Key Genes In The Myocardialization Of Proximal Cardiac Outflow Tract Septum In Cx43 Knockout Mice

Conotruncal anomaly is composed of a group of complex congenital heart diseases,which is one of the leading causes of death for young children.Cardiac proximal outflow tract(OFT) septation plays an essential role in the cardiac conotruncal development.Connexin43 knockout(Cx43KO) mice die at birth with conotruncal heart malformations,outflow obstructions and coronary anomalies.These striking phenotypes suggest an important role for Cx43 in the outflow tract morphogenesis and development.Cx43 is now known to be the most widely expressed gap junction protein in mammals,and is a primary component of intercellular gap junction channels in cardiac tissue.Myocardialization plays an essential role in the development of proximal cardiac outflow tract septum.The genes that regular myocardialization and the molecular mechanisms involved in myocardialization are research hotspots.Previous study found that the process of myocardialization was delayed in Cx43KO mice,which indicates a complicated molecular mechanism involved in the pathogenesis.In this study we used Cx43KO mice to investigate changes of gene expression in the cardiac outflow tract tissues, and to elucidate the genes involving in the pathogenesis of pulmonary outflow obstruction.Partâ… Morphological Features during Myocardialization of Cardiac Proximal Outflow Septum in Cx43 Knockout MiceObjectiveTo investigate the morphological features of conotruncal region in the process of myocardialization of proximal cardiac OFT septum in Cx43KO mice.Methods The objects were C₅₇BL₆ mice of ED11.5 to ED15.5 by mating of 2-month old heterozygous Cx43 knockout mice,which will provide Cx43KO homozygotes (Cx43-/-),Cx43KO heterozygotes(Cx43+/-) and wild-type(Cx43+/+) genotyped by PCR method.Micro dissection and HE staining were used to examine the structures of hearts.The expression ofα-SCA was detected by immunohistochemistry.Results1.Cx43-/- mice died within 24h after birth with a swelling and blockage of the conotruncal region,which led to the obstruction of OFT.No apparent malformations were observed in Cx43+/- and Cx43+/+ mice.2.HE staining showed plenty of abnormal tissues forming in this region,with malformation of many pouch-like cavities,which was known as intertrabecμlar pouches in the right ventricular chamber of Cx43-/- mouse hearts.It was also shown that narrowing and obstruction of the right ventricular outflow tract was manifested.This was associated with distinct thinning of the compact myocardium.3.In the wild-type hearts,the proximal OFT cushions began to fuse at ED11.5,and completed at ED 13.5.Myocardialization started at ED 12.5 and completed at ED15.5.The process of Myocardialization showed a tendency of ingrowth of the myocardium form the outside septum into the mesenchymal OFT septum which was shown by immunostaining ofα-SCA.The expression ofα-SCA in the proximal OFT septum was obviously delayed in Cx43-/- predominantly from ED13.5 to ED15.5.No apparent abnormalities were observed in Cx43+/- mice.Conclusions1.Cx43-/- mice are characterized by malformations of the conotruncal region.2.Cx43-/- mice exhibits the delayed myocardialization in the proximal OFT septum.Partâ…¡Differentially Expressed Genes in the Cardiac Outflow Tract Tissues of Cx43 Knockout Mice ObjectiveTo investigate changes of gene expression in the cardiac outflow tract(OFT) in Cx43KO mouse embryos,and to elucidate the genes in the pathogenesis of outflow obstruction.MethodsThe cDNA was retrotranscripted from RNA extracted from OFT tissues of both Cx43-/- and Cx43-/- mouse embryos at ED14.5.The biotin-labeled cRNA derived from the transcription of cDNA was fragmented as probes.The probes were hybridized with Affymetrix Mouse Genome 430 2.0 Array.Gene Array Scanner was used to screen the signals of hybridization and the expression of genes was detected.Results1.Among the differentially expressed genes,there were 143 upregulated and 235 downregnlated in Cx43-/- OFT tissues as compared with Cx43+/+ hearts. Functions of proteins encoded by the altered genes encompassed all functional categories,with largest percentage in genes involved in regulation of transcription,cell cycle,etc.2.Among the differentially expressed genes in the Cx43-/- hearts were those related to TGFβ/ BMP signaling pathway and Ssr1,Ptk2,bmpr2,which are related to conotruncal defects.Conclusions1.Among those differentially expressed genes in Cx43-/- hearts,some may play role in the development of the OFT.2.Genes related with TGFβ/BMP signaling pathway and Ssr1,Ptk2,bmpr2,which are differentially expressed in Cx43-/- OFT tissue,may be involved in the pathogenesis of pulmonary outflow obstruction. Partâ…¢Verification of differentially expressed genes by Realtime PCRObjectiveTo identify the mRNA expression levels of 5 genes related with TGFβ/BMP pathway and Ssr1,Ptk2,bmpr2 which are related to conotruncal defects by Realtime quantitative RT-PCR at ED14.5.MethodsThe cDNA was retrotranscripted from RNA extracted from OFT tissues of both Cx43-/- and Cx43+/+ mouse embryos at ED14.5.The 5 genes related with TGFβ/ BMP pathway and Ssr1,Ptk2,bmpr2 was identified by Realtime quantitative RT-PCR at ED14.5.Results1.The real time PCR analysis of genes including TGFβ2,Ssr1,RhoA,Bmp6 and Ptk2 indicated upregulated,which showed excellent concordance with the gene expression levels indicated by the microarray analysis.2.The real time PCR analysis of genes including Bmpr2,Bmp2 and smad6 indicated downregulated,which showed excellent concordance with the gene expression levels indicated by the microarray analysis.ConclusionsThe real time PCR analysis of 8 genes including TGFβ2,Ssr1,RhoA,Bmp6, Ptk2,Bmpr2,Bmp2 and smad6 shows excellent concordance with the gene expression levels indicated by the microarray analysis.The process of Myocardialization of proximal cardiac OFT septum was delayed in Cx43KO mice,indicating that Cx43 may play an important role in the process of myocardialization.Gene chip array was used to find some genes related with the development of OFT.Differentially expressed genes related with TGFβ/BMP pathway and Ssr1,Ptk2,bmpr2 which are related to conotruncal defects may have close relationship with the obstruction of cardiac OFT in Cx43KO mice.