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and -
isoforms
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
PKC
but had no effect on PKC
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 PKC isoform is important in mediating the protection.
hypoxia; cultured cardiomyocytes
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