Abstract
Changes in cardiac ryanodine receptor (RyR2) phosphorylation are thought to be important regulatory and disease related post-translational protein modifications. The extent of RyR2 phosphorylation is mainly determined by the balance of the activities of protein kinases and phosphatases, respectively. Increased protein phosphatase-1 (PP1) activity has been observed in heart failure (HF), but the regulatory role of this enzyme on intracellular Ca2+ handling remains poorly understood. To determine the physiological and pathophysiological significance of increased PP1 activity, we investigated the effect of the PP1 catalytic subunit on Ca2+ sparks in permeabilized cardiomyocytes. We used wild-type (WT) and transgenic mice in which the highly phosphorylated site RyR2-S2808 has been ablated to investigate its involvement in RyR2 modulation. In WT myocytes, where cytosolic Ca2+ was clamped at 60nM, 2U/ml of PP1 initially increased Ca2+ spark frequency (CaSpF) by 2.2-fold, followed by a second phase during which CaSpF returned to control. Spark mass was decreased, but due to the high CaSpF, spark-mediated leak was increased by PP1. This was accompanied by depletion of the sarcoplasmic reticulum (SR) Ca2+ stores, as determined by application of caffeine. Changes in Ca2+ release and SR Ca2+ load were prevented by 5uM of okadaic acid, an inhibitor of PP1. S2808A mutant myocytes showed lower resting CaSpF compared to WT (1.18±0.23 vs 2.3±0.35 sparks/100um/s) and 2U/ml of PP1 failed to generate changes in CaSpF as well as in SR Ca2+ load. A higher concentration of PP1 (10U/ml) increased CaSpF 4-fold compared to control in WT, and 2.8-fold in S2808A cells, indicating a concentration-dependence. Our results suggest that increased intracellular PP1 activity stimulates RyR2-mediated SR Ca2+ release and that de-phosphorylation of RyR2-S2808 and at least one not yet identified phosphorylation site may be important in RyR2 modulation.
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