Protein kinase C contributes to the maintenance of contractile force in human ventricular cardiomyocytes.
Prolonged Ca(2+) stimulations often result in a decrease in contractile force of isolated, demembranated human ventricular cardiomyocytes, whereas intact cells are likely to be protected from this deterioration. We hypothesized that cytosolic protein kinase C (PKC) contributes to this protection. Prolonged contracture (10 min) of demembranated human cardiomyocytes at half-maximal Ca(2+) resulted in a 37 +/- 5% reduction of active force (p < 0.01), whereas no decrease (2 +/- 3% increase) was observed in the presence of the cytosol (reconstituted myocytes). The PKC inhibitors GF 109203X and Gö 6976 (10 micromol/liter) partially antagonized the cytosol-mediated protection (15 +/- 5 and 9 +/- 2% decrease in active force, p < 0.05). Quantitation of PKC isoform expression revealed the dominance of the Ca(2+)-dependent PKCalpha over PKCdelta and PKCepsilon (189 +/- 31, 7 +/- 3, and 7 +/- 2 ng/mg protein, respectively). Ca(2+) stimulations of reconstituted human cardiomyocytes resulted in the translocation of endogenous PKCalpha, but not PKCbeta1, delta, and epsilon from the cytosol to the contractile system (PKCalpha association: control, 5 +/- 3 arbitrary units; +Ca(2+), 39 +/- 8 arbitrary units; p < 0.01, EC(50,Ca) = 645 nmol/liter). One of the PKCalpha-binding proteins were identified as the thin filament regulatory protein cardiac troponin I (TnI). Finally, the Ca(2+)-dependent interaction between PKCalpha and TnI was confirmed using purified recombinant proteins (binding without Ca(2+) was only 28 +/- 18% of that with Ca(2+)). Our data suggest that PKCalpha translocates to the contractile system and anchors to TnI in a Ca(2+)-dependent manner in the human heart, contributing to the maintenance of contractile force.
Molnár
Andrea
A
Division of Clinical Physiology, Institute of Cardiology, Faculty of Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary.
Borbély
Attila
A
Czuriga
Dániel
D
Ivetta
Siket M
SM
Szilágyi
Szabolcs
S
Hertelendi
Zita
Z
Pásztor
Eniko T
ET
Balogh
Agnes
A
Galajda
Zoltán
Z
Szerafin
Tamás
T
Jaquet
Kornelia
K
Papp
Zoltán
Z
Edes
István
I
Tóth
Attila
A
D000328:Adult; D002118:Calcium / Q000494:pharmacology; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D006352:Heart Ventricles / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008297:Male; D009119:Muscle Contraction; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000201:enzymology*; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D013379:Substrate Specificity; D019210:Troponin I / Q000378:metabolism
D007527:Isoenzymes; D019210:Troponin I; D011493:Protein Kinase C; D002118:Calcium