Functional effects of protein kinase C-mediated myofilament phosphorylation in human myocardium.
OBJECTIVE
In human heart failure beta-adrenergic-mediated protein kinase A (PKA) activity is down-regulated, while protein kinase C (PKC) activity is up-regulated. PKC-mediated myofilament protein phosphorylation might be detrimental for contractile function in cardiomyopathy. This study was designed to reveal the effects of PKC on myofilament function in human myocardium under basal conditions and upon modulation of protein phosphorylation by PKA and phosphatases.


METHODS
Isometric force was measured at different [Ca(2+)] in single permeabilized cardiomyocytes from non-failing and failing human left ventricular tissue. Basal phosphorylation of myofilament proteins and the influence of PKC, PKA, and phosphatase treatments were analyzed by one- and two-dimensional gel electrophoresis, Western immunoblotting, and ELISA.


RESULTS
Troponin I (TnI) phosphorylation at the PKA sites was decreased in failing compared to non-failing hearts and correlated well with myofilament Ca(2+) sensitivity (pCa(50)). Incubation with the catalytic domain of PKC slightly decreased maximal force under basal conditions, but not following PKA and phosphatase pretreatments. PKC reduced Ca(2+) sensitivity to a larger extent in failing (DeltapCa(50)=0.19+/-0.03) than in non-failing (DeltapCa(50)=0.08+/-0.01) cardiomyocytes. This shift was reduced, though still significant, when PKC was preceded by PKA, while PKA following PKC did not further decrease pCa(50). Protein analysis indicated that PKC phosphorylated PKA sites in human TnI and increased phosphorylation of troponin T, while myosin light chain phosphorylation remained unaltered.


CONCLUSIONS
In human myocardium PKC-mediated myofilament protein phosphorylation only has a minor effect on maximal force development. The PKC-mediated decrease in Ca(2+) sensitivity may serve to improve diastolic function in failing human myocardium in which PKA-mediated TnI phosphorylation is decreased.
van der Velden
Jolanda
J
Laboratory for Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands. j.vandervelden@vumc.nl
Narolska
Nadiya A
NA
Lamberts
Regis R
RR
Boontje
Nicky M
NM
Borbély
Attila
A
Zaremba
Ruud
R
Bronzwaer
Jean G F
JG
Papp
Zoltan
Z
Jaquet
Kornelia
K
Paulus
Walter J
WJ
Stienen
Ger J M
GJ
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000328:Adult; D001696:Biomechanical Phenomena; D002118:Calcium / Q000378:metabolism; D002311:Cardiomyopathy, Dilated / Q000201:enzymology*; D016022:Case-Control Studies; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D032383:Myocytes, Cardiac / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism* / Q000494:pharmacology; D019210:Troponin I / Q000378:metabolism
D019210:Troponin I; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D002118:Calcium