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

<jats:p> We sought to understand the effect of a transient exposure of cardiac myocytes to H <jats:sub>2</jats:sub> O <jats:sub>2</jats:sub> at a concentration that did not induce apoptosis. Myocytes were exposed to 30 μmol/L H <jats:sub>2</jats:sub> O <jats:sub>2</jats:sub> for 5 minutes followed by 10 U/mL catalase for 5 minutes to degrade the H <jats:sub>2</jats:sub> O <jats:sub>2</jats:sub> . Cellular superoxide was measured using dihydroethidium. Transient exposure to H <jats:sub>2</jats:sub> O <jats:sub>2</jats:sub> 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 <jats:italic>p</jats:italic> -(trifluoromethoxy)phenyl-hydrazone and when calcium uptake by the mitochondria was inhibited with Ru360. We investigated the L-type Ca <jats:sup>2+</jats:sup> channel ( <jats:italic>I</jats:italic> <jats:sub>Ca-L</jats:sub> ) as a source of calcium influx. Nisoldipine, an inhibitor of <jats:italic>I</jats:italic> <jats:sub>Ca-L</jats:sub> , 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 <jats:sub>2</jats:sub> O <jats:sub>2</jats:sub> . The response of <jats:italic>I</jats:italic> <jats:sub>Ca-L</jats:sub> to β-adrenergic receptor stimulation was used as a functional reporter because decreasing intracellular H <jats:sub>2</jats:sub> O <jats:sub>2</jats:sub> alters the sensitivity of <jats:italic>I</jats:italic> <jats:sub>Ca-L</jats:sub> to isoproterenol. H <jats:sub>2</jats:sub> O <jats:sub>2</jats:sub> increased the <jats:italic>K</jats:italic> <jats:sub>0.5</jats:sub> required for activation of <jats:italic>I</jats:italic> <jats:sub>Ca-L</jats:sub> 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 <jats:sub>2</jats:sub> O <jats:sub>2</jats:sub> . We propose that extracellular H <jats:sub>2</jats:sub> O <jats:sub>2</jats:sub> is associated with an increase in superoxide from the mitochondria caused by an increase in Ca <jats:sup>2+</jats:sup> influx from <jats:italic>I</jats:italic> <jats:sub>Ca-L</jats:sub> . The effect persists because a positive feedback exists among increased basal channel activity, elevated intracellular calcium, and superoxide production by the mitochondria. </jats:p>