Role of mTOR in reduction of oxygen-glucose deprivation and restoration-induced injury to isolated cardiomyocytes by hypoxic preconditioning in neonatal rats

Abstract

Objective To evaluate the role of mammalian target of rapamycin （mTOR ） in reduction of oxygen-glucose deprivation and restoration （OGD/R ）-induced injury to isolated cardiomyocytes by hypoxic preconditioning （HPC） in neonatal rats .Methods Primary cardiomyocytes were obtained from neonatal Sprague-Dawley rats （1-3 days after birth ） ,and seeded in 96-well plates at the density of 1 × 105 cells/ml （200μl/well ） . The cells were randomly divided into 6 groups （ n=6 each ） using a random number table：control group （group C） ,rapamycin group （group R） ,group OGD/R ,rapamycin＋OGD/R group （group R＋OGD/R）;group HPC＋OGD/R ,and rapamycin＋HPC＋OGD/R group （group R＋HPC＋OGD/R） .Rapamycin （final concentration 20 nmol/L） was added to the culture media and the cells were then incubated for 1 h in group R .In R＋OGD/R group ,rapamycin （final concentration 20 nmol/L ） was added to the culture media ,the cells were then incubated for 1 h ,and then OGD/R was performed .In HPC＋OGD/R group ,OGD/R was performed immediately after HPC was completed .In R＋HPC＋OGD/R group ,rapamycin （final concentration 20 nmol/L） was added to the culture media ,the cells were then incubated for 1 h , and then HPC and OGD/R were performed . All the cells were subjected to OGD for 2 h followed by restoration of O2-glucose supply for 3 h to induce OGD/R .HPC was induced by 3 cycles of 10 min hypoxia followed by 30 min reoxygenation .The cell viability （using MTT assay ） and lactic＆nbsp;dehydrogenase （LDH） activity in the culture medium were determined .Results Compared with group C ,the cell viability was significantly decreased , and LDH activities in the culture medium were increased in OGD/R , R＋OGD/R ,HPC＋OGD/R and R＋ HPC＋OGD/R groups （ P0.05） .The cell viability was significantly higher ,and LDH activity in the culture medium were lower in HPC＋OGD/R group than in OGD/R group ,and in R＋HPC＋OGD/R group than in HPC ＋ OGD/R group （ P〈 0.05 ） .Conclusion mTOR is involved in HPC-induced attenuation of OGD/R injury to isolated cardiomyocytes in neonatal rats . Key words: Anoxia; Ischemic preconditioning; Myocardial reperfusion injury; Mammalian target of rapamycin