Expression Of Connexin43 Between Neonatal Rat Cardiomyocytes And Rat Skeletal Myoblasts In Coculture Of Vitro

Mature myocardial cells are terminally differentiated cells, once necrosis, they can't regenerate .Although we can improve symptoms of myocardial ischemia by treating after myocardial infarction (MI) , myocardial necrosis can't be reversed and finally heart failure happened, resulted in death. Therefore, for curing cardiovascular disease, people have been searching a kind of cell to substitute necrotic myocardial cells. In recent years, it has brought new hope for the treatment of myocardial infarction by stem cell transplantation. In a great deal of studies about cell transplantation , skeletal myoblasts have been paid close attention, because they have many desirable advantages, such as high tolerance to ischemia;high proliferative potential; high yield of myoblasts ; autologous transplantation, avoiding immune rejection reaction; no unlimited proliferation or tumor development after cell-grafting. The success of repairing damaged myocardium depended on the anatomical and functional integration between the engrafted cells and the resident cardiomyocytes. If delaying passage or cutting down concentrations of serum , monocultured L6 cells would differentiate into multinucleated myotubes. Expression of Cx43 protein was downregulated significantly. Cx43 is the major gap junction protein of cardiomyocytes. Gap junctional intercellular communication is a special membrane structure.It forms by junction protein between the two adjacent cells. Gap junction was mediated by gap junctional intercellular communication (GJIC).It plays an important role in cell metabolism , electric signal transmission, environmental stability, proliferation , differentiation and other physiological processes. Electric signal transduction between myocardial cells depends on the integrity of structure and function of gap junctional protein. At present, many studies showed that gap junctions were detected at the interface between adjacent cells of an coculture model or after grafting; transient calcium current was detected; the cardiac-like sodium current could form. All these implied that the engrafted cells may forme good gap junction with the resident cardiomyocytes,and achieve synchronous contraction. But many scholars hold opposite views. They suggested that it should not form functional gap couplings between cells . Serious arrhythmias would happen after grafting. At present, there were many different findings in the field. Our aim was to investigate expression of Cx43 and formation of functional gap coupling between cardiomyocytes and rat skeletal myoblasts through immunocytochemistry, transmission electron microscopy, Western Blotting .The present study was divided into three parts.1 The proliferation , differentiation characteristics and expression of Cx43 of L6 cells.The characteristics of L6 cells was examined by HE staining.The expression of Cx43 following the formation of myotubes was detected through Western Blotting.L6 cells were spindle-shaped in normal medium . They began to fuse into multinucleated myotubes after maintained for 3 days in differentiation medium. The results of Western Blotting showed that the expression of Cx43 protein was downregulated compared with the control group,and downregulated significantly with increasing of time. (comparison of every both groups at different time point, both P<0.05).2 The primary culture and identification of neonatal rat cardiomyocytes. 1-3-day-old SD neonatal rat, degermed with75% alcohol, operated the chest to take the ventricle out and cut into small pieces of 1mm3, dispersed by trypsin and EDTA, purified cardiomyocytes through differentially preplated and add to 0.1mmoL/LBrdu. Under the conditions of 37℃, 5% CO2,cardiomyocytes culture were maitained in DMEM medium supplemented with 10% fetal bovine serum, penicillin 100 ug / mL, streptomycin 100μg/mL. Brdu was removed after incubated for 48 hours, and then the medium was changed every 2 days. At 72 hours, observation with inverted microscope: cardiomyocytes were fusiform, polygonal, star, irregular shape and so on .they touched with each other by extended pseudopods, intertwined into a network, emerged into a piece and beated synchronously at a frequency of 60-120 times / minutes.The immunocytochemical results in Sarcomeric Actin antibodies showed that the shape of cardiomyocytes were irregular with extended pseudopods. Cytoplasm appeared brown granular material.3 Expression of connexin43 between neonatal rat cardiomyocytes and rat skeletal myoblasts in coculture We investigated the labelling concentration and time of DAPIof L6 cells;the expression of Cx43 and the formation of gap junction between cardiomyocytes and rat skeletal myoblasts in coculture through immunocytochemistry, Western Blotting techniques, transmission electron microscopy.L6 cells were labled with DAPI at different concentration of 15μg/mL, 25μg/ mL, 50μg/ mL, for 4h, 8h, 12h.Finally we selected the appropriate concentration and time according to cells growth status and fluorescence quench. The results showed that the cells status were best when they were labled for 8 hours at a concentration of 25μg/ mL.It could meet the requirement of study.The immunocytochemical results showed that L6 cells nuclei were labled in blue. Coculture 6 days later, the sarcomeric actin was positive expression in cardiomyocytes, Cx43 of L6 cells was positive expression.There were Cx43 expression both L6-Cx43 cells group and L6 cells group at the interface between adjacent cells. The level of Cx43 expression was detected by western Blotting.The results of western Blotting showed that the expression of Cx43 in both coculture groups was upregulated significantly compared with the cardiomyocytes group, L6 cells group, L6-Cx43 cells group, L6 cells of conditional medium of cardiomyocytes group. Cx43 expression of L6-Cx43 cells coculture group was increased significantly than L6 cells cocultured group (comparison of every both groups , P<0.05, there is significant difference).The expression of Cx43 in L6 cells group, L6-Cx43 cells group, L6 cells of conditional medium of cardiomyocytes group downregulated significantly with increasing of time (take 3,6,9 days).The results of TEM showed that the parallel array of actin filaments, intercalated disc of cardiomyocytes could be seen.There were intermediate junction-like between cardiomyocytes and skeletal myoblasts,but no gap junction was found. Conclusion1 L6 cells were spindle-shaped cultured in DMEM medium containing 15% FBS. 3days later, L6 cells fused into myotubes cultured in differentiation medium. Cx43 expression was downregulated.2 The immunocytochemical results showed that expression of Cx43 could be watched at the interface between adjacent cardiomyocytes and skeletal myoblasts in coculture.3 The results of western Blotting further suggested the expression of Cx43 was upregulated significantly in both coculture groups,but L6-Cx43 cells group had more obvious upregulation. But the expression of Cx43 had no obvious change in conditional medium of cardiomyocytes.These implied expression of Cx43 could be promoted in coculture. The level of Cx43 expression could be better promoted with gene of exogenous connexin43, direct cell-cell contacts was necessary.4 Under TEM , intermediate junction-like between cardiomyocytes and skeletal myoblasts was found,but no gap junction was found, it may be related to the different site of the sections.