Hypoxia reoxygenation induces premature senescence in neonatal SD rat cardiomyocytes

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 degrees C in a humidified atmosphere of 5% CO2 and 95% air. The hypoxic cultures were incubated in a modular incubator chamber filled with 1% O-2, 5% CO2, and balance N-2 for 6 h. The reoxygenated cultures were subjected to 1% O-2 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. beta-Galactosidase activity was determined by using a senescence beta-galactosidase Staining Kit. p16(INK4a) 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 ELISA(plus) 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). 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.