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Research ArticleArticle

Hypoxia-Reoxygenation-Induced Apoptosis in Cultured Adult Rat Myocytes and the Protective Effect of Platelets and Transforming Growth Factor-β1

B. C. Yang, D. S. Zander and J. L. Mehta
Journal of Pharmacology and Experimental Therapeutics November 1999, 291 (2) 733-738;
B. C. Yang
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D. S. Zander
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J. L. Mehta
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Abstract

The outcome of myocardial ischemia-reperfusion has been partially attributed to the degree of apoptosis in cardiomyocytes. Aggregating platelets by release of transforming growth factor-β1 (TGF-β1) protect the isolated heart against ischemia-reperfusion injury and preserve myocardial TGF-β1 content. To gain more insight into the modulation of hypoxia-reoxygenation-induced injury (apoptosis and necrosis) to myocytes by TGF-β1 and aggregating platelets, cultured adult rat myocytes were exposed for 48 or 72 h to hypoxia alone, or to hypoxia followed by 3 h of reoxygenation. Apoptosis in the cells was determined by in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining and DNA fragmentation on gel electrophoresis. Hypoxia alone caused a time-dependent increase in myocyte apoptosis (number of apoptotic cells: 19 ± 3% at 48 h and 39 ± 5% at 72 h compared with 5 ± 1% in control cells, based on a 500-cell count). Three hours of reoxygenation after 48 h of hypoxia further increased the number of apoptotic cells (34 ± 8 versus 19 ± 3% in hypoxia for 48 h), but reoxygenation after 72 h of hypoxia did not additionally increase the number of apoptotic cells, perhaps because of extensive cell necrosis on prolonged hypoxia. Forty-eight hours of hypoxia followed by 3 h of reoxygenation also resulted in a decrease in Bcl-2 and an increase in Fas protein level. Incubation of myocytes with either recombinant TGF-β1 (0.5–5 ng/ml) or aggregated platelet supernatant (from 2–3 × 107 platelets/ml, containing ∼0.5 ng/ml of TGF-β1) markedly (P < .01) decreased the number of apoptotic cells after hypoxia-reoxygenation. Incubation with TGF-β1 also reduced myocyte necrosis as evident from lactate dehydrogenase release and trypan blue dye exclusion. These data demonstrate that hypoxia-reoxygenation results in apoptosis and necrosis in cultured adult rat myocytes; this can be attenuated by TGF-β1. Similarity of data with TGF-β1 and aggregated platelet supernatant suggests that platelet-mediated cardioprotection during hypoxia-reoxygenation may relate in part to the release of TGF-β1.

Footnotes

  • Send reprint requests to: J.L. Mehta, M.D., Ph.D., Professor of Medicine and Physiology, University of Florida, Department of Medicine, Box 100277, JHMHC, Gainesville, FL 32610. E-mail:mehta{at}medmac.ufl.edu

  • ↵1 This work was supported in part by a Merit Review Award from the Department of Veterans Affairs.

  • Abbreviations:
    TGF-β1
    transforming growth factor-β1, TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling
    K-H
    Krebs-Henseleit
    LDH
    lactate dehydrogenase
    • Received May 4, 1999.
    • Accepted July 13, 1999.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 291 (2)
Journal of Pharmacology and Experimental Therapeutics
Vol. 291, Issue 2
1 Nov 1999
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Research ArticleArticle

Hypoxia-Reoxygenation-Induced Apoptosis in Cultured Adult Rat Myocytes and the Protective Effect of Platelets and Transforming Growth Factor-β1

B. C. Yang, D. S. Zander and J. L. Mehta
Journal of Pharmacology and Experimental Therapeutics November 1, 1999, 291 (2) 733-738;

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Research ArticleArticle

Hypoxia-Reoxygenation-Induced Apoptosis in Cultured Adult Rat Myocytes and the Protective Effect of Platelets and Transforming Growth Factor-β1

B. C. Yang, D. S. Zander and J. L. Mehta
Journal of Pharmacology and Experimental Therapeutics November 1, 1999, 291 (2) 733-738;
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