Inhibition of hypoxia/reoxygenation-induced apoptosis in metallothionein-overexpressing cardiomyocytes.
To study possible mechanisms for metallothionein (MT) inhibition of ischemia-reperfusion-induced myocardial injury, cardiomyocytes isolated from MT-overexpressing transgenic neonatal mouse hearts and nontransgenic controls were subjected to 4 h of hypoxia (5% CO2-95% N2, glucose-free modified Tyrode's solution) followed by 1 h of reoxygenation in MEM + 20% fetal bovine serum (FBS) (5% CO2-95% air), and cytochrome c-mediated caspase-3 activation apoptotic pathway was determined. Hypoxia/reoxygenation-induced apoptosis was significantly suppressed in MT-overexpressing cardiomyocytes, as measured by both terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling and annexin V-FITC binding. In association with apoptosis, mitochondrial cytochrome c release, as determined by Western blot, was observed to occur in nontransgenic cardiomyocytes. Correspondingly, caspase-3 was activated as determined by laser confocal microscopic examination with the use of FITC-conjugated antibody against active caspase-3 and by enzymatic assay. The activation of this apoptotic pathway was significantly inhibited in MT-overexpressing cells, as evidenced by both suppression of cytochrome c release and inhibition of caspase-3 activation. The results demonstrate that MT suppresses hypoxia/reoxygenation-induced cardiomyocyte apoptosis through, at least in part, inhibition of cytochrome c-mediated caspase-3 activation.
Wang
G W
GW
Department of Medicine, University of Louisville, Louisville, Kentucky 40292, USA.
Zhou
Z
Z
Klein
J B
JB
Kang
Y J
YJ
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D003574:Cytochrome c Group / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology; D000860:Hypoxia / Q000378:metabolism / Q000473:pathology; D020287:In Situ Nick-End Labeling; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008668:Metallothionein / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D008928:Mitochondria / Q000201:enzymology; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000201:enzymology*; D015428:Myocardial Reperfusion Injury / Q000378:metabolism* / Q000473:pathology; D009206:Myocardium / Q000166:cytology*
D003574:Cytochrome c Group; D008668:Metallothionein; D007770:L-Lactate Dehydrogenase; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases