Aims During β-adrenergic receptor (β-AR) stimulation, phosphorylation of cardiomyocyte ryanodine receptors by protein kinases may contribute to an increased diastolic Ca2+ spark frequency. Regardless of prompt activation of protein kinase A during β-AR stimulation, this appears to rely more on activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII), by a not yet identified signalling pathway. The goal of the present study was to identify and characterize the mechanisms which lead to CaMKII activation and elevated Ca2+ spark frequencies during β-AR stimulation in single cardiomyocytes in diastolic conditions. Methods and results Confocal imaging revealed that β-AR stimulation increases endogenous NO production in cardiomyocytes, resulting in NO-dependent activation of CaMKII and a subsequent increase in diastolic Ca2+ spark frequency. These changes of spark frequency could be mimicked by exposure to the NO donor GSNO and were sensitive to the CaMKII inhibitors KN-93 and AIP. In vitro, CaMKII became nitrosated and its activity remained increased independent of Ca2+ in the presence of GSNO, as assessed with biochemical assays. Conclusions β-AR stimulation of cardiomyocytes may activate CaMKII by a novel direct pathway involving NO, without requiring Ca2+ transients. This crosstalk between two established signalling pathways may contribute to arrhythmogenic diastolic Ca2+ release and Ca2+ waves during adrenergic stress, particularly in combination with cardiac diseases. In addition, NO-dependent activation of CaMKII is likely to have repercussions in many cellular signalling systems and cell type
