Nitroxyl-Mediated Disulfide Bond Formation Between Cardiac Myofilament Cysteines Enhances Contractile Function

Abstract

Rationale:In the myocardium, redox/cysteine modification of proteins regulating Ca2+cycling can affect contraction and may have therapeutic value. Nitroxyl (HNO), the one-electron-reduced form of nitric oxide, enhances cardiac function in a manner that suggests reversible cysteine modifications of the contractile machinery.Objective:To determine the effects of HNO modification in cardiac myofilament proteins.Methods and Results:The HNO-donor, 1-nitrosocyclohexyl acetate, was found to act directly on the myofilament proteins, increasing maximum force (Fmax) and reducing the concentration of Ca2+for 50% activation (Ca50) in intact and skinned cardiac muscles. The effects of 1-nitrosocyclohexyl acetate are reversible by reducing agents and distinct from those of another HNO donor, Angeli salt, which was previously reported to increase Fmaxwithout affecting Ca50. Using a new mass spectrometry capture technique based on the biotin switch assay, we identified and characterized the formation by HNO of a disulfide-linked actin–tropomyosin and myosin heavy chain–myosin light chain 1. Comparison of the 1-nitrosocyclohexyl acetate and Angeli salt effects with the modifications induced by each donor indicated the actin–tropomyosin and myosin heavy chain–myosin light chain 1 interactions independently correlated with increased Ca2+sensitivity and force generation, respectively.Conclusions:HNO exerts a direct effect on cardiac myofilament proteins increasing myofilament Ca2+responsiveness by promoting disulfide bond formation between critical cysteine residues. These findings indicate a novel, redox-based modulation of the contractile apparatus, which positively impacts myocardial function, providing further mechanistic insight for HNO as a therapeutic agent.