Induction of necrosis but not apoptosis after anoxia and reoxygenation in isolated adult cardiomyocytes of rat

Objectives: Apoptosis is one feature of myocardial damage after ischemia-re perfusion, but the causes for its induction are unclear. The present study was undertaken to investigate whether apoptosis in cardiomyocytes is direct ly initiated by their sub-lethal injury that results from ischemia-reperfus ion. Methods: Ischemia was simulated on isolated ventricular cardiomyocytes of adult rats by anoxia in a glucose free medium, pH 6.4. Induction of apo ptosis was detected by (1) DNA laddering of genomic DNA, (2) TUNEL positive cells (terminal deoxynucleotidyl transferase-mediated-UTP nick end labelli ng) and (3) annexinV-fluorescein isothiocyanate (annexinV-FITC) binding to cells under exclusion of propidium iodide. Necrotic cells were identified b y (1) staining with both annexinV-FITC and propidium iodide, (2) unspecific DNA degradation and (3) enzyme release. Results: Simulated ischemia caused a >75% loss of high-energy phosphates within 2 h, which was reversible upo n reoxygenation at pH 7.4. Even after 18 h of simulated ischemia, creatine phosphate contents recovered to 55.2+/-7.3% of control within 1 h. Apoptosi s could be induced by UV irradiation (80 J/m(2)), H2O2 and the NO-donor N2- acetyl-S-nitroso-D,L-penicillinaminamide. In contrast to this, simulated is chemia and reoxygenation could not induce apoptosis in the cells, but with prolonged ischemia more cells became necrotic. After 18 hours of simulated ischemia and 4 h of reoxygenation 41.2+/-10.2% myocytes were necrotic (vs. 6.3+/-4.3% of control) and only 1.7+/-0.5% (vs. 8.7+/-4.6% of control) were apoptotic. The percentage of necrotic cells correlated with an increase in lactate dehydrogenase release from 9.9+/-0.6% (of total activity) of normo xic controls to 37.9+/-5.1% after 18 h of simulated ischemia and 12 h of re oxygenation. Conclusions: Simulated ischemia-reoxygenation causes necrosis of isolated cardiomyocytes but is not sufficient for induction of apoptosis . (C) 1999 Elsevier Science B.V. All rights reserved.