Abstract
Aim:
To investigate whether hypoxia reoxygenation induces premature senescence in neonatal Sprague-Dawley (SD) rat cardiomyocytes.
Methods:
Cardiomyocytes were isolated from neonatal SD rat heart and identified by immunohistochemistry. The control cultures were incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. The hypoxic cultures were incubated in a modular incubator chamber filled with 1% O2, 5% CO2, and balance N2 for 6 h. The reoxygenated cultures were subjected to 1% O2 and 5% CO2 for 6 h, then 21% oxygen for 4, 8, 12, 24, and 48 h, respectively. Cell proliferation was determined using bromodeoxyuridine labeling. The ultrastructure of cardiomyocytes was observed by using an electron microscope. β-Galactosidase activity was determined by using a senescence β-galactosidase Staining Kit. p16INK4a and telomerase reverse transcriptase (TERT) mRN A levels were measured by real time quantitative PCR. TERT protein expression was determined by immunohistochemistry. Telomerase activities were assayed by using the Telo TAGGG Telomerase PCR ELISAplus kit.
Results:
The initial cultures consisted of pure cardiomyocytes identified by immunohistochemistry. The proportion of BrdU positive cells was reduced significantly in the hypoxia reoxygenation-treated group (P<0.01). Under the condition of hypoxia reoxygenation, mitochondrial dehydration appeared; p16INK4a and TERT mRNA levels, β-galactosidase activity, TERT protein expression and telomerase activities were all significantly increased (P<0.01 or P<0.05).
Conclusion:
These data indicate that premature senescence could be induced in neonatal SD rat cardiomyocytes exposed to hypoxia reoxygenation. Although TERT significantly increased, it could not block senescence.
Similar content being viewed by others
Article PDF
References
Hayflick L, Moorhead PS . The serial cultivation of human diploid cell strains. Exp Cell Res 1961; 25: 585–621.
Von Zglinicki T, Schewe C . Mitochondrial water loss and aging of cells. Cell Biochem Funct 1995; 13: 181–7.
Chen Q, Ames BN . Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroblast F65 cells. Proc Natl Acad Sci USA 1994; 91: 4130–4.
Toussaint O, Fuchs SY, Ronai ZA, Isoyama S, Yuko N, Petronilli V, et al. Reciprocal relationships between the resistance to stresses and cellular aging. Ann N Y Acad Sci 1998; 851: 450–65.
Saretzki G, Feng J, von Zglinicki T, Villeponteau B . Similar gene expression pattern in senescent and hyperoxic-treated fibroblasts. J Gerontol A Biol Sci Med Sci 1998; 53: B438–42.
Yang NC, Hu ML . The limitations and validities of senescence associated-beta-galactosidase activity as an aging marker for human foreskin fibroblast Hs68 cells. Exp Gerontol 2005; 40: 813–9.
Kang MK, Shin KH, Yip FK, Park NH . Normal human oral keratinocytes demonstrate abnormal DNA end joining activity during premature senescence. Mech Ageing Dev 2005; 26: 475–9.
Unterluggauer H, Hampel B, Zwerschke W, Jansen-Durr P . Senescence-associated cell death of human endothelial cells: the role of oxidative stress. Exp Gerontol 2003; 38: 1149–60.
Van der Loo B, Fenton MJ, Erusalimsky JD . Cytochemical detection of a senescence-associated beta-galactosidase in endothelial and smooth muscle cells from human and rabbit blood vessels. Exp Cell Res 1998; 241: 309–15.
Reincke BS, Rosson GB, Oswald BW, Wright CF . INI1 expression induces cell cycle arrest and markers of senescence in malignant rhabdoid tumor cells. J Cell Physiol 2003; 194: 303–13.
Meng A, Wang Y, Van Zant G, Zhou D . Ionizing radiation and busulfan induce premature senescence in murine bone marrow hematopoietic cells. Cancer Res 2003; 63: 5414–9.
Chimenti C, Kajstura J, Torella D, Urbanek K, Heleniak H, Colussi C, et al. Senescence and death of primitive cells and myocytes lead to premature cardiac aging and heart failure. Circ Res 2003; 93: 604–13.
Ball AJ, Levine F . Telomere-independent cellular senescence in human fetal cardiomyocytes. Aging Cell 2005; 4: 21–30.
Huang Y, Hickey RP, Yeh JL, Liu D, Dadak A, Young LH, et al. Cardiac myocyte-specific HIF-1 alpha deletion alters vascularization, energy availability, calcium flux, and contractility in the normoxic heart. FASEB J 2004; 18: 1138–40.
Troen BR . The biology of aging. Mt Sinai J Med 2003; 70: 3–22.
Minamino T, Miyauchi H, Yoshida T, Ishida Y, Yoshida H, Komuro I . Endothelial cell senescence in human atherosclerosis: role of telomere in endothelial dysfunction. Circulation 2002; 105: 1541–4.
Colgin LM, Reddel RR . Telomere maintenance mechanisms and cellular immortalization. Curr Opin Genet Dev 1999; 9: 97–103.
Holzmann B, Nekarda H, Siewert JR . Telomere length and human telomerase reverse transcriptase expression as markers for progression and prognosis of colorectal carcinoma. J Clin Oncol 2004; 22: 1807–14.
Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB, et al. Extension of life-span by introduction of telomerase into normal human cells. Science 1998; 279: 349–52.
Finkel T, Holbrook NJ . Oxidants, oxidative stress and the biology of ageing. Nature 2000; 408: 239–47.
Zhang X, Li J, Sejas DP, Pang Q . Hypoxia-reoxygenation induces premature senescence in FA bone marrow hematopoietic cells. Blood 2005; 106: 75–85.
Takeuchi H, Bilchik A, Saha S, Turner R, Wiese D, Tanaka M, et al. c-MET expression level in primary colon cancer: a predictor of tumor invasion and lymph node metastases. Clin Cancer Res 2003; 9: 1480–8.
Vasa M, Breitschopf K, Zeiher AM, Dimmeler S . Nitric oxide activates telomerase and delays endothelial cell senescence. Circ Res 2000; 87: 540–2.
Von Zglinicki T, Marzabadi MR, Roomans GM . Water and ion distributions in myocytes cultured under oxidative stress mimic changes found in the process of aging. Mech Ageing Dev 1991; 58: 49–60.
Soonpaa MH, Kim KK, Pajak L, Franklin M, Field LJ . Cardiomyocyte DNA synthesis and binucleation during murine development. Am J Physiol 1996; 40: H2183–9.
Kawamata N, Inagaki N, Mizumura S, Sugimoto KJ, Sakajiri S, Ohyanagi-Hara M, et al. Methylation status analysis of cell cycle regulatory genes (pINK4a, pl5INK4B, p21Waf1/Cip1, p27Kip1 and p73) in natural killer cell disorders. Eur J Haematol 2005; 74: 424–9.
Tevelev A, Byeon IJ, Selby T, Ericson K, Kim HJ, Kraynov V, et al. Tumor suppressor p16INK4a: structural characterization of wild type and mutant proteins by NMR and circular dichroism. Biochemistry 1996; 35: 9475–87.
Sherr CJ . Cancer cell cycles. Science 1996; 274: 1672–7.
Ausserlechner MJ, Obexer P, Geley S, Kofler R . G1 arrest by p16INK4a uncouples growth from cell cycle progression in leukemia cells with deregulated cyclin E and c-Myc expression. Leukemia 2005; 19: 1051–7.
Mouton RE, Venable ME . Ceramide induces expression of the senescence histochemical marker, beta-galactosidase, in human fibroblasts. Mech Ageing Dev 2000; 113: 169–81.
Stockschlader MA, Schuening FG, Graham TC, Storb R . Transplantation of retrovirus-transduced canine keratinocytes expressing the beta-galactosidase gene. Gene Ther 1994; 1: 317–22.
Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA 1995; 92: 9363–7.
Serrano M, Blasco MA . Putting the stress on senescence. Curr Opin Cell Biol 2001; 13: 748–53.
Antropova YG, Bryantsev AL, Kalinina NI, Il'inskaya OP, Tararak EM . Proliferative activity and expression of cyclin-dependent kinase inhibitor p21WAF1 and p53 protein in endothelial cells of human aorta during replicative aging invitro. Bull Exp Biol Med 2002; 134: 81–3.
Minamino T, Mitsialis SA, Kourembanas S . Hypoxia extends the life span of vascular smooth muscle cells through telomerase activation. Mol Cell Biol 2001; 21: 3336–42.
Seimiya H, Tanji M, Oh-Hara T, Tomida A, Naasani I, Tsuruo T . Hypoxia up-regulates telomerase activity via mitogen-activated protein kinase signaling in human solid tumor cells. Biochem Biophys Res Commun 1999; 260: 365–70.
Meyerson, M, Counter CM, Eaton EN, Ellisen LW, Steiner P, Caddle SD, et al. hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell 1997; 90: 785–95.
Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB, et al. Extension of life-span by introduction of telomerase into normal human cells. Science 1998; 279: 349–52.
Hirotaka N, Toshihide N, Satoru K, Masaki I, Jerry WS, Keiichi I . Hypoxia-inducible factor 1 mediates upregulation of telomerase (hTERT). Mol Cell Biol 2004; 24: 6076–83.
Naka K, Tachibana A, Ikeda K, Motoyama N . Stress-induced premature senescence in hTERT-expressing ataxia telangiectasia fibroblasts. J Biol Chem 2004; 279: 2030–7.
Martin JA, Klingelhutz AJ, Moussavi-Harami F, Buckwalter JA . Effects of oxidative damage and telomerase activity on human articular cartilage chondrocyte senescence. J Gerontol A Biol Sci Med Sci 2004; 59: 324–37.
Iwasa H, Han J, Ishikawa F . Mitogen-activated protein kinase p38 defines the common senescence-signalling pathway. Genes Cells 2003; 8: 131–44.
Bulavin DV, Saito S, Hollander MC, Sakaguchi K, Anderson CW, Appella E, et al. Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV radiation. EMBO J 1999; 18: 6845–54.
Sanchez-Prieto R, Rojas JM, Taya Y, Gutkind JS . A role for the p38 mitogen-acitvated protein kinase pathway in the transcriptional activation of p53 on genotoxic stress by chemotherapeutic agents. Cancer Res 2000; 60: 2464–72.
Author information
Authors and Affiliations
Corresponding author
Additional information
Project supported by National Natural Science Foundation of China (No 30671073).
Rights and permissions
About this article
Cite this article
Zhang, Fx., Chen, Ml., Shan, Qj. et al. Hypoxia reoxygenation induces premature senescence in neonatal SD rat cardiomyocytes. Acta Pharmacol Sin 28, 44–51 (2007). https://doi.org/10.1111/j.1745-7254.2007.00488.x
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1111/j.1745-7254.2007.00488.x
Keywords
This article is cited by
-
c-Jun N-terminal kinase signaling in cellular senescence
Archives of Toxicology (2023)
-
Sedimentary facies and sequence stratigraphy of the Silurian at Tabei uplift, Tarim Basin, China
Arabian Journal of Geosciences (2016)
-
Hydrocarbon charging of the Ordovician reservoirs in Tahe-Lunnan area, China
Science China Earth Sciences (2013)
-
Relationship between the later strong gas-charging and the improvement of the reservoir capacity in deep Ordovician carbonate reservoir in Tazhong area, Tarim Basin
Chinese Science Bulletin (2009)