Effects Of C3G Knockdown On Rat Post-infarct Myocardial Remodeling And Its Underlying Mechanism

Objective:To investigate the effects of C3G[Crk SH3-domain-binding guanine nucleotide exchange factor]knockdown on rat post-infarct myocardial remodeling and its underlying mechanism.Methods:The non-target(NT CRISPR/Cas9),C3G CRISPR/Cas9 plasmids were constructed and packaged into lentiviruses respectively.C3G CRISPR/Cas9 lentiviruses were screened.In vivo,the myocardial infarction model of rats were constructed by ligating the left anterior descending coronary artery.The rats were divided into Sham + NT CRISPR/Cas9,Sham + C3G CRISPR/Cas9,MI + NT CRISPR/Cas9 and MI + C3G CRISPR/Cas9 groups.In vitro,the H9C2 cardiomyocytes and myocardial fibroblasts were infected with lentiviruses respectively.These cells treated with hypoxia were used as hypoxia model.The experiments were divided into NT CRISPR/Cas9,C3G CRISPR/Cas9,NT CRISPR/Cas9 + Hypoxia,and C3G CRISPR/Cas9 + Hypoxia groups.The cardiac structure and function of rats were tested by Doppler ultrasonography at 1 week and 12 weeks after myocardial infarction.After 12 weeks of myocardial infarction,the rats were sacrificed and the hearts and weight were collected.The histological changes were observed in the hematoxylin-eosin staining.Myocardial collage fibers were determined by Sirius red staining.The C3G mRNA and protein were detected by RT-PCR and immunohistochemistry.Relevant proteins were tested by Western blot.Cell proliferative rate was examined by CCK-8.Apoptotic rate were determined by flow cytometry and TUNEL.Results:In vivo,compared with the sham + NT CRISPR/Cas9 and sham + C3G CRISPR/Cas9 groups,the expression of C3G mRNA and protein,p-ERK1/2 and Bcl-2 proteins were increased(P<0.05),while the Bax protein was decreased(P<0.05),cardiomyocyte apoptosis and myocardial collage fibers were increased(P<0.05)and cardiac structural remodeling and function deteriorated in the MI + NT CRISPR/Cas9 and MI+ C3G CRISPR/Cas9 groups respectively.Compared with the sham + NT CRISPR/Cas9 and MI + NT CRISPR/Cas9 groups,the expression of C3G mRNA and protein,p-ERK1/2 and Bcl-2 proteins were decreased(P<0.05),while the Bax protein was increased(P<0.05),cardiomyocyte apoptosis and myocardial collage fibers were decresed(P<0.05)and the cardiac structural remodeling and function improved in the sham + C3G CRISPR/Cas9 and MI + C3G CRISPR/Cas9 groups respectively.In vitro,the expression of C3G mRNA and protein were absent in the C3G CRISPR/Cas9 and C3G CRISPR/Cas9 + Hypoxia groups.Compared with the NT CRISPR/Cas9 and NT CRISPR/Cas9 + Hypoxia groups,the expression of p-ERK1/2 and Bcl-2 proteins and cell proliferative rate were decreased(P<0.05),while the expressed Bax protein and the apoptotic rate were increased(P<0.05)in the C3G CRISPR/Cas9 and C3G CRISPR/Cas9+ Hypoxia groups.Compared with the NT CRISPR/Cas9 group,the expression of C3G mRNA and protein and p-ERK1/2 and Bcl-2 proteins and cell proliferative rate were decreased(P<0.05),while the expressed Bax protein and apoptotic rate were increased(P<0.05)in the NT CRISPR/Cas9 + Hypoxia group.Compared with the C3G CRISPR/Cas9 group,the expression of p-ERK1/2 and Bcl-2 proteins and cell proliferative rate were decreased(P<0.05),while the expressed Bax protein and the apoptotic rate were increased(P<0.05)in the C3G CRISPR/Cas9 +Hypoxia group.Conclusions:C3G knockdown can regulate the proliferation and apoptosis of H9C2 cardiomyocytes and myocardial fibroblasts and improve the cardiac remodeling after myocardial infarction through regulation of p-ERK1/2,Bcl-2 and Bax.