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Circ Res. 2004 Mar 5;94(4):478-86. Epub 2003 Dec 29.

NADH oxidase activity of rat cardiac sarcoplasmic reticulum regulates calcium-induced calcium release.

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1
Department of Molecular Biosciences, Northern California Health Care System, University of California, Davis, Calif 95616, USA.

Abstract

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 [3H](trifluoromethyl)diazirinyl-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.

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