Inhibition of phospholipase C-γ1 augments the decrease in cardiomyocyte viability by H2O2

The present study was conducted to examine the role of a major cardiac phospholipase C ( PLC) isozyme, PLC-gamma(1), in cardiomyocytes during oxidative stress. Left ventricular cardiomyocytes were isolated by collagenase digestion from adult male Sprague-Dawley rats (250 - 300 g) and treated with 20, 50, and 100 mu M H2O2 for 15 min. A concentration-dependent (up to 50 mu M) increase in the mRNA level and membrane protein content of PLC-gamma(1) was observed with H2O2 treatment. Furthermore, PLC-gamma(1) was activated in response to H2O2, as revealed by an increase in the phosphorylation of its tyrosine residues. There was a marked increase in the phosphorylation of the antiapoptotic protein Bcl-2 by H2O2; this change was attenuated by a PLC inhibitor, U-73122. Although both protein kinase C (PKC)-delta and -epsilon protein contents were increased in the cardiomyocyte membrane fraction in response to H2O2, PKC-epsilon activation, unlike PKC-delta, was attenuated by U-73122 (2 mu M). Inhibition of PKC-epsilon with inhibitory peptide (0.1 mu M) prevented Bcl-2 phosphorylation. Moreover, different concentrations (0.05, 0.1, and 0.2 mu M) of this peptide augmented the decrease in cardiomyocyte viability in response to H2O2. In addition, a decrease in cardiomyocyte viability, as assessed by trypan blue exclusion, due to H2O2 was also seen when cells were pretreated with U-73122 and was as a result of increased apoptosis. It is therefore suggested that PLC-gamma(1) may play a role in cardiomyocyte survival during oxidative stress via PKC-epsilon and phosphorylation of Bcl-2.