Mechanism Of Myocardial Cell Damage Induced By Coxsackievirus B3 Structural Protein VP2

Objective:To validate the pathogenic mechanism of structural protein VP2 of Coxsackies virus(CVB3)group B on myocardial cells briefly,providing a theoretical basis for studying the pathogenesis of viral myocarditis infected by CVB3.Methods:1.Construction of combinant adenovirus vetor: After the VP2 gene was amplified by RT-PCR and the APEX2 gene was amplified by PCR,the PCR product was inserted into the plasmid of pCAG.Then we constructed and packed the recombinational adenovirus vector pAdTrack-2Strep-Flag-VP2 and pAdTrack-2Strep-Flag-VP2-APEX2,obtaining the adenovirus VP2(Ad-VP2)and adenovirus VP2-APEX2(Ad-VP2-APEX2).Finally,the adenovirus titer was measured by RT-PCR.2.The rat heart tissues of neonatal within 1-3 days were taken and digested with 0.1%Trypsin-EDTA Solution.The pulsating Neonatal Rat Ventricular Cardiomyocytes(NRVCs)were collected by differential attachment for 2 hours.3.The changes of morphology were observed by microscope after NRVCs were infected with Ad-VP2(MOI=10).4.The cell activity was detected by CCK8 assay when H9C2 cells infected with Ad-VP2(MOI=10)at 0 h,24 h,48 h and 72 h.5.NRVCs were infected with Ad-VP2-APEX2(MOI=10),and proteins interacted with VP2 were screened by APEX assay.6.The apoptosis rates of H9C2 cells were detected by Annexin V/FITC method when H9C2 cells infected with Ad-VP2(MOI=10)at 24 h,48 h,72 h.7.The Reactive oxygen level was measured by DCFH-DA method when H9C2 cells infected with Ad-VP2(MOI=10)at 0 h,24 h,48 h,72 h.8.NRVCs were infected by Ad-VP2(MOI=10)and total cellular proteins were collected at different time points,and the expression of S100A6,c-Myc and ?-Catenin were detected by Western blot.Results:1.CVB3 VP2 were expressed successfully in recombinant adenovirus.2.After NRVCs were infected with Ad-VP2 for 5 days,a typical cytopathetic effect was observed.The results of CCK8 assay revealed that cell activity decreased compared with the Mock group.3.41 host proteins,which might interact with VP2 protein,were identified by APEX2.And S100A6 was further characterized to interact with VP2 by immunofluorescence and immunoprecipitation assay.4.Flow cytometry analysis revealed that the apoptosis rate increased after H9C2 cells infected with Ad-VP2 at 24 h,48 h and 72 h compared with the Mock group.5.After H9C2 cells were infected with Ad-VP2 for 0 h,24 h,48 h and 72 h,intracellular ROS levels were increased,and the ROS level reached the peak in 48 h compared with the Mock group.6.With the prolongation of Ad-VP2 infection time,gradually increasing expressions of S100A6,c-Myc and ?-catenin were detected in total protein,respectively.Conclusions:It was confirmed that CVB3 VP2 may induce cytopathic effects in myocardial cells through the following two ways: VP2 interacts with the calcium-binding protein S100A6 and activates the ?-catenin/c-myc axis.It also leads to intracellular oxidative stress injury that leads to myocardial cells injury and apoptosis.