Abstract 575: Both Phosphorylation and Dephosphorylation of Ryanodine Receptor Enhance Sarcoplasmic Reticulum Calcium Leak in Canine Ventricular Myocytes

Abstract

In recent years, there has been an increase of emphasis on the possibility that alterations in phosphorylation of the cardiac ryanodine receptor (RyR2) may contribute to the defective intracellular Ca release in heart failure (HF). However, the specific mechanisms proposed by different laboratories remain controversial. We studied the effects of exogenous and endogenous kinases and phosphatases on sarcoplasmic reticulum (SR) Ca release by measuring [Ca] changes in the cytosolic and SR luminal ([Ca]SR) compartments, using the Ca indicators rhod-2 and fluo-5N, respectively, in permeabilized canine ventricular myocytes. Application of PP1 (10 u/ml) led to an acute increase in the frequency of spontaneous Ca release events (Ca sparks) followed by a decline in intra-SR [Ca] consistent with the possibility that activation of RyR2s contributes to unloading of the SR by releasing Ca. Indeed, PP1 caused a marked acceleration of the RyR2-mediated SR Ca leak directly measured as time-dependent decline of [Ca]SR in the presence of the inhibitor of the SR CaATPase (SERCA2), thapsigargin. Application of cAMP (20 uM) and PKA (10 u/ml) resulted in a significant increase in Ca spark frequency that was paralleled by an increase in [Ca]SR. Direct measurements of the RyR2-mediated leak in the presence of thapsigargin revealed a small but significant acceleration in the leak following cAMP or PKA application, implying that the potentiation of sparks in the absence of thapsigargin may involve direct effects upon RyR2s in addition to effects mediated by SERCA2-dependent increase in [Ca]SR. As determined by a phosphospecific antibody, RyR2 phosphorylation on Ser-2809 was increased by cAMP and reduced by PP1. Collectively, these results suggest that under baseline phosphorylation conditions, both phosphorylation and dephosphorylation stimulate RyR2 activity resulting in an increased SR Ca leak. Considering the multimeric nature of the RyR2 and the presence of multiple sets of phophorylation sites, these results can be rationalized by suggesting that the effects of kinases and phosphotases on RyR2 function are mediated by different degrees of RyR2 phosphorylation at a certain set of sites (e.g. Ser-2809) or by phosphorylation/dephosphorylation of different sets of sites.