The Wnt Connexin43 Expression And Function Of The Impact

Being a member of connexin family, Connexin43 plays a crucial role in cardiac development. Studies in mouse pointed out the abnormal expression of Cx43 could result in cardiac anomalies, mainly with right outflow tract. The impairment of Cx43 may be an important reason for Congenital Malformation of Heart.Cx43 may be one of the leading causes of cardiac anomalies. It has been suggestted that the abnormal migration of cells, including neural crest cells and cardiac myocytes, may contribute to these processes.The Wnt signaling pathways is though to be functionally conserved in vertebrates and invertebrates and plays an important role in cell proliferation, differentiation, organogenesis and oncogenesis. And several members of the Wnt-frizzled signal transduction pathways were found to be expressed during cardiac development in vertebrates.Wnt 1 of the Wnt 1 family member signaling has been shown to be an important modulator of Cx43-dependent intercellular coupling in the heart. The Wnt-1/β-catenin signaling pathway could change the expression of Cx43, which affected the structure and function of gap junctional intercellular communication. Few reports are concerned about Wnt-3a, which is another member of the family.To clarify the molecular mechanism that the expression of Cx43 may control the growth and several phenotypes of neural crest cells and cardiac myocytes by regulation of Wn-3at, and my be involved in the CHM, we designed in vitro experiments.In first part, we constructed high-expressed and low-expressed eukaryotic vector of Wnt-3a.Then we chose PC12 and H9c2 cells to take further research. Rat adrenal pheochromocytoma(PC12) cell line is a well established model for the nerve system research. Rat heart cell-line H9c2 cells were also good models. Then we detected the expression profile of Cx43 in cell models. The Reverse transcription-polymerase chain action (RT-PCR), Western blot and immunocytochemistry were performed, and the method of Transwell migration assay was taken in the experiments. The results indicated that mRNA and protein level of Cx43 were increased in the cells high expressed Wnt-3a.Ferthermore, The migration ability of those cells were also improved compared to the non-transfected cells As for the situation in the cells low expressed Wnt-3a group , mRNA,protein level of Cx43 and the migration ability were reduced in siWnt-3a-l and siWnt-3a-2. Thus, the dysfunction of Cx43 expression could induce the change for migration ability of those cells.Then we investigated the regulation of Wnt signaling pathway to Cx43 and severalphenotypes of Rat heart cell-line H9c2 cells. To investigate the change of the intracellular [Ca²⁺], we examined it by LSCM with fluorescent dye Fluo-3/AM labeling cells, and [Ca²⁺] changes were represented by fluorescent intensities. The expression levels of two genes related to cardiac development were also detected by RT-PCR. The results of MTT assay and FCM analysis indicated that more Wnt-3a⁺-H9c2 cells enter into G2-phase and the proliferation rate of the cells increased significantly compared to the non-transfected cells..Ferthermore, The intracellular [Ca²⁺] of those cells were also improved compared to the non-transfected cells As for the situation in the cells low expressed Wnt-3a group, intracellular [Ca²⁺] were reduced in pSilencer/siWnt-3a-1 and pSilencer/siWnt-3a-2. The two genes (GATA4,Nkx2.5) related to cardiac development were even increased in the Wnt-3a⁺-H9c2 cells, while those of siWnt-3a-1-H9c2 and siWnt-3a-2-H9c2 were all reduced compared to the control.Being the important part in Wnt signaling pathway, the expression ofβ-catenin took responsibility for the proliferation and migration of the cell. Furthermore, the lack ofβ-catenin in the intercellular junction of cardiac myocytes would result in the impairment of the whole structure in heart. It's reported that the relationship betweenβ-catenin and cytoplasmic membrane was so important that it was responsible for the intercellular junction. The relation ofβ-catenin and Cx43 could reveal the assumption that the regulation of Cx43 and the change of cell migration by Wnt-3a. In this paper we detected Cx43-β-catenin interaction by coimmunoprecipitaton, and observed the location of them both in Wnt-3a overexpressed and low expressed H9c2 cells by immunofluorescence analysis. The results showed that.β-catenin existed widely in cytoplasm, nuclera, even near the cytoplasmic membrane in Cx43 high expressed H9c2 cells. The expression ofβ-catenin were reduced greatly and located only near the cytoplasmic membrane in Cx43 low expressed H9c2 cells. There was direct interaction between them by coimmunoprecipitaton assay. All these indicated that the change of Cx43 may affect the location ofβ-catenin. Cooperation effect of several aspects changed the migration of the cell together.For all the above, this research work verified the regulation of Wnt-3a on Cx43 and the corresponding change of cell phenotypes. And thus offers potential opportunities for further therapeutic interventions.