p21 WAF1 and hypoxia/reoxygenation-induced premature senescence of H9c2 cardiomyocytes.
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% CO(2), balancing the incubator chamber with N(2) 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.
Department of Cardiology, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China.
Dan
D
Wang
Yu-Zhen
YZ
Zhang
Bing
B
Yang
Feng-Xiang
FX
Zhang
Ming-Yong
MY
Cao
Cheng
C
Wang
Ming-Long
ML
Chen
D000818:Animals; D002453:Cell Cycle; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000502:physiology*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism*; D000860:Hypoxia / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000502:physiology*; D010100:Oxygen / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D001616:beta-Galactosidase / Q000378:metabolism
D050759:Cyclin-Dependent Kinase Inhibitor p21; D034741:RNA, Small Interfering; D001616:beta-Galactosidase; D010100:Oxygen