The Effect Of ROCK1 And ROCK2 Silencing By ShRNA On Apoptosis Induced By Hypoxia In Rat Cardiomyocyte

OBJECTIVEThe model of cardiomyocyte apoptosis was prepared through simulation of hypoxia environment in vitro. ShRNAs(short hairpin RNA) targeting ROCK1 and ROCK2 were transfected into rat cardiomyocytes to downregulate the expression of ROCK1 and ROCK2.Cardiomyocyte apoptosis was observed. The effect of ROCK1 and ROCK2 on apoptosis induced by hypoxia and its signaling mechanism were studied in rat cardiomyocyte.METHODS1.Rat cardiomyocytes were cultured primarily and identified by using antibody targetingα-actin of striated muscle.2.Cardiomyocytes were subjected to hypoxia for different time:0 hours,1 hours,3 hours,6 hours and 9 hours. Then, the protein of ROCK1 and ROCK2 was detected by Western Blotting to confirm the time with the highest expression of ROCK1 and ROCK2.3.Three recombinant plasmids of ROCK1-shRNA and ROCK2-shRNA were constructed respectively and identified.4.ROCK1-shRNA and ROCK2-shRNA were transfected transiently into cells by liposome.After 48 hours, the protein of ROCK1 and ROCK2 was isolated and detected through Western Blotting.The shRNAs with the best silencing efficiency were selected from these shRNAs.5.ROCK1-shRNA and ROCK2-shRNA with the best silencing efficiency were transfected into cardiomyocytes. After 48 hours, these cells were subjected to hypoxia. There were five groups in the experiment:Blank Control Group, Hypoxia Group, Hypoxia+Negative Control shRNA Group, Hypoxia+ROCK1-shRNA Group, Hypoxia+ROCK2-shRNA Group. The expression of green fluorescent protein was observed through fluorescence microscopy.6.Followed by treatment with transfection and hypoxia, cardiomyocyte beat frequency and rhythm were assessed using microscopy, the content of lactate dehydrogenase(LDH) in cell culture fluid was detected by automatic biochemical analyzer, cell survival rate was determined with MTT, cell apoptosis rate was assessed by using flow cytometry, Western Blotting was used to determine the expression of Caspase3,p-PI3K and PI3K.RESULTS1.It was confirmed that rat cardiomyocytes were cultured successfully.2.The expression of ROCK1 and ROCK2 was the highest in cardiomyocytes subjected to hypoxia for 6 hours.3.The transfection of both ROCK1-shRNAs and ROCK2-shRNAs inhibited the expression of ROCK1 and ROCK2 obviously (P<0.05 or P<0.01).Among these shRNAs, ROCK1-shRNA1 and ROCK2-shRNA2 have the best silencing efficiency (P<0.05). The silencing efficiency of ROCK1-shRNA1 was 82.15%±7.53%,while ROCK2-shRNA2 was 89.42%±5.81%..4. Green fluorescences were observed in the cells transfected with shRNA, indicating that these recombinant plasmids were transfected successfully.5.Compared to Blank Control group, hypoxia made the beat frequency of cardiomyocyte slow and the rhythm disorder (P<0.01); compared to Hypoxia group, the beat frequency of Hypoxia+ROCK1-shRNA Group and Hypoxia+ ROCK2-shRNA Group was elevated obviously,and the rhythm was more regular (P <0.05 or P<0.01)6.Compared to Blank Control group, hypoxia increased the content of LDH (P <0.01); compared to Hypoxia group, the content of LDH of Hypoxia+ ROCK1-shRNA Group and Hypoxia+R0CK2-shRNA Group was inhibited obviously (P<0.01)7.Compared to Blank Control group, hypoxia decreased cell survival rate (P <0.05); compared to Hypoxia group, the cell survival rate of Hypoxia+ ROCKl-shRNA Group and Hypoxia+ROCK2-shRNA Group was elevated obviously (P<0.05)8.Compared to Blank Control group, hypoxia increased cell apoptosis rate (P <0.01);compared to Hypoxia group, the cell apoptosis rate of Hypoxia+ ROCK1-shRNA Group and Hypoxia+ROCK2-shRNA Group was inhibited obviously (P<0.05 or P<0.01)9.Compared to Blank Control group, the expression of Caspase3 was induced by hypoxia, while the expression of p-PI3K was inhibited by hypoxia (P<0.05 or P <0.01); compared to Hypoxia group, the expression of Caspase3 in Hypoxia+ ROCK1-shRNA Group and Hypoxia+ROCK2-shRNA Group was inhibited obviously, and the expression of p-PI3K was elevated obviously(P<0.05 or P<0.01). The expression of PI3K wasn't changed.CONCLUSIONS1.Hypoxia can up-regulate the expressions of ROCK1 and ROCK2 in cardiomyocyte 2.The expression of ROCK1 and ROCK2 in cardiomyocyte can be inhibited effectively by the transfection of ROCK1-shRNA and ROCK2-shRNA.3.ROCK1 and ROCK2 are involved in the process of cardiomyocyte apoptosis induced by hypoxia.4.It may be through Caspase3 and p-PI3K that apoptosis is influenced by ROCK1 and ROCK2.