The Modulation Of Dkk3 And Wif1 On The Transgenic Mice Of CTnT^R141W

Objective To analysis the modulation of Dkk3 and Wifl on the transgenic mice of cTnTR141WMethods The temporal expression patterns of Dkk3 and Wifl in the heart of WT mice were analyzed by reverse transcriptional polymerase chain reaction (RT-PCR), while the spatial expression of Dkk3 were detected by Whole-Mount RNA in situ hybridization and immunohistochemical staining. The expression of Dkk3 and Wifl in both hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) mice were analyzed using gene expression microarray analysis of cTnTR92Q and cTnTR141w myocardium and confirmed by RT-PCR. The expression of Dkk3 and Wifl in myocardial injury mice model induced by adriamycin were analyzed by RT-PCR. The transgenic vector was constructed by inserting the mouse Dkk3 and human Wifl gene into the down stream of a-MHC promoter respectively. The transgenic mice were created by the method of microinjection. The genotype of transgenic mice of cTnTR141W, Dkk3, Dkk3×cTnTR141W, Wifl and Wifl xcTnTR141wwas detected by PCR, the expression level of the Dkk3 and Wif1 gene was determined by Western blotting. Pathologic changes were observed by light microscopy and transmission electronic microscopy. The cardiac structure and function were analyzed with M-mode echocardiography. Survival data of the experimental mice were recorded. The genes of sarcomeric proteins, cytoskeletal proteins, calcium-regulation proteins, extracellular matrix proteins and the genes associated with signal transduction were analyzed by RT-PCR. The accumulation ofβ-catenin were determined by Western Blot in cTnTR141W, Dkk3, Dkk3×cTnTR141W, Wifl and Wifl xcTnTR141w transgenic mice. The expression constructs for cTnTR141w, Dkk3 and Wifl was generated by cloning a PCR-amplified full-length cDNA fragment into pcDNA3.1+vector. H9c2 cell line was transfected with cTnTR141W, Dkk3, Dkk3×cTnTR141W, Wif1 and WiflxcTnTR141W constructs using Lipofectamine 2000. Immunofluorescence was used to examine localization ofβ-catenin in CMV-Dkk3 and CMV-Wifl cell line.Results The spatial expression of Dkk3 and Wif1 in WT mice show a tissue-specific characteristic. The expression of Dkk3 and Wifl gene were detected in the heart of WT mice with a high level, and the highest level of Dkk3 shows at the birth of mouse, while Wifl at the embryonic stage. Gene expression microarray analysis and RT-PCR revealed that a higher expression of Dkk3 and Wif1 in DCM mice, but different changes in HCM mice. The expression of Dkk3 incresed in injury mice model induced by adriamycin compared with control group. C57BL/6J transgenic mice carrying the Dkk3, Wif1, Dkk3×cTnTR141W and Wif1×cTnTR141W genes were all established. The heart of Dkk3 transgenic mice showed hypertrophic ventricular wall, reduced ventricular chamber, diastolic dysfunction compared with that of wild type. Hypertrophic cardiomyocytes, myocyte disarray of the Dkk3 transgenic mice were observed under the transmission electronic microscope. All the pathological changes of Dkk3 transgenic mice display a similar pathologic phenotype to light hypertrophic cardiomyopathy. The Dkk3×cTnTR141W transgenic mice showed a thicker ventricular wall, smaller ventricular chamber, improved diastolic function, elongated and lysed myofrils when compared with cTnTR141w transgenic mice. The expression of hypertrophy markers, Nppa and Nppb, were consistent with the phenotype of Dkk3 transgenic mice. The genes of sarcomeric proteins, Myot and Cnn1, were decreased in Dkk3 transgenic mice, while calcium-regulation proteins, Atp2a2, showed a slight increasion. The genes of extracellular matrix proteins, as Collal and Col3a1, cytoskeletal proteins, as Acta1, Itga8 and Itgblbp3, were all increased in the Dkk3×cTnTR141W transgenic mice, which hinted a remodeling of myocardial arrangement. An accumulation ofβ-catenin was seen both in Dkk3 transgenic mice and CMV-Dkk3 cell line. However the heart of Wif1 transgenic mice showed thinner ventricular wall, enlarged ventricular chamber, systolic dysfunction compared with that of the wild type. Slight elongated myofrils and expanded sarcoplasmic reticulum of the Wif1 transgenic mice were observed under the transmission electronic microscope. All the pathological changes of Wif1 transgenic mice display a similar pathologic phenotype to light dilated cardiomyopathy. The Wif1 xcTnTR141W transgenic mice showed a enlarger ventricular chamber, even worse systolic function, diffused, elongated and lysed myofrils, much lysosome when compared with cTnTR141W transgenic mice. The expression of hypertrophy markers, Nppa and Nppb, were consistent with the phenotype of Wif1 transgenic mice. The genes of extracellular matrix proteins, as Collal and Col3al, the genes of cytoskeletal proteins, as Acta1 and Itgb1bp3, were strongly decreased in Wif1 transgenic mice, which were increased in cTnTR141W transgenic mice. The gene of calcium-regulation protein, Sln, was shown to be decreased, which indicated that Wif1 may have a effection on calcium transportation. The genes of sarcomeric proteins, Myot and Cnn1, were decreased in Wif1 transgenic mice, furthermore, Myot was shown to be decreased significantly in Wif1×cTnTR141W transgenic mice. Wif1 inhibited the accumulation ofβ-catenin both in Wif1 transgenic mice and CMV-Wif1 cell line. Immature death occurred after 4 months of age and the immature death rate was 15% before 10 months of age in the cTnTR141w mice, while Wif1×cTnTR141W transgenic mice was 20%. Dkk3, Wif1 and Dkk3×cTnTR141W transgenic mice showed a low immature death compared with that of cTnTR141W transgenic mice, but higer than that of WT mice.Conclusions The Dkk3 transgenic mouse displayed a similar pathologic phenotype with the light hypertrophic cardiomyopathy, and it shown an improvement on the pathologic phenotype of cTnTR141W transgenic mice, while the Wif1 transgenic mouse displayed a similar pathologic phenotype with the light dilated cardiomyopathy, and it shown an even worse pathologic phenotype as cTnTR141w transgenic mice had. The Dkk3 and Wif1 shown a different effects on the cardiomyopathy of cTnTR141w transgenic mice, the difference on regulation ofβ-catenin maybe the cause of the former. The Dkk3 and Wif1 maybe important modifer genes, which caused the myocyte differentiation in a different way and play roles in the HCM and DCM, and it maybe a new strategy to treat the cardiomyopathy. Objective To generate the systemic expression Dkk3 transgenic mice, a model for the study of its function and effects on the development and maintain of the bone tissues.Methods The expression pattern of Dkk3 in C57BL/6J mice was observed by tissue in situ hybridization. The transgenic vector was constructed by inserting the murine Dkk3 gene into the down stream of CMV promoter. The transgenic mice were created by the method of microinjection. The genotype of transgenic line was identified by PCR and the expression level of the gene was determined by RT-PCR and Western blot. The growth of the in cell epiphyseal plate was detected by the BrdU incorporationResults The endogenous Dkk3 was commonly expressed in mouse, and especially expressed in a higher level in the tissues of bone, heart and brain. Two lines of C57BL/6J transgenic mice with high level of Dkk3 were established. The growth of cells in the epiphyseal plate was inhibited in Dkk3 transgenic mice comparing with that of wild type.Conclusions The systemic expression Dkk3 transgenic mouse was successfully established and the transgenic Dkk3 inhibited the cell growth of the epiphyseal plate. The transgenic mouse could be an useful animal model for study of Dkk3 on bone. objective To generate the heart-specific LMNAE82K expression transgenic mice and an animal model for the study of its effects on cardiomyopathy.Methods The transgenic vector was constructed by inserting the human LMNAE82K gene into the down stream of a-MHC promoter. The transgenic mice were created by the method of microinjection. The genotype of transgenic line was identified by PCR and the expression level of the gene was determined by Western Blot. The pathologic changes were analyzed with echocardiography.Results Two lines of C57BL/6J transgenic mice with high levels of LMNAE82K expression were established. The heart of LMNAE82K transgenic mice showed thinner ventricular wall and enlarged ventricular chamber compared with that of the wild type. The ejection fraction (EF%) and Fractionl shortening (FS%) of the transgenic mice were decreased than those of the wild type mice.Conclusions The expression of mutant LMNAE82K gene in heart caused thin ventricular wall and ventricular chamber enlargement, which was similar with human family dilated cardiomyopathy caused by LMNAE82K mutation. The transgenic mouse could be an useful animal model for the research of LMNAE82K caused DCM.