ITRAQ-based Comparative Proteomics Of Mice Ischemic Myocardial Injury Model And Mechanism Research Of Vitamin D Binding Protein Involved In Myocardial Injury

Objective: To find early changes of comparative proteomics responsible for LV remodeling after acute myocardial infarction(AMI)and provide the new idea for myocardial protection after AMI.1)Adopt the technology with isobaric tags for relative and absolute quantitation(iTRAQ)based on proteomics,investigatingand choosing the performing of proteome changes in mice AMI model in high volume and efficiency.2)In mice ischemic myocardial injury model,the validation of candidate target protein expression was processed by molecular biology method.3)In cell model,target protein functional verification was processed by gene transfection technology.Methods:(1)Male C57B/6J mice were divided into 4groups: Sham-3d,Sham-7d,MI-3d and MI-7d.The left coronary artery was identified and ligated to construct mice AMI model.After modelling,the method of transthoracic echocardiography,hemodynamic assessment and HE staining were adopted to assess cardiac function and ventricular remodeling.Protein extracted and relative quantification by iTRAQ technology and bioinformatics analysis were performed to identify differentially expressed protein subcellular localization and functionfor the preliminary analysis.(2)Further,in animal tissues,pathologic histology were adapoted for detection,assessing the situation of cardiac function and ventricular remodeling,differentially expressed proteins were described their subcellular localization,the process of cell biology and molecular biology function.(3)In mice ischemic myocardial injury model,pathohistologicalexamination were performed to assess performance of early myocardial fibrosis after AMI.And qRT-PCR and Western Blot were adopted to assess mRNA and protein expression levels of candidate target protein.In hypoxia/reoxygenation myocardial cell injury model,the expressions of VDBP were observed in different point(0h,6h,12 h,24h,48 h and 72h).Then,we used MTS,Tunel,Annexin V/PI and flow cytometry techniques to assess apoptosis in VDBP overexpression H9C2 cardiomyocytes by plasmid transient transfection.Further,we used qRT-PCR and western blot techniques to verify VDBP signaling pathway involved in the early process of myocardial injury.Results: 1)After construct mice AMI model,IS%in MI-3d and MI-7d groups were 45.29±7.52%and 44.83±7.36%.Compared with Sham groups,LVW/TL increased(P<0.001);compared with Sham-7d group,LW/TL increased in MI-7d group(P<0.001).Compared with Sham-3d group,LVESD、LVEDD and ExLVDd increased significantly in MI-3d group;compared with Sham-7d group,LVESD、LVEDD and ExLVDd increased significantly,LVW were thinner,and FS%decrease significantly by echocardiography(P<0.001).Compared with Sham-3d group,SBP,MAP,LVSP,LVDP,dP/dtmax and dP/dtmindcreased in MI-3d group(P<0.05);compared with Sham-7d group,SBP、LVhinnerDP 、 dP/dtmax and dP/dtmindcreased in MI-7d group by hemodynamic assessment(P<0.01).Hematoxylin and eosin(H&E)staining images of heart tissue samples from C57B/6J mice after surgery and sham-operation indicated that the animal model was successfully constructed.Left ventricular histology for MI-3d showed lots of necrotic myocardial cells,a glut of inflammatory cells,missed regular striated pattern in myocardial infarction area compared with sham-3d.Left ventricular histology in MI-7d showed myocardial swelling,myocardial fibers necrosis and interstitial edema in myocardial infarction area.A total of 2540 detected protein groupsby iTRAQ technique.Of the 2540 protein groups,776 were considered as significantly differentially accumulated proteins by iTRAQ technique.Gene ontology annotation of the 776 DAPs were obtained by Blast2 GO and the enriched GO terms were determined by agriGO.Wnt signal pathway,ERS signal pathway,VDBP signal pathway and actin signal pathway were identified involving in ventricular remodeling in AMI mice.2)Masson staining images of heart tissue samples from C57B/6J mice after surgery indicated myocardial fibrosis increased in MIgroups compared with Sham-groups.Furthermore,histological analysis of type I and III collagen content showed collagen content of two types increased(P<0.01)after AMI.Compared with Sham-3d group,relative expressions of LDH、cTNT and BNP increased compared with MI-3d group(P<0.01).Compared with Sham groups,relative expressions of LDH、cTNT and BNP increased in MI groups(P<0.05).Candidate target proteins in Wnt signal pathway,ERS signal pathway,VDBP signal pathway and actin signal pathway were activated in MI-3d and MI-7d groups.The protein expression of VDBP,VDR and GSN increased obviously in MI groups compared with Sham groups(P<0.001).3)Hypoxia/reoxygenation myocardial cell injury model was constructed successfully using cell line H9C2 to further validate the expression pattern of VDBP during acute myocardial infarction.The percent of cell survival decreased gradually at 6h,12 h,24h,48 h and 72 h after Hypoxia/reoxygenation treatment.Consistent with the mRNA levels,the protein levels of VDBP were significantly higher at 6h and 12 h of OGD treatment.Construction of VDBP overexpression H9C2 cardiomyocytes by plasmid transient transfection to assess cardiomyocytes injury by MTS,Tunel and flow cytometry.Cell apoptosis increased in VDBP overexpression H9C2 cardiomyocytes by MTS,Tunel and Annexin V/PI.Relative VDR expression decreased obviously(P<0.01)and relative GSN,Caspase3 expression increased(P<0.001)by qRT-PCR.Western Blot also verified these results.Conclusion: In this study,we used iTRAQ technique to identify and screen differential proteins,according to the verification by molecular biological technique,observing the process ofwnt signal pathway,ERS signal pathway,VDBP signal pathway and actin signal pathway involving in ventricular remodeling in AMI mice.Then,we used construction of VDBP overexpression H9C2 cardiomyocytes by plasmid transient transfection to verify the mechanism of VDBP overexpression injuring myocardial cell.And we found VDR expression decreased and GSN expression increased in VDBP overexpression H9C2 cardiomyocytes.Last,we verified VDBP involved in myocardial apoptosis by caspase 3 by construction of VDBP overexpression in H9C2 cardiomyocytes.Our data provide new insights into proteome changes after AMI and indicate thatVDBP might involving in left ventricle remodeling and cardiac dysfunction after AMI.