The Efficacy And Mechanism Of Recombinant VEGF Plasmid On Primary Cardiomyocytes And Role Of 7T MR Quantitative Assessment Myocardial Tissue Characterization In A Rat Model After Myocardial Infarction

Part one The Efficacy and Mechanism of pcDNA 3.1（+）/VEGF121 on Primary CardiomyocytesAim:To investigate the transfection and expression of pcDNA 3.1（+）/VEGF121 recombinant plasmid in cardiomyoctes apoptosis.Methods:Cardiomyocytes were incubated with media either control vehicle or other treatments(pcDNA 3.1（+）/VEGF121,inhibitor of Ca²⁺,10μmol/Linhibitor of CaSR,inhibitor of calpain),the treatments were performed with pcDNA 3.1（+）/VEGF121.Western-blot was used to detect the expression of VEGF gene after transfection.MTT assay and LDH leakage rate were used to detect cardiomyocytes injury.Apoptosis was evaluated by TUNEL assay and Annexin V-FITC conjugated with PI Staining.At last,CaSR,tBid as well as AIF were evaluated by Western-blot,respectively.Results:There were significantly increased of VEGF protein expression in cardiomyocytes after transfection.pcDNA 3.1（+）/VEGF121 decreased the expression of CaSR、calpain、AIF as well as tBid.Calpain,Bid cleavage and caspase-3 activity were inhibited by NPS2390 and Calpeptin but not CdCl2.Furthermore,treatment with NPS2390 not only attenuated the tBid but also the calpain protein expression.It showed the effect on inhibit the proferation of fibroblast after VEGF gene transfection.Conclusion:VEGF protein expression can be obtained in the cardiomyocytes after pcDNA 3.1（+）/VEGF121 transfection,which decreased apoptosis in rat cardiomyocytes through inhibition of CaSR and calpain then promotion of tBid and AIF release,thereby the CaSR/calpain as a potential promising target of intervention in human heart damage.Part two Role of 7T MR Quantitative Assessment Myocardial Tissue Characterization in a Rat Model of Myocardial infarctionAim:To investigate quantitative assessment myocardial tissue characterization in a rat model of myocardial infarction based on 7T MR.Methods:20 SD rats were randomly divided into 2 groups as follows:the sham group（n=10）and the model group of myocardial infarction by left anterior descending branch ligation（LAD）（n=10）.Both of these animal groups were imaged on a 70 Tesla system with Cine-imaging,T2-mapping and late gadolinium enhancement images（LGE）after 24-h.Finally,the rats were sacrificed,and the infarct-core size（IS）and AAR were identified using TTC)and HE staining,respectively.Results:The AAR was defined by novel faster T2-mapping,which was significantly higher than that of the sham group.On a slice-by-slice basis,there was no remarkable T2 value in the T2maps in the entire heart of the sham group（P>0.05）.No significant difference was observed in infarction-core size（IS）defined by LGE and TTC staining in the model group,and there was no significant difference in AAR defined by T2-mapping and HE staining.T2-mapping can also characterize the IMH as a phenomenon resulting from the area of hypointensity in the hyperintensity involving infarct-core zone.Conclusion:Quantification of salvaged myocardial size（SMZ）and intramyocardial hemorrhage（IMH）is feasible in rat models after myocardial infarction on 7T MRI,as well as quantification of IMH.Part three The efficacy of gene transfer of pcDNA 3.1（+）/VEGF121 into a rat model of infarction by 7T MRIAim:The study aimed to investigate the efficacy of gene transfer of pcDNA 3.1（+）/VEGF121 into a rat model of infarction by 7T MR.Methods:24 SD rats were random divided into 2 groups:Model group（underwent myocardial infarction by ligation of LAD）,pcDNA 3.1（+）/VEGF121 group（underwent with VEGF gene infusing into coronary artery at the some time of ligation LAD）.LV functional parameters were evaluated by Cine-CMR.All animals were imaged and detected by AAR,MIC,SMZ on a 7T MRI with Cine-imaging,T2-mapping and late gadolinium enhancement images（LGE）at 24h,48h,72h,7day.Finally,the rats were sacrificed,and the infarct-core size（IS）and AAR were identified using TTC),Masson,HE staining,as well as immunohistochemistry respectively.At last,VEGF,CaSR and caspase-3were evaluated by Western blot.Results:The VEGF gene was confirmed by Western-blot analysis.Cine MRI demonstrated pcDNA 3.1（+）/VEGF121 group decreased MIC and SMZ compared with Model group,MIC and SMZ were also decreased time-dependently in both groups.But there were not significantly differences in day-7 on AAR and T2 value.Furthermore,CaSR and caspase-3 protein expression were significantly decreased in gene group.7 days later,the CD31 positive cells and collagen hyperplasia were positive lower by anti-CD31 stain in gene group compared with model group.Conlusion:It confirmed the efficacy to transfter pcDNA 3.1（+）/VEGF121 into myocardium.The rat model can be measured with 7T MRI using Cine-MRI,T2-mapping and LGE,provided that SMZ and MIC were adjusted.pcDNA 3.1（+）/VEGF 121 protect myocardium maybe related with decresed apoptosis,increased capillary number,as well as decreased collagen expression.