Cardiovascular research · 1999
Induction of necrosis but not apoptosis after anoxia and reoxygenation in isolated adult cardiomyocytes of rat.
СтатьяПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Taimor G, Lorenz H, Hofstaetter B, Schlüter K D, Piper H M
Аннотация
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.
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.