Journal
CIRCULATION RESEARCH
Volume 100, Issue 7, Pages 1036-1044Publisher
LIPPINCOTT WILLIAMS & WILKINS
DOI: 10.1161/01.RES.0000263010.19273.48
Keywords
hydrogen peroxide; mitochondria; L-type Ca2+ channel
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We sought to understand the effect of a transient exposure of cardiac myocytes to H2O2 at a concentration that did not induce apoptosis. Myocytes were exposed to 30 mu mol/L H2O2 for 5 minutes followed by 10 U/mL catalase for 5 minutes to degrade the H2O2. Cellular superoxide was measured using dihydroethidium. Transient exposure to H2O2 caused a 66.4% increase in dihydroethidium signal compared with controls exposed to only catalase, without activation of caspase 3 or evidence of necrosis. The increase in dihydroethidium signal was attenuated by the mitochondrial inhibitors myxothiazol or carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone and when calcium uptake by the mitochondria was inhibited with Ru360. We investigated the L-type Ca2+ channel (ICa-L) as a source of calcium influx. Nisoldipine, an inhibitor of ICa-L, attenuated the increase in superoxide. Basal channel activity increased from 5.4 to 8.9 pA/pF. Diastolic calcium was significantly increased in quiescent and contracting myocytes after H2O2. The response of ICa-L to beta-adrenergic receptor stimulation was used as a functional reporter because decreasing intracellular H2O2 alters the sensitivity of ICa-L to isoproterenol. H2O2 increased the K-0.5 required for activation of ICa-L by isoproterenol from 5.8 to 27.8 nmol/L. This effect and the increase in basal current density persisted for several hours after H2O2. We propose that extracellular H2O2 is associated with an increase in superoxide from the mitochondria caused by an increase in Ca2+ influx from ICa-L. The effect persists because a positive feedback exists among increased basal channel activity, elevated intracellular calcium, and superoxide production by the mitochondria.
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