CaMKII-dependent phosphorylation of RyR2 promotes targetable pathological RyR2 conformational shift

Abstract

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.

High-Throughput Screens to Discover Small-Molecule Modulators of Ryanodine Receptor Calcium Release Channels

“…The dysregulation of RyR that promotes this leaky state of the channel is not only associated with disease causing mutations in RyR, but also altered post-translational modifications that have been implicated in altering binding of RyR stabilizers, CaM and FKBP12/12.6 (1,3,4,6,10–12). Moreover, excessive post-translational modifications are typically thought as key players in late-stage pathology.…”

Maternal obesity impairs fetal cardiomyocyte contractile function in sheep

“…Increased intracellular Ca 2+ levels suggest Ca 2+ overload in MO fetal cardiomyocytes, which may be associated with impaired activity of the Ca 2+ -release channel. Studies have shown that phosphorylation by PKA of RyR2 at Ser 2808 and phosphorylation by CaMKII of RyR2 at Ser 2814 can reduce the stability of the Ca 2+ release channel RyR2 and lead to a Ca 2+ leak from the SR associated with cardiac contractility dysfunction and heart failure (70)(71)(72)(73)(74). Our data show increased activity of PKA and CaMKII and high phosphorylation levels at both Ser 2808 and Ser 2814 of RyR2, which further confirm impaired contractile function in MO fetal cardiomyocytes at the molecular level.…”

β-Adrenergic induced SR Ca 2+ leak is mediated by an Epac-NOS pathway

“…Cardiac myocytes were isolated from New Zealand white rabbits and C57BL6 mice using retrograde Langendorff perfusion using Liberase TM (0.075 mg/mL, Roche) and Trypsin (0.0138%, Gibco) (37°C) as previously described [26]. All procedures were approved by the University of California Davis Institutional Animal Care and Use Committee (IACUC) in accordance with the NIH Guide for the Care and Use of Laboratory Animals.…”