Abstract
In heart failure (HF), arrhythmogenic spontaneous sarcoplasmic reticulum (SR) Ca2+ release and afterdepolarizations in cardiac myocytes have been linked to abnormally high activity of ryanodine receptors (RyR2s) associated with enhanced phosphorylation of the channel. However, the specific molecular mechanisms underlying RyR2 hyperphosphorylation in HF remain poorly understood. The objective of the current study was to test the hypothesis that the enhanced expression of muscle-specific microRNAs (miRNAs) underlies the HF-related alterations in RyR2 phosphorylation in ventricular myocytes by targeting phosphatase activity localized to the RyR2. We studied hearts isolated from canines with chronic HF exhibiting increased left ventricular (LV) dimensions and decreased LV contractility. qRT-PCR revealed that the levels of miR-1 and miR-133, the most abundant muscle-specific miRNAs, were significantly increased in HF myocytes compared with controls (2- and 1.6-fold, respectively). Western blot analyses demonstrated that expression levels of the protein phosphatase 2A (PP2A) catalytic and regulatory subunits, which are putative targets of miR-133 and miR-1, were decreased in HF cells. PP2A catalytic subunit mRNAs were validated as targets of miR-133 by using luciferase reporter assays. Pharmacological inhibition of phosphatase activity increased the frequency of diastolic Ca2+ waves and afterdepolarizations in control myocytes. The decreased PP2A activity observed in HF was accompanied by enhanced Ca2+/calmodulin-dependent protein kinase (CaMKII)-mediated phosphorylation of RyR2 at sites Ser-2814 and Ser-2030 and increased frequency of diastolic Ca2+ waves and afterdepolarizations in HF myocytes compared with controls. In HF myocytes, CaMKII inhibitory peptide normalized the frequency of pro-arrhythmic spontaneous diastolic Ca2+ waves. These findings suggest that altered levels of major muscle-specific miRNAs contribute to abnormal RyR2 function in HF by depressing phosphatase activity localized to the channel, which in turn, leads to the excessive phosphorylation of RyR2s, abnormal Ca2+ cycling, and increased propensity to arrhythmogenesis.
Highlights
Heart failure (HF) remains a leading cause of mortality and approximately 50% of HF patients die suddenly as a result of ventricular tachyarrhythmias stemming from the increased propensity of ventricular myocytes to generate delayed and/or early afterdepolarizations [1,2]
We have investigated the possible link between enhanced predisposition of ventricular myocytes toward pro-arrhythmic spontaneous Ca2+ release and altered expression levels of two major muscle-specific miRNAs, miR-1 and miR-133 in a canine model of chronic HF
Our main finding is that the increased expression of miR-1 and miR-133 observed in HF was associated with reduced levels of phosphatase 2A (PP2A) regulatory
Summary
Heart failure (HF) remains a leading cause of mortality and approximately 50% of HF patients die suddenly as a result of ventricular tachyarrhythmias stemming from the increased propensity of ventricular myocytes to generate delayed and/or early afterdepolarizations [1,2]. The generation of arrhythmogenic afterdepolarizations in HF has been linked to extrasystolic spontaneous Ca2+ release from the sarcoplasmic reticulum (SR) [3,4,5,6,7]. It is believed that the increased occurrence of spontaneous Ca2+ release in ventricular myocytes corresponds to the abnormally high activity of ryanodine receptors (RyR2s), the SR Ca2+ release channels [8,9]. Enhanced phosphorylation can be explained by the increased activity of kinases and/or the decreased activity of phosphatases in the vicinity of the channel.
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