Metabolic Regulation of Sodium Channels

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DESCRIPTION (provided by applicant): Despite extensive research and novel treatments, conditions associated with deranged cardiac metabolism such as heart failure (HF) or ischemia are still associated with a substantial risk of arrhythmic sudden death. Cardiac injury from many causes is associated with altered metabolism and downregulation of the cardiac sodium channel (SCN5A). Recently, data demonstrated that the SCN5A was substantially and immediately modulated by pyridine nucleotides. Physiologically relevant elevations in intracellular NADH resulted in a rapid decrease in INa in both HEK cells and cardiomyocytes that was large enough to be clinically significant. The immediacy of the NADH effect on reducing INa and the lack of change in mRNA abundances under various experimental conditions suggested that the effect of NADH was post-transcriptional. Internally or externally applied NAD+ antagonized the downregulation of current seen with a rise of internal NADH. The finding that the balance of oxidized and reduced pyridine nucleotides regulates the Na+ current suggests that the metabolic state of myocytes may influence INa. The results identify a heretofore unknown regulation of cardiac Na+ channels that may help explain the link between metabolism and arrhythmic risk and may suggest that NAD+ could lessen arrhythmic risk resulting from reduced INa. This application proposes to extend these findings to better understand the mechanism whereby changes in pyridine nucleotides cause SCN5A regulation and to establish the relevance of these changes to arrhythmogenesis in myopathy models. Specific Objectives. Specific aim 1: To determine the mechanism by which NADH acts on the Na+ channel to mediate downregulation of INa. These experiments will differentiate between two leading hypotheses for how NADH mediates its effects on the Na+ channel, either by direct action on the channel complex or by causing channel isolation from the sarcolemma. Insight into Na+ channel regulation may allow mitigation of arrhythmic risk associated with low INa states. Specific aim 2: To determine which proteins are modified in the mitochondrial electron transport chain (ETC) by NADH and NAD+. As we have recently shown, the effects of NADH and NAD+ on mitochondrial ROS production are kinase dependent. Our preliminary data show that NADH activates mitochondrial ROS production via PKC, and NAD+ prevents this via PKA. Using ETC inhibitors and activators, we localized the source of ROS to either complexes I or III.17 We propose to use proteomic techniques to establish if PKA and PKC phosphorylate these complexes and whether that phosphorylation results in alteration of mitochondrial respiration, complex activity, or ROS production to better understand the mechanisms regulating mitochondrial ROS production. Specific aim 3: To determine whether NAD+ can mitigate reduced INa in ischemic and nonischemic cardiomyopathy models. Our preliminary data suggests that NAD+ may serve to mitigate arrhythmic risk in states where INa is decreased. We will test this hypothesis in two cardiomyopathy models as preliminary data for NAD+ use in humans.