Cnn1 And Meox1 Gene A Preliminary Study Of The Regulatory Mechanisms Of Cardiac Development And Cardiomyopathy

Background Cardiovascular disease has become the top killer of human health. Compare expression changes of cardiac gene of hypertrophic cardiomyopathy and dilated cardiomyopathy transgenic mice using gene expression microarray analysis, to research important modifier genes involved in regulation from gene mutation to the pathogenesis, which is important to understand the mechanism, treatment and drug research of cardiomyopathy. Gene expression microarray analysis showed that the expression levels of Meoxl gene in the heart tissues of cTnTR92Q and cTnTR141W transgenic mice were improved compared with that of wild-type mice, which suggests that Meox1 that encodes a member of a subfamily of non-clustered, diverged, antennapedia-like homeobox-containing genes may be involved in regulation of cardiomyopathy. This article is to establish the heart-specific expression Meox1 transgenic mice and to investigate its effect on the development of heart and cardiomyopathy.Methods The expression levels of Meox1 in the heart tissues of both hypertrophic cardiomyopathy (HCM, cTnTR92Q) and dilated cardiomyopathy (DCM, cTnTR141W) mice were analyzed using gene expression microarray analysis and confirmed by reverse transcriptional polymerase chain reaction (RT-PCR) and western blot. The temporal expression patterns of Meox1 in the heart of WT mice were analyzed by western blot. The transgenic plasmid was constructed by inserting the murine Meox1 gene into the down-stream ofα-MHC promoter. The transgenic mice were generated by the method of microinjection. The genotype of transgenic lines was identified by PCR, and the expression levels of the Meoxl gene in the transgenic mice were detected by western blot. The pathologic and functional changes of heart were analyzed with echocardiography and light microscopy. The hypertrophy markers, Nppa, Nppb, Co11a1, Co13a1, Acta1, and Atp2a2, and the transcription factors, GATA-4, NKX2-5, MEF2C, were analyzed by RT-PCR. Survival datas of the experimental mice were recorded.Results Gene expression microarray analysis and RT-PCR revealed that a higher expression level of Meox1 in HCM mice and DCM mice. The results of western blot showed that the expression of Meox1 was only detected in the heart of one-week wide-type mice, but its expression was up-regulated in the adult mice with HCM mice and DCM mice. The heart-specific transgenic C57BL/6J mice were established and used to analyze its effect on the heart with echocardiography. Compared with the wild type mice, both of the two lines of Meox1 transgenic mice showed significantly heart remodeling with increased left ventricular systolic diameter (15.6%or 24.2%, P<0.01, n = 16),increased left ventricular diastolic diameter (7.2% or 12.8%, P<0.01, n=16), increased systolic volume(36.8% or 65.7%, P<0.01, n=16), and increased diastolic volume(18.2% or 33.8%, P<0.01, n=16). Compared with the wild type mice, both of the two lines of Meoxl transgenic mice showed weaker heart function with decreased ejection fraction (6.6% or 9.3%, P<0.05, n= 16), and decreased fraction shortening (9.4% or 12.3%, P<0.05, n= 16). The heart of the Meoxl transgenic mice exhibited an enlarged ventricular chamber compared with that of the wild type mice under the light microscope. The expressions of hypertrophy markers, Nppb, Co11a1, Co13a1 and Acta1 were increased, however Atp2a2 was decreased in the heart of transgenic mice, there was no change with Nppa. The expressions of transcription factor GATA-4, NKX2-5, MEF2C were increased in the heart of transgenic mice. There was no death until 9 months of age both in the Meoxl transgenic mice and widetype mice.Conclusions The expression level of Meoxl was up-regulated during cardiomyopathy. The Meoxl transgenic mice display a similar pathologic phenotype with the light dilated cardiomyopathy. It suggests that we established the heart-specific expression Meox1 transgenic mice successfully, Meoxl may be one of modifier genes which enhance pathogenesis of cardiomyopathy and the heart-specific expression Meox1 transgenic mice maybe a useful animal model for developmet of DCM and drug research. Objective To establish the heart-specific CNN1 expression transgenic mice and to investigate its effect on the development of heart and cardiomyopathy.Methods The expression of h1 calponin in the heart tissues of both hypertrophic cardiomyopathy (cTnTR92Q) and dilated cardiomyopathy (cTnTR141W) mice were analyzed by western blot. The temporal expression patterns of h1 calponin in the heart of WT mice were analyzed by western blot. The transgenic plasmid was constructed by inserting the human CNN1 gene into the down-stream ofα-MHC promoter. The transgenic mice were generated by the method of microinjection. The genotype of transgenic lines was identified by PCR, and the expression levels of the h1 calponin.were detected by western blot. The pathologic and functional changes of heart were analyzed with echocardiography. The pathologic changes were observed by microscopy.Results The results of western blot showed that the expression of h1 calponin was detected in the heart of different-age-wide-type mice, but its expression was down-regulated in the mice with DCM. The heart-specific transgenic C57BL/6J mice were established and used to analyze its effect on heart with echocardiography. Compared with the wild type mice, CNN1 transgenic mice showed significantly heart remodeling with increased left ventricular systolic diameter (28%, P<0.01, n= 12), increased left ventricular diastolic diameter (16.2%, P<0.01, n= 12), decreased left ventricular posterior wall during systolic (15.7%, P<0.01, n= 12), and decreased left ventricular posterior wall during diastole (21%, P<0.01, n= 12). Compared with the wild type mice, CNN1 transgenic mice showed weaker heart function with decreased Ejection fraction (11.5%, P＜0.01, n= 12), and decreased fraction shortening (14.6%, P <0.05, n= 12). The heart of the CNN1 transgenic mouse exhibited an enlarged ventricular chamber when compared with that of the wild type mouse under the light microscope. There are no death until 9 months of age both of CNN1 transgenic mice and widetype mice.Conclusions Over-expression of CNN1 in the heart of the transgenic mice caused dilated cardiomyopathy. It suggested that CNN1 may be one of modifier genes which enhance pathogenesis of cardiomyopathy. Objective To design and construct miRNA expression vector targeting mouse Meoxl gene, and establish the heart specific Meox1 gene silence mice by the method of microinjection for function study of Meoxl gene with RNA interference technology.Methods Amplify pLKO.1-shRNA-Meoxl plasmid (Open Biosystems, United States, TRCN0000070603-TRCN0000070607). Transfect 293T cell, compare the silence effect of the five plasmids by the RT-PCR to choose two better silence plasmids. According to Invitrogen's BLOCK-iT TM PolⅡmiR RNAi Expression Vector Kits Manual and informations of silence plasmids, synthesize the specific pre-miRNA single strand DNA oligos for mouse Meox1 gene containing palindromic cohesive end, then via annealing and ligating with pcDNATM 6.2-GW/EmGFP-miR in order, the two kinds of miRNA expression vectors are constructed which included pre-miRNA-1 and pre-miRNA-2 separately. Then the pre-miRNA-1 and pre-miRNA-2 are combined using the Chaining technology of the-vector. After the recombined plasmids were cotransfected into 293T cells with the constructed recombined plasmid of pcDNA3.1(+)-Meox1, the suppression effect targeting Meox1 is identified by RT-PCR. Amplify EmGFP-Meox1-miR1+2 by PCR, then inserte it into down-stream of a-MHC promoter, sequence and correct, then lineearized with NotⅠand purified with SephedexG50. Inject the DNA fragmentα-MHC-EmGFP-Meox1-miR1+2 which was adjusted to 5 ng/μL into C57BL/6J mice with microinjection, to prepare Meoxl gene silence mice. The genotype of transgenic lines was identified by PCR, and the expression levels of the GFP protein were detected by western blot, the expression levels of the Meox1 gene were detected by RT-PCR.Results The miRNA expression plasmids were successfully constructed by the treatment of specific restriction enzymes and identification of sequencing analysis, and they could effectively inhibit the mRNA expressing of Meoxl from the recombined plasmid of pcDNA3.1(+)-Meox1 in vitro. The heart-specific Meox1 gene silence mice were established, and the GFP protein of one line of Meox1 gene silence mice is increased and Meox1 gene was decreaed compaired with the wide-type mice.Conclusion The effective miRNA expression vector targeting mouse Meox1 has been successfully constructed, and the heart-specific Meox1 gene silence mice were successfully established. It can be used to cross with the DCM and HCM models to investigate the function of Meox1 gene on the development of cardiomyopathy.