Hypoxia reoxygenation induces premature senescence in neonatal SD rat cardiomyocytes
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) 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; 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.
Keywords:
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) 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; 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.