Glutathione is a cofactor for H2O2-mediated stimulation of Ca2+-induced Ca2+ release in cardiac myocytes

Reactive oxygen species are known to cause attenuation of cardiac muscle contraction. This attenuation is usually preceded by transient augmentation of twitch amplitude as well as cytosolic Ca2+. The present study examines the role of an endogenous antioxidant, glutathione in the mechanism of H2O2-mediated augmentation of Ca2+ release from the sarcoplasmic reticulum. Whole-cell patch-clamped single rat ventricular myocytes were dialyzed with the Cs+-rich internal solution containing 200 microM fura-2 and 2 mM glutathione (reduced form). After equilibration of the myocyte with intracellular dialyzing solution, Ca2+ current-induced Ca2+ release from the sarcoplasmic reticulum was monitored. Rapid perfusion with H2O2 (100 microM or 1 mM) for 20 s inhibited Ca2+ current, but enhanced the intracellular Ca2+ transients for 3-4 min. Thus, the efficacy of Ca2+-induced Ca2+ release mechanism was augmented in 71% of myocytes (n = 7). This enhancement ranged between 1.5- to threefold as the concentrations of H2O2 were raised from 100 microM to 1 mM. If glutathione were excluded from the patch pipette or replaced with glutathione disulfide, the enhancement of Ca2+-induced Ca2+ release was seen in only a minority (20%) of the myocytes. H2O2 exposure did not increase the basal intracellular Ca2+ levels, suggesting that the mechanism of H2O2 action was not mediated by inhibition of the sarcoplasmic reticulum Ca2+ uptake or activation of passive Ca2+ leak pathway. H2O2-mediated stimulation of Ca2+-induced Ca2+ release was also observed in myocytes dialyzed with dithiothreitol (0.5 mM). Therefore, reduced thiols support the action of H2O2 to enhance the efficacy of Ca2+-induced Ca2+ release, suggesting that redox reactions might regulate Ca2+ channel-gated Ca2+ release by the ryanodine receptor.