S-nitrosylation of CaMKII Cys273 suppresses CaMKII activation and mobility within cardiac myocytes.

Calcium/calmodulin-dependent protein kinase II delta (CaMKIIδ) is a central regulator of cardiac excitation-contraction coupling and is chronically activated in multiple cardiac pathologies. Nitrosylation has been implicated in cardiac cell signaling and function in health and disease. We tested how S-nitrosylation at two known CaMKII cysteines alter kinase activation conformation and intracellular mobility in cardiac myocytes. We directly monitored in real time, the CaMKII activation state in myocytes with GSNO treatment- before or after activation of CaMKII. Active CaMKII conformation was assessed using the fluorescent biosensor Camui and fluorescence lifetime values, and cellular mobility was assessed by fluorescence recovery after photobleach (FRAP) in CaMKII-GFP and movement away from the sarcomeric Z-line. To test the influence of each known S-nitrosylation site we used CaMKII in which each site was mutated (Cys273Ser and Cys290Ala) in Camui and CaMKII-GFP. The results reveal that in intact ventricular cardiomyocytes S-nitrosylation of CaMKII at Cys273 prior to electrical field stimulation (and β-adrenergic activation) suppresses CaMKIIδ activation, and mobility, effects that are suppressed in Cys273Ser CaMKIIδ. In contrast S-nitrosylation of CaMKIIδ Cys290 was critical for GSNO-induced activation and mobility. Thus, baseline S-nitrosylation at Cys273 limits CaMKIIδ activation and mobility, while S-nitrosylation at C290 when the kinase is activated enhances activation and mobility. Thus, prevailing nitrosylation conditions in cardiac myocytes can modulate the activation and mobility of CaMKII with respect to its targets.