title: Oxidation of ryanodine receptor (RyR) and calmodulin enhance Ca release and pathologically alter, RyR structure and calmodulin affinity.
abstract: Oxidative stress may contribute to cardiac ryanodine receptor (RyR2) dysfunction in heart failure (HF) and arrhythmias. Altered RyR2 domain-domain interaction (domain unzipping) and calmodulin (CaM) binding affinity are allosterically coupled indices of RyR2 conformation. In HF RyR2 exhibits reduced CaM binding, increased domain unzipping and greater SR Ca leak, and dantrolene can reverse these changes. However, effects of oxidative stress on RyR2 conformation and leak in myocytes are poorly understood. We used fluorescent CaM, FKBP12.6, and domain-peptide biosensor (F-DPc10) to measure, directly in cardiac myocytes, (1) RyR2 activation by hydrogen peroxide (H2O2)-induced oxidation, (2) RyR2 conformation change caused by oxidation, (3) CaM-RyR2 and FK506-binding protein (FKBP12.6)-RyR2 interaction upon oxidation, and (4) whether dantrolene affects 1-3. H2O2 was used to mimic oxidative stress. H2O2 significantly increased the frequency of Ca(2+) sparks and spontaneous Ca(2+) waves, and dantrolene almost completely blocked these effects. H2O2 pretreatment significantly reduced CaM-RyR2 binding, but had no effect on FKBP12.6-RyR2 binding. Dantrolene restored CaM-RyR2 binding but had no effect on intracellular and RyR2 oxidation levels. H2O2 also accelerated F-DPc10-RyR2 association while dantrolene slowed it. Thus, H2O2 causes conformational changes (sensed by CaM and DPc10 binding) associated with Ca leak, and dantrolene reverses these RyR2 effects. In conclusion, in cardiomyocytes, H2O2 treatment markedly reduces the CaM-RyR2 affinity, has no effect on FKBP12.6-RyR2 affinity, and causes domain unzipping. Dantrolene can correct domain unzipping, restore CaM-RyR2 affinity, and quiet pathological RyR2 channel gating. F-DPc10 and CaM are useful biosensors of a pathophysiological RyR2 state.
authors.affiliation: Department of Pharmacology University of California, Davis, CA, USA; Department of Medicine and Clinical Science, Division of Cardiology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan.
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Oda
authors.affiliation: Department of Pharmacology University of California, Davis, CA, USA; Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Department of Pharmacology University of California, Davis, CA, USA.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Uchinoumi
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA.
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
authors.affiliation: Department of Pharmacology University of California, Davis, CA, USA.
authors.forename: Ye
authors.initials: Y
authors.lastname: Chen-Izu
authors.affiliation: Department of Medicine and Clinical Science, Division of Cardiology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan.
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Kato
authors.affiliation: Department of Medicine and Clinical Science, Division of Cardiology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Department of Medicine and Clinical Science, Division of Cardiology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Yano
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Razvan L
authors.initials: RL
authors.lastname: Cornea
authors.affiliation: Department of Pharmacology University of California, Davis, CA, USA. Electronic address: dmbers@ucdavis.edu.
authors.forename: Donald M
authors.initials: DM
authors.lastname: Bers
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D002147:Calmodulin / Q000378:metabolism*; D002478:Cells, Cultured; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007700:Kinetics; D032383:Myocytes, Cardiac / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D022021:Tacrolimus Binding Proteins / Q000378:metabolism
chemical_list: D002147:Calmodulin; D019837:Ryanodine Receptor Calcium Release Channel; D006861:Hydrogen Peroxide; D022021:Tacrolimus Binding Proteins; C403484:tacrolimus binding protein 1B; D002118:Calcium
keywords: Calmodulin; Dantrolene; FKBP12.6; Reactive oxygen species; Ryanodine receptor