title: Protein changes contributing to right ventricular cardiomyocyte diastolic dysfunction in pulmonary arterial hypertension.
abstract: BACKGROUND
  Right ventricular (RV) diastolic function is impaired in patients with pulmonary arterial hypertension (PAH). Our previous study showed that elevated cardiomyocyte stiffness and myofilament Ca(2+) sensitivity underlie diastolic dysfunction in PAH. This study investigates protein modifications contributing to cellular diastolic dysfunction in PAH.
  
  
  RESULTS
  RV samples from PAH patients undergoing heart-lung transplantation were compared to non-failing donors (Don). Titin stiffness contribution to RV diastolic dysfunction was determined by Western-blot analyses using antibodies to protein-kinase-A (PKA), Cα (PKCα) and Ca(2+)/calmoduling-dependent-kinase (CamKIIδ) titin and phospholamban (PLN) phosphorylation sites: N2B (Ser469), PEVK (Ser170 and Ser26), and PLN (Thr17), respectively. PKA and PKCα sites were significantly less phosphorylated in PAH compared with donors (P<0.0001). To test the functional relevance of PKA-, PKCα-, and CamKIIδ-mediated titin phosphorylation, we measured the stiffness of single RV cardiomyocytes before and after kinase incubation. PKA significantly decreased PAH RV cardiomyocyte diastolic stiffness, PKCα further increased stiffness while CamKIIδ had no major effect. CamKIIδ activation was determined indirectly by measuring PLN Thr17phosphorylation level. No significant changes were found between the groups. Myofilament Ca(2+) sensitivity is mediated by sarcomeric troponin I (cTnI) phosphorylation. We observed increased unphosphorylated cTnI in PAH compared with donors (P<0.05) and reduced PKA-mediated cTnI phosphorylation (Ser22/23) (P<0.001). Finally, alterations in Ca(2+)-handling proteins contribute to RV diastolic dysfunction due to insufficient diastolic Ca(2+) clearance. PAH SERCA2a levels and PLN phosphorylation were significantly reduced compared with donors (P<0.05).
  
  
  CONCLUSIONS
  Increased titin stiffness, reduced cTnI phosphorylation, and altered levels of phosphorylation of Ca(2+) handling proteins contribute to RV diastolic dysfunction in PAH.
authors.affiliation: Department of Pulmonology, VU University Medical Center/Institute for Cardiovascular Research, Amsterdam, The Netherlands (S.R., D.S.G.B., N.W., H.J.B., A.V.N., F.S.M.) Department of Physiology, VU University Medical Center/Institute for Cardiovascular Research, Amsterdam, The Netherlands (S.R., L.H., N.W., G.S., C.O., M.G., J.V., F.S.M.).
authors.forename: Silvia
authors.initials: S
authors.lastname: Rain
authors.affiliation: Department of Pulmonology, VU University Medical Center/Institute for Cardiovascular Research, Amsterdam, The Netherlands (S.R., D.S.G.B., N.W., H.J.B., A.V.N., F.S.M.).
authors.forename: Denielli da Silva Goncalves
authors.initials: Dda S
authors.lastname: Bos
authors.affiliation: Department of Physiology, VU University Medical Center/Institute for Cardiovascular Research, Amsterdam, The Netherlands (S.R., L.H., N.W., G.S., C.O., M.G., J.V., F.S.M.) Department of Cardiology, VU University Medical Center/Institute for Cardiovascular Research, Amsterdam, The Netherlands (L.H.).
authors.forename: M Louis
authors.initials: ML
authors.lastname: Handoko
authors.affiliation: Department of Pulmonology, VU University Medical Center/Institute for Cardiovascular Research, Amsterdam, The Netherlands (S.R., D.S.G.B., N.W., H.J.B., A.V.N., F.S.M.) Department of Physiology, VU University Medical Center/Institute for Cardiovascular Research, Amsterdam, The Netherlands (S.R., L.H., N.W., G.S., C.O., M.G., J.V., F.S.M.).
authors.forename: Nico
authors.initials: N
authors.lastname: Westerhof
authors.affiliation: Department of Physiology, VU University Medical Center/Institute for Cardiovascular Research, Amsterdam, The Netherlands (S.R., L.H., N.W., G.S., C.O., M.G., J.V., F.S.M.) Department of Physics and Astronomy, VU University Medical Center/Institute for Cardiovascular Research, Amsterdam, The Netherlands (G.S.).
authors.forename: Ger
authors.initials: G
authors.lastname: Stienen
authors.affiliation: Department of Physiology, VU University Medical Center/Institute for Cardiovascular Research, Amsterdam, The Netherlands (S.R., L.H., N.W., G.S., C.O., M.G., J.V., F.S.M.).
authors.forename: Coen
authors.initials: C
authors.lastname: Ottenheijm
authors.affiliation: Department of Physiology, VU University Medical Center/Institute for Cardiovascular Research, Amsterdam, The Netherlands (S.R., L.H., N.W., G.S., C.O., M.G., J.V., F.S.M.).
authors.forename: Max
authors.initials: M
authors.lastname: Goebel
authors.affiliation: Faculté de Médecine, Université Paris-Sud, Le Kremlin-Bicêtre, France (P.D., C.G., M.H.) Inserm U999, LabEx LERMIT, Centre Chirurgical Marie Lannelongue, Le Plessis Robinson, France (P.D., C.G., M.H.).
authors.forename: Peter
authors.initials: P
authors.lastname: Dorfmüller
authors.affiliation: Faculté de Médecine, Université Paris-Sud, Le Kremlin-Bicêtre, France (P.D., C.G., M.H.) Inserm U999, LabEx LERMIT, Centre Chirurgical Marie Lannelongue, Le Plessis Robinson, France (P.D., C.G., M.H.).
authors.forename: Christophe
authors.initials: C
authors.lastname: Guignabert
authors.affiliation: Faculté de Médecine, Université Paris-Sud, Le Kremlin-Bicêtre, France (P.D., C.G., M.H.) Inserm U999, LabEx LERMIT, Centre Chirurgical Marie Lannelongue, Le Plessis Robinson, France (P.D., C.G., M.H.) Service d'Anatomie Pathologique, Centre Chirurgical Marie Lannelongue, Le Plessis Robinson, France (M.H.) Assistance Publique-Hôspitaux de Paris, Service de Pneumologie, Département Hôspital Universitaire, Thorax innovation, (DHU-TORINO), Hôpital Bicêtre, France (M.H.).
authors.forename: Marc
authors.initials: M
authors.lastname: Humbert
authors.affiliation: Department of Pulmonology, VU University Medical Center/Institute for Cardiovascular Research, Amsterdam, The Netherlands (S.R., D.S.G.B., N.W., H.J.B., A.V.N., F.S.M.).
authors.forename: Harm-Jan
authors.initials: HJ
authors.lastname: Bogaard
authors.affiliation: Muscle Research Unit, Discipline of Anatomy & Histology, Bosch Institute, The University of Sydney, Sydney, Australia (C.R.).
authors.forename: Cris Dos
authors.initials: CD
authors.lastname: Remedios
authors.affiliation: Sarver Molecular Cardiovascular Research Program, Department of Physiology, University of Arizona, Tucson, AZ, The Netherlands (C.S., C.G.H., H.L.G.).
authors.forename: Chandra
authors.initials: C
authors.lastname: Saripalli
authors.affiliation: Sarver Molecular Cardiovascular Research Program, Department of Physiology, University of Arizona, Tucson, AZ, The Netherlands (C.S., C.G.H., H.L.G.).
authors.forename: Carlos G
authors.initials: CG
authors.lastname: Hidalgo
authors.affiliation: Sarver Molecular Cardiovascular Research Program, Department of Physiology, University of Arizona, Tucson, AZ, The Netherlands (C.S., C.G.H., H.L.G.).
authors.forename: Henk L
authors.initials: HL
authors.lastname: Granzier
authors.affiliation: Department of Pulmonology, VU University Medical Center/Institute for Cardiovascular Research, Amsterdam, The Netherlands (S.R., D.S.G.B., N.W., H.J.B., A.V.N., F.S.M.).
authors.forename: Anton
authors.initials: A
authors.lastname: Vonk-Noordegraaf
authors.affiliation: Department of Physiology, VU University Medical Center/Institute for Cardiovascular Research, Amsterdam, The Netherlands (S.R., L.H., N.W., G.S., C.O., M.G., J.V., F.S.M.) ICIN - The Netherlands Heart Institute, Amsterdam, The Netherlands (J.V.).
authors.forename: Jolanda
authors.initials: J
authors.lastname: van der Velden
authors.affiliation: Department of Pulmonology, VU University Medical Center/Institute for Cardiovascular Research, Amsterdam, The Netherlands (S.R., D.S.G.B., N.W., H.J.B., A.V.N., F.S.M.) Department of Physiology, VU University Medical Center/Institute for Cardiovascular Research, Amsterdam, The Netherlands (S.R., L.H., N.W., G.S., C.O., M.G., J.V., F.S.M.).
authors.forename: Frances S
authors.initials: FS
authors.lastname: de Man
mesh_terms: D000328:Adult; D015153:Blotting, Western; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000032:analysis / Q000502:physiology; D016022:Case-Control Studies; D064211:Connectin / Q000032:analysis / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis / Q000502:physiology; D005260:Female; D006352:Heart Ventricles / Q000737:chemistry / Q000503:physiopathology; D006801:Humans; D006976:Hypertension, Pulmonary / Q000503:physiopathology*; D008297:Male; D032383:Myocytes, Cardiac / Q000737:chemistry* / Q000502:physiology; D010766:Phosphorylation; D051571:Protein Kinase C-alpha / Q000032:analysis / Q000502:physiology; D019210:Troponin I / Q000502:physiology; D018497:Ventricular Dysfunction, Right / Q000503:physiopathology*
chemical_list: D064211:Connectin; D019210:Troponin I; D017868:Cyclic AMP-Dependent Protein Kinases; D051571:Protein Kinase C-alpha; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2
keywords: diastole; pulmonary heart disease