The Role Of Cx43 Gene In Myocardialization Of Cardiac Proximal Outflow Tract Septum In Mice

Conotruncal anomaly is composed of a group of complex congenital heart diseases, which is one of the leading causes of death for children. Proximal outflow tract (OFT) septation plays an essential role in the cardiac conotruncal development. Proximal OFT septum separates the OFT at the semilunar level and beneath semilunar level. During the development of proximal OFT septum, myocardium gradually takes place of the mesenchyme of endocardial cushions, which is called "Myocardialization". Myocardialization is one of the considerable events during OFT development, and aberrant myocardialization could result in disordered OFT structurally and functionally. Therefore, to elucidate the myocardialization process could be helpful to reveal the pathogenesis mechanisms of OFT malformations. Some cellular and molecular mechanisms which involve the myocardialization have been proposed in recent years. Among these cells and molecules, cardiac neural crest cells (NCC), apoptosis signaling pathways, and transforming growth factor (TGF) β2 have attracted researchers. Recent studies demonstrate that connexin (Cx) 43 plays a crucial role in conotruncal development. Both the Cx43 knockout (KO) and Cx43 overexpression mice exhibited right OFT malformations and obstruction, and in which cardiac NCC may involved. Hence, in this study we used Cx43 KO mice to explore the role of Cx43 in myocardialization and therefore further reveal the pathogenesis mechanisms of the OFT malformations in Cx43 KO mice.Part I Normal myocardialization process of cardiac outflowtract septum in miceObjectiveTo investigate the spatiotemporal pattern of myocardialization and the possible roles of cardiac NCC and apoptosis in myocardialization process.MethodsThe objects were C57/BL6 mice of embryonic day (E) 11.5 to 1 day after birth by the mating of 2-month old adult. a-Sarcomeric Actin (a-SCA), activator protein-2a (AP-2a), and Active Caspase-3 were detected by immunohistochemistry. Apoptotic cells were identified by TUNEL method.Results1. The fusion of proximal OFT endocardial cushions began at El 1.5, and completed at E13.5; Myocardialization started at E12.5 and completed at E15.5. During this process the myocardium extended by ingrowth from the outside septum into the mesenchymal OFT septum.2. A few of AP-2a positive cells in proximal OFT septum were observed at E12.5 and El3.5, and it disappeared from ED14.5 onwards.3. Apoptosis took place mainly at E12.5 and E13.5, and the distribution of the apoptotic cells was similar to that of the AP-2a positive cells.Conclusion1. The myocardialization of proximal OFT in mice started at El2.5 and completed atE15.5.2. During myocardialization process, myocardium extended by ingrowth from the outside septum into the mesenchymal OFT septum.3. The apoptosis of OFT septum cells may involve in the myocardialization process based on their spatiotemporal correlation.4. The neural crest cells are likely to contribute to myocardialization by their apoptosis because that the distribution of the apoptotic cells was similar to that of the AP-2a positive cells.Part II Myocardialization of cardiac proximal OFT septum and its mechanism in Cx43 KO miceObjectiveTo demonstrate the role of Cx43 in the process of myocardialization of cardiac proximal OFT septum.MethodsThe objects were C57/BL6 mice of El 1.5 to 1 day after birth by the mating of 2 month old heterozygous mice, which included Cx43 KO 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 of the a-SCA, Smooth Muscle a-Actin (a-SMA), AP-2a and Active Caspase-3 were detected by immunohistochemistry. AP-2a mRNA was detected by in situ hybridization.Results1. Cx43-/- mice died within 24h after birth with a swelling and blockage of the conotruncal region, which led to the obstruction of OFT and enlargement of right ventricle. HE staining showed plenty of abnormal tissues in this region forming many pouches. No apparent malformations were observed in Cx43+/- and Cx43+/+ mice.2. The expression of a-SCA in the proximal OFT septum was delayed obviously in Cx43-/- predominantly at El3.5 and El4.5.3. The expression of a-SMA in the OFT in Cx43+/- and Cx43-/- was stronger than that of Cx43+/+ mice, and mostly located in the hyperplastic conotruncal region especially at El3.5-El5.5 in Cx43-/- mice. The expression could still be observed at the birth day in Cx43-/- mice, which was not observed in Cx43+/+ mice.4. The expression of AP-2a and AP-2a mRNA at E13.5 increased in Cx43-/- and abnormally located in the proximal OFT septum.5. The apoptotic cells existed in the proximal OFT septum of Cx43+/+ during E12.5 to E15.5 particularly at E13.5. But there were less apoptotic cells observed in Cx43+/-, and even less in Cx43-/-.Conclusion1. Cx43 KO mice are characterized by hyperplasia in conotruncal region.2. Cx43 KO mice exhibited a delayed myocardialization.3. The expression of a-SMA could not disappear normally, which results in the developmental immaturity of the cardiomyocytes.4. The abnormal distribution of cardiac NCC and the decreased apoptosis are likely to contribute to the aberrant myocardialization in mice with Cx43 defects.5. Cx43 KO mice could be an animal model to study the myocardialization process.