Effects And Mechanism Of Deubiquitinase BRCC36 On DNA Damage Repair And Myocardial Remodeling After Myocardial Infarction In Mice

ObjectiveTo explore the role of deubiquitinase BRCC36 protein in DNA damage repair and myocardial remodeling after myocardial infarction in mice and its possible mechanism.Methods1)We have constructed a transgenic mice model of cardiomyocyte specific over-expression of BRCC36(a-MHC-BRCC36)utilizing commercial services.We used PCR and Western blot to identify the positive mice.2)A classical model of myocardial infarction was established in mice by ligation of the left anterior descending branch of the coronary artery(LAD).The experiment was divided into four groups:Wide type Sham group,Wide type MI group,Transgenic sham group,and transgenic MI group.3)After myocardial infarction,we continued to feed the mice for 4 weeks.The survival rate of mice in each group was calculated,the cardiac function of mice was detected by echocardiography,and the infarct range of the mice was calculated by TTC staining.4)The apoptosis ratio of cardiomyocyte was detected by TUNEL staining and the expression of apoptosis related proteins was determined by Western blot.5)The expression of connective tissue growth factor(CTGF)and periosteal protein(Periostin)mRNA in extracellular matrix were measured by reall-time quantitative PCR(Real-time PCR).Myocardial fibrosis were detected by Sirius red staining and Masson trichrome staining method.The levels of Smad3 phosphorylation and the expression of collage-? in the extracellular matrix were determined by Western blot analysis.6)The expression of yH2AX and RAD51 level after MI were determined by Western blot analysis.Results1)The genotypes of transgenic mice were identified by PCR,and the positive mice were screened.The expression level of protein was detected by Western blot.It was found that the expression of BRCC36 protein in transgenic mice was significantly increased compared those in the wild type mice.2)Eelectrocardiogram analysis were carried out after myocardial infarction,and the ST segment was significantly elevated.Twenty-eight days after myocardial infarction,the survival rate of the mice in the transgenic operation group was significantly higher than that in the wild type mice(79%vs 53%).The area of myocardial ischemia was detected by TTC staining in mice after LAD operation.Compared with the wild type MI group,the area of myocardial ischemia was significantly reduced in the transgenic operation group.3)Compared with the wild type MI group,the cardiac function of the mice in the transgenic operation group was obviously improved.28 days after the LAD operation,the heart of the mice in the wild type MI group was significantly dilated compared with that in the transgenic MI group.The heart weight ratio of the mice in the wild type MI group was significantly higher than that in the transgenic MI group.4)By Sirius red staining and Masson staining,we found that myocardial fibrosis in mice was significantly increased.Compared with the wild type mice,the myocardial fibrosis of transgenic mice was significantly reduced.Through real-time quantitative PCR(Real-time PCR),we found that compared with wild type mice,the expression of connective tissue growth factor(CTGF)and periostin(Periostin)mRNA in transgenic operation group were significantly decreased.Also,the expression of collagen ? in extracellular matrix in TG MI group was significantly lower than that in WT MI group,and the expression of P-Smad3 was also significantly lower than that in WT MI group.5)Western blot detection showed that the expression of anti-apoptotic protein Bcl-2,p-Bad and Mcl-1 in transgenic surgical group were significantly higher than these in wild type operation group,and the expression of Pro-apoptotic protein puma and caspase-3 were significantly lower than that in wild type transgenic mice.6)Compared with the wild-type operation group,the expression of DNA damage marker yH2AX was significantly decreased,while the expression of DNA damage repair factor RAD51 increased significantly in the transgenic operation group by Western blot.ConclusionSpecific expressing BRCC36 in cardiomyocyte can significantly improve ventricular remodeling and improve cardiac function after myocardial infarction,which may be related to inhibition of TGF-?/Smad3 signaling and enhancement of DNA damage repair.