Activation of endogenous protein phosphatase 1 enhances the calcium sensitivity of the ryanodine receptor type 2 in murine ventricular cardiomyocytes

Potenza, Duilio Michele; Janicek, Radoslav; Fernandez Tenorio, Miguel; Niggli, Ernst (2020). Activation of endogenous protein phosphatase 1 enhances the calcium sensitivity of the ryanodine receptor type 2 in murine ventricular cardiomyocytes. The journal of physiology, 598(6), pp. 1131-1150. The Physiological Society 10.1113/JP278951

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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 Ca2+ 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 Ca2+ 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 Ca2+ spark frequency by two-fold, followed by depletion of the sarcoplasmic reticulum Ca2+ store. Similarly, PDP3 transiently increased spark frequency and decreased sarcoplasmic reticulum Ca2+ load. RyR2 Ca2+ sensitivity, which was assessed by Ca2+ 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 Ca2+ 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.

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