Abstract
Previous studies using transgenic mice in which metallothionein (MT) was overexpressed only in the heart have demonstrated that MT protects from oxidative cardiac injury induced by doxorubicin (DOX), an important anticancer agent. MT cardioprotection is associated with its antiapoptotic effect. The present study was undertaken to test the hypothesis that MT suppresses DOX-induced apoptosis through inhibition of mitochondrial cytochrome c release and caspase-3 activation. Primary cultures of cardiomyocytes isolated from the hearts of transgenic neonatal mice and nontransgenic controls were treated with DOX at a clinically relevant concentration (1.0 μM) for varying time periods. Apoptosis was detected in nontransgenic cardiomyocyte cultures by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and Annexin V-fluorescein isothiocyanate binding. This apoptotic effect was significantly suppressed in the MT-overexpressing transgenic cardiomyocytes. Western blot analysis revealed that DOX caused mitochondrial cytochromec release. Furthermore, caspase-3 activation was observed. The activation of this apoptotic pathway by DOX was dramatically inhibited in the MT-overexpressing cardiomyocytes. To elucidate the role of reactive oxygen species (ROS) in the activation of the cytochrome c-mediated caspase-3 activation pathway, the intracellular levels of ROS and their localization were detected by fluorescent confocal microscopy. Mitochondrial ROS concentrations were dramatically elevated by DOX in nontransgenic cardiomyocytes. This elevation was completely inhibited almost in the MT-overexpressing cardiomyocytes. Thus, these results demonstrate that MT suppresses DOX-induced apoptosis in cardiomyocytes through, at least in part, inhibition of the cytochrome c-mediated apoptotic pathway.
Footnotes
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This work was supported in part by National Institutes of Health Grants CA68125 and HL59225, an Established Investigator Award (9640091 N) from the American Heart Association National Center, a research grant from the Jewish Hospital Foundation, Louisville, KY (Y.J.K.), and a research grant from National Institutes of Health Grant HL66358 (J.B.K.). Y.J.K. is a University scholar of the University of Louisville. This work was presented in part at the 39th Annual Meeting of the Society of Toxicology held in Philadelphia, Pennsylvania, March 19–23, 2000.
- Abbreviations:
- MT
- metallothionein
- DOX
- doxorubicin
- MAPK
- mitogen-activated protein kinase
- ROS
- reactive oxygen species
- DCFDA
- dichlorofluorescein diacetate
- DCF
- dichlorofluorscein
- FITC
- fluorescein isothiocyanate
- CMXRos
- Mito Tracker Red CMXRos
- TUNEL
- terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling
- PBS
- phosphate-buffered saline
- LDH
- lactate dehydrogenase
- TG
- thioglycerol
- CHAPS
- 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
- Received November 2, 2000.
- Accepted April 11, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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