Changes Of Expression Of Genes Related With Galpha13 Pathway During Myocardialization Of 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 suggested 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. Gap junctions are membrane channels that serve as major conduits for cell-cell communication by mediating the intercellular passage of ions and small molecules. In addition, Cx43 plays an essential role in heart development. Our 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.PartⅠMorphological 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 cardiac proximal OFT septum in Cx43KO mice.MethodsThe objects were C57/BL6 mice of ED11.5 to ED15.5 by the mating of 2-month old heterozygous Cx43 knockout mice, which will provide Cx43KO homozygotes (Cx43-/-), heterozygotes (Cx43+/-) and wild-types (Cx43+/+) genotyped by PCR method. Microdissection 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 intertrabecular pouches in the right ventricular chamber of Cx43-/- mice 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 endocardial cushions began to fuse at ED11.5, which completed at ED13.5. Myocardialization started at ED12.5 and completed at ED15.5. The process of myocardialization showed a tendency of ingrowth of the myocardium from 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.PartⅡDifferentially Expressed Genes Related with Galpha13 Pathway in the Cardiac Outflow Tract Tissues of Cx43 Knockout MiceObjectiveTo investigate changes of gene expression in the cardiac outflow tract (OFT) in Cx43KO mouse embryos, and to elucidate the genes involving in the pathogenesis of pulmonary outflow obstruction.MethodsThe cDNA was retrotranscriped from RNA extracted from OFT tissues of both Cx43-/- and Cx43+/+ mice 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. The mRNA expression levels of 4 genes related with Galpha13 pathway were identified by real time quantitative RT-PCR from ED11.5 to ED15.5.Results1. Among the differentially expressed genes, there were 287 upregulated and 199 downregulated 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 Galpha13 signaling pathway.3. The real time PCR analysis of genes including RhoA, Rac1, Calm1 and Rock-1 showed excellent concordance with the gene expression levels indicated by the microarray analysis.Conclusions1. Among those differentially expressed genes in Cx43-/- hearts, some might may play role in the development of the OFT.2. Genes related with Galpha13 signaling pathway differentially expressed in Cx43-/- OFT tissue may be involved in the pathogenesis of pulmonary outflow obstruction.PartⅢContribution of Rock-1 in the Process of Myocardialization of Cardiac Proximal Outflow Tract in Mice ObjectiveThis study aimed to observe the expression of Rock-1 in the development of cardiac outflow tract, and to investigate its role in the process of myocardilization of proximal outflow tract septum both in Cx43-/- and Cx43+/+ mice.MethodsThe objects were Cx43-/- and Cx43+/+ mice from ED11.5 to ED15.5. The Expression of Rock-1 andα-SCA were detected by immunohistochemical analysis. Rock-1 mRNA expression was examined by reverse-transcription and real time RT-PCR at the same time.Results1. The expression of Rock-1 protein was mainly limited in the proximal outflow tract from ED11.5 to ED15.5. Its expression pattern was similar with that ofα-SCA, which represented the process of myocardialization. Rock-1 mRNA was detected by real time RT-PCR at a lower level at ED11.5, and reached peak at ED13.5 and ED14.5, which was consistent with the tendency of myocardialization.2. During the process of myocardialization, which was delayed in the Cx43-/- mice as compared with Cx43+/+ mice, especially from ED13.5 to ED15.5, the expression level of Rock-1 was obviously downregulated by both immunohistochemical analysis and real time RT-PCR.Conclusions1. Rock-1 may play an important role in the process of myocardialization of cardiac proximal outflow tract septum.2. The downregulated expression of Rock-1 may have some relationship with the delayed myocardialization of cardiac OFT septum in the Cx43KO mice.SummaryThe process of myocardialization of cardiac proximal OFT septum was delayed in Cx43KO mice, indicating that Cx43 may play an important roles 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 Galph13 pathway may have some relationship with the obstruction of OFT in Cx43KO mice. Rock-1 may play an important role in the process of myocardialization, and the downregulated expression of Rock-1 may be involved with the delayed myocardialization in the Cx43KO mice.