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Calmodulin (CaM) is a Ca-binding protein that binds to, and can directly inhibit cardiac ryanodine receptor calcium release channels (RyR2). Animal studies have shown that RyR2 hyperphosphorylation reduces CaM binding to RyR2 in failing hearts, but data are lacking on how CaM regulates human RyR2 and how this regulation is affected by RyR2 phosphorylation. Physiological concentrations of CaM (100 nM) inhibited the diastolic activity of RyR2 isolated from failing human hearts by ~50% but had no effect on RyR2 from healthy human hearts. Using FRET between donor-FKBP12.6 and acceptor-CaM bound to RyR2, we determined that CaM binds to RyR2 from healthy human heart with a K d  = 121 ± 14 nM. Ex-vivo phosphorylation/dephosphorylation experiments suggested that the divergent CaM regulation of healthy and failing human RyR2 was caused by differences in RyR2 phosphorylation by protein kinase A and Ca-CaM-dependent kinase II. Ca 2+ -spark measurements in murine cardiomyocytes harbouring RyR2 phosphomimetic or phosphoablated mutants at S2814 and S2808 suggest that phosphorylation of residues corresponding to either human RyR2-S2808 or S2814 is both necessary and sufficient for RyR2 regulation by CaM. Our results challenge the current concept that CaM universally functions as a canonical inhibitor of RyR2 across species. Rather, CaM's biological action on human RyR2 appears to be more nuanced, with inhibitory activity only on phosphorylated RyR2 channels, which occurs during exercise or in patients with heart failure., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Rationale: Heart failure is a multimodal disorder, of which disrupted Ca 2+ homeostasis is a hallmark. Central to Ca 2+ homeostasis is the major cardiac Ca 2+ release channel - the ryanodine receptor (RyR2) - whose activity is influenced by associated proteins, covalent modification and by Ca 2+ and Mg 2+ . That RyR2 is remodelled and its function disturbed in heart failure is well recognized, but poorly understood., Objective: To assess Ca 2+ and Mg 2+ regulation of RyR2 from left ventricles of healthy, cystic fibrosis and failing hearts, and to correlate these functional changes with RyR2 modifications and remodelling., Methods and Results: The function of RyR2 from left ventricular samples was assessed using lipid bilayer single-channel measurements, whilst RyR2 modification and protein:protein interactions were determined using Western Blots and co-immunoprecipitation. In all failing hearts there was an increase in RyR2 activity at end-diastolic cytoplasmic Ca 2+ (100nM), a decreased cytoplasmic [Ca 2+ ] required for half maximal activation (K a ) and a decrease in inhibition by cytoplasmic Mg 2+ . This was accompanied by significant hyperphosphorylation of RyR2 S 2808 and S 2814 , reduced free thiol content and a reduced interaction with FKBP12.0 and FKBP12.6. Either dephosphorylation of RyR2 using PP1 or thiol reduction using DTT eliminated any significant difference in the activity of RyR2 from healthy and failing hearts. We also report a subgroup of RyR2 in failing hearts that were not responsive to regulation by intracellular Ca 2+ or Mg 2+ ., Conclusion: Despite different aetiologies, disrupted RyR2 Ca 2+ sensitivity and biochemical modification of the channel are common constituents of failing heart RyR2 and may underlie the pathological disturbances in intracellular Ca 2+ signalling., (Copyright © 2017 Elsevier Ltd. All rights reserved.)