title: Crucial role for Ca2(+)/calmodulin-dependent protein kinase-II in regulating diastolic stress of normal and failing hearts via titin phosphorylation.
abstract: BACKGROUND
  Myocardial diastolic stiffness and cardiomyocyte passive force (F(passive)) depend in part on titin isoform composition and phosphorylation. Ca(2+)/calmodulin-dependent protein kinase-II (CaMKII) phosphorylates ion channels, Ca(2+)-handling proteins, and chromatin-modifying enzymes in the heart, but has not been known to target titin.
  
  
  OBJECTIVE
  To elucidate whether CaMKII phosphorylates titin and modulates F(passive) in normal and failing myocardium.
  
  
  RESULTS
  Titin phosphorylation was assessed in CaMKIIδ/γ double-knockout (DKO) mouse, transgenic CaMKIIδC-overexpressing mouse, and human hearts, by Pro-Q-Diamond/Sypro-Ruby staining, autoradiography, and immunoblotting using phosphoserine-specific titin-antibodies. CaMKII-dependent site-specific titin phosphorylation was quantified in vivo by mass spectrometry using stable isotope labeling by amino acids in cell culture mouse heart mixed with wild-type (WT) or DKO heart. F(passive) of single permeabilized cardiomyocytes was recorded before and after CaMKII-administration. All-titin phosphorylation was reduced by >50% in DKO but increased by up to ≈100% in transgenic versus WT hearts. Conserved CaMKII-dependent phosphosites were identified within the PEVK-domain of titin by quantitative mass spectrometry and confirmed in recombinant human PEVK-fragments. CaMKII also phosphorylated the cardiac titin N2B-unique sequence. Phosphorylation at specific PEVK/titin N2B-unique sequence sites was decreased in DKO and amplified in transgenic versus WT hearts. F(passive) was elevated in DKO and reduced in transgenic compared with WT cardiomyocytes. CaMKII-administration lowered F(passive) of WT and DKO cardiomyocytes, an effect blunted by titin antibody pretreatment. Human end-stage failing hearts revealed higher CaMKII expression/activity and phosphorylation at PEVK/titin N2B-unique sequence sites than nonfailing donor hearts.
  
  
  CONCLUSIONS
  CaMKII phosphorylates the titin springs at conserved serines/threonines, thereby lowering F(passive). Deranged CaMKII-dependent titin phosphorylation occurs in heart failure and contributes to altered diastolic stress.
authors.affiliation: Department of Cardiovascular Physiology, Ruhr University Bochum, Bochum, Germany.
authors.forename: Nazha
authors.initials: N
authors.lastname: Hamdani
authors.forename: Judith
authors.initials: J
authors.lastname: Krysiak
authors.forename: Michael M
authors.initials: MM
authors.lastname: Kreusser
authors.forename: Stefan
authors.initials: S
authors.lastname: Neef
authors.forename: Cristobal G
authors.initials: CG
authors.lastname: Dos Remedios
authors.forename: Lars S
authors.initials: LS
authors.lastname: Maier
authors.forename: Markus
authors.initials: M
authors.lastname: Krüger
authors.forename: Johannes
authors.initials: J
authors.lastname: Backs
authors.forename: Wolfgang A
authors.initials: WA
authors.lastname: Linke
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001696:Biomechanical Phenomena; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D003187:Compliance; D064211:Connectin; D003971:Diastole / Q000502:physiology; D006333:Heart Failure / Q000201:enzymology* / Q000503:physiopathology; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000201:enzymology / Q000502:physiology; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000502:physiology
chemical_list: D064211:Connectin; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins; C577285:TTN protein, human; D010769:Phosphothreonine; D010768:Phosphoserine; D011494:Protein Kinases; C579730:titin protein, mouse; C517660:CAMK2D protein, human; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; C517673:Camk2d protein, mouse; C517677:Camk2d protein, rat; C517674:Camk2g protein, mouse