Abstract 5302: Control of Reversible Phosphorylation of Ryanodine Receptor by MiR-1 : Mechanism of Cardiac Arrhythmia

Abstract

MicroRNAs are small endogenous noncoding RNAs that regulate protein expression by hybridization to imprecise complementary sequences of target mRNAs. Changes in abundance of muscle-specific microRNA, miR-1 , have been implicated in cardiac disease, including arrhythmia and heart failure. However, the specific molecular targets and cellular mechanisms involved in the action of miR-1 in the heart are only beginning to emerge. In this study we investigated the effects of increased expression of miR-1 on excitation-contraction coupling and Ca cycling in adult rat ventricular myocytes by using methods of electrophysiology, confocal Ca imaging and quantitative immunoblotting. Adenoviral-mediated overexpressions of miR-1 in myocytes resulted in a marked increase in the amplitude of the inward Ca current and flatten cytosolic Ca transients voltage dependency. The frequency of spontaneous Ca sparks recorded in intact resting cells was enhanced, while the sarcoplasmic reticulum Ca content was reduced in miR-1 - overexpressing myocytes as compared with controls. In the presence of beta-adrenergic receptor agonist isoproterenol, rhythmically paced miR-1 -overexpressing myocytes exhibited spontaneous arrhythmogenic oscillations of intracellular Ca, events that occurred only rarely in control myocytes. The effects of miR-1 were completely reversed by the CaMKII inhibitor KN93. Immunological analysis with phospho-specific antibodies showed that while phosphorylation of phospholamban was not altered, miR-1 overexpression increased phosphorylation of the ryanodine receptor at Ser-2814 (CaMKII) but not at Ser-2808 (PKA). Overexpression of miR-1 was accompanied by a selective decrease in expression of the protein phosphatase PP2A regulatory subunit B56alpha involved in PP2A targeting to specialized subcellular domains. We conclude that miR-1 through translational inhibition of this mRNA target, causes CaMKII-dependent hyperphosphorylation of RyR2 via disrupting localization of PP2A activity to this channel, enhances RyR2 activity, and promotes arrhythmogenic SR Ca release. This mechanism could contribute to induction of arrhythmia in disease states accompanied by elevated miR-1 . This research has received full or partial funding support from the American Heart Association, AHA National Center.