Inhibition of phospholipase C-γ1augments the decrease in cardiomyocyte viability by H2O2

Abstract

The present study was conducted to examine the role of a major cardiac phospholipase C (PLC) isozyme, PLC-γ1, 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 μM H2O2for 15 min. A concentration-dependent (up to 50 μM) increase in the mRNA level and membrane protein content of PLC-γ1was observed with H2O2treatment. Furthermore, PLC-γ1was 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)-δ and -ε protein contents were increased in the cardiomyocyte membrane fraction in response to H2O2, PKC-ε activation, unlike PKC-δ, was attenuated by U-73122 (2 μM). Inhibition of PKC-ε with inhibitory peptide (0.1 μM) prevented Bcl-2 phosphorylation. Moreover, different concentrations (0.05, 0.1, and 0.2 μM) 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 H2O2was also seen when cells were pretreated with U-73122 and was as a result of increased apoptosis. It is therefore suggested that PLC-γ1may play a role in cardiomyocyte survival during oxidative stress via PKC-ε and phosphorylation of Bcl-2.