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

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-β 1 (TGF-β 1 ) protect the isolated heart against ischemia-reperfusion injury and preserve myocardial TGF-β 1 content. To gain more insight into the modulation of hypoxia-reoxygenation-induced injury (apoptosis and necrosis) to myocytes by TGF-β 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-β 1 (0.5–5 ng/ml) or aggregated platelet supernatant (from 2–3 × 10 7 platelets/ml, containing ∼0.5 ng/ml of TGF-β 1 ) markedly ( P 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-β 1 . Similarity of data with TGF-β 1 and aggregated platelet supernatant suggests that platelet-mediated cardioprotection during hypoxia-reoxygenation may relate in part to the release of TGF-β 1 .