Morphology And Electrophysiology Study Of The Junction Between Cocultured Skeletal Myoblasts And Cardiac Myocytes In-vitro

Abundant clinical trial and animal research have demonstrated that skeletal myoblasts could survive among normal and infarcted myocardium and improve the function of damaged heart. Although clinical trial has been done in this field, the mechanism is still unclear. One of the key problems is whether the base of electronic conduction between transplanted myoblasts and myocytes is through intercellular contact or some effective connection such as gap junction. Previous research about the connection between myoblasts and myocytes are mainly morphological, with limiting data on electrophysiology. In this study, the myoblasts and myocytes of newborn SD rat were co-cultured in vitro, immuno-histochemistry, RT-PCR, transmission electroscopy were used to investigate morphologically the possible connection between the two cells consecutively; and Multiple Electrodes Array (MEA) was applied to investigate the electro-activity among the cells. Western blot was also used to investigate the possible affecting factors.Part I Cell obtainment, culture and co-cultureObjective Establish an in vitro co-culture model of skeletal myoblasts and cardiac myocytes. Methods Cardiac myocytes were obtained by multi-step digestion from newly born dayl-2 SD rats (n=8-12). Myoblast was got by one step digestion. Cell was purified by adhesion-seperation technique and cocultured in DMEM containing 20% fetal bovine serum. Phase contrast microscope was used for observation and calculation. Growth curve was thus made. Results Cells fusion and maturation were not observed among the myoblasts which were co-cultured with myocytes for 7 days in DMEM containing 20% fetal bovine serum. The growth speed of myoblasts which co-cultured with myocyte was faster than those cultured alone. Conclusion Myoblasts and myocytes could be co-cultured in DMEM containing 20% fetal bovine serum. The model was fit for our further study. Myocytes might improve the cell proliferation of myoblasts but has little effect on its differentiation and maturation.Part II Morphology Study of Co-cultured Skeletal Myoblasts and Cardiac MyocytesObjective Investigate the morphological evidence for the connection of co-cultured myoblasts and myocytes. Methods Cell suspension of rat myocytes and myoblasts were prepared in DMEM (containing 20% fetal bovine serum)with the ratio 4:1. Cadherin, Connexin43, NCAM were stained by ABC-AEC staining when the cells were co-cultured for 2,4,7,14 days respectively. The staining results were studied under microscope. Cells were observed by transmission electroscope on day 4,7,10,14. Results Connexin43 was expressed in mypblasts from day 4 and increased with cultural duration among the co-cultured myoblasts and myocytes. The expression of connexin43 was higher in the cell membrane of myoblasts which contacted with cardiac myocytes. The expression of cadherin also increased with the prolongation of co-culture time but less significant than that of connexin43. The expression of NCAM which suggests the differentiation and maturation of myoblasts specifically remained lowly two weeks after co-culture. Transmission electroscope revealed that myoblasts remained mono-nucleus cell 2 weeks after co-cultured with myocytes, and cell fusion and maturation was not observed. Connection similar to gap junction was observed to exist between myoblasts and myocytes. Conclusion Material bases and possibility for the existence of the connection similar to gap junction do exist among the in vitro co-cultured skeletal myoblast and cardiac myocytes.Part III Junction Related Protein Expression among Skeletal Myoblasts and Cardiac Myocytes: RT-PCR StudyObjective The expression of junction related protein Connexin43, Cadherin, NCAM among co-cultured myoblast and myocyte were quantified. Methods 3 groups were set as followed: experiment group (myocyte and myoblast were co-cultured with ratio 4:1); control group 1 (myoblast was cultured alone with same cell account); control group 2 (myocyte was cultured alone with same cell account). Cells were digested andgathered at day 2,4,7,14 after cultured and RT-PCR was applied. Results were compared among the 3 groups. Results Connexin43 was expressed by the co-cultured myoblast and the expression increased with the prolongation of culture time. The expression of Cadherin was not increased significantly. The expression of NCAM remained low level and increased only after 2 weeks of cocultured. Conclusion The myoblasts "myocytelized" in some characters when co-cultured with myocytes: the fusion and maturation was delayed, the expression of connexin43 was increased and it is quite possible that gap junction was formed between myocytes and skeletal myoblasts. Part IV Western blot Analysis of the Affecting Factors on the " Myocytelization " of Skeletal MyoblastsObjective The affecting factors of the increase of connexin43 expression in myoblasts were investigated. Methods Experimental groups and control groups were set. Experimental group 1: myocytes and myoblasts were co-cultured with the ratio 4:1, isoproterenol was added in 25nM. Experimental group 2: myoblasts were cultured alone with same cell account, the culture medium of myocyte was added daily (cultured under the myocytes' circumstance). Experiment group 3: myoblasts was cultured alone with same cell account, the culture medium of control group 1 was added daily (cultured under the co-culture's circumstance). Control group 1: the same as experiment group 1 except the isoproterenol. Control group 2: myoblasst were cultured alone with same cell account. Control group 3: myocytes were cultured alone with same cell account. Each group was repeated 4 times and the culture medium was changed daily. Cells were digested on day 5 and counted. The cells were gathered after centrifugal deposition and western blot was applied. Results The expression of connexin43 was similar between experiment group and control group. The expression of NCAM was similar among experiment group 1,2,3 and control group 1 which were all at low level. The expression of NCAM was the highest in control group 2 (myoblasts cultured alone). The average cell count in experiment group 2,3 and control group 2 was (5.15 ± 0.3) ×10~6, (4.90 ± 0.33) ×10~6, (2.32 ± 0.18) ×10~6 respectively. The cell count increased 5 times in experiment group 2 and 3, increased 2 times in control group 2. P value >0.05 when comparing the increasement of cellcount between experiment group 2 and 3 by T test while P<0.01 when comparing with control group. Conclusion The myoblasts displayed the increasment of cell count and delay of cell differentiation and maturation under the circumstance of myocyte or co-culture, the expression of connexin 43 was not increased which suggested that the contaction with myocyte is one of the most important factors affecting the differentiation of myoblast. The expression of connexin43 was not increased through increasing electronic or mechanical stimulation lonely. The affection of myocytes to myoblasts under co-culture circumstance might be the combination of multi-factors. Part V Multi Electrodes Array (MEA) electrophysiological studyObjective The characters of the transmission of electronic message between co-cultured myoblasts and myocytes were investigated to help judging the type of the cell connection. Methods 3 groups were set. Experiment group 1: The myocytes and myoblasts were co-cultured in MEA. Experiment group 2: The myocytes and myoblasts were co-cultured for 2 weeks ( transferred 4-5 times during the 2 weeks and new myocytes was added to keep the ratio 4:1). Then the cells were co-cultured with myocytes in MEA. Control group: Myocytes was cultured in MEA with same amount. Myoblasts was marked by live cell fluorescence staining PKH26 Red. MEA system was connected and results were recorded. Sub-thresh hold electronic stimulation was given and the electronic activity was recorded and analyzed. Results The range of electronic message in experiment group 1 was lower than experiment group 2, which was 1/5 of the latter. That of experiment 2 was similar to control group. The cells in experiment group 2 and control group connected in network after 24 hours of culture and beated spontaneously and synchronizingly. Synchronizingly beat net work was formed 5 days after cultured in experiment group 1. The average transmision speed of electronic activity in experiment group 1 was (2.1±0.6cm/s, n=5) which was slower than that of experiment group 2 (6.8±3.1cm/s, n=8, p=0.053) and control group (9.3±3.8cm/s, n=11, P=0.003) . The local transmision speed of electronic activity was not uniform in experiment group 1 and was related to the flurescent distribution of myoblasts. There was no significant difference between experient group 2 and control group (P=0. 26), the transmision speed was fast and uniform.The time interval of electronic message was even more uniform and the...