title: Ca2+/Calmodulin-dependent protein kinase II phosphorylation of ryanodine receptor does affect calcium sparks in mouse ventricular myocytes.
abstract: Previous studies in transgenic mice and with isolated ryanodine receptors (RyR) have indicated that Ca2+-calmodulin-dependent protein kinase II (CaMKII) can phosphorylate RyR and activate local diastolic sarcoplasmic reticulum (SR) Ca2+ release events (Ca2+ sparks) and RyR channel opening. Here we use relatively controlled physiological conditions in saponin-permeabilized wild type (WT) and phospholamban knockout (PLB-KO) mouse ventricular myocytes to test whether exogenous preactivated CaMKII or endogenous CaMKII can enhance resting Ca2+ sparks. PLB-KO mice were used to preclude ancillary effects of CaMKII mediated by phospholamban phosphorylation. In both WT and PLB-KO myocytes, Ca2+ spark frequency was increased by both preactivated exogenous CaMKII and endogenous CaMKII. This effect was abolished by CaMKII inhibitor peptides. In contrast, protein kinase A catalytic subunit also enhanced Ca2+ spark frequency in WT, but had no effect in PLB-KO. Both endogenous and exogenous CaMKII increased SR Ca2+ content in WT (presumably via PLB phosphorylation), but not in PLB-KO. Exogenous calmodulin decreased Ca2+ spark frequency in both WT and PLB-KO (K0.5 approximately 100 nmol/L). Endogenous CaMKII (at 500 nmol/L [Ca2+]) phosphorylated RyR as completely in <4 minutes as the maximum achieved by preactivated exogenous CaMKII. After CaMKII activation Ca2+ sparks were longer in duration, and more frequent propagating SR Ca2+ release events were observed. We conclude that CaMKII-dependent phosphorylation of RyR by endogenous associated CaMKII (but not PKA-dependent phosphorylation) increases resting SR Ca2+ release or leak. Moreover, this may explain the enhanced SR diastolic Ca2+ leak and certain triggered arrhythmias seen in heart failure.
authors.affiliation: Department of Physiology and Cardiovascular Institute, Loyola University Chicago, Stritch School of Medicine, Maywood, IL 60153, USA.
authors.forename: Tao
authors.initials: T
authors.lastname: Guo
authors.forename: Tong
authors.initials: T
authors.lastname: Zhang
authors.forename: Ruben
authors.initials: R
authors.lastname: Mestril
authors.forename: Donald M
authors.initials: DM
authors.lastname: Bers
mesh_terms: D000818:Animals; D002118:Calcium / Q000502:physiology*; D002135:Calcium-Binding Proteins / Q000172:deficiency / Q000235:genetics; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002147:Calmodulin / Q000502:physiology*; D004789:Enzyme Activation; D006352:Heart Ventricles / Q000201:enzymology; D007700:Kinetics; D051379:Mice; D018345:Mice, Knockout; D032342:Muscle Cells / Q000201:enzymology / Q000502:physiology*; D010766:Phosphorylation; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000502:physiology; D016276:Ventricular Function; D000099211:Phospholamban
chemical_list: D002118:Calcium; D002135:Calcium-Binding Proteins; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin; D019837:Ryanodine Receptor Calcium Release Channel; D000099211:Phospholamban