Activation of endogenous protein phosphatase 1 enhances the calcium sensitivity of the ryanodine receptor type 2 in murine ventricular cardiomyocytes. (14th February 2020)

• Record Type: Journal Article
• Authors: Potenza, Duilio Michele; Janicek, Radoslav; Fernandez‐Tenorio, Miguel; Niggli, Ernst
• Is Part Of: Journal of physiology. Volume 598:Number 6(2020)
• Page Start: 1131
• Page End: 1150
• Publication Date: 2020-02-14
• Subjects: Ca2+‐induced Ca2+ release -- cardiac muscle -- de‐phosphorylation -- excitation–contraction coupling -- protein phosphatase -- ryanodine receptor 2; Physiology -- Periodicals; 612.005
• DOI: 10.1113/JP278951
• Languages: English
• ISSNs: 0022-3751

Abstract

Abstract : Key points: Increased protein phosphatase 1 (PP‐1) activity has been found in end stage human heart failure. Although PP‐1 has been extensively studied, a detailed understanding of its role in the excitation–contraction coupling mechanism, in normal and diseased hearts, remains elusive. The present study investigates the functional effect of the PP‐1 activity on local Ca 2+ release events in ventricular cardiomyocytes, by using an activating peptide (PDP3) for the stimulation of the endogenous PP‐1 protein. We report that acute de‐phosphorylation may increase the sensitivity of RyR2 channels to Ca 2+ in situ, and that the RyR2‐serine2808 phosphorylation site may mediate such a process. Our approach unmasks the functional importance of PP‐1 in the regulation of RyR2 activity, suggesting a potential role in the generation of a pathophysiological sarcoplasmic reticulum Ca 2+ leak in the diseased heart. Abstract: Changes in cardiac ryanodine receptor (RyR2) phosphorylation are considered 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 (PP‐1) activity has been observed in heart failure, although the regulatory role of this enzyme on intracellular Ca 2+ handling remains poorly understood. To determine the physiological and pathophysiological significance of increased PP‐1 activity, we investigated how the PP‐1 catalytic subunit (PP‐1c) alters Ca 2+ sparks in permeabilized cardiomyocytes and we also applied a PP‐1‐disrupting peptide (PDP3) to specifically activate endogenous PP‐1, including the one anchored on the RyR2 macromolecular complex. We compared wild‐type and transgenic mice in which the usually highly phosphorylated site RyR2‐S2808 has been ablated to investigate its involvement in RyR2 modulation (S2808A +/+ ). In wild‐type myocytes, PP‐1 increased Ca 2+ spark frequency by two‐fold, followed by depletion of the sarcoplasmic reticulum Ca 2+ store. Similarly, PDP3 transiently increased spark frequency and decreased sarcoplasmic reticulum Ca 2+ load. RyR2 Ca 2+ sensitivity, which was assessed by Ca 2+ spark recovery analysis, was increased in the presence of PDP3 compared to a negative control peptide. S2808A +/+ cardiomyocytes did not respond to both PP‐1c and PDP3 treatment. Our results suggest an increased Ca 2+ sensitivity of RyR2 upon de‐phosphorylation by PP‐1. Furthermore, we have confirmed the S2808 site as a target for PP‐1 and as a potential link between RyR2s modulation and the cellular response. Key points: Increased protein phosphatase 1 (PP‐1) activity has been found in end stage human heart failure. Although PP‐1 has been extensively studied, a detailed understanding of its role in the excitation–contraction coupling mechanism, in normal and diseased hearts, remains elusive. The present study investigates the functional effect of the PP‐1 activity on local Ca 2+ release events in ventricular cardiomyocytes, by using an activating peptide (PDP3) for the stimulation of the endogenous PP‐1 protein. We report that acute de‐phosphorylation may increase the sensitivity of RyR2 channels to Ca 2+ in situ, and that the RyR2‐serine2808 phosphorylation site may mediate such a process. Our approach unmasks the functional importance of PP‐1 in the regulation of RyR2 activity, suggesting a potential role in the generation of a pathophysiological sarcoplasmic reticulum Ca 2+ leak in the diseased heart.