Abstract 226: Nitroxyl Activates SERCA in Cardiac Myocytes via Reversible S-Glutathiolation of Cysteine-674
Background: Oxidative modifications of sarcoplasmic reticulum Ca2+-ATPase (SERCA) regulate enzyme activity in vascular smooth muscle. In myocardium, nitroxyl anion (HNO) generated from Angeli’s salt (AS) exerts inotropic/lusitropic effects that are associated with activation of SERCA via a redox-sensitive mechanism. We tested the hypothesis that HNO activates SERCA via oxidative modifcation of cysteine 674.
Methods/Results: In adult rat ventricular myocytes, AS (500μM; 15 min) increased maximal SERCA activity 3-fold from 1.5 ±0.4 to 4.9 ±0.4 nmol Ca2+/min/mg protein (p<0.001; n=3). HNO decreased SERCA biotinylated-iodoacetamide (BIAM) labeling by 27 ± 3% (p<0.0001; n=14), indicative of oxidative thiol modification. HNO caused an 18 ± 4% increase in SERCA S-glutathiolation (p<0.05; n=5) as detected by immunoprecipitation of SERCA followed by immunoblotting with an antibody against glutathione. S-glutathiolation was prevented by HNO washout or adenoviral overexpression of glutaredoxin-1 (GRX). Adenoviral overexpression of SERCA in which cysteine 674 was mutated to serine (C674S) prevented the HNO-induced decrease in SERCA BIAM-labeling. Likewise, overexpression of either GRX or the C674S SERCA mutant prevented HNO-stimulated SERCA activation. Preincubation with the NO scavenger carboxy-PTIO (200μM, 15min) or the protein kinase G (PKG) inhibitor ODQ (10μM, 15min) had no effect on HNO-stimulated SERCA activation. In parallel studies in isolated adult rat ventricular myocytes, HNO increased contractility, and this effect was prevented by overexpression of the C674S mutant.
Conclusion: HNO causes activation of SERCA in cardiac myocytes via S-glutathiolation of cysteine 674. This effect of HNO is reversible, and does not appear to be mediated by nitric oxide. Thus, S-glutathiolation of SERCA cysteine 674 by HNO may participate in the regulation of myocardial function.