Connexin43 Gene And Pathogenesis Of Congenital Heart Disease

Congenital heart disease (CHD) is one of the common congenital defects in human and the pathogenesis of CHD has already been more highlighted. Scientists have been working hard to find out the causes of CHD in order to prevent the occurrence of CHD beforehand and improve the quality of population. Hereditary factors have been recognized in process of research and mutations of some genes controlling human heart development maybe the molecular basis of CHD. Connexin43 (Cx43), the major gap junction protein in the mammalian heart, makes up gap junction channels that mediate the cell-to-cell diffusion of ions, small metabolites, and small cell signaling molecules. Recently, a series of animal models suggest that Cx43 plays an important role in the morphogenesis of heart, especially the pulmonary conotruncal region. However, the role of Cx43 gene in the development of CHD in human being is still unclear. Hence, in this study Cx43 gene knockout mice was used to observe the heart morphology and further reveal the pathogenesis mechanism of the heart malformations in Cx43 gene defect mouse model. Cx43 gene coding sequence was also detected in a large number of Chinese Han patients with CHD to identify the association of Cx43 gene mutation with CHD. Furthermore, Cx43 expression in the myocardium was detected in some patients with CHD to illustrate the potential mechanism of CHD.Part I Cardiac Morphology and Expression of Associated Genes in Cx43 Gene Knockout MiceObjectiveTo observe cardiac morphology of Cx43 knockout (Cx43KO) mice and investigate the spatio-temporal expression of Cx40 and Cx45 in Cx43KO mice. Materials and methodsThe objects were offsprings of mice of ED13.5 to 1 day after birth by the mating of 2-month-old heterozygous Cx43KO mice, which included Cx43KO homozygotes(Cx43-/-), heterozygotes (Cx43+/-) and wild types (Cx43+/+) genotyped by PCR method. C57BL6 mice were used as control. Micro-dissection and HE staining were used to examine the structures of the mouse hearts. Immuno-histochemistry was used to detect Cx40 and Cx45 expression in the myocardium of Cx43KO mice from ED10.5 tol5.5. Results1. Cx43-/- mice died within 24hr after birth with a swelling and blockage of the conotruncal region, which led to the obstruction of outlet of right ventricle and enlargement of right ventricle. In control C57BL6 mice did not exhibit any heart malformations.2. HE staining showed that the homozygous Cx43KO mice had severe right ventricular outflow tract obstruction (RVOTO). In contrast, C57BL6 mice did not exhibit any heart malformations.3. With the wide type mouse fetal heart development, Cx40 expression was detected in right ventricle in ED11.5. Subsequently it was distributed in atria and ventricles. Peak expression of Cx40 was in ED14.5, then it faded. After birth it was only detected in atria and His-Purkinje conductive system. Cx45 expression was detected in inflow tract, outflow tract and atrio-ventricular cushion in ED10.5. Then it faded and distributed in outflow tract. After birth it was detected only in interventricular septum. Cx40 and Cx45 expression in Cx43KO mouse fetal hearts were obviously weakened as compared with wild type (P<0.05).Conclusions1. Cx43 gene defect is obviously associated with abnormal heart morphogenesis, especially in the conotruncal outflow region.2. Lower expression of Cx40 and Cx45 in Cx43KO mouse myocardium maybe the potential mechanism of heart malformations.Part II Mutations of Cx43 in Patients with Congenital HeartDiseaseObjectiveTo detect the mutations of Cx43 gene in patients with congenital heart disease (CHD), and illustrate the relationship between Cx43 gene mutations and CHD in the human being. Materials and methodsTwo hundred and fifty patients with CHD were recruited in this study, among which 182 patients had a variety of complex heart defects and 68 patients had simple heart defects, such as atrial septal defect (ASD), ventricular septal defect (VSD), pulmonary stenosis (PS) and patent ductus arteriosus (PDA). Forty children without CHD were selected as control. Samples were obtained from peripheral blood in all subjects, and from myocardial tissue in some patients. The entire coding region of Cx43 was amplified by PCR with the primers F1 and R3. A small portion of an intron in the 5' untranslated region of the gene (the coding region contains no introns) was designed to exclude any contribution from a processed pseudogene. Then the PCR products were purified and directly sequenced by using sequencing primers F1, F2, F3, R1, R2 and R3. Sequencing products were run on ABI 3700 DNA sequencer and both coding and the antisense strand of the Cx43 gene were sequenced. ResultsThe entire Cx43 coding sequences of all subjects were analyzed and compared with human Cx43 coding sequence. No Cx43 mutation was found except for a single nucleotide polymorphism (SNP) in a patient with TOF. Conclusions1. Our results indicate that Cx43 gene mutations may be very rare in patients with CHD.2. The morphogenesis of the human heart is a very complex process, in which a lot of genes are involved. Cx43 may take an important role in the signal pathway for the heart development.Part III Expression of Cx43 in Patients with Tetralogy of FallotObjectiveTo detect expression Cx43 in patients with tetralogy of Fallot (TOF), whichwould give some clues to pathogenesis of the disease. Materials and methodsSixteen patients with TOF were recruited in this study and 4 patients with VSD and 1 patient with primary pulmonary hypertension were selected as control. The myocardial samples were obtained from right ventricle outlet of hearts. They were analyzed by RT-PCR. Moreover, immuno-histochemistry was used to observe Cx43 expression in the myocardium. Results1. More Cx43 in mRNA level was observed as compared with controls.2. Plenty of Cx43 protein was expressed in the myocardium in patients with TOF, while no expression of Cx43 was observed in the control.Conclusions1. The higher expression of Cx43 in the myocardium indicates that the turbulence of Cx43 spatio-temporal expression in heart morphogenesis in patients with TOF.2. The abnormal expression of Cx43 in the myocardium gives a clue to the potential pathogenesis of TOF.SummaryCx43 gene is involved in heart morphogenesis with a complicated delicate mechanism. However, Cx43 gene mutations rarely exist in patients with CHD. Cx43 gene defect may cause turbulence of some related genes in the process of cardiomorphogenesis and lead to heart malformations.