title: Length-dependent activation is modulated by cardiac troponin I bisphosphorylation at Ser23 and Ser24 but not by Thr143 phosphorylation.
abstract: 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.
authors.affiliation: Laboratory for Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Paul J M
authors.initials: PJ
authors.lastname: Wijnker
authors.forename: Vasco
authors.initials: V
authors.lastname: Sequeira
authors.forename: D Brian
authors.initials: DB
authors.lastname: Foster
authors.forename: Yuejin
authors.initials: Y
authors.lastname: Li
authors.forename: Cristobal G
authors.initials: CG
authors.lastname: Dos Remedios
authors.forename: Anne M
authors.initials: AM
authors.lastname: Murphy
authors.forename: Ger J M
authors.initials: GJ
authors.lastname: Stienen
authors.forename: Jolanda
authors.initials: J
authors.lastname: van der Velden
mesh_terms: D002118:Calcium / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009200:Myocardial Contraction / Q000502:physiology; D032383:Myocytes, Cardiac / Q000378:metabolism*; D009210:Myofibrils / Q000187:drug effects / Q000502:physiology; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D010769:Phosphothreonine / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D012518:Sarcomeres / Q000502:physiology; D019210:Troponin I / Q000378:metabolism*
chemical_list: D019210:Troponin I; D010769:Phosphothreonine; D010768:Phosphoserine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D002118:Calcium
keywords: myofilament function; protein phosphorylation; troponin I