Hypoxia-reoxygenation-induced apoptosis in cultured adult rat myocytes and the protective effect of platelets and transforming growth factor-beta(1).
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-beta(1) (TGF-beta(1)) protect the isolated heart against ischemia-reperfusion injury and preserve myocardial TGF-beta(1) content. To gain more insight into the modulation of hypoxia-reoxygenation-induced injury (apoptosis and necrosis) to myocytes by TGF-beta(1) 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-beta(1) (0.5-5 ng/ml) or aggregated platelet supernatant (from 2-3 x10(7) platelets/ml, containing approximately 0.5 ng/ml of TGF-beta(1)) markedly (P<.01) decreased the number of apoptotic cells after hypoxia-reoxygenation. Incubation with TGF-beta(1) 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-beta(1). Similarity of data with TGF-beta(1) and aggregated platelet supernatant suggests that platelet-mediated cardioprotection during hypoxia-reoxygenation may relate in part to the release of TGF-beta(1).
Yang
B C
BC
Department of Medicine, University of Florida, College of Medicine, Gainesville, Florida 32610, USA.
Zander
D S
DS
Mehta
J L
JL
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002478:Cells, Cultured; D004587:Electrophoresis, Agar Gel; D019254:Genes, bcl-2 / Q000502:physiology; D006352:Heart Ventricles / Q000473:pathology*; D020287:In Situ Nick-End Labeling; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009336:Necrosis; D010974:Platelet Aggregation / Q000502:physiology*; D011233:Precipitin Tests; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000473:pathology*; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000145:classification / Q000502:physiology*; D014343:Trypan Blue / Q000493:pharmacokinetics; D019014:fas Receptor / Q000378:metabolism
D016212:Transforming Growth Factor beta; D019014:fas Receptor; D007770:L-Lactate Dehydrogenase; D014343:Trypan Blue