title: Inhibition of phospholipase C-gamma 1 augments 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-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 microM H2O2 for 15 min. A concentration-dependent (up to 50 microM) 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 microM). Inhibition of PKC-epsilon with inhibitory peptide (0.1 microM) prevented Bcl-2 phosphorylation. Moreover, different concentrations (0.05, 0.1, and 0.2 microM) 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.
authors.affiliation: Department of Human Nutritional Sciences, Faculty of Human Ecology, University of Manitoba, and Institute of Cardiovascular Sciences, St. Boniface Hospital Research Centre, Winnipeg, Canada.
authors.forename: Rabban
authors.initials: R
authors.lastname: Mangat
authors.forename: Tushi
authors.initials: T
authors.lastname: Singal
authors.forename: Naranjan S
authors.initials: NS
authors.lastname: Dhalla
authors.forename: Paramjit S
authors.initials: PS
authors.lastname: Tappia
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006352:Heart Ventricles / Q000166:cytology; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008297:Male; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000201:enzymology; D009336:Necrosis; D016877:Oxidants / Q000494:pharmacology*; D018384:Oxidative Stress; D051966:Phospholipase C gamma / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D051744:Protein Kinase C-epsilon / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D016277:Ventricular Function, Left / Q000187:drug effects
chemical_list: D004791:Enzyme Inhibitors; D016877:Oxidants; D019253:Proto-Oncogene Proteins c-bcl-2; D012313:RNA; D006861:Hydrogen Peroxide; D051744:Protein Kinase C-epsilon; D051966:Phospholipase C gamma