Abstract 2635: Phosphorylation of Ryanodine Receptor by Both PKA and CAMKII is Required for Induction of Arrhythmogenic Disturbances in Myocyte Ca Cycling Caused by miR-1
We recently demonstrated that muscle-specific microRNA, miR-1, increases myocyte arrhythmogenic potential by increasing phosphorylation of the ryanodine receptor (RyR2) by disrupting localization of PP2A phosphatase activity to the RyR2. In the present study, we investigated the specific contributions of CAMKII and PKA to the effects of miR-1 by combining functional approaches (electrophysiology and Ca imaging) with measurements of RyR2 phosphorylation using antibodies to the PKA site S2808, and the CAMKII site, S2814. Adenoviral-mediated overexpression of miR-1 increased frequency of spontaneous Ca waves and delayed after depolarizations in cells challenged with isoproterenol (ISO). RyR2 phosphorylation at S2808 was low at baseline conditions and increased following ISO treatment, while phosphorylation at S2814 was already maximal under baseline. ISO enhanced SERCA-mediated Ca uptake and elevated SR Ca content through phosphorylation of PLB. To determine which of these factors (i.e. increased RyR2 PKA-phosphorylation or increased SR Ca content) mediated the increased arrhythmogenic potential in miR-1 cells, we coinfected myocytes with constructs for expression of miR-1 and of a PLB mutant variant that increases SERCA-mediated SR Ca uptake by displacing endogenous PLB from SERCA (dnPLB). Myocytes coexpressing miR-1 and dnPLB (miR-1xdnPLB) did not exhibit enhanced predisposition to arrhythmia in the absence of ISO despite the fact that their SR Ca content was as high as in cells expressing miR-1 alone in the presence of ISO. Notably, treatment of the miR-1xdnPLB myocytes with either PKA or CAMKII inhibitors completely abolished increased arrhythmogenic activity without a significant effect on the SR Ca content. We conclude that neither CAMKII nor PKA phosphorylation of RyR2 alone is sufficient to produce the changes in RyR2 activity that underlies the arrhythmogenic disturbances in Ca handling caused by miR-1. Simultaneous phosphorylation of RyR2 by PKA and CAMKII appears to be necessary for this effect. In addition to delineating the mechanisms of the pro-arrhythmic effect of miR-1 our study has a broader implication by helping to reconcile existing controversies about the role and mechanisms of RyR2 phosphorylation in cardiac disease.