Liu Man, Shi Guangbin, Yang Kai-Chien, Gu Lianzhi, Kanthasamy Anumantha G, Anantharam Vellareddy, Dudley Samuel C
Division of Cardiology, Department of Medicine, The Warren Alpert Medical School of Brown University, Lifespan Cardiovascular Institute, Providence, Rhode Island.
Division of Cardiology, Department of Medicine, The Warren Alpert Medical School of Brown University, Lifespan Cardiovascular Institute, Providence, Rhode Island; Graduate Institute of Pharmacology, National Taiwan University School of Medicine, Taipei City, Taiwan.
Heart Rhythm. 2017 Mar;14(3):440-447. doi: 10.1016/j.hrthm.2016.12.026. Epub 2016 Dec 15.
The reduced form of nicotinamide adenine dinucleotide (NADH) increases in cardiomyopathy, activates protein kinase C (PKC), up-regulates mitochondrial reactive oxygen species (mitoROS), and down-regulates the cardiac Na channel (Na1.5).
The purpose of this study was to determine how NADH signals down-regulation of Na1.5.
Isolated mouse cardiomyocytes were used for patch-clamp recording and for monitoring mitoROS with MitoSOX Red. HEK293 cells were used for transient transfections. HEK293 cells stably expressing human Na1.5 were used for single channel recording, whole-cell patch-clamp recording, activity measurements of phospholipase C and phospholipase D (PLD), channel protein purification, and co-immunoprecipitation with PKC isoforms. HL-1 cells were used for mitochondria isolation.
NADH enhanced PLD activity (1.6- ± 0.1-fold, P <.01) and activated PKCδ. Activated PKCδ translocated to mitochondria and up-regulated mitoROS (2.8- ± 0.3-fold, P <.01) by enhancing the activities of mitochondrial complexes I, II, and IV (1.1- to 1.5-fold, P <.01). PKCδ also interacted with Na1.5 to down-regulate Na current (I). Reduction in I by activated PKCδ was prevented by antioxidants and by mutating the known PKC phosphorylation site S1503. At the single channel level, the mechanism of current reduction by PKC and recovery by protein kinase A was a change in single channel conductance.
NADH activated PKCδ by enhancing PLD activity. PKCδ modulated both mitoROS and Na1.5. PKCδ elevated mitoROS by enhancing mitochondrial oxidative phosphorylation complex activities. PKCδ-mediated channel phosphorylation and mitoROS were both required to down-regulate Na1.5 and alter single channel conductance.
烟酰胺腺嘌呤二核苷酸（NADH）的还原形式在心肌病中增加，激活蛋白激酶C（PKC），上调线粒体活性氧（mitoROS），并下调心脏钠通道（Na1.5）。
本研究旨在确定NADH如何信号传导导致Na1.5下调。
分离的小鼠心肌细胞用于膜片钳记录和用MitoSOX Red监测mitoROS。HEK293细胞用于瞬时转染。稳定表达人Na1.5的HEK293细胞用于单通道记录、全细胞膜片钳记录、磷脂酶C和磷脂酶D（PLD）活性测量、通道蛋白纯化以及与PKC同工型的免疫共沉淀。HL-1细胞用于线粒体分离。
NADH增强了PLD活性（1.6±0.1倍，P<.01）并激活了PKCδ。激活的PKCδ转位至线粒体并通过增强线粒体复合物I、II和IV的活性（1.1至1.5倍，P<.01）上调mitoROS（2.8±0.3倍，P<.01）。PKCδ还与Na1.5相互作用以下调钠电流（I）。抗氧化剂和突变已知的PKC磷酸化位点S1503可阻止激活的PKCδ导致的I降低。在单通道水平，PKC导致电流降低和蛋白激酶A使其恢复的机制是单通道电导的改变。
NADH通过增强PLD活性激活PKCδ。PKCδ调节mitoROS和Na1.5。PKCδ通过增强线粒体氧化磷酸化复合物活性升高mitoROS。PKCδ介导的通道磷酸化和mitoROS都是下调Na1.5和改变单通道电导所必需的。
