p21WAF1 and hypoxia/reoxygenation-induced premature senescence of H9c2 cardiomyocytes

Abstract

We have previously reported on hypoxia/reoxygenation-induced premature senescence in neonatalrat cardiomyocytes. In this research, we investigated the effects of p21WAF1 (p21) in hypoxia/reoxygenation-inducedsenescence, using H9c2 cells. A plasmid overexpressing wild type p21WAF1 and a plasmid expressing smallhairpin RNA (shRNA) targeting p21WAF1 were constructed, and transfected into H9c2 cells to control the p21expression. Hypoxia/reoxygenation conditions were 1% O2 and 5% CO2, balancing the incubator chamber withN2 for 6 h (hypoxia 6 h), then 21% oxygen for 8 h (reoxygenation 8 h). Cell cycle was examined using flowcytometry. Senescence was assessed using b-galactosidase staining. The expression of p53, p21, p16INK4a, andcyclin D1 was assayed using Western blotting. At hypoxia 6 h, cells overexpressing p21 had a larger G1 distribution,stronger b-galactosidase activity, and lower cyclin D1 expression compared to control cells, while the oppositeresults and higher p53 expression were obtained in p21-knockdown cells. At reoxygenation 8 h, p21-silencedcells had a smaller percentage of G1 cells, weaker b-galactosidase activity and lower 16INK4a expression, andhigher cyclin D1 expression, but the overexpression group showed no difference. Taken together, this data impliesthat p21WAF1 is important for the hypoxia phase, but not the reoxygenation phase, in the H9c2 senescenceprocess. (Folia Histochemica et Cytobiologica 2011, Vol. 49, No. 3, 445–451)

Keywords

• H9c2
• hypoxia reoxygenation
• p21WAF1
• b-galactosidase
• senescence