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Transient exposure to extracellular hydrogen peroxide is associated with a persistent increase in intracellular calcium and superoxide from the mitochondria in ventricular myocytes without apoptosis

We examined the effects of a transient hydrogen peroxide (H₂O₂) exposure on myocyte function at a concentration insufficient to cause apoptosis or necrosis. Myocytes were exposed to 30 μM H₂O₂ for 5 min followed by 10U/ml catalase for 5 min to degrade the H₂O₂. Cellular superoxide was measured using dihydroethidium (DHE). Exposure to H₂O₂ caused a 66.1% increase in DHE signal (n=45, p<0.05) compared to controls exposed to catalase only (n=8) without activation of caspase 3 or evidence of necrosis. The increase in DHE was attenuated when cells were exposed to the mitochondrial inhibitor myxothiazol (7nM, n=14) and when calcium uptake by the mitochondria was inhibited with 2μM Ru360 (n=5). We investigated the L-type Ca²⁺ channel (I_Ca-L) as a source of calcium influx. The increase in superoxide could be attenuated when I_Ca-L was inhibited with 2 μM nisoldipine (n=9). Basal channel activity was significantly increased from 5.4pA/pF (n=7) to 8.9pA/pF (n=25) after H₂O₂. The response of the channel to β-adrenergic receptor stimulation was used as a functional reporter for changes in cellular production of reactive oxygen species since a decrease in cellular H₂O₂ is associated with altered sensitivity of the channel to β-adrenergic receptor stimulation. After H₂O₂, the K_0.5 for activation of the channel by isoproterenol was significantly increased from 5.8 to 27.8nM. This effect and the increase in basal activity persisted for several hours after H₂O₂. In addition, intracellular calcium was persistently elevated with a two fold increase from a resting calcium of 24nM (n=5, p<0.05). We propose that extracellular H₂O₂ is associated with an increase in mitochondrial-derived superoxide via calcium influx from I_Ca-L. The effect persists because a positive feedback exists between increased basal channel activity, elevated intracellular calcium and superoxide production by the mitochondria. This may represent a mechanism for cardiovascular pathology that involves elevated calcium and reactive oxygen species.