Spatio-temporal Expression Of Bmp2 And Bmpr2 During Myocardialization Of Cardiac Proximal Outflow Tract Septum In Cx43 Knockout Embryonic Mice

The prevalence of congenital heart diseases (CHD) in newborn is 6 to 10 percent approximately. Conotruncal anomaly is composed of a group of complex congenital heart diseases, which make up 33 to 38 percent of all CHD. Heart development is the result of spatio-temporal combination of a serial of reticular mechanism. Cardiac proximal outflow tract (OFT) septation plays an essential role in the cardiac conotruncal development. Myocardialization plays an essential role in the development of proximal cardiac outflow tract septum. Cx43 is a primary component of intercellular gap junction channels in cardiac tissue. Previous study found that the process of myocardialization was delayed in Cx43KO mice, and there is different expression of the coding genes of Bone Morphogenetic Protein(Bmp) 2 and Bone Morphogenetic Protein Receptor(Bmpr)2. In this study, we used Cx43KO mice to investigate Spatio-temporal expression of Bmp2 and Bmpr2 in myocardialization of cardiac proximal outflow tract septum in Cx43 knockout embryonic mice, discover the relation between this two genes and myocardialization abnormality, and to elucidate pathogeny of conotruncal anomaly in Cx43KO mice.Part I Morphological Features during Myocardialization of Cardiac Proximal Outflow Tract 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.MethodsThe objects were C57BL6 mice of ED11.5 to ED16.5 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 and the structures of proximal outflow tract septum. The expression of a-SCA was detected by immunohistochemistry.Results1. 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.2. In the Cx43+/+ hearts, the proximal OFT cushions began to fuse at ED11.5, and 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 form the outside septum into the mesenchymal OFT septum which was shown by immunostaining of a-SCA. The expression ofα-SCA in the proximal OFT septum was delayed in Cx43-/- predominantly from ED13.5 to ED15.5 and completed at ED16.5, The expression of a-SCA in the proximal OFT septum in Cx43+/- is between Cx43+/+ and Cx43-/-.Conclusions1. Cx43-/- mice are characterized by malformations of the conotruncal region.2. Cx43-/- mice exhibits the delayed myocardialization in the proximal OFT septum, comleted at ED 16.5.PartⅡContribution of Bmp2 and Bmpr2 during the Process of Myocardialization of Cardiac Proximal Outflow Tract Septum in Cx43 Knockout MiceObjectiveTo observe the spatio-temporal expression of bone morphogenetic protein (Bmp)2 and bone morphogenetic protein reporter (Bmpr)2 in proximal outflow tract (OFT) septum in connexin (Cx) 43 knockout(KO) mice and investigate its relationship with the abnormal myocardialization of cardiac proximal OFT septum in Cx43 knockout embryonic mice.MethodsObjects were C57/BL6 mice of ED11.5 to ED16.5, which included Cx43 homozygotes (Cx43-/-), heterozygotes (Cx43+/-) and wild-types (Cx43+/+) genotyped by PCR method. Bmp2 and a-sarcomeric acti (a-SCA) were detected by immunohistochemistry.Results1. From ED11.5 to ED13.5, Bmp2 expression was not detected in proximal OFT septum of Cx43+/+fetal heart. From ED14.5 to ED16.5, Bmp2 expression was detected in the myocardial cell of OFT septum of Cx43+/+ fetal heart, without the mesenchymal cell which has not completed myocardialization. From ED11.5 to ED14.5, Bmp2 expression was not detected in proximal OFT septum of Cx43+/- fetal heart. From ED15.5 to ED16.5, Bmp2 expression was detected in the myocardial cell of OFT septum of Cx43+/- fetal heart, and Bmp2 expression was also detected slightly in the mesenchymal cell which has not completed myocardialization. From ED11.5 to ED 14.5, Bmp2 expression was not detected in proximal OFT septum of Cx43-/- fetal heart. From ED15.5 to ED16.5, Bmp2 expression was detected in the myocardial cell of OFT septum of Cx43-/- fetal heart, and Bmp2 expression was also detected obviously in the mesenchymal cell which has not completed myocardialization.Other than this, Bmp2 expression was also detected in the other cardiac tissues. From ED11.5 to ED13.5, Bmp2 expression was detected in both cardiac atrium and epicardium of the Cx43+/+ fetal heart, and the expression amount increased gradually. Bmp2 expression was not detected in the trabeculum of Cx43+/+ fetal heart from ED11.5 to ED12.5, and then Bmp2 was detected slightly at ED13.5, finally, Bmp2 was detected obviously from ED 14.5 to ED16.5. Bmp2 expression of cardiac atrium, epicardium, endocardium, trabeculum, ventricular muscle of Cx43+/- and Cx43-/- is the same as that of Cx43+/+.2. From ED11.5 to ED13.5, Bmpr2 expression was not detected in proximal OFT septum of Cx43+/+ fetal heart. From ED 14.5 to ED 16.5, Bmpr2 expression was detected in the myocardial cell of OFT septum of Cx43+/+ fetal heart, without the mesenchymal cell which has not completed myocardialization. From ED11.5 to ED 14.5, Bmpr2 expression was not detected in proximal OFT septum of Cx43+/- fetal heart. From ED15.5 to ED16.5, Bmpr2 expression was detected in the myocardial cell of OFT septum of Cx43+/- fetal heart, and Bmpr2 expression was also detected slightly in the mesenchymal cell which has not completed myocardialization. From ED11.5 to ED14.5, Bmpr2 expression was not detected in proximal OFT septum of Cx43-/- fetal heart. From ED15.5 to ED 16.5, Bmpr2 expression was detected in the myocardial cell of OFT septum of Cx43-/- fetal heart, and Bmpr2 expression was also detected obviously in the mesenchymal cell which has not completed myocardialization. Other than this, Bmpr2 expression was also detected in the other cardiac tissues. From ED11.5 to ED13.5, Bmpr2 expression was detected in both cardiac atrium and epicardium of the Cx43+/+ fetal heart, and the expression amount increased gradually. Bmpr2 expression was not detected in the trabeculum of Cx43+/+ fetal heart from ED11.5 to ED12.5, and then Bmpr2 was detected slightly at ED13.5, finally, Bmpr2 was detected obviously from ED14.5 to ED16.5. Bmpr2 expression of cardiac atrium, epicardium, endocardium, trabeculum, ventricular muscle of Cx43+/- and Cx43-/- is the same as that of Cx43+/+.Conclusions1. The expression of Bmp2 and Bmpr2 in OFT septum is almost coincidence, they may play a coordination role in the abnormal myocardialization of cardiac proximal outflow tract septum in Cx43 knockout embryonic mice.2. The expression of Bmp2 and Bmpr2 in OFT septum of Cx43+/- and Cx43-/- fetal heart is abnormal and delayed. Bmp2 and Bmpr2 may involve in the abnormal myocardialization of cardiac proximal outflow tract septum in Cx43 knockout embryonic mice, they may mediate the process of cells proliferation.SummaryThe 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. Immunohistochemistry was used to reveal the intimate relation between two genes (Bmp2&Bmpr2) and spatio-temporal process. Bmp2 and Bmpr2 may involve in the abnormal myocardialization of cardiac proximal outflow tract septum in Cx43 knockout embryonic mice, they may mediate the process of cells proliferation.