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 ?-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.
Abnormalities in cardiac sodium channels cause inherited arrhythmias syndromes complicated by sudden death and are present in acquired heart conditions with sudden death such as heart failure. NAD+ and related cellular metabolites important for cellular energy and redox metabolism control posttranslational modifications on the cardiac sodium currents. This application will determine how metabolism alters sodium channel structure and function, and whether manipulation of the NAD+ metabolome can be used to increase sodium currents and prevent arrhythmias.
