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Am J Physiol Heart Circ Physiol 281: H404-H410, 2001;
0363-6135/01 $5.00
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Vol. 281, Issue 1, H404-H410, July 2001

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

Huiping Liu, Bradley C. McPherson, and Zhenhai Yao

Department of Anesthesia and Critical Care, The University of Chicago, Chicago, Illinois 60637

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-end labeling assay, and DNA-laddering electrophoresis. Apoptosis increased with 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 reoxygenation; none was detected during simulated ischemia. Preconditioning with 10 min of simulated ischemia reduced necrosis (18 ± 6%, n = 8) but had no effect on apoptosis. However, three 1-min cycles of simulated ischemia separated by 5 min of reoxygenation reduced necrosis and apoptosis similarly. The protein kinase C (PKC) inhibitors Go6976 (0.1 µM) or chelerythrene (4 µM) abolished the effect of preconditioning. Preconditioning selectively activated PKCepsilon but had no effect on PKCdelta and on total PKC enzyme activity. Preconditioning protected against necrosis and apoptosis, but the preconditioning ischemia required for blocking apoptosis was less than that for reducing necrosis. Activation of PKCepsilon isoform is important in mediating the protection.

hypoxia; cultured cardiomyocytes


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