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(Circulation Research. 2007;100:1036.)
© 2007 American Heart Association, Inc.

Cellular Biology

Transient Exposure to Hydrogen Peroxide Causes an Increase in Mitochondria-Derived Superoxide As a Result of Sustained Alteration in L-Type Ca²⁺ Channel Function in the Absence of Apoptosis in Ventricular Myocytes

Helena M. Viola, Peter G. Arthur, Livia C. Hool

From the School of Biomedical, Biomolecular and Chemical Sciences, University of Western Australia, Crawley; and Western Australian Institute for Medical Research, Crawley.

Correspondence to Dr Livia Hool, Physiology M311, School of Biomedical, Biomolecular and Chemical Sciences, University of Western Australia, 35 Stirling Hwy, Crawley, WA, 6009, Australia. E-mail lhool{at}cyllene.uwa.edu.au

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 µmol/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 (I_Ca-L) as a source of calcium influx. Nisoldipine, an inhibitor of I_Ca-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 I_Ca-L to ß-adrenergic receptor stimulation was used as a functional reporter because decreasing intracellular H₂O₂ alters the sensitivity of I_Ca-L to isoproterenol. H₂O₂ increased the K_0.5 required for activation of I_Ca-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 I_Ca-L. The effect persists because a positive feedback exists among increased basal channel activity, elevated intracellular calcium, and superoxide production by the mitochondria.

Key Words: hydrogen peroxide • mitochondria • L-type Ca²⁺ channel