Regulation of sarcoplasmic reticulum Ca2+ release by cytosolic glutathione in rabbit ventricular myocytes

Of the major cellular antioxidant defenses, glutathione (GSH) is particularly important in maintaining the cytosolic redox potential. Whereas the healthy myocardium is maintained at a highly reduced redox state, it has been proposed that oxidation of GSH can affect the dynamics of Ca2+-induced Ca2+ release. In this study, we used multiple approaches to define the effects of oxidized glutathione (GSSG) on ryanodine receptor (RyR)-mediated Ca2+ release in rabbit ventricular myocytes. To investigate the role of GSSG on sarcoplasmic reticulum (SR) Ca2+ release induced by the action potential, we used the thiol-specific oxidant diamide to increase intracellular GSSG in intact myocytes. To more directly assess the effect of GSSG on RyR activity, we introduced GSSG within the cytosol of permeabilized myocytes. RyR-mediated Ca2+ release from the SR was significantly enhanced in the presence of GSSG. This resulted in decreased steady-state diastolic [Ca2+]SR, increased SR Ca2+ fractional release, and increased spark- and non-spark-mediated SR Ca2+ leak. Single-channel recordings from RyR's incorporated into lipid bilayers revealed that GSSG significantly increased RyR activity. Moreover, oxidation of RyR in the form of intersubunit crosslinking was present in intact myocytes treated with diamide and permeabilized myocytes treated with GSSG. Blocking RyR crosslinking with the alkylating agent N-ethylmaleimide prevented depletion of SR Ca2+ load induced by diamide. These findings suggest that elevated cytosolic GSSG enhances SR Ca2+ leak due to redox-dependent intersubunit RyR crosslinking. This effect can contribute to abnormal SR Ca2+ handling during periods of oxidative stress.