Protein kinase C contributes to the maintenance of contractile force in human ventricular cardiomyocytes

Abstract

Prolonged Ca2+ stimulations often result in a decrease in contractile force of isolated, demembranated human ventricular car-diomyocytes, 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 Ca2+ 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 μmol/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 Ca2+-dependent PKCα over PKCδ and PKCε (189 ± 31, 7 ± 3, and 7 ± 2 ng/mg protein, respectively). Ca2+ stimulations of reconstituted human cardiomyocytes resulted in the translocation of endogenous PKCα, but not PKCβ1, δ, and ε from the cytosol to the contractile system (PKCα association: control, 5 ± 3 arbitrary units; +Ca 2+,39 ± 8 arbitrary units; p < 0.01, EC50,ca = 645 nmol/liter). One of the PKCα-binding proteins were identified as the thin filament regulatory protein cardiac troponin I (Tnl). Finally, the Ca 2+-dependent interaction between PKCα and Tnl was confirmed using purified recombinant proteins (binding without Ca2+ was only 28 ± 18% of that with Ca2+). Our data suggest that PKCα translocates to the contractile system and anchors to TnI in a Ca2+ -dependent manner in the human heart, contributing to the maintenance ofcontractile force. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.