Evaluating NAD Supplementation as a Novel Treatment for Arrhythmias

通过心脏Na+通道Nav1.5的内向去极化Na+电流（INa），由 SCN 5A在调节心脏中的肌细胞的动作电位中起关键作用。Nav1.5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SCN 5A功能缺失突变导致遗传性心律失常 综合征，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中的更改还可以 在常见的获得性病症如心力衰竭中加重心律失常。 NAD+和NADH是心肌生物能量学和氧化还原状态的关键调节剂，并且NAD+是一种重要的代谢酶。 调节乙酰化的去乙酰化酶所需的底物。我们的实验室报告说， NAD+/NADH依赖性酶甘油-3磷酸脱氢酶-1样（GPD 1-L）通过以下途径引起BrS： 改变Nav1.5膜运输。我们和其他人随后表明，NAD+增加细胞系中的INa 和心肌细胞，至少部分是通过活性氧（ROS）和PKC- 在细胞内Nav1.5结构域III-IV接头中介导的磷酸化。我们设计了Gpd 1 l- 靶向具有降低的INa、传导疾病、心律失常和改变的Nav1.5 PTM的小鼠。 烟酰胺核苷（NR）是一种高生物利用度的NAD+前体，可增加异源细胞中的INa。 表达系统和肌细胞，并缩短小鼠的QRS持续时间。机制， NAD+补充改变Nav1.5膜运输和INa仍不清楚。此外，如何 尚未研究多个PTM以协调方式改变Nav1.5和INa。 该提议的中心假设是细胞代谢改变NAD+代谢组， 通过PTM和作用于Nav1.5的结合蛋白的协调相互作用调节Nav1.5 结构域III-IV接头。为了验证这一假设，我们将1）确定NAD+前体和抑制剂如何 使用心肌细胞改变NAD+代谢组、氧化还原状态、Nav1.5 PTM、INa和心律失常 2）确定NAD+前体是否以协调的方式作用于Nav1.5。 通过SIRT 1介导的去乙酰化、PKC介导的磷酸化和α-辅肌动蛋白2的结构域III-IV接头 结合;和3）鉴定由GPD 1-L调节的Nav1.5 PTM。 该提案的主要目标是确定NAD+代谢影响 Nav1.5的表达和功能。最终，我们希望确定是否增加膜 Nav1.5与NAD+前体或其他代谢调节剂可预防遗传性Na+ 经络缺乏综合征和更常见的疾病，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.