧物种(ROS)和PKC的变化- 胞内NaV1.5结构域III-IV连接子的介导磷酸化。我们已经设计了Gpd1l- 降低INA、传导性疾病、心律失常和改变NaV1.5 PTM的目标小鼠。 烟酰胺核苷(NR)是一种生物利用度高的NAD前体，可增加异种NAD中的INA 表达系统和心肌细胞，并缩短QRS时限。机制(S) NAD补充改变了NaV1.5膜的运输，而INA仍不清楚。此外，如何 多个PTM以协调的方式改变NaV1.5和INA还没有研究。 这一提议的中心假设是，细胞代谢改变了NAD代谢体， PTM与作用于NaV1.5的结合蛋白的协调相互作用调节NaV1.5 结构域III-IV连接子。为了验证这一假设，我们将1)确定NAD前体和抑制剂如何 利用心肌细胞修饰NAD代谢体、氧化还原状态、NaV1.5 PTMS、Ina和心律失常 和小鼠模型；2)确定NAD前体在NaV1.5中是否以协调的方式起作用 结构域III-IV通过SIRT1介导的脱乙酰化、PKC介导的磷酸化和α-Actinin 2 结合；3)鉴定受GPD1-L调控的NaV1.5 PTM。 该提案的主要目标是确定NAD新陈代谢影响的机制 NaV1.5的表达和功能。最终，我们希望确定是否增加了膜 NAD前体或其他代谢调节剂的NaV1.5可预防遗传性Na+的心律失常 经络不足综合征和更常见的情况，如心力衰竭。

The inward depolarizing Na+ current (INa) through the cardiac Na+ channel Nav1.5, encoded by SCN5A, plays a critical role in regulating the action potential of myocytes in the heart. Nav1.5 post- translational modifications (PTMs) and binding proteins can alter channel abundance and/or electrophysiological properties. Loss of function mutations in SCN5A cause inherited arrhythmia syndromes including Brugada syndrome (BrS) and conduction system disease by changes in transcription, translation, channel properties and membrane trafficking. Alterations in Nav1.5 can also exacerbate arrhythmias in common acquired conditions such as heart failure. NAD+ and NADH are critical regulators of myocardial bioenergetics and redox state, and NAD+ is a required substrate for sirtuins that regulate acetylation. Our laboratory reported that mutations in the NAD+/NADH dependent enzyme Gylcerol-3 Phosphate Dehydrogenase-1 Like (GPD1-L) cause BrS by altering Nav1.5 membrane trafficking. We and others then showed that NAD+ increases INa in cell lines and cardiac myocytes, at least in part through changes in reactive oxygen species (ROS) and PKC- mediated phosphorylation in the intracellular Nav1.5 Domain III-IV linker. We have engineered Gpd1l- targeted mice that have decreased INa, conduction disease, arrhythmias and altered Nav1.5 PTMs. Nicotinamide Riboside (NR), a highly bioavailable NAD+ precursor, increases INa in heterologous expression systems and myocytes, and shortens QRS duration in mice. The mechanism(s) by which NAD+ supplementation alters Nav1.5 membrane trafficking and INa remains unclear. In addition, how multiple PTMs alter Nav1.5 and INa in a coordinated manner has not been studied. The central hypothesis of this proposal is that cellular metabolism alters the NAD+ metabolome, regulating Nav1.5 by coordinated interactions of PTMs and binding proteins that act at the Nav1.5 Domain III-IV linker. To test this hypothesis, we will 1) Determine how NAD+ precursors and inhibitors modify the NAD+ metabolome, redox state, Nav1.5 PTMs, INa and arrhythmias using cardiac myocytes and mouse models; 2) Determine whether NAD+ precursors act in a coordinated manner at the Nav1.5 Domain III-IV linker via SIRT1-mediated deacetylation, PKC-mediated phosphorylation and α-actinin 2 binding; and 3) Identify the Nav1.5 PTMs modulated by GPD1-L. The primary goal of this proposal is to identify the mechanisms by which NAD+ metabolism affects Nav1.5 expression and function. Ultimately, we hope to determine whether increasing membrane Nav1.5 with NAD+ precursors or other metabolic modulators can prevent arrhythmias in inherited Na+ channel deficiency syndromes and in more common conditions such as heart failure.