S-Nitrosylation Decreases Ca2+ Sensitivity and Actomyosin ATPase Activity of Contractile Proteins in Cardiac Myofibrils

Abstract

Increases in muscle nitric-oxide (NO) production can be buffered by reaction with intracellular glutathione, forming S-nitrosoglutathione (GSNO). GSNO has been shown to S-nitrosylate Cys thiols of cardiac contractile proteins in vivo and in vitro, but effects on maximal force, thin-filament Ca2+ sensitivity and actomyosin ATPase activity are unknown. Here, we analyzed the targets of S-nitrosylation in mouse cardiac contractile proteins, and examined the effects of these modifications on function in myocytes and skinned cardiac myofibrils. S-Nitrosylation and denitrosylation were detected using resin-assisted capture (SNO-RAC) and targets for S-nitrosylation were identified by quantitative LC-MS/MS. Isolated cardiomyocytes treated with S-nitrosocysteine (CysNO, 500μM, 10min) showed an increase in total protein-SNO, followed by progressive denitrosylation (30-60min with CysNO). At 10min, CysNO dose-dependently increased S-nitrosylation of specific Cys thiols in myosin heavy chain, actin, TnC, TnI, myosin-binding protein C and other muscle proteins. Myofibril thin-filament Ca2+ sensitivity decreased (P 0.05). Loss of Ca2+ sensitivity was partially reversed by the denitrosation agent, ascorbate. Relaxation kinetics of skinned fibers, as measured by flash photolysis, were also significantly reduced by100μM GSNO (k1,15.33 to 11.68/s; k2 2.33 to 0.87/s; fit to double exponential). Maximal myofibrillar ATPase activity (pCa 5.0) was also dose-dependently inhibited (8, 15, 30%) by 50, 100 and 500μM GSNO, an effect that was reversed by ascorbate. The findings suggest that S-nitrosylation of regulatory Cys thiol(s) can reversibly modulate cardiac muscle contraction and may be able to protect the heart by reducing myosin ATPase demand without affecting the maximal force of contraction.