title: PKCα-specific phosphorylation of the troponin complex in human myocardium: a functional and proteomics analysis.
abstract: OBJECTIVE
  Protein kinase Cα (PKCα) is one of the predominant PKC isoforms that phosphorylate cardiac troponin. PKCα is implicated in heart failure and serves as a potential therapeutic target, however, the exact consequences for contractile function in human myocardium are unclear. This study aimed to investigate the effects of PKCα phosphorylation of cardiac troponin (cTn) on myofilament function in human failing cardiomyocytes and to resolve the potential targets involved.
  
  
  RESULTS
  Endogenous cTn from permeabilized cardiomyocytes from patients with end-stage idiopathic dilated cardiomyopathy was exchanged (∼69%) with PKCα-treated recombinant human cTn (cTn (DD+PKCα)). This complex has Ser23/24 on cTnI mutated into aspartic acids (D) to rule out in vitro cross-phosphorylation of the PKA sites by PKCα. Isometric force was measured at various [Ca(2+)] after exchange. The maximal force (Fmax) in the cTn (DD+PKCα) group (17.1±1.9 kN/m(2)) was significantly reduced compared to the cTn (DD) group (26.1±1.9 kN/m(2)). Exchange of endogenous cTn with cTn (DD+PKCα) increased Ca(2+)-sensitivity of force (pCa50 = 5.59±0.02) compared to cTn (DD) (pCa50 = 5.51±0.02). In contrast, subsequent PKCα treatment of the cells exchanged with cTn (DD+PKCα) reduced pCa50 to 5.45±0.02. Two PKCα-phosphorylated residues were identified with mass spectrometry: Ser198 on cTnI and Ser179 on cTnT, although phosphorylation of Ser198 is very low. Using mass spectrometry based-multiple reaction monitoring, the extent of phosphorylation of the cTnI sites was quantified before and after treatment with PKCα and showed the highest phosphorylation increase on Thr143.
  
  
  CONCLUSIONS
  PKCα-mediated phosphorylation of the cTn complex decreases Fmax and increases myofilament Ca(2+)-sensitivity, while subsequent treatment with PKCα in situ decreased myofilament Ca(2+)-sensitivity. The known PKC sites as well as two sites which have not been previously linked to PKCα are phosphorylated in human cTn complex treated with PKCα with a high degree of specificity for Thr143.
authors.affiliation: Laboratory for Physiology, Institute for Cardiovascular Research, VU Medical Center, Amsterdam, The Netherlands ; Johns Hopkins Bayview Proteomics Center, Department of Medicine, School of Medicine, Johns Hopkins University, Baltimore, Maryland, United States of America.
authors.forename: Viola
authors.initials: V
authors.lastname: Kooij
authors.forename: Pingbo
authors.initials: P
authors.lastname: Zhang
authors.forename: Sander R
authors.initials: SR
authors.lastname: Piersma
authors.forename: Vasco
authors.initials: V
authors.lastname: Sequeira
authors.forename: Nicky M
authors.initials: NM
authors.lastname: Boontje
authors.forename: Paul J M
authors.initials: PJ
authors.lastname: Wijnker
authors.forename: Connie R
authors.initials: CR
authors.lastname: Jiménez
authors.forename: Kornelia E
authors.initials: KE
authors.lastname: Jaquet
authors.forename: Cris
authors.initials: C
authors.lastname: dos Remedios
authors.forename: Anne M
authors.initials: AM
authors.lastname: Murphy
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Van Eyk
authors.forename: Jolanda
authors.initials: J
authors.lastname: van der Velden
authors.forename: Ger Jm
authors.initials: GJ
authors.lastname: Stienen
mesh_terms: D002311:Cardiomyopathy, Dilated / Q000378:metabolism*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009206:Myocardium / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism*; D009210:Myofibrils / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D040901:Proteomics; D014336:Troponin / Q000378:metabolism*
chemical_list: D014336:Troponin; D011493:Protein Kinase C