The Role Of Bim And PI3K/AKT In The Hypothermia-induced Cardiomyocytes Damage

Objective:To explore the effect of hypothermia on the cardiomyocytes apoptosis and the level of LDH activity, as well as the role of pro-apoptosis protein Bim and PI3K/AKT signal-transduction pathway.Methods:l.The hearts of 1to3-day-old SD neonatal rats were harvested and then digested using 0.08% trypsin and 1.00%Ⅱcollagenase. Adding 0.1mM 5'-Brdu to suspension to inhibit fibroblast cell's growth after 2h adhesion. The cells were seeded in the 35mm culture plate according to the density of 1×106/ml.When the cardio-myocytes were joined together and synchronous pulsation, then the experiment was began.2.To construct the apoptosis model of cardiomyocytes, the myocytes were exposed in vitro to 4℃cold environment for 60min, then cultivated on 37℃CO2 incubator for 0h,4h,8h,12h,16h,24h. Choosing the time point when Bim began to express, the inhibitor of PI3K/AKT, LY294002 was added to cardiomyocytes before hypothermia intervention. Then, the related parameters were measured again.3.The apoptosis rate was measured by flow cytometer; LDH activity was determined with automatic biochemistry analyzer. Expressions of Bim and PI3K were determined by Western blotting.Results:1.Immediately after cold stress, the apoptosis rate and the LDH activity in hypothermia group were significantly increased compared to the control group (20.01±1.43vs6.79±1.50,p<0.05,43.30±2.50vs18.13±1.57,p<0.05 respectively). The apoptosis rate was increased following the cultivated time after rewarming which was conformity to the changes of LDH. 2.The expressions of Bim in the cardiomyocytes after cold stress increased compared to the control (P<0.05).The Bim expressions in all control groups were not statistically significant.3. The apoptosis rate in LY294002 group was significantly increased compared to the pure hypothermia group(P<0.01). p-PI3K expression was increased in the pure hypothermia group than in the LY294002 and control groups.There was no significant difference in PI3K expression in all groups (P>0.05).Conclusion:Cardiomyoctyes apoptosis could be induced by hypothermia. The Bim was likely to participate the apoptosis process and played the role through PI3K/AKT signal-transduction pathway.