Screening Of Dilated Cardiomyopathy Transgenic Mouse Model Of Cardiomyopathy Modifier Genes

Objective To establish the transgenic mouse of cTnT^R141W gene to make an animal model of dilated cardiomyopathy.Methods A transgenic plasmid was constructed by inserting the cTnT^R141W gene driven by theα-MHC promoter and the genotyping of the transgenic mice was detected by the polymerase chain reaction(PCR).The copy number of the transgenic gene was determined by Realtime PCR and the expression level of the gene was determined with Northern blotting and confirmed by HpaⅡrestriction endonuclease digestion Of reverse transcriptional PCR product made with a pair of primers amplifying both the transgenic and endogenous gene.Pathologic changes were observed by light microscopy and transmission electronic microscopy and analyzed with echocardiography.The localization of the mutant human cTnT protein was detected by Immunohistochemistry.The hypertrophy markers,Nppa,Nppb,Actal, and Atp2a2 were analyzed by reverse transcriptional PCR.Survival data of the experimental mice were recorded.Results Transgenic mice carrying the cTnT^R141W mutation were established and the copy number of the transgenic gene was found to be 15,20 and 59 in the three respective lines identified.The expression level of the cTnT^R141W was 1.5-to 2.0-fold that of the endogenous cTnT gene.The heart of the cTnT^R141W transgenic mouse exhibited a thinner ventricular wall and an enlarged ventricular chamber when compared with these aspects of the wild type mouse under the light microscope.Interstitial fibrosis was also observed in the 4 months old cTnT^R141W heart tissue.In the transgenic heart tissue,the elongated and lysed myofrils were observed under the transmission electronic microscope.The mutant human cTnT protein was'assembled in the sarcomere of the transgenic heart.The ejection fraction (EF%),fractional shortening(FS%) and movement of the ventricular wall were significantly decreased in the cTnT^R141W mice.Furthermore,the LV volume was increased,and EF%and FS%were decreased continuously in the age range of 2 to 8 months.Immature death occurred after 4 months of age and the immature death rate was 11.1%before 8 months of age in the cTnT^R141W mice.Nppb was shown to be increased 29%,and Actal increased 4 fold,however Atp2a2 was decreased 58% in transgenic heart.Conclusions The expression of mutant cTnT^R141W in the mouse heart caused ventricular chamber enlargement,systolic dysfunction,myocardial hypertrophy,and interstitial fibrosis.This mouse model can display a similar pathologic phenotype with the human DCM.Our results suggest that the cTnT^R141W gene is a causal factor for DCM and that the cTnT^R141W transgenic mouse is a useful animal model for the study of human DCM. Objective To compare the transgenic mice of cTnT^R92Q gene and cTnT^R141W gene and screen the modifier genes in the pathogenesis of hypertrophic cardiomyopathy and dilated cardiomyopathy.Methods The cardiac structure and function of the transgenic mice of cTnT^R92Q and cTnT^R141W were compared with M-mode echocardiography and by light microscopy and transmission electronic microscopy oberservation.The localization of the mutant human cTnT protein was detected by Immunohistochemistry.The gene expression patterns of cTnT^R92Q and cTnT^R141W hearts were analyzed using Gene expression microarray analysis and confirmed by reverse transcriptional PCR.Results The cTnT^R920 transgenic mice showed hypertrophic ventricular wall, reduced ventricular chamber,myocyte disarray,interstitial fibrosis,hypertrophic cardiomyocytes,increased Ejection fraction(EF%) and increased Fractional shortening(FS%) and decreased LV volume.However the cTnT^R141W transgenic heart exhibited a thinner ventricular wall,an enlarged ventricular chamber, interstitial fibrosis,elongated and lysed myofrils,decreased EF%,decreased FS% and increased LV volume.The two different mutant human cTnT proteins were also showed different localization in the sarcomere.Gene expression microarray analysis revealed that more than 1200 genes were showed opposite regulation compared the two transgenic hearts.The opposite regulated genes included the genes of sarcomeric proteins,cytoskeletal proteins,calcium-regulation proteins,ion channel proteins and extracellular matrix proteins,the genes associated with signal transduction and unknown genes.Thirty of them were verified by RT-PCR.Conclusions The expression of mutant cTnT^R92Q and cTnT^R141W iri the mouse heart caused opposite pathologic phenotypes.The gene expression microarry analysis using the same genetic background mice models indicated that a certain number of genes were regulated in an opposite way.The extracellular factor genes and receptor genes,as Gas5,Riokl,Askl,Meoxl,Ryk,Dkk3,and Wif-1 were strongly showed different expression in the opposite model hearts.These results suggest that those genes could be the important modifer genes,which caused the myocyte differentiation in a different way and play role in the HCM and DCM. Objective To investigate the function of the heparin-binding EGF-like growth factor (HB-EGF) on heart.Methods The expression level of HB-EGF was detected by Western blotting between the wild type and DCM transgenic hearts.The pathologic changes of DCM transgenic heart were observed under the microscope,and the expression of collagenⅠand collagenⅢwas detected by Realtime PCR.The HB-EGF transgenic mice were produced by microinjection method,and the expression of HB-EGF was detected with PCR and Western blotting.The histology of the HB-EGF transgenic heart was observed,and the expression of collagenⅠand collagenⅢwas also detected.The primary mouse cardiac fibroblasts were cultured,then stimulated with the recombinant human HB-EGF.The proliferation of primary mouse cardiac fibroblasts was examined by BrdU staining.The expression of collagenⅠand collagenⅢwas detected by Realtime PCR,and the phorsphoralation of ERK1/2 and JNK were detected by western blotting.The structure and function of the HB-EGF transgenic heart were analyzed with M-mode echocardiography.The proliferation of cardiomyocytes of HB-EGF transgenic mice was examined by BrdU staining.The total cell number of heart was counted according to the genomic DNA amount from the heart tissue.The phosphoraltion sites of ERK1/2 and JNK in wild type and transgenic hearts were detected by two-dimensional gel electrophoresis.The recombinant human HB-EGF stimulated the H9c2(2-1) cells,and then the phosphoraltion of ERK 1/2 and JNK were detected by westem blotting.Results The expression level of HB-EGF was decreased in the wild type heart with aging while the expression level of HB-EGF was increased in the heart with development of the DCM following increased interstitial content and increased collagenⅠand collagenⅢby 1.7-fold and 1.6-fold.The HB-EGF transg'enic C57BL/6J mouse was established for invetagation of the effect of HB-EGF on heart, which showed increased interstitial fibrosis,increased expression of collagenⅠand collagenⅢby 1.8-fold.Isolated the mouse cardiac fibroblasts and stimulated with the recombinant human HB-EGF,the primary cardiac fibroblasts increased the expression of collagenⅠand collagenⅢfor 30 min and proliferated for 24h after stimulation.A strong phosphorylation both of ERK1/2 and JNK was also induced by the stimulati6n at 10 min.Compared with the wild type heart,the HB-EGF transgenic heart showed enlarged ventricular chamber with increased proliferation of cardiomyocytes and resulted in the increasing of total cell number of heart.The phosphoraltion sites of ERK1/2 and JNK in wild type and transgenic hearts were different.While the rat cardiac myocytes cell line,H9c2(2-1) cell,was also showed a strong phosphorylation of ERK1/2 and JNK following the HB-EGF sitimaulation for 10 min.Conclusions The results indicated that HB-EGF was involed in the development of the DCM.The HB-EGF increased the interstitial fibrosis and the expression of collagenⅠand collagenⅢ.The HB-EGF was also stimulated the proliferation of the myocytes and resulted in the increasing of total cell number of heart.The results suggest that the HB-EGF play roles in heart through the MAPKs signaling pathway. Objective The transgenic mice of DsRed-Express gene was constructed for establishment of the mouse model,which will be used for in vivo fluorescence imaging analysis.Methods The transgenic plasmid was constructed by inserting the DsRed-Express gene under the control of chickenβ-actin powerful promoter.The transgenic mice were genotyped by PCR.The expression of the DsRed-Express gene was observed under in vivo fluorescence imaging system and fluorescent microscope.Results Three lines of DsRed-Express transgenic C57BL/6J mice were established. The red fluorescent protein was expressed in a certain number of tissues,such as heart,liver,spleen,-pancreas,kidney,lung and uterus in the DsRed-Express transgenic mouse.Conclusions The red fluorescent expression analysis indicated that the DsRed-Express transgenic mouse was established and it will be an important mouse model for in vivo fluorescence imaging analysis. Objective The nervous tissues specific expressed green fluorescent protein transgenic mouse was to establish and to make an animal model for the research of nervous system by fluorescent imaging.Methods The transgenic plasmid was constructed by inserting the EGFP gene under the control of PDGF B-chain promoter and the transgenic C57BL/6J mice was established by microinjection method.The transgenic mice were genotyped by PCR. The total of 48 pairs of freezing sections of transgenic mice were prepared in the sagittal plane,and the every two adjacent sections were observed under microscopes and compared with H&E staining and fluorescent imaging.Results One transgenic line with high levels of green fluorescent protein expression was detected from eight transgenic lines.Green fluorescent protein expression was observed in the tissues of cerebrum,hippocampus,thalamencephalon,cerebellum, and brain stem.Conclusions The results indicated that a brain tissue specific EGFP transgenic mouse line was established.The results suggested that it will be an available animal model for the research of nervous tissues by fluorescent imaging.