Diastolic calcium (Ca) leak via cardiac ryanodine receptors (RyR2) can cause arrhythmias and heart failure (HF). Ca/calmodulin (CaM)-dependent kinase II (CaMKII) is upregulated and more active in HF, promoting RyR2-mediated Ca leak by RyR2-Ser2814 phosphorylation. Here, we tested a mechanistic hypothesis that RyR2 phosphorylation by CaMKII increases Ca leak by promoting a pathological RyR2 conformation with reduced CaM affinity. Acute CaMKII activation in wild-type RyR2, and phosphomimetic RyR2-S2814D (vs. non-phosphorylatable RyR2-S2814A) knock-in mouse myocytes increased SR Ca leak, reduced CaM-RyR2 affinity, and caused a pathological shift in RyR2 conformation (detected via increased access of the RyR2 structural peptide DPc10). This same trio of effects was seen in myocytes from rabbits with pressure/volume-overload induced HF. Excess CaM quieted leak and restored control conformation, consistent with negative allosteric coupling between CaM affinity and DPc10 accessible conformation. Dantrolene (DAN) also restored CaM affinity, reduced DPc10 access, and suppressed RyR2-mediated Ca leak and ventricular tachycardia in RyR2-S2814D mice. We propose that a common pathological RyR2 conformational state (low CaM affinity, high DPc10 access, and elevated leak) may be caused by CaMKII-dependent phosphorylation, oxidation, and HF. Moreover, DAN (or excess CaM) can shift this pathological gating state back to the normal physiological conformation, a potentially important therapeutic approach.
Bibliographical noteFunding Information:
This work was supported by: (1) NIH P01-80101 (DMB), R01-HL092097 (DMB and RLC), R01-HL089598 (XHTW), R01-HL091947 (XHTW), R01-HL117641 (XHTW), and R41-HL129570 (XHTW); (2) the Uehara Memorial Foundation (HU); and (3) the American Heart Association 14SDG20080008 (NL), 15GRNT25610022 (RLC), and 13EIA14560061 (XHTW).
- Calcium/calmodulin-dependent protein kinase II
- Fluorescence resonance energy transfer
- Ryanodine receptor