Abstract 495: Ryanodine Receptor Phosphorylation in Intact Rat Ventricular Cardiomyocytes: Caution with Phosphorylation Site-Specific Antibodies
The cardiac Ryanodine Receptor (RyR) controls SR Ca release during excitation-contraction. Its large cytoplasmic domain is a scaffold for proteins that modulate RyR function by phospho-/ dephosphorylation. Evidence suggests that changes in RyR function in heart failure contribute to arrhythmias and reduced contractility. Three phosphorylation sites have been identified so far (S2808, S2814 and more recently S2030). We thoroughly studied RyR phosphorylation in intact cardiomyocytes using 2 or 3 site-specific antibodies for each site. We found that for S2808 results were clearly antibody-dependent. When immunoprecipitated RyR was partially dephosphorylated with alkaline phosphatase, only 1 of 3 antibodies revealed the decrease in phosphorylation at S2808, while one showed an increase! Using only validated antibodies, RyR is partially phosphorylated in quiescent myocytes at S2808 (54±5% of total RyR) and S2814 (16±4%). Baseline phosphorylation at both sites could be decreased by incubation with Staurosporine, EGTA and Thapsigargin, but not the CaMKII inhibitor KN93, indicating continuous phosphorylation by a Ca2+ dependent kinase other than CaMKII. Iso (0.1 μ M, 15 min) saturates phosphorylation at S2808 and this increase can be blunted by PKA inhibitors PKI/H89 but not KN93. Iso increases S2814 only when cells are simultaneously electrically stimulated and this is attenuated by KN93. The RyR appears to be unphosphorylated at S2030 in quiescent myocytes and high concentrations of Iso are necessary to partially phosphorylate this site. A dose-response curve to the phosphatase inhibitor okadaic acid in combination with in vitro application of purified phosphatases reveals that phosphatase 1 is the main phosphatase dephosphorylating S2808/S2814, but phosphatase 2a may also dephosphorylate S2814. RyR phosphorylation is complex, but very important in understanding RyR functional modulation and altered function in physiological and pathological states such as heart failure and arrhythmogenesis. Cautious semi-quantitative analysis using phospho-specific antibodies can help in understanding molecular mechanisms.