title: Hypoxia reoxygenation induces premature senescence in neonatal SD rat cardiomyocytes.
abstract: OBJECTIVE
  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 degree centigrade in a humidified atmosphere of 5% CO(2) and 95% air. The hypoxic cultures were incubated in a modular incubator chamber filled with 1% O(2), 5% CO(2), and balance N2 for 6 h. The reoxygenated cultures were subjected to 1% O(2) and 5% CO(2) 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. beta-Galactosidase activity was determined by using a senescence beta-galactosidase Staining Kit. p16( INK4a ) and telomerase reverse transcriptase (TERT) mRNA 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; p16( INK4a ) and TERT mRNA levels, beta-galactosidase activity, TERT protein expression and telomerase activities were all significantly increased (P< 0.01 or P< 0.05).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Cardiology, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China.
authors.forename: Feng-Xiang
authors.initials: FX
authors.lastname: Zhang
authors.forename: Ming-Long
authors.initials: ML
authors.lastname: Chen
authors.forename: Qi-Jun
authors.initials: QJ
authors.lastname: Shan
authors.forename: Jian-Gang
authors.initials: JG
authors.lastname: Zou
authors.forename: Chun
authors.initials: C
authors.lastname: Chen
authors.forename: Bing
authors.initials: B
authors.lastname: Yang
authors.forename: Dong-Jie
authors.initials: DJ
authors.lastname: Xu
authors.forename: Yu
authors.initials: Y
authors.lastname: Jin
authors.forename: Ke-Jiang
authors.initials: KJ
authors.lastname: Cao
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D015687:Cell Hypoxia; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000096:biosynthesis / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D010100:Oxygen / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D019098:Telomerase / Q000096:biosynthesis / Q000235:genetics; D013997:Time Factors; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16; D012333:RNA, Messenger; D019098:Telomerase; D001616:beta-Galactosidase; D010100:Oxygen