The Expression Of ROCK In Cardiomyocyte Exposed To Hypoxia And Its Role In Cardiomyocyte Apoptosis

OBJECTIVETo investigate the expression of ROCK at different hypoxia phase and explore its role in the mechanism of apoptosis of myocardial cell through the establishment of cardiomyocytes model in vitrolMETHODS1. Cardiomyocytes from 1-3-day-old neonatal rats were prepared with 0.08% trypsin and 1.00% II collagenase digestion, suspension by the differential adhesion 2h, was added 0.1mM 5'-Brdu to inhibit fibroblast cell's growth, apply cell density of 1×106/ml seeded in 35mm culture dishes, until the cardiomycytes converged to synchronous pulsation, set about the experiment.2. Established myocardial cell apoptosis model ,prepared the simulated hypoxia liquid which have been deoxygenated by bubbling with the mixture of 95% nitrogen and 5% carbon dioxcide for 1 hour. Replaced normal medium with hypoxia liquid, exposed cells to it for 1h,3h,6h,9h respectively. Normal Cardiomyocytes were regarded as control group.3. Selected the time when ROCK-1 and ROCK-2 expression is the highest, put the Erk5 blocking agent into hypoxia liquid, then detected related indicators.4. The cell apoptosis and viability were detected by flow cytometry and MTS respectively. The expression of ROCK-1, ROCK-2, Erk5,p-Erk5, Caspase-3, PI3k, p-Pi3k at different hypoxia phase were detected by western blotting.RESULTS1. After being exposed to hypoxia liquid for 1h, 3h, 6h, 9h respectively, the rate of myocyte apoptosis were as follows : 8.76±1.51%, 15.36±2.34%, 26.50±3.43%, 41.96±4.22%.Compared with the control group(2.6±0.34%) (P<0.01); survival rates were as follows :93.20±4.12%, 86.14±3.10%, 75.53±7.25%,60.21±6.75%, Compared with the control group (97.60±1.12)% (P <0.05).2. After 1h ,ROCK-1 and ROCK-2 began to rise, reached its peak at 3-6 hours, at 9 hours began to decrease, which were significantly higher than the control group (P <0.05).3. After 1h ,Caspase-3 began to rise, which was sustained expression in a high state at following observed time, all compared with the control group (P <0.01).4. There was no significace of the expression of Pi3k in the course of hypoxia.But after 1h , p-Pi3k began to rise, reached its peak at 3 hours, at 6 hours began to decrease, at 9 hours almost can't be detected.There were significantly statistical significance between the two groups (P <0.05).5. After 1h , Erk5 in hypoxia liquid began to rise, reached its peak at 3-6 hours, at 9 hours began to decrease, all compared with the control group (P <0.05).6. Put Erk5 blocking agent PD98059 (final concentration 20umol / L) into liquid,pretreated for 2h.cardiomyocytes were treated in hypoxia liquid for 6h , myocardial cell apoptosis compared with the group of hypoxia 6 hours was significantly higher (P <0.01), the survival rate compared with hypoxia 6 hours group decreased significantly (P <0.01). the expression of ROCK-1 and ROCK-2 were also significantly higher than hypoxia 6h group (P <0.05).CONCLUSION1. After hypoxia,the expression of ROCK-1 and ROCK-2 in hypoxia-ischemia liquid began to rise,and are in parallel correlation with cardiomyocyte apoptosis, and may be involved in myocardial apoptosis which induced by hypoxia.2. ROCKs are involved in the process of hypoxia-induced cardiomyocyte apoptosis , which may play an important role by inhibiting the p-PI3K pathways.3. Erk5 perform anti-apoptosis effection through reducing the activation of ROCKs4. ROCKs can promote cardiac myocyte apoptosis through activating Caspase-3 expression.