Effectes And Mechanism Of Dock180 Knockout Recombinant Lentivirus On Ischemic Cardiomyopathy After Myocardial Infarction In Rats

Objective: To investigate the effects of dedicator of cytokinesis 1(Dock180)knockout recombinant lentivirus on ischemic cardiomyopathy after myocardial infarction in rats and their mechanisms.Methods: A single guide RNA(sgRNA)targeting rat Dock180 gene was designed and constructed using CRISPR/Cas9 system.A plasmid contained above sgRNA was packaged into lentivirus.Spregue Dawley(SD)male rats with ligation of the left anterior descending coronary artery were used as myocardial infarction(MI)model in vivo.Myocardium infected by this lentivirus,then SD rats were divided into sham+ negative lentivirus group(sham+Cas9),sham+Dock180 knockout group,MI+ negative lentivirus group(MI+Cas9)and MI+ Dock180 knockout group.Rat derived H9C2 cardiomyocyte was also infected by this lentivirus in vitro,a single clone of H9C2 cardiomyocyte with Dock180 knockout was established.Cardiomyocytes were treated with hypoxia,then Cardiomyocytes were divided into negative lentivirus group(Cas9),Dock180 knockout group,negative lentivirus hypoxia group(Cas9+hypoxia)and Dock180 knockout hypoxia group.Cardiac structure and function of SD rats was examined by Doppler ultrasonography at 12 weeks after operation,then rats were sacrificed and the cardiac specimens were collected.Hematoxylin-eosin staining,Sirius red staining and TUNEL were used to observe the histological changes in myocardium.The expression of Dock180 mRNA and protein were detected by RT-PCR and immunohistochemistry.Relevant proteins were detected by Western blot.The cell proliferation rate of the H9C2 cardiomyocytes was determined by MTT,and the apoptotic rate was measured by flow cytometry.Results: Compared with sham+Cas9 group and MI+Cas9 group,Dock180 mRNA and protein,p-ERK1/2 and Bcl-2 proteins expression were reduced(P<0.05),while Bax protein expression were increased(P<0.05),cardiac structure and function deteriorated,cell interstitial collagen volume fraction(CVF)and myocardial apoptosis were worsen(P<0.05)in sham+Dock180 knockout group and MI+ Dock180 knockout group respectively.In addition,Dock180 mRNA and protein were absent in Dock180 knockout group and Dock180 knockout hypoxia group.Compared with Cas9 group and Cas9+hypoxia group,p-ERK1/2 and Bcl-2 protein expression and cell proliferation were reduced(P<0.05),while Bax protein expression and cell apoptosis were increased(P<0.05)in Dock180 knockout group and Dock180 knockout hypoxia group respectively.Conclusions: Dock180 knockout recombinant lentivirus can inhibit cardiomyocytes survival and promoted apoptosis and cardiac pathologic ventricular remodeling,thus aggravated ischemic cardiomyopathy deteriorated after myocardial infarction via p-ERK1/2,Bcl-2 and Bax.