Cherednichenko Gennady, Zima Aleksey V, Feng Wei, Schaefer Saul, Blatter Lothar A, Pessah Isaac N
Department of Molecular Biosciences, Northern California Health Care System, University of California, Davis, Calif 95616, USA.
Circ Res. 2004 Mar 5;94(4):478-86. doi: 10.1161/01.RES.0000115554.65513.7C. Epub 2003 Dec 29.
NADH and Ca2+ have important regulatory functions in cardiomyocytes related to excitation-contraction coupling and ATP production. To elucidate elements of these functions, we examined the effect of NADH on sarcoplasmic reticulum (SR) Ca2+ release and the mechanisms of this regulation. Physiological concentrations of cytosolic NADH inhibited ryanodine receptor type 2 (RyR2)-mediated Ca2+-induced Ca2+ release (CICR) from SR membranes (IC50=120 micromol/L) and significantly lowered single channel open probability. In permeabilized single ventricular cardiomyocytes, NADH significantly inhibited the amplitude and frequency of spontaneous Ca2+ release. Blockers of electron transport prevented the inhibitory effect of NADH on CICR in isolated membranes and permeabilized cells, as well as on the activity of RyR2 channels reconstituted in lipid bilayer. An endogenous NADH oxidase activity from rat heart copurified with SR enriched with RyR2. A significant contribution by mitochondria was excluded as NADH oxidation by SR exhibited >9-fold higher catalytic activity (8.8 micromol/mg protein per minute) in the absence of exogenous mitochondrial complex I (ubiquinone) or complex III (cytochrome c) electron acceptors, but was inhibited by rotenone and pyridaben (IC50=2 to 3 nmol/L), antimycin A (IC50=13 nmol/L), and diphenyleneiodonium (IC50=28 micromol/L). Cardiac junctional SR treated with 3Hdiazirinyl-pyridaben specifically labeled a single 23-kDa PSST-like protein. These data indicate that NADH oxidation is tightly linked to, and essential for, negative regulation of the RyR2 complex and is a likely component of an important physiological negative-feedback mechanism coupling SR Ca2+ fluxes and mitochondrial energy production.
NADH和Ca2+在心肌细胞中对兴奋-收缩偶联和ATP生成具有重要的调节功能。为阐明这些功能的要素，我们研究了NADH对肌浆网（SR）Ca2+释放的影响及其调节机制。胞质NADH的生理浓度抑制了2型兰尼碱受体（RyR2）介导的Ca2+诱导的Ca2+从SR膜释放（CICR）（IC50 = 120微摩尔/升），并显著降低单通道开放概率。在透化的单个心室心肌细胞中，NADH显著抑制自发Ca2+释放的幅度和频率。电子传递阻滞剂可防止NADH对分离膜和透化细胞中CICR的抑制作用，以及对脂质双分子层中重组的RyR2通道活性的抑制作用。来自大鼠心脏的内源性NADH氧化酶活性与富含RyR2的SR共纯化。排除了线粒体的显著贡献，因为在没有外源性线粒体复合物I（泛醌）或复合物III（细胞色素c）电子受体的情况下，SR对NADH的氧化表现出>9倍更高的催化活性（每分钟8.8微摩尔/毫克蛋白质），但被鱼藤酮和哒螨灵（IC50 = 2至3纳摩尔/升）、抗霉素A（IC50 = 13纳摩尔/升）和二苯碘鎓（IC50 = 28微摩尔/升）抑制。用[3H]（三氟甲基）二氮杂萘基哒螨灵处理的心脏连接SR特异性标记了一种单一的23 kDa PSST样蛋白。这些数据表明，NADH氧化与RyR2复合物的负调节紧密相关且至关重要，并且可能是耦合SR Ca2+通量和线粒体能量生成的重要生理负反馈机制的一个组成部分。
