NADH and Ca 2ϩ 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) Ca 2ϩ release and the mechanisms of this regulation. Physiological concentrations of cytosolic NADH inhibited ryanodine receptor type 2 (RyR2)-mediated Ca 2ϩ -induced Ca 2ϩ release (CICR) from SR membranes (IC 50 ϭ120 mol/L) and significantly lowered single

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... l open probability. In permeabilized single ventricular cardiomyocytes, NADH significantly inhibited the amplitude and frequency of spontaneous Ca 2ϩ 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 Ͼ9-fold higher catalytic activity (8.8 mol/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 (IC 50 ϭ2 to 3 nmol/L), antimycin A (IC 50 ϭ13 nmol/L), and diphenyleneiodonium (IC 50 ϭ28 mol/L). Cardiac junctional SR treated with [ 3 H](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 Ca 2ϩ fluxes and mitochondrial energy production. (Circ Res. 2004;94:478-486.) Cardiac and skeletal muscle SR membrane fractions enriched in terminal cisternae (junctional SR) were isolated from Sprague-