GW24-e2990 The expression of rock in cardiomyocte exposed to hypoxia and its role in cardiomyocte injury

Objectives Using cultured myocardial cells in vitro and establishing virtual hypoxia environment of cardiomyocyte apoptosis model, to observe the expression of Rock in myocyte exposed to different hypoxia phase and its role in the mechanism of apoptosis. Methods 1. Cardiomyocytes from the hearts of 1-3-day-old neonatal rats were prepared. 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 hours. Then exposed to it for 1 h,3 h,6 h,9 h, respectively. Select the time when Rock-1and Rock-2 expression the highest, put the PI3K/AKT blocking agent and Erk5 blocking agent into hypoxia liquid respectively, and then detect related indicators. 3. The cell apoptosis and viability were detected by flow cytometry and MTS respectively. The expression of Rock-1, Rock-2, p-Erk5, Eek5, Caspase-3, PI3k, p-Pi3k, GSK3β at different hypoxia phase were detected by western blotting. Results 1. After exposed to hypoxia liquid for 1 h, 3 h, 6 h, 9 h respectively, the rate of myocyte apoptosis increased and survival rates decreased. After 1 h, Rock-1, Rock-2 and Erk5 in hypoxia-ischaemia liquid began to rise, reached its peak in 3-6 hours, in 9 hours began to decrease, which were significantly higher than the control group (P < 0.01); Caspase-3, Gsk3β in hypoxia-ischaemia liquid, began to rise, which was sustained expression in a high state at following observed time. There was no significace of the expression of Pi3k in the course of hypoxia-ischaemia which was no statistical significance. But After 1 h, p-Pi3k in hypoxia-ischaemia liquid began to rise, reached its peak in 6 hours, in 9 hours began to decrease, which were significantly statistical significance between the two groups (P < 0.05); 3. Put PI3K/AKT blocking agent LY294002 (final concentration 20 umol / L) into liquid, pretreatment for 2h, and then, 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 group decreased significantly (P < 0.01). The expression of Caspase-3, Rock-1 and Rock-2 were also significantly higher compared with hypoxia 6 h group (P < 0.05) 4. Put Erk5 blocking agent LY294002 (final concentration 20 umol/L) into liquid, pretreatment for 2 h, and then, cardiomyocytes were treated in hypoxia liquid for 6 h, myocardial cell apoptosis compared with the group of hypoxia 6 hours was significantly higher (P < 0.01), the survival rate compared with hypoxia group decreased significantly (P < 0.01). The expression of Rock-1 and Rock-2 were also significantly higher compared with hypoxia 6h group. Conclusions 1. Rock-1 and Rock-2 are involved in the process of hypoxia-induced cardiomyocyte apoptosis, which may play an important role by inhibiting the PI3K/AKT pathways. 2. Erk5 perform anti-apoptosis effection through reducing the activation of Rocks. 3. Hypoxia can activate Gsk3β, Caspase-3, thereby increasing cardiac myocyte apoptosis. Hypoxia can promote cardiac myocyte apoptosis through activating Gsk3β and Caspase-3 expression.