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

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / metabolism
  • Amino Acid Substitution
  • Calcium / metabolism
  • Cardiomyopathy, Dilated / metabolism
  • Cardiomyopathy, Dilated / physiopathology
  • Cyclic AMP-Dependent Protein Kinases / metabolism*
  • Female
  • Heart Failure / metabolism
  • Heart Failure / physiopathology
  • Humans
  • In Vitro Techniques
  • Isometric Contraction / physiology
  • Male
  • Middle Aged
  • Mutagenesis, Site-Directed
  • Myocardial Contraction / physiology
  • Myocytes, Cardiac / metabolism*
  • Phosphorylation
  • Protein Kinase C / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Serine / chemistry
  • Troponin I / chemistry*
  • Troponin I / genetics
  • Troponin I / metabolism*

Substances

  • Recombinant Proteins
  • Troponin I
  • Serine
  • Cyclic AMP-Dependent Protein Kinases
  • Protein Kinase C
  • Adenosine Triphosphatases
  • Calcium