title: Protein kinase G modulates human myocardial passive stiffness by phosphorylation of the titin springs.
abstract: The sarcomeric titin springs influence myocardial distensibility and passive stiffness. Titin isoform composition and protein kinase (PK)A-dependent titin phosphorylation are variables contributing to diastolic heart function. However, diastolic tone, relaxation speed, and left ventricular extensibility are also altered by PKG activation. We used back-phosphorylation assays to determine whether PKG can phosphorylate titin and affect titin-based stiffness in skinned myofibers and isolated myofibrils. PKG in the presence of 8-pCPT-cGMP (cGMP) phosphorylated the 2 main cardiac titin isoforms, N2BA and N2B, in human and canine left ventricles. In human myofibers/myofibrils dephosphorylated before mechanical analysis, passive stiffness dropped 10% to 20% on application of cGMP-PKG. Autoradiography and anti-phosphoserine blotting of recombinant human I-band titin domains established that PKG phosphorylates the N2-B and N2-A domains of titin. Using site-directed mutagenesis, serine residue S469 near the COOH terminus of the cardiac N2-B-unique sequence (N2-Bus) was identified as a PKG and PKA phosphorylation site. To address the mechanism of the PKG effect on titin stiffness, single-molecule atomic force microscopy force-extension experiments were performed on engineered N2-Bus-containing constructs. The presence of cGMP-PKG increased the bending rigidity of the N2-Bus to a degree that explained the overall PKG-mediated decrease in cardiomyofibrillar stiffness. Thus, the mechanically relevant site of PKG-induced titin phosphorylation is most likely in the N2-Bus; phosphorylation of other titin sites could affect protein-protein interactions. The results suggest that reducing titin stiffness by PKG-dependent phosphorylation of the N2-Bus can benefit diastolic function. Failing human hearts revealed a deficit for basal titin phosphorylation compared to donor hearts, which may contribute to diastolic dysfunction in heart failure.
authors.affiliation: Physiology and Biophysics Unit, University of Muenster, Germany.
authors.forename: Martina
authors.initials: M
authors.lastname: Krüger
authors.forename: Sebastian
authors.initials: S
authors.lastname: Kötter
authors.forename: Anika
authors.initials: A
authors.lastname: Grützner
authors.forename: Patrick
authors.initials: P
authors.lastname: Lang
authors.forename: Christian
authors.initials: C
authors.lastname: Andresen
authors.forename: Margaret M
authors.initials: MM
authors.lastname: Redfield
authors.forename: Elke
authors.initials: E
authors.lastname: Butt
authors.forename: Cris G
authors.initials: CG
authors.lastname: dos Remedios
authors.forename: Wolfgang A
authors.initials: WA
authors.lastname: Linke
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D064211:Connectin; D016384:Consensus Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D006152:Cyclic GMP / Q000502:physiology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000502:physiology*; D004285:Dogs; D004548:Elasticity; D054144:Heart Failure, Diastolic / Q000378:metabolism*; D006352:Heart Ventricles / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D009210:Myofibrils / Q000187:drug effects / Q000648:ultrastructure; D009569:Nitric Oxide / Q000502:physiology; D010766:Phosphorylation; D020033:Protein Isoforms / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012518:Sarcomeres / Q000378:metabolism / Q000648:ultrastructure; D013329:Structure-Activity Relationship; D020257:Ventricular Remodeling / Q000502:physiology
chemical_list: D064211:Connectin; D009124:Muscle Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; C577285:TTN protein, human; D009569:Nitric Oxide; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP