Beta‐adrenergic stimulation causes rapid NOS1‐dependent S‐nitrosylation of cardiac proteins, associated with increased contractility

Neuronal nitric oxide synthase (NOS1) contributes to cardiac inotropism, but the mechanisms remain controversial. We used the isolated rat heart preparation and biotin‐switch assay, to test the hypothesis that β‐adrenergic stimulation activates NOS1, inducing protein S‐nitrosylation and this process enhances contractility. Isoprotenerol (ISO, 10nM, 3‐min) increased cardiac contractility (88±9%) and S‐nitrosylation (45±14%) of a broad range of proteins. NOS1 inhibitors S‐methyl‐L‐thiocitrulline (SMTC) and N ω ‐propyl‐l‐arginine (LNPA), and a nitroxyl radical inhibitor of the nitrosylation reaction (Tempol), decreased basal contractility (−15±5%, −34±3%, −14±8% respectively) and S‐nitrosylation (−52±8%, −25±13%, −60±6%). SMTC, LNPA and Tempol reduced the inotropic response (by 35–50%) and the increase in protein S‐nitrosylation induced by ISO (by 35–75%). Inhibition of NOS1 did not alter ISO‐induced phospholamban phosphorylation. In addition, SMTC treatment reduced endogenous S‐nitrosylation of SERCA2 by 92%, associated with a 30% reduction in (dP/dt)min, an index of relaxation. Unlike total proteins, ISO did not modify SERCA2 S‐nitrosylation either in control or during NOS1 inhibition. These results indicate that β‐adrenergic stimulation induces rapid NOS1‐mediated protein S‐nitrosylation, and this process associates with increased cardiac contractility. Fondecyt 1090757