title: Differential changes in titin domain phosphorylation increase myofilament stiffness in failing human hearts.
abstract: OBJECTIVE
  Titin-based myofilament stiffness is defined by the expression levels of the cardiac titin-isoforms, N2B and N2BA, and by phosphorylation of the elastic titin domains N2-B unique sequence (N2-Bus) and PEVK. Phosphorylation of the N2-Bus by cGMP-dependent protein kinase (PKG) or cAMP-dependent protein kinase (PKA) decreases titin stiffness, whereas phosphorylation of the PEVK-domain by PKC increases it. We aimed to identify specific sites within the N2-Bus phosphorylated by PKA and PKG and to determine whether differential changes in titin domain phosphorylation could affect passive stiffness in human failing hearts.
  
  
  RESULTS
  Using mass spectrometry, we identified seven partly conserved PKA/PKG-targeted phosphorylation motifs in human and rat N2-Bus. Polyclonal antibodies to pSer4185, pSer4010, and pSer4099 in the N2-Bus, and to pSer11878 in the PEVK-region were used to quantify titin-domain phosphorylation by western blot analyses of a set of human donor and failing hearts with similar titin-isoform composition. Passive tension determined in skinned human myocardial fibre preparations was significantly increased in failing compared with donor hearts, notably at shorter sarcomere lengths where titin contributes most to total passive tension. Phosphorylation of Ser4185, Ser4010, and Ser4099 in the N2-Bus was significantly reduced in failing hearts, whereas phosphorylation of Ser11878 in the PEVK-region was increased compared with donor hearts.
  
  
  CONCLUSIONS
  We conclude that hypo-phosphorylation of the N2-Bus and hyper-phosphorylation of the PEVK domain can act complementary to elevate passive tension in failing human hearts. Differential changes in titin-domain phosphorylation may be important to fine-tune passive myocardial stiffness and diastolic function of the heart.
authors.affiliation: Department of Cardiovascular Physiology, Heinrich Heine University Düsseldorf, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Kötter
authors.forename: Laurence
authors.initials: L
authors.lastname: Gout
authors.forename: Marion
authors.initials: M
authors.lastname: Von Frieling-Salewsky
authors.forename: Anna Eliane
authors.initials: AE
authors.lastname: Müller
authors.forename: Stefan
authors.initials: S
authors.lastname: Helling
authors.forename: Katrin
authors.initials: K
authors.lastname: Marcus
authors.forename: Cristobal
authors.initials: C
authors.lastname: Dos Remedios
authors.forename: Wolfgang A
authors.initials: WA
authors.lastname: Linke
authors.forename: Martina
authors.initials: M
authors.lastname: Krüger
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D064211:Connectin / Q000737:chemistry / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000502:physiology; D006333:Heart Failure / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D032383:Myocytes, Cardiac / Q000378:metabolism*; D009210:Myofibrils / Q000502:physiology*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D051381:Rats
chemical_list: D064211:Connectin; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases
keywords: Connectin; Diastolic dysfunction; Myocardial stiffness; Passive tension; Phospho-specific antibodies