Journal
CIRCULATION RESEARCH
Volume 94, Issue 4, Pages 478-486Publisher
LIPPINCOTT WILLIAMS & WILKINS
DOI: 10.1161/01.RES.0000115554.65513.7C
Keywords
ryanodine receptors; cardiac SR NADH oxidase; rotenone
Funding
- NHLBI NIH HHS [HL062231] Funding Source: Medline
- NIAMS NIH HHS [R01 AR43140] Funding Source: Medline
- NIEHS NIH HHS [P01 ES11269, P42 ES05707] Funding Source: Medline
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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 mumol/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 mumol/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 mumol/L). Cardiac junctional SR treated with [H-3]( 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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