Direct CaMKII Activation by Endogenous Nitric Oxide Modulates Calcium Spark Frequency in Cardiomyocytes

Abstract

During β-adrenergic stimulation of cardiomyocytes, phosphorylation of the ryanodine receptors (RyRs) by protein kinases may contribute their increased open probability. We previously reported that β-adrenergic stimulation (β-ARS) with isoproterenol (Iso) increases the Ca2+ spark frequency by about 4-fold in quiescent, voltage-clamped cardiomyocytes. Kinase inhibitors (KN-93, AIP, H89) and nitric oxide synthase (NOS) inhibitors (L-NAME, AAAN) indicated that CaMKII and NOS were involved in the change of spark frequency.Here we show a new mechanism where CaMKII is activated by nitric oxide (NO). using confocal NO imaging with DAF-2 we observed a significant (∼19%) raise in endogenous NO production matching the time-course of the increase in spark frequency upon β-ARS. In contrast, this increase in NO was not observed in the presence of L-NAME, confirming that the DAF-2 fluorescence was reflecting NO and not ROS. In parallel experiments, bypassing the classical β-ARS pathway by application of a NO donor, increased the frequency of sparks to the same extent as during β-ARS with Iso. This increase induced by NO was blocked by the CaMKII inhibitor AIP, suggesting that NO first activated CaMKII, which subsequently phosphorylated the RyRs. Biochemical in-vitro CaMKII activity assays confirmed direct activation by NO, in a Ca2+ independent manner. CaMKII has been reported to be activated by ROS. However, control experiments revealed that ROS were not involved in the examined pathway. Neither incubation of the cells with SOD mimetics or ROS scavengers (Mn-TBAP, TIRON) prevented the increase in spark frequency upon Iso application.Based on these findings we conclude that endogenously produced NO activates CaMKII, contributing to the regulation of RyR Ca2+ sensitivity and Ca2+ spark frequency upon β-adrenergic stimulation of cardiomyocytes. Supported by SNF