Preconditioning attenuates apoptosis and necrosis: role of protein kinase C epsilon and -delta isoforms

Preconditioning reduces cardiomyocyte necrosis in vivo and in vitro, but it is unknown whether preconditioning blocks apoptosis. We wanted to compare the effects of preconditioning on necrosis and apoptosis in cardiomyocytes. Necrosis was detected with propidium iodide, and apoptosis was quantified by three complementary techniques: flow cytometry, TdT-mediated dUTP nick-e nd labeling assay, and DNA-laddering electrophoresis. Apoptosis increased w ith simulated ischemia time (6 h, 19 +/- 1%; 12 h, 27 +/- 2%; 18 h, 40 +/- 4%; 24 h, 54 +/- 4%; and 36 h, 83 +/- 4%; n = 6 for each group). Simulated ischemia and reoxygenation contributed equally to apoptosis (12-h ischemia, 27 +/- 2%, n = 6; 12-h ischemia and 12-h reoxygenation, 51 +/- 4%, n = 6; and 24-h ischemia, 54 +/- 5%, n = 8). Necrosis occurred primarily during re oxygenation; none was detected during simulated ischemia. Preconditioning w ith 10 min of simulated ischemia reduced necrosis (18 +/- 6%, n = 8) but ha d no effect on apoptosis. However, three 1-min cycles of simulated ischemia separated by 5 min of reoxygenation reduced necrosis and apoptosis similar ly. The protein kinase C (PKC) inhibitors Go6976 (0.1 muM) or chelerythrene (4 muM) abolished the effect of preconditioning. Preconditioning selective ly activated PKC epsilon but had no effect on PKC delta and on total PKC en zyme activity. Preconditioning protected against necrosis and apoptosis, bu t the preconditioning ischemia required for blocking apoptosis was less tha n that for reducing necrosis. Activation of PKC epsilon isoform is importan t in mediating the protection.