title: Cardiac myocyte Z-line calmodulin is mainly RyR2-bound, and reduction is arrhythmogenic and occurs in heart failure.
abstract: BACKGROUND
  Calmodulin (CaM) associates with cardiac ryanodine receptor type-2 (RyR2) as an important regulator. Defective CaM-RyR2 interaction may occur in heart failure, cardiac hypertrophy, and catecholaminergic polymorphic ventricular tachycardia. However, the in situ binding properties for CaM-RyR2 are unknown.
  
  
  OBJECTIVE
  We sought to measure the in situ binding affinity and kinetics for CaM-RyR2 in normal and heart failure ventricular myocytes, estimate the percentage of Z-line-localized CaM that is RyR2-bound, and test cellular function of defective CaM-RyR2 interaction.
  
  
  RESULTS
  Using fluorescence resonance energy transfer in permeabilized myocytes, we specifically resolved RyR2-bound CaM from other potential binding targets and measured CaM-RyR2 binding affinity in situ (Kd=10-20 nmol/L). Using RyR2(ADA/+) knock-in mice, in which half of the CaM-RyR2 binding is suppressed, we estimated that >90% of Z-line CaM is RyR2-bound. Functional tests indicated a higher propensity for Ca2+ wave production and stress-induced ventricular arrhythmia in RyR2(ADA/+) mice. In a post-myocardial infarction rat heart failure model, we detected a decrease in the CaM-RyR2 binding affinity (Kd≈51 nmol/L; ≈3-fold increase) and unaltered RyR2 affinity for the FK506-binding protein FKBP12.6 (Kd~0.8 nmol/L).
  
  
  CONCLUSIONS
  CaM binds to RyR2 with high affinity in cardiac myocytes. Physiologically, CaM is bound to >70% of RyR2 monomers and inhibits sarcoplasmic reticulum Ca2+ release. RyR2 is the major binding site for CaM along the Z-line in cardiomyocytes, and dissociating CaM from RyR2 can cause severe ventricular arrhythmia. In heart failure, RyR2 shows decreased CaM affinity, but unaltered FKBP 12.6 affinity.
authors.affiliation: From the Department of Pharmacology (Y.Y., T.G., T.O., H.U., A.A.K., D.M.B.), and Molecular and Cellular Cardiology Division, Department of Medicine (L.C., A.A.K.), University of California, Davis, CA; Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC (A.C., G.M.); and Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN (B.R.F., R.L.C.).
authors.forename: Yi
authors.initials: Y
authors.lastname: Yang
authors.forename: Tao
authors.initials: T
authors.lastname: Guo
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Oda
authors.forename: Asima
authors.initials: A
authors.lastname: Chakraborty
authors.forename: Le
authors.initials: L
authors.lastname: Chen
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Uchinoumi
authors.forename: Anne A
authors.initials: AA
authors.lastname: Knowlton
authors.forename: Bradley R
authors.initials: BR
authors.lastname: Fruen
authors.forename: Razvan L
authors.initials: RL
authors.lastname: Cornea
authors.forename: Gerhard
authors.initials: G
authors.lastname: Meissner
authors.forename: Donald M
authors.initials: DM
authors.lastname: Bers
mesh_terms: D000818:Animals; D001145:Arrhythmias, Cardiac / Q000209:etiology* / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology; D020013:Calcium Signaling; D002147:Calmodulin / Q000378:metabolism*; D004195:Disease Models, Animal; D031541:Fluorescence Resonance Energy Transfer; D006333:Heart Failure / Q000150:complications* / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology; D007700:Kinetics; D051379:Mice; D008822:Mice, Transgenic; D032383:Myocytes, Cardiac / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D051381:Rats; D017207:Rats, Sprague-Dawley; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D022021:Tacrolimus Binding Proteins / Q000378:metabolism
chemical_list: D002147:Calmodulin; D019837:Ryanodine Receptor Calcium Release Channel; D022021:Tacrolimus Binding Proteins; C403484:tacrolimus binding protein 1B
keywords: arrhythmias, cardiac; fluorescence resonance energy transfer; heart failure; ryanodine receptor calcium release channel