Length-dependent activation is modulated by cardiac troponin I bisphosphorylation at Ser23 and Ser24 but not by Thr143 phosphorylation

Am J Physiol Heart Circ Physiol. 2014 Apr 15;306(8):H1171-81. doi: 10.1152/ajpheart.00580.2013. Epub 2014 Feb 28.


Frank-Starling's law reflects the ability of the heart to adjust the force of its contraction to changes in ventricular filling, a property based on length-dependent myofilament activation (LDA). The threonine at amino acid 143 of cardiac troponin I (cTnI) is prerequisite for the length-dependent increase in Ca(2+) sensitivity. Thr143 is a known target of protein kinase C (PKC) whose activity is increased in cardiac disease. Thr143 phosphorylation may modulate length-dependent myofilament activation in failing hearts. Therefore, we investigated if pseudo-phosphorylation at Thr143 modulates length dependence of force using troponin exchange experiments in human cardiomyocytes. In addition, we studied effects of protein kinase A (PKA)-mediated cTnI phosphorylation at Ser23/24, which has been reported to modulate LDA. Isometric force was measured at various Ca(2+) concentrations in membrane-permeabilized cardiomyocytes exchanged with recombinant wild-type (WT) troponin or troponin mutated at the PKC site Thr143 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 incubation with exogenous PKA. Pseudo-phosphorylation of Thr143 increased myofilament Ca(2+) sensitivity compared with WT without affecting LDA in failing and donor cardiomyocytes. Subsequent PKA treatment enhanced the length-dependent shift in Ca(2+) sensitivity after WT and 143D exchange. Exchange with Ser23/24 variants demonstrated that pseudo-phosphorylation of both Ser23 and Ser24 is needed to enhance the length-dependent increase in Ca(2+) sensitivity. cTnI pseudo-phosphorylation did not alter length-dependent changes in maximal force. Thus phosphorylation at Thr143 enhances myofilament Ca(2+) sensitivity without affecting LDA, while Ser23/24 bisphosphorylation is needed to enhance the length-dependent increase in myofilament Ca(2+) sensitivity.

Keywords: myofilament function; protein phosphorylation; troponin I.

Publication types

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

MeSH terms

  • Calcium / pharmacology
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • Female
  • Humans
  • Male
  • Middle Aged
  • Myocardial Contraction / physiology
  • Myocytes, Cardiac / metabolism*
  • Myofibrils / drug effects
  • Myofibrils / physiology
  • Phosphorylation
  • Phosphoserine / metabolism*
  • Phosphothreonine / metabolism*
  • Protein Kinase C / metabolism
  • Sarcomeres / physiology
  • Troponin I / metabolism*


  • Troponin I
  • Phosphothreonine
  • Phosphoserine
  • Cyclic AMP-Dependent Protein Kinases
  • Protein Kinase C
  • Calcium