Involvement of Protein Kinase C and RhoA in Protease-Activated Receptor 1-Mediated F-Actin Reorganization and Cell Growth in Rat Cardiomyocytes

Protease-activated receptor 1 (PAR1) that can be activated by serine proteinases such as thrombin has been demonstrated to contribute to the development of cardiac remodeling and hypertrophy after myocardial injury. Here, we investigated the mechanisms by which PAR1 leads to hypertrophic cardiomyocyte growth using cultured rat neonatal ventricular myocytes. PAR1 stimulation with thrombin (1 U/ml) or a synthetic agonist peptide (TFLLR-NH2, 50 mu M) for 48 h induced an increase in cell size and myofibril formation associated with BNP (brain natriuretic peptide) production. This actin reorganization assessed by fluorescein isothiocyanate (FITC)-conjugated phalloidin staining appeared at 1 h after PAR1 stimulation, and this response was reduced by a protein kinase C (PKC) inhibitor, chelerythrine, inhibitors of Rho (simvastatin) and Rho-associated kinase (ROCK) (Y-27632), but not by pertussis toxin (PTX). By Western blot analysis, translocation of PKC alpha or PKC epsilon from the cytosol to membrane fractions was observed in cells stimulated with thrombin or TFLLR-NH2 for 2 - 5 min. In addition, PAR1 stimulation for 3 - 5 min increased the level of active RhoA. Furthermore, inhibitors of PKC and ROCK and Rho abrogated PAR1-mediated increase in cell size. Depletion of PKC alpha or PKC epsilon by specific small interfering RNA also suppressed both actin reorganization and cell growth. These results suggest that PAR1 stimulation of cardiomyocytes induces cell hypertrophy with actin cytoskeletal reorganization through activation of PKC alpha and PKC epsilon isoforms and RhoA via PTX-insensitive G proteins.