CaMKII and Heart Failure Promote a Pathological Ryanodine Receptor Conformation that Reduces Calmodulin Binding and Enhances SR Ca2+ Leak

Abstract

Increased diastolic SR Ca2+ (Ca) leak via cardiac ryanodine receptors (RyR2) can cause cardiac arrhythmias and dysfunction in heart failure (HF). RyR2 phosphorylation (at Ser2814) by Ca/calmodulin-dependent protein kinase II (CaMKII) is thought to be a critical promoter of leaky RyR2 in HF and arrhythmias. A conformational change in RyR2 (“domain unzipping”) observed in HF can increase SR Ca leak. Calmodulin (CaM) quiets RyR2 gating, and CaM binding affinity for RyR2 is reduced in HF. Here, we tested the hypothesis that CaMKII-dependent phosphorylation of RyR2 increases Ca leak by favoring the same unzipped-conformation that exhibits reduced CaM-RyR2 binding affinity. Using non-phosphorylatable RyR2-S2814A and phosphomimetic RyR2-S2814D knock-in mice, fluorescence resonance energy transfer (FRET) was measured to directly detect binding of fluorescently labeled DPc10 (RyR2 unzipping peptide), FK506-binding protein12.6, and CaM to RyR2 in permeabilized cardiomyocytes. In S2814D vs. S2814A: (1) Ca sparks and waves were increased; (2) CaM-RyR2 binding affinity was reduced 3-fold, but FRETmax was unaltered; and (3) F-DPc10 accessibility was 2-fold faster, reflecting unzipped-conformation. Dantrolene (1 μM), which stabilizes the zipped conformation: (1) restored normal CaM-RyR2 binding affinity; (2) reduced DPc10 access (zipped-conformation); and (3) reduced frequency of Ca sparks and waves in S2814D. Furthermore, in rabbit non-ischemic HF myocytes, in which RyR2 is more highly phosphorylated by CaMKII, vs. age-matched control: (1) The CaM-RyR2 binding affinity was reduced by 2.5-fold; and (2) F-DPc10 accessibility was 1.5-fold faster. Dantrolene also restored CaM-RyR2 binding affinity and kept zipped conformation in rabbit HF. We conclude that CaMKII-dependent phosphorylation of RyR2 promotes a pathological RyR2 conformation with reduced CaM binding affinity and elevated Ca leakiness. Dantrolene reverses this conformational change and restores normal CaM binding affinity in S2814D mouse and rabbit HF.