© 1999 by European Society of Cardiology
Copyright © 1999, European Society of Cardiology
Induction of necrosis but not apoptosis after anoxia and reoxygenation in isolated adult cardiomyocytes of rat
Physiologisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany
* Corresponding author. Tel.: +49-641-994-7243; fax: +49-641-994-7239; e-mail: gerhild.taimor@physiologie.med.uni-giessen.de
Objectives: Apoptosis is one feature of myocardial damage after ischemia–reperfusion, but the causes for its induction are unclear. The present study was undertaken to investigate whether apoptosis in cardiomyocytes is directly initiated by their sub-lethal injury that results from ischemia–reperfusion. Methods: Ischemia was simulated on isolated ventricular cardiomyocytes of adult rats by anoxia in a glucose free medium, pH 6.4. Induction of apoptosis was detected by (1) DNA laddering of genomic DNA, (2) TUNEL positive cells (terminal deoxynucleotidyl transferase-mediated-UTP nick end labelling) and (3) annexinV–fluorescein isothiocyanate (annexinV–FITC) binding to cells under exclusion of propidium iodide. Necrotic cells were identified by (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 upon 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. Apoptosis could be induced by UV irradiation (80 J/m2), H2O2 and the NO-donor N2-acetyl-S-nitroso-D,L-penicillinaminamide. In contrast to this, simulated ischemia 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±4.4% 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 normoxic controls to 37.9±5.1% after 18 h of simulated ischemia and 12 h of reoxygenation. Conclusions: Simulated ischemia-reoxygenation causes necrosis of isolated cardiomyocytes but is not sufficient for induction of apoptosis.
KEYWORDS Apoptosis; Anoxia; Cell culture; Energy-metabolism; Necrosis
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