Phosphorylation-Dependent Interactome of Ryanodine Receptor Type 2 in the Heart.

David Y. Chiang,Sung Y. Jung,Guoliang Wang,Arjen Scholten,Meng C. Wang,Albert J. R. Heck,Satadru Lahiri,Jason Karch,Xander H. T. Wehrens

doi:10.3390/proteomes9020027

Abstract

: BackgroundHyperphosphorylation of the calcium release channel/ryanodine receptor type 2 (RyR2) at serine 2814 (S2814) is associated with multiple cardiac diseases including atrial fibrillation and heart failure. Despite recent advances, the molecular mechanisms driving pathological changes associated with RyR2 S2814 phosphorylation are still not well understood. Methods: Using affinity-purification coupled to mass spectrometry (AP-MS), we investigated the RyR2 interactome in ventricles from wild-type (WT) mice and two S2814 knock-in mutants: the unphosphorylated alanine mutant (S2814A) and hyperphosphorylated mimic aspartic acid mutant (S2814D). Western blots were used for validation. Results: In WT mouse ventricular lysates, we identified 22 proteins which were enriched with RyR2 pull-down relative to both IgG control and no antibody (beads-only) pull-downs. Parallel AP-MS using WT, S2814A, and S2814D mouse ventricles identified 72 proteins, with 20 being high confidence RyR2 interactors. Of these, 14 had an increase in their binding to RyR2 S2814A but a decrease in their binding to RyR2 S2814D. We independently validated three protein hits, Idh3b, Aifm1, and Cpt1b, as RyR2 interactors by western blots and showed that Aifm1 and Idh3b had significantly decreased binding to RyR2 S2814D compared to WT and S2814A, consistent with MS findings. Conclusion: By applying state-of-the-art proteomic approaches, we discovered a number of novel RyR2 interactors in the mouse heart. In addition, we found and defined specific alterations in the RyR2 interactome that were dependent on the phosphorylation status of RyR2 at S2814. These findings yield mechanistic insights into RyR2 regulation which may guide future drug designs.

Highlights

Ryanodine receptor type 2 (RyR2) is the main intracellular calcium (Ca2+ ) channel which resides on the sarcoplasmic reticulum (SR) in cardiomyocytes, responsible for excitation–contraction (EC) coupling
Using whole ventricles harvested from WT, S2814 knock-in mutants: the unphosphorylated alanine mutant (S2814A), and S2814D mice, we immunoprecipitated ryanodine receptor type 2 (RyR2) across these samples in parallel and validated the efficiency of the pull-down by western blotting (Figure 1B)
We found and defined specific alterations in the RyR2 interactome that were dependent on the phosphorylation status of RyR2 at one single calmodulin-dependent protein kinase II (CaMKII) phosphorylation site, serine 2814 (S2814), which has previously been implicated in several cardiac diseases

Summary

Introduction

Ryanodine receptor type 2 (RyR2) is the main intracellular calcium (Ca2+ ) channel which resides on the sarcoplasmic reticulum (SR) in cardiomyocytes, responsible for excitation–contraction (EC) coupling. Dysregulation of RyR2 by a number of mechanisms can lead to inappropriate Ca2+ release during diastole (“RyR2 leak”), which is associated with several cardiac diseases including catecholaminergic polymorphic ventricular tachycardia, atrial fibrillation, and heart failure [2,3,4,5,6,7]. We and others have shown that hyperphosphorylation of RyR2 at serine 2814 (S2814) by Ca2+ /calmodulin-dependent protein kinase II (CaMKII) contributes to these RyR2 leaks and is implicated in both atrial fibrillation and heart failure; by contrast, genetic ablation of S2814 (mutating to an alanine, S2814A) rescues disease phenotypes in mice [3,4,5]. Despite the elucidation of these and other pathogenic mechanisms involving RyR2, there is still a lack of viable therapeutic options targeting RyR2 function [2]

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Discussion

Conclusion