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

Paul J M Wijnker, Vasco Sequeira, E. Rosalie Witjas-Paalberends, Darren Brian Foster, Cristobal G. Dos Remedios, Anne M Murphy, Ger J M Stienen, Jolanda Van Der Velden

Research output: Contribution to journalArticle

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 Ca2+-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 Ca2+-sensitivity at physiological Ca2+- concentrations is relatively large phosphorylation of Ser42/44 may result in a decrease of force and associated ATP utilization in the human heart.

Original languageEnglish (US)
Pages (from-to)11-21
Number of pages11
JournalArchives of Biochemistry and Biophysics
Volume554
DOIs
StatePublished - Jul 15 2014

Fingerprint

Phosphorylation
Cyclic AMP-Dependent Protein Kinases
Cardiac Myocytes
Protein Kinase C
Troponin
Chemical activation
Adenosine Triphosphatases
Myocardium
D-Aspartic Acid
Troponin I
Alanine
Muscle
Rodentia
Heart Failure
Adenosine Triphosphate
Membranes
Costs and Cost Analysis
Costs

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

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. / Wijnker, Paul J M; Sequeira, Vasco; Witjas-Paalberends, E. Rosalie; Foster, Darren Brian; Dos Remedios, Cristobal G.; Murphy, Anne M; Stienen, Ger J M; Van Der Velden, Jolanda.

In: Archives of Biochemistry and Biophysics, Vol. 554, 15.07.2014, p. 11-21.

Research output: Contribution to journalArticle

Wijnker, Paul J M ; Sequeira, Vasco ; Witjas-Paalberends, E. Rosalie ; Foster, Darren Brian ; Dos Remedios, Cristobal G. ; Murphy, Anne M ; Stienen, Ger J M ; Van Der Velden, Jolanda. / 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. In: Archives of Biochemistry and Biophysics. 2014 ; Vol. 554. pp. 11-21.
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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 Ca2+-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 Ca2+-sensitivity at physiological Ca2+- concentrations is relatively large phosphorylation of Ser42/44 may result in a decrease of force and associated ATP utilization in the human heart.",
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AU - Wijnker, Paul J M

AU - Sequeira, Vasco

AU - Witjas-Paalberends, E. Rosalie

AU - Foster, Darren Brian

AU - Dos Remedios, Cristobal G.

AU - Murphy, Anne M

AU - Stienen, Ger J M

AU - Van Der Velden, Jolanda

PY - 2014/7/15

Y1 - 2014/7/15

N2 - 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 Ca2+-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 Ca2+-sensitivity at physiological Ca2+- concentrations is relatively large phosphorylation of Ser42/44 may result in a decrease of force and associated ATP utilization in the human heart.

AB - 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 Ca2+-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 Ca2+-sensitivity at physiological Ca2+- concentrations is relatively large phosphorylation of Ser42/44 may result in a decrease of force and associated ATP utilization in the human heart.

KW - Cardiomyocyte

KW - Myofilament function

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KW - Protein phosphorylation

KW - Troponin I

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