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Arch Biochem Biophys. 2014 Jul 15;554:11-21. doi: 10.1016/j.abb.2014.04.017. Epub 2014 May 9.

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.

Author information

1
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.
2
Laboratory for Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: v.sequeiraoliveira@vumc.nl.
3
Laboratory for Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: e.paalberends@vumc.nl.
4
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.
5
Muscle Research Unit, Bosch Institute, The University of Sydney, Sydney, Australia. Electronic address: cris.dosremedios@sydney.edu.au.
6
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.
7
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.
8
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.

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.

KEYWORDS:

Cardiomyocyte; Myofilament function; Protein kinase C; Protein phosphorylation; Troponin I

PMID:
24814372
PMCID:
PMC4121669
DOI:
10.1016/j.abb.2014.04.017
[Indexed for MEDLINE]
Free PMC Article

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