Transient Exposure to Hydrogen Peroxide Causes an Increase in Mitochondria-Derived Superoxide As a Result of Sustained Alteration in L-Type Ca ²⁺ Channel Function in the Absence of Apoptosis in Ventricular Myocytes

Abstract

We sought to understand the effect of a transient exposure of cardiac myocytes to H 2 O 2 at a concentration that did not induce apoptosis. Myocytes were exposed to 30 μmol/L H 2 O 2 for 5 minutes followed by 10 U/mL catalase for 5 minutes to degrade the H 2 O 2 . Cellular superoxide was measured using dihydroethidium. Transient exposure to H 2 O 2 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 Ca 2+ 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 H 2 O 2 . The response of I Ca-L to β-adrenergic receptor stimulation was used as a functional reporter because decreasing intracellular H 2 O 2 alters the sensitivity of I Ca-L to isoproterenol. H 2 O 2 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 H 2 O 2 . We propose that extracellular H 2 O 2 is associated with an increase in superoxide from the mitochondria caused by an increase in Ca 2+ 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.