p21 WAF1 and hypoxia/reoxygenation-induced premature senescence of H9c2 cardiomyocytes

Abstract

We have previously reported on hypoxia/reoxygenation-induced premature senescence in neonatal rat cardiomyocytes. In this research, we investigated the effects of p21^WAF1 (p21) in hypoxia/reoxygenation-induced senescence, using H9c2 cells. A plasmid overexpressing wild type p21^WAF1 and a plasmid expressing small hairpin RNA (shRNA) targeting p21^WAF1 were constructed, and transfected into H9c2 cells to control the p21 expression. Hypoxia/reoxygenation conditions were 1% O2 and 5% CO₂, balancing the incubator chamber with N₂ for 6 h (hypoxia 6 h), then 21% oxygen for 8 h (reoxygenation 8 h). Cell cycle was examined using flow cytometry. Senescence was assessed using β-galactosidase staining. The expression of p53, p21, p16^INK4a, and cyclin D1 was assayed using Western blotting. At hypoxia 6 h, cells overexpressing p21 had a larger G1 distribution, stronger β-galactosidase activity, and lower cyclin D1 expression compared to control cells, while the opposite results and higher p53 expression were obtained in p21-knockdown cells. At reoxygenation 8 h, p21-silenced cells had a smaller percentage of G1 cells, weaker β-galactosidase activity and lower 16^INK4a expression, and higher cyclin D1 expression, but the overexpression group showed no difference. Taken together, this data implies that p21^WAF1 is important for the hypoxia phase, but not the reoxygenation phase, in the H9c2 senescence process. (Folia Histochemica et Cytobiologica 2011, Vol. 49, No. 3, 445–451)