Role of thiol modifications in modulating SERCA activity in myocardial regulation

The sarcoplasmic reticulum Ca2+-ATPase (SERCA) is a critical determinant of cardiac excitation-contraction coupling that controls the diastolic sequestration of Ca2+ into the SR, thereby providing the workload of Ca2+ available for contraction. Oxidant signaling can cause reversible modifications of cysteine thiols in SERCA that activate the enzyme, while exposure to more intense oxidative stress can cause irreversible modifications that impair enzyme function. Prior studies in vascular smooth muscle cells suggest that cysteine-674 may be a critical site of SERCA modification. The aim of this study was to elucidate the mechanism by which oxidants regulate SERCA activity in cardiomyocytes. The specific oxidative modifications of SERCA that are associated with different reactive oxygen and nitrogen species including hydrogen peroxide (H2O2), nitroxyl (HNO), and peroxynitrite (ONOO-), and their effects on myocyte Ca2+ handling and contractile function, were determined. In work described in Aim 1, H2O2 was shown to cause inactivation of SERCA involving irreversible cysteine-674 sulfonylation. Mutation of cysteine-674 of SERCA not only prevented H2O2-induced thiol oxidation and SERCA inactivation, but also attenuated myocyte dysfunction induced by H2O2, including decreases in cell shortening and Ca2+ transient amplitude, as well as prolongation of cell relaxation and diastolic Ca2+ sequestration. Data presented in Aim 2 shows that HNO reversibly activates SERCA via S-glutathiolation of cysteine-674. Mutation of cysteine-674 prevented HNO-induced thiol oxidation and SERCA activation. Cysteine-674 was also implicated in mediating HNO-stimulated myocyte contractility and Ca2+ re-uptake by SERCA. This effect of HNO did not depend on ONOO-, NO, PKG or phospholamban. Aim 3 presents data that ONOO- exerts bi-directional, concentration-dependent regulation of SERCA in cardiomyocytes. A low concentration of ONOO - (50muM) activated SERCA via glutaredoxin-1 (GRX)-sensitive S-glutathiolation of cysteine-674, whereas a high concentration of ONOO- (500muM) caused SERCA inactivation that involved cysteine-674 sulfonylation. Low and high concentrations of ONOO- also caused bi-directional effects on myocyte intracellular Ca2+ reuptake and contractility.;In summary, in cardiomyocytes specific oxidative modifications of cysteine-674 may either stimulate or inhibit SERCA function thereby regulating myocardial function. Activation by HNO or low concentrations of ONOO- involves reversible cysteine-674 S-glutathiolation, may be relevant to physiological regulation, and may thus have therapeutic potential. Inhibition of SERCA by H2O2 or high concentrations of ONOO- involves irreversible cysteine-674 sulfonylation and may participate in the pathophysiology of conditions associated with high oxidant levels such as myocardial failure and ischemia/reperfusion.