title: Phosphorylation of protein kinase C sites Ser42/44 decreases Ca(2+)-sensitivity and blunts enhanced length-dependent activation in response to protein kinase A in human cardiomyocytes.
abstract: Protein kinase C (PKC)-mediated phosphorylation of troponin I (cTnI) at Ser42/44 is increased in heart failure. While studies in rodents demonstrated that PKC-mediated Ser42/44 phosphorylation decreases maximal force and ATPase activity, PKC incubation of human cardiomyocytes did not affect maximal force. We investigated whether Ser42/44 pseudo-phosphorylation affects force development and ATPase activity using troponin exchange in human myocardium. Additionally, we studied if pseudo-phosphorylated Ser42/44 modulates length-dependent activation of force, which is regulated by protein kinase A (PKA)-mediated cTnI-Ser23/24 phosphorylation. Isometric force was measured in membrane-permeabilized cardiomyocytes exchanged with human recombinant wild-type troponin or troponin mutated at Ser42/44 or Ser23/24 into aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. In troponin-exchanged donor cardiomyocytes experiments were repeated after PKA incubation. ATPase activity was measured in troponin-exchanged cardiac muscle strips. Compared to wild-type, 42D/44D decreased Ca(2+)-sensitivity without affecting maximal force in failing and donor cardiomyocytes. In donor myocardium, 42D/44D did not affect maximal ATPase activity or tension cost. Interestingly, 42D/44D blunted the length-dependent increase in Ca(2+)-sensitivity induced upon PKA-mediated phosphorylation. Since the drop in Ca(2+)-sensitivity at physiological Ca(2+)-concentrations is relatively large phosphorylation of Ser42/44 may result in a decrease of force and associated ATP utilization in the human heart.
authors.affiliation: Laboratory for Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands; Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. Electronic address: p.wijnker@vumc.nl.
authors.forename: Paul J M
authors.initials: PJ
authors.lastname: Wijnker
authors.affiliation: Laboratory for Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: v.sequeiraoliveira@vumc.nl.
authors.forename: Vasco
authors.initials: V
authors.lastname: Sequeira
authors.affiliation: Laboratory for Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: e.paalberends@vumc.nl.
authors.forename: E Rosalie
authors.initials: ER
authors.lastname: Witjas-Paalberends
authors.affiliation: Department of Pediatrics/Division of Cardiology, Johns Hopkins University School of Medicine, Ross Bldg 1144/720 Rutland Avenue, Baltimore, MD 21205, USA. Electronic address: dbrianfoster@jhmi.edu.
authors.forename: D Brian
authors.initials: DB
authors.lastname: Foster
authors.affiliation: Muscle Research Unit, Bosch Institute, The University of Sydney, Sydney, Australia. Electronic address: cris.dosremedios@sydney.edu.au.
authors.forename: Cristobal G
authors.initials: CG
authors.lastname: dos Remedios
authors.affiliation: Department of Pediatrics/Division of Cardiology, Johns Hopkins University School of Medicine, Ross Bldg 1144/720 Rutland Avenue, Baltimore, MD 21205, USA. Electronic address: murphy@jhmi.edu.
authors.forename: Anne M
authors.initials: AM
authors.lastname: Murphy
authors.affiliation: Laboratory for Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands; Department of Physics and Astronomy, VU University, Amsterdam, The Netherlands. Electronic address: g.stienen@vumc.nl.
authors.forename: Ger J M
authors.initials: GJ
authors.lastname: Stienen
authors.affiliation: Laboratory for Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands; ICIN-Netherlands Heart Institute, Utrecht, The Netherlands. Electronic address: j.vandervelden@vumc.nl.
authors.forename: Jolanda
authors.initials: J
authors.lastname: van der Velden
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D019943:Amino Acid Substitution; D002118:Calcium / Q000378:metabolism; D002311:Cardiomyopathy, Dilated / Q000378:metabolism / Q000503:physiopathology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D005260:Female; D006333:Heart Failure / Q000378:metabolism / Q000503:physiopathology; D006801:Humans; D066298:In Vitro Techniques; D007537:Isometric Contraction / Q000502:physiology; D008297:Male; D008875:Middle Aged; D016297:Mutagenesis, Site-Directed; D009200:Myocardial Contraction / Q000502:physiology; D032383:Myocytes, Cardiac / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D019210:Troponin I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D019210:Troponin I; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D000251:Adenosine Triphosphatases; D002118:Calcium
keywords: Cardiomyocyte; Myofilament function; Protein kinase C; Protein phosphorylation; Troponin I