Increased Levels of Micrornas miR-1 and miR-133 in Failing Heart Underlie Dissociation of Phosphatase Activity from RyR2 Complex Resulting in Enhanced RyR2 CaMKII-Dependent Phosphorylation and Cardiac Arrhythmias

Abstract

Increased propensity of ventricular myocytes to arrhythmogenic spontaneous SR Ca release and afterdepolarizations in heart failure (HF) has been linked to abnormally high activity of RyR2. Growing evidence supports hyperphosphorylation of RyR2 at the CaMKII site S-2814 as a potential mechanism for altered RyR2 function. However, the specific molecular mechanisms underlying RyR2 hyperphosphorylation remain poorly understood. MicroRNAs are small noncoding RNAs that regulate protein expression by interfering with mRNAs of target genes. We recently reported that 2-fold overexpession of microRNA miR-1 enhances CaMKII-dependent RyR2 phosphorylation by disrupting protein phosphatase 2A scaffolding to the RyR2, resulting in increased activity of the channel and Ca-dependent afterdepolarizations in myocytes. In the present study, we used a canine model of nonischemic HF to test the hypothesis that the HF-related alterations in RyR2 phosphorylation levels are caused by a decrease in phosphatase activity localized to RyR2 due to enhanced expression of two most abundant muscle-specific microRNAs miR-1 and miR-133. qRT-PCR studies revealed that the levels of miR-1 and miR-133 were significantly increased in HF myocytes compared to controls (2 and 1.6 fold accordingly). Western blotting showed that PP2A regulatory (b56alpha) and catalytic subunits, specific targets of miR-1 and miR-133 validated by luciferase-reporter assay, were decreased in HF cells. Analysis using phospho-specific antibodies confirmed that RyR2 phosphorylation at Ser-2814 was significantly increased in HF myocytes compared to controls. CaMKII inhibitory peptide reduced the frequency of spontaneous Ca waves in paced current-clamped HF myocytes to low control values. These finding suggest that altered levels of major muscle-specific microRNAs contribute to abnormal RyR2 function in HF by depressing localized phosphatase activity to the channel, thus leading to excessive phosphorylation of RyR2s.