title: Effect of troponin I Ser23/24 phosphorylation on Ca2+-sensitivity in human myocardium depends on the phosphorylation background.
abstract: Protein kinase A (PKA)-mediated phosphorylation of Ser23/24 of cardiac troponin I (cTnI) causes a reduction in Ca(2+)-sensitivity of force development. This study aimed to determine whether the PKA-induced modulation of the Ca(2+)-sensitivity is solely due to cTnI phosphorylation or depends on the phosphorylation status of other sarcomeric proteins. Endogenous troponin (cTn) complex in donor cardiomyocytes was partially exchanged (up to 66+/-1%) with recombinant unphosphorylated human cTn and in failing cells similar exchange was achieved using PKA-(bis)phosphorylated cTn complex. Cardiomyocytes immersed in exchange solution without complex added served as controls. Partial exchange of unphosphorylated cTn complex in donor tissue significantly increased Ca(2+)-sensitivity (pCa(50)) to 5.50+/-0.02 relative to the donor control value (pCa(50)=5.43+/-0.04). Exchange in failing tissue with PKA-phosphorylated cTn complex did not change Ca(2+)-sensitivity relative to the failing control (pCa(50)=5.60+/-0.02). Subsequent treatment of the cardiomyocytes with the catalytic subunit of PKA significantly decreased Ca(2+)-sensitivity in donor and failing tissue. Analysis of phosphorylated cTnI species revealed the same distribution of un-, mono- and bis-phosphorylated cTnI in donor control and in failing tissue exchanged with PKA-phosphorylated cTn complex. Phosphorylation of myosin-binding protein-C in failing tissue was significantly lower compared to donor tissue. These differences in Ca(2+)-sensitivity in donor and failing cells, despite similar distribution of cTnI species, could be abolished by subsequent PKA-treatment and indicate that other targets of PKA are involved the reduction of Ca(2+)-sensitivity. Our findings suggest that the sarcomeric phosphorylation background, which is altered in cardiac disease, influences the impact of cTnI Ser23/24 phosphorylation by PKA on Ca(2+)-sensitivity.
authors.affiliation: Laboratory for Physiology, Institute for Cardiovascular Research, VU University Medical Center, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. v.kooij@vumc.nl
authors.forename: Viola
authors.initials: V
authors.lastname: Kooij
authors.forename: Martina
authors.initials: M
authors.lastname: Saes
authors.forename: Kornelia
authors.initials: K
authors.lastname: Jaquet
authors.forename: Ruud
authors.initials: R
authors.lastname: Zaremba
authors.forename: D Brian
authors.initials: DB
authors.lastname: Foster
authors.forename: Anne M
authors.initials: AM
authors.lastname: Murphy
authors.forename: Cris
authors.initials: C
authors.lastname: Dos Remedios
authors.forename: Jolanda
authors.initials: J
authors.lastname: van der Velden
authors.forename: Ger J M
authors.initials: GJ
authors.lastname: Stienen
mesh_terms: D000704:Analysis of Variance; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D006801:Humans; D009206:Myocardium / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism; D010766:Phosphorylation; D012518:Sarcomeres / Q000378:metabolism; D019210:Troponin I / Q000378:metabolism*; D000099163:Myosin Binding Protein C
chemical_list: D002118:Calcium; D002352:Carrier Proteins; D019210:Troponin I; D000099163:Myosin Binding Protein C