Progressive apoptotic cell death triggered by transient oxidative insult in H9c2 rat ventricular cells: a novel pattern of apoptosis and the mechanisms.
Many pathophysiological processes are associated with oxidative stress and progressive cell death. Oxidative stress is an apoptotic inducer that is known to cause rapid cell death. Here we show that a brief oxidative insult (5-min exposure to 400 microM H(2)O(2)), although it did not kill H9c2 rat ventricular cells during the exposure, triggered an intracellular death cascade leading to delayed time-dependent cell death starting from 1 h after the insult had been withdrawn, and this post-H(2)O(2) cell death cumulated gradually, reaching a maximum level 8 h after H(2)O(2) withdrawal. By comparison, sustained exposure to H(2)O(2) caused complete cell death within a narrow time frame (2 h). The time-dependent post-H(2)O(2) cell death was typical of apoptosis, both morphologically (cell shrinkage and nuclear condensation) and biochemically (DNA fragmentation, extracellular exposure of phosphatidylserines, and caspase-3 activation). A dichlorofluorescein fluorescent signal showed a time-dependent endogenous increase of reactive oxygen species (ROS) production, which was almost abolished by inhibition of the mitochondrial electron transport chain. Application of antioxidants (vitamin E or DTT) before H(2)O(2) addition or after H(2)O(2) withdrawal prevented the H(2)O(2)-triggered progressive ROS production and apoptosis. Sequential appearance of events associated with activation of the mitochondrial death pathway was found, including progressive dissipation of mitochondrial membrane potential, cytochrome c release, and late activation of caspase-3. In conclusion, transient oxidative stress triggers an intrinsic program leading to self-sustained apoptosis in H9c2 cells via cumulative production of mitochondrial ROS and subsequent activation of the mitochondrial death pathway. This pattern of apoptosis may contribute to the progressive and long-lasting cell loss in some degenerative diseases.
Han
Hong
H
Research Center, Montreal Heart Institute, 5000 Belanger East, Montreal, Quebec, Canada H1T 1C8.
Long
Hong
H
Wang
Huizhen
H
Wang
Jingxiong
J
Zhang
Yiqiang
Y
Wang
Zhiguo
Z
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D045304:Cytochromes c / Q000378:metabolism; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007150:Immunohistochemistry; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009206:Myocardium / Q000166:cytology* / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014810:Vitamin E / Q000494:pharmacology
D000975:Antioxidants; D016877:Oxidants; D017382:Reactive Oxygen Species; D014810:Vitamin E; D045304:Cytochromes c; D006861:Hydrogen Peroxide; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; C505410:Casp3 protein, rat; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases