Simultaneous Phosphorylation of RyR2 by PKA and Camkii is Required for Induction of CA-Dependent Arrhythmia Caused by MIR-1 Overexpression

We recently showed that disruption of localization of PP2A phosphatase activity to the ryanodine receptor (RyR2) complex by overexpression of the muscle-specific microRNA, miR-1, stimulates excitation-contraction coupling and results in increased arrhythmogenic potential in cardiac myocytes. In the present study, we examined the role of PKA and CAMKII as mediators of the effects miR-1 on Ca signaling and arrhythmogenesis using cellular electrophysiology and Ca imaging complemented with quantitative measurements of RyR2 phosphorylation at PKA site S-2808, and at CAMKII site S-2814. Adenovirally-mediated 2-fold overexpression of miR-1 resulted in 10-fold increase in arrhythmogenic potential measured as a frequency of spontaneous Ca waves and DADs in myocytes exposed to 100 nM isoproterenol (ISO). Quantitative imminoblotting using site-directed phosphospecific-antibodies showed that RyR2 phosphorylation in miR-1 overexpressing cells was low at S-2808 under basal conditions. Exposure of myocytes to ISO maximized phosphorylation at S-2808. Phosphorylation at S-2814 under basal conditions was maximal and did not further increase in the presence of ISO. Additionally, ISO increased SERCa-mediated SR Ca uptake and SR Ca load through phosphorylation of phospholamban (PLB). To define which of these factors (increased CaSR content or increased RyR2 PKA phosphorylation) mediated the increased arrhythmogenic potential we infected myocytes with viral constructs of a dominant-negative PLB mutant that accelerates SERCa-mediated SR Ca uptake by displacing endogenous PLB from SERCa. Myocytes coexpressing miR-1 and dnPLB did not exhibit enhanced predisposition to Ca-dependent arrhythmia in the presence of ISO despite maximal SR Ca load. Importantly treatment of cells with either PKA or CAMK inhibitors completely abolished increased arrhythmogenic activity. We conclude that neither CAMKII nor PKA phosphorylation alone is sufficient to produce the changes in RyR2 activity that underlies the arrhythmogenic disturbances caused by miR-1 overexpression.