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

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 Ca2+ 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 Ca2+ 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 Ca2+ 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 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.

Too much of a good thing? Establishing a role of excessive RyR2 dephosphorylation in heart disease

“…As described above, Potenza et al . (2020) convincingly demonstrate that excessive RyR2 dephosphorylation by PP‐1 can acutely enhance calcium leak in wild‐type mice, which is in contrast to previous reports of a protective role of increased PP‐1 activity by reducing RyR2‐mediated calcium leak and arrhythmias in a setting of heart failure (Fischer et al . 2018).…”

“…Looking forward, it will be important to explore the interplay between PP-1 and the kinases throughout the progression of disease and determine when PP-1 may have beneficial or detrimental effects on RyR2 function. No doubt the work of Potenza et al (2020) will further fuel the efforts of researchers to consolidate the complexities of the relationship between RyR2 function and its phosphorylation state in cardiac pathophysiology.…”

“…While the primary kinases implicated in phosphorylation of RyR2 have been well established, the nature of the specific phosphatase(s) responsible for dephosphorylating each site in vivo, until now, was less clear. In this issue of the Journal of Physiology, Potenza et al (2020) establish protein phosphatase 1 (PP-1) as an endogenous regulator of RyR2 phosphorylation at one of these key sites. They additionally demonstrate that excessive dephosphorylation of RyR2 by PP-1, much like excessive phosphorylation, results in enhanced calcium leak, further supporting the idea that RyR2 requires an intermediate level of phosphorylation to function correctly.…”

“…This theme appears to also be maintained in the context of RyR2 dephosphorylation and the role of protein phosphatases. As described above, Potenza et al (2020) convincingly demonstrate that excessive RyR2 dephosphorylation by PP-1 can acutely enhance calcium leak in wild-type mice, which is in contrast to previous reports of a protective role of increased PP-1 activity by reducing RyR2-mediated calcium leak and arrhythmias in a setting of heart failure (Fischer et al 2018). Rather than lessening the impact of these opposing findings, this highlights the need to further understand the intricacy of RyR2 dephosphorylation as a mechanism contributing to calcium dysfunction in cardiac disease.…”

“…In this issue of the Journal of Physiology , Potenza et al . (2020) establish protein phosphatase 1 (PP‐1) as an endogenous regulator of RyR2 phosphorylation at one of these key sites. They additionally demonstrate that excessive dephosphorylation of RyR2 by PP‐1, much like excessive phosphorylation, results in enhanced calcium leak, further supporting the idea that RyR2 requires an intermediate level of phosphorylation to function correctly.…”