Metabolic Regulation of Sodium Channels

描述(由申请人提供)：尽管有广泛的研究和新的治疗方法，但与心脏代谢紊乱相关的疾病，如心力衰竭(HF)或缺血，仍与心律失常猝死的巨大风险有关。多种原因引起的心脏损伤与新陈代谢改变和心脏钠通道(SCN5A)下调有关。最近的数据表明，SCN5A基本上并立即受到吡啶核苷酸的调控。生理上相关的细胞内NADH升高导致HEK细胞和心肌细胞中INA的迅速下降，其幅度大到足以具有临床意义。在不同的实验条件下，NADH对降低INA的作用是即刻的，并且在mRNA丰度上没有变化，这表明NADH的作用是转录后的。内服或外用NAD可拮抗内源性NADH升高引起的电流下调。氧化和还原的吡啶核苷酸平衡调节Na电流的发现提示，心肌细胞的代谢状态可能影响INA。这些结果确认了一种迄今未知的对心肌钠通道的调节，这可能有助于解释新陈代谢与心律失常风险之间的联系，并可能表明NAD可以降低因INA减少而导致的心律失常风险。这项应用建议扩展这些发现，以更好地理解吡啶核苷酸的变化导致SCN5A调节的机制，并在肌病模型中建立这些变化与心律失常发生的相关性。具体目标。具体目的1：确定NADH作用于钠通道介导INA下调的机制。这些实验将区分两种主要的假说，即NADH如何通过直接作用于通道复合体或通过导致通道与肌膜隔离来调节其对钠通道的影响。对钠通道调节的洞察可能有助于减轻与低INa状态相关的心律失常风险。具体目的2：确定线粒体电子传递链(ETC)中哪些蛋白质被NADH和NAD修饰。我们最近发现，NADH和NAD对线粒体ROS产生的影响是依赖于激酶的。我们的初步数据显示，NADH通过PKC激活线粒体ROS的产生，而NAD通过PKA阻止这一过程。使用ETC抑制剂和激活剂，我们将ROS的来源定位为复合体I或III。17我们建议使用蛋白质组学技术来确定PKA和PKC是否磷酸化这些复合体，以及这种磷酸化是否导致线粒体呼吸、复合体活性或ROS产生的改变，以更好地了解调控线粒体ROS产生的机制。具体目标3：确定NAD是否能减轻缺血性和非缺血性心肌病模型中INA的减少。我们的初步数据表明，在INA降低的状态下，NAD可能有助于减轻心律失常的风险。我们将在两个心肌病模型中测试这一假设，作为NAD用于人类的初步数据。 公共卫生相关性：尽管有广泛的研究和新的治疗方法，但与心脏代谢紊乱相关的疾病，如心力衰竭或缺血，仍与猝死的巨大风险有关。氧化和还原的吡啶核苷酸的平衡调节钠电流的发现表明，心脏的代谢状态影响钠电流。我们的结果确定了一种迄今未知的心脏钠通道调节，这可能有助于解释新陈代谢和猝死之间的联系，并可能表明NAD可以降低由于钠电流减少而导致的死亡风险。

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. PUBLIC HEALTH RELEVANCE: Despite extensive research and novel treatments, conditions associated with deranged cardiac metabolism such as heart failure or ischemia are still associated with a substantial risk of sudden death. The finding that the balance of oxidized and reduced pyridine nucleotides regulates the sodium current suggests that the heart metabolic state influences sodium current. Our results identify a heretofore unknown regulation of cardiac sodium channels that may help explain the link between metabolism and sudden death and may suggest that NAD+ could lessen death risk resulting from reduced sodium current.