Morphological and molecular characterization of adult cardiomyocyte apoptosis during hypoxia and reoxygenation.
Apoptosis has been implicated in ischemic heart disease, but its mechanism in cardiomyocytes has not been elucidated. In this study, we investigate the effects of hypoxia and reoxygenation in adult cardiomyocytes and the molecular mechanism involved in cardiomyocyte apoptosis. Morphologically, reoxygenation induced rounding up of the cells, appearance of membrane blebs that were filled with marginated mitochondria, and ultrastructural findings characteristic of apoptosis. Reoxygenation (18 hours of reoxygenation after 6 hours of hypoxia) and prolonged hypoxia (24 hours of hypoxia) resulted in a 59% and 51% decrease in cellular viability, respectively. During reoxygenation, cell death occurred predominantly via apoptosis associated with appearance of cytosolic cytochrome c and activation of caspase-3 and -9. However, nonapoptotic cell death predominated during prolonged hypoxia. Both caspase inhibition and Bcl-2 overexpression during reoxygenation significantly improved cellular viability through inhibition of apoptosis but had minimal effect on hypoxia-induced cell death. Bcl-2 overexpression blocked reoxygenation-induced cytochrome c release and activation of caspase -3 and -9, but caspase inhibition alone did not block cytochrome c release. These results suggest that apoptosis predominates in cardiomyocytes after reoxygenation through a mitochondrion-dependent apoptotic pathway, and Bcl-2 prevents reoxygenation-induced apoptosis by inhibiting cytochrome c release from the mitochondria and prevents activation of caspase-3 and -9.
Kang
P M
PM
Cardiovascular Division, Beth Israel Deaconess Medical Center and Department of Medicine, Harvard Medical School, Boston, MA 02215, USA.
Haunstetter
A
A
Aoki
H
H
Usheva
A
A
Izumo
S
S
D000332:Aerobiosis; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D061945:Caspase Inhibitors; D015687:Cell Hypoxia / Q000502:physiology*; D003574:Cytochrome c Group / Q000378:metabolism; D005260:Female; D019254:Genes, bcl-2; D006321:Heart / Q000502:physiology*; D006801:Humans; D008929:Mitochondria, Heart / Q000378:metabolism / Q000648:ultrastructure; D009206:Myocardium / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D003574:Cytochrome c Group; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone