Hypoxia reoxygenation induces premature senescence in neonatal SD rat cardiomyocytes.
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.
Zhang
Feng-Xiang
FX
Department of Cardiology, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China.
Chen
Ming-Long
ML
Shan
Qi-Jun
QJ
Zou
Jian-Gang
JG
Chen
Chun
C
Yang
Bing
B
Xu
Dong-Jie
DJ
Jin
Yu
Y
Cao
Ke-Jiang
KJ
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
D019941:Cyclin-Dependent Kinase Inhibitor p16; D012333:RNA, Messenger; D019098:Telomerase; D001616:beta-Galactosidase; D010100:Oxygen