title: Protein kinase C alpha and epsilon phosphorylation of troponin and myosin binding protein C reduce Ca2+ sensitivity in human myocardium.
abstract: Previous studies indicated that the increase in protein kinase C (PKC)-mediated myofilament protein phosphorylation observed in failing myocardium might be detrimental for contractile function. This study was designed to reveal and compare the effects of PKCalpha- and PKCepsilon-mediated phosphorylation on myofilament function in human myocardium. Isometric force was measured at different [Ca2+] in single permeabilized cardiomyocytes from failing human left ventricular tissue. Activated PKCalpha and PKCepsilon equally reduced Ca2+ sensitivity in failing cardiomyocytes (DeltapCa50 = 0.08 +/- 0.01). Both PKC isoforms increased phosphorylation of troponin I- (cTnI) and myosin binding protein C (cMyBP-C) in failing cardiomyocytes. Subsequent incubation of failing cardiomyocytes with the catalytic subunit of protein kinase A (PKA) resulted in a further reduction in Ca2+ sensitivity, indicating that the effects of both PKC isoforms were not caused by cross-phosphorylation of PKA sites. Both isozymes showed no effects on maximal force and only PKCalpha resulted in a modest significant reduction in passive force. Effects of PKCalpha were only minor in donor cardiomyocytes, presumably because of already saturated cTnI and cMyBP-C phosphorylation levels. Donor tissue could therefore be used as a tool to reveal the functional effects of troponin T (cTnT) phosphorylation by PKCalpha. Massive dephosphorylation of cTnT with alkaline phosphatase increased Ca2+ sensitivity. Subsequently, PKCalpha treatment of donor cardiomyocytes reduced Ca2+ sensitivity (DeltapCa50 = 0.08 +/- 0.02) and solely increased phosphorylation of cTnT, but did not affect maximal and passive force. PKCalpha- and PKCepsilon-mediated phosphorylation of cMyBP-C and cTnI as well as cTnT decrease myofilament Ca2+ sensitivity and may thereby reduce contractility and enhance relaxation of human myocardium.
authors: 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: Viola
authors: V
authors: Kooij
authors: Nicky
authors: N
authors: Boontje
authors: Ruud
authors: R
authors: Zaremba
authors: Kornelia
authors: K
authors: Jaquet
authors: Cris
authors: C
authors: dos Remedios
authors: Ger J M
authors: GJ
authors: Stienen
authors: Jolanda
authors: J
authors: van der Velden
mesh_terms: D000293:Adolescent; D000328:Adult; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D002478:Cells, Cultured; D005260:Female; D006333:Heart Failure / Q000201:enzymology*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009206:Myocardium / Q000201:enzymology*; D010766:Phosphorylation; D051571:Protein Kinase C-alpha / Q000378:metabolism*; D051744:Protein Kinase C-epsilon / Q000378:metabolism*; D019210:Troponin I / Q000378:metabolism; D020107:Troponin T / Q000378:metabolism; D055815:Young Adult; D000099163:Myosin Binding Protein C
chemical_list: D002118:Calcium; D002352:Carrier Proteins; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D019210:Troponin I; D020107:Troponin T; D000099163:Myosin Binding Protein C