Myocardial Differentiation Of P19 Cells Induced By Exogenous Expression Of Nkx2-5 Gene

Objective: The cardiomyocytes in the mature individual of the mammal are terminal differentiation cells. Generally, the injured cells can be replaced by fibrous scar instead of cardiomyocytes. The lack of and decrease in number of cardiomyocytes are responsible for drop of cardiac contractile function which further result in heart disease such as heart failure. Recent studies show that cardiac stem cells in adult myocardium have a very small quantity and can not repair the damaged myocardium. As to heart transplantation therapy, there are some restrictions of donor, immunogenicity and expensive cost. So, many researchers have now focused on cell transplantation in the treatment of myocardial necrosis to increase the number of functional cardiomyocytes. Animal tests have confirmed that transplantation of bone marrow-derived mesenchymal stem cells, newborn cardiomyocytes, or even skeletal muscle cells can be certain extent improved the cardiac function. However, there are still many problems to be solved in cell transplantation research. One of the primary problems is the low transformation efficiency of above-mentioned cells into cardiomyocytes and could not get numerous contractile cardiomyocytes through such cell transplantation for cell transplantation. Therefore study on the mechanism in the process of myocardial differentiation will be of great significance. Many researchers speculated the processes of the myocardial differentiation begin from the start of a certain or some key genes, usually the protein products of these genes are transcription factors, they can make a downstream target genes cascade activated until completion of the differentiation.Homeobox gene Nkx2-5, also known as the cardiac-specific homeobox gene (Csx), is one of the earliest transcription factor expressed in the cardiogenesis of all vertebrates. It is also one of largely-researched transcription factors which are closely related to heart development and myocardial differentiation. Nkx2-5 is a homologous of Drosophila tinman gene which is found earlier and required for the development of Drosophila heart. And it is a member of NK2 homeobox gene family. The gene plays an important role in the complex development process of heart. Many studies showed that the expression of Nkx2-5 is extremely important for the formation of embryo cardiomyocytes. In Xepopus embryos, the over-expression of Nkx2-5 caused an increase in heart size due to increasing number of cardiomyocytes. The injection of Nkx2-5 into zebrafish embryos led to larger hearts and increased numbers in cardiomyocytes. Transplanting the cells which express Nkx2-5 gene into the area outside of cardiogenic area can cause the expression and transcription of cardiac markers. Foreigner reports said that expression of Nkx2-5 in P19 cells or P19CL6 cells can promote myocardial differentiation, improve the expression of cardiac-specific gene and protect cardiomyocytes against stress-caused damage. Researches have confirmed that Nkx2-5 gene regulate the expression of many genes, such as Atrial natriuretic peptide, cardiacα-actin, cardiac ankyrin repeat protein, A1 adenosine receptor, calreticulin and connexin40, etc. These genes encoding some important structural proteins regulate myocardial characteristics. The results show that Nkx2-5 owns the function of transcriptional regulation of the cardiac procedures. But whether expression of Nkx2-5 could produce contractile cardiomyocytes from mammalian stem cells or induce non-cardiac cells into cardiomyocyte are controversial. And domestically, the relevant reports about the role of Nkx2-5 on myocardial differentiation were fewer. Thus the present study was to carry researches about this issue.P19 embryonic carcinoma cells were induced from male mice C3H/HE teratoma. It remained the characteristics of embryonic stem cells after several passages in vitro. P19 cells can be induced into a variety of cells, including cardiomyocyte. In the process of myocardial differentiation from P19 cells to cardiomyocytes, the expression of cell development-related gene and electrophysiological characteristics were similar to the normal myocardial development of mice. Also the cells are easy to carry out gene manipulation, it has been considered as a good model in vitro on the study of the development of cardiomyocyte.Accordingly, the proposed research plans to introduce Nkx2-5 gene into P19 cells, and selected out the P19 cells which stable expressing of Nkx2-5 gene. At the same time, its myocardial differentiation without inductor is observed. Furthermore, with 5-aza induction, the myocardial differentiation of P19 cells that expressing exogenous Nkx2-5 gene was detected under monolayer culture condition. Our aim is to clarify some laws in the process of myocardial differentiation and to provide effective strategy for transforming various stem cells into cardiomyocytes.Methods:1 The construction and identification of eukaryotic expression vector pEGFP-N1-Nkx2-5The plasmid pEFSA-HA-Nkx2-5 was presented by Dr. Hiroshi Akazawa from Japan. We transformed it into competent E. coli DH5αby the conventional CaCl2 method. After being amplified and extracted, the plasmid pEFSA-HA-Nkx2-5 was identified with XbaI and HindⅢdigestion. Using pEFSA-HA-Nkx plasmid as a template, the full length of Nkx2-5 gene was amplified with PCR. Then PCR products were subjected to agarose gel electrophoresis and target fragment was recovered from the agarose gel. Using amplified full length Nkx2-5 gene as a template, Nkx2-5 Coding sequence was amplified with PCR. Then target fragment was recovered by agarose gel electrophoresis. The Coding sequence of Nkx2-5 and vector pEGFP-N1 were digested by BamHI and XhoI overnight and recovered, respectively. Then the two were ligated at 16℃overnight. The ligation product was transformed into competent E. coli DH5αcells and coated to the LB medium plate containing kanamycin, cultured inverted at 37℃for 16~24 h. Picked a single positive bacteria, the recombinant plasmid were extracted for colony bacterial PCR identification and BamHI, XhoI digestion identification. At the same time, the recombinant plasmid was sent to Shanghai Sangon Co., Ltd. for sequencing identification.2 Exogenous expression of Nkx2-5 gene induced the aggregated P19 cells to differentiate toward the cardiomyocyteThe experiment contains two groups, transfected group and non-transfected group. In transfected group, before transfection, our preliminary experiment found the appropriate cell seeding density, the best transfection system, and the appropriate G418 selection concentration. Using the cationic liposome reagent, Lipofectamine 2000, the plasmid pEGFP-N1-Nkx2-5 were transfected into P19 cells. The transfected cells were passaged at the ratio of 1:10 after 24 hours at the end of transfection. Two weeks later after stable selection by G418, the survival cells were suspension cultured for 4 days to form aggregates. The aggregates were transferred to the Petri dish for adherent culture. At 4th day, 8th day, 12th day and 16th day of the adherent culture, the cells were collected. The expression of desmin,α-sarcomeric actin and cTnT was detected with immunocytochemical and immunofluorescence methods. Also, RT-PCR was used to detect the expression of GATA-4,α-MHC and ANP gene. Western blot was used to detect the expression ofα-sarcomeric actin and cTnT protein. At 16th day of the adherent culture, the ultrastructural changes of the cells were observed. Non-transfected P19 cells were aggregated directly. The detection index and sampling time of non-transfected group were same as that of transfected group.3 5-azacytidine induced monolayer cultured P19 cells with exogenous expression Nkx2-5 gene to differentiate toward cardiomyocyteThe Experiment contains two groups, experimental group and control group. Experimental group, 5-aza induced monolayer cultured P19 cells which expressed Nkx2-5 gene constantly. Control group, 5-aza induced monolayer cultured P19 cells. Under the monolayer culture condition, the cells of experimental group and control group were induced by 5-aza with a concentration of 1μmol/L. At 4th day, 8th day, 12th day and 16th day after induction, the expression ofα-sarcomeric actin and cTnT was detected with immunocytochemical and immunofluorescence methods. Also, RT-PCR was used to detect the expression of GATA-4,α-MHC and ANP gene. Western blot was used to detect the expression ofα-sarcomeric actin and cTnT protein. The ultrastructural changes of cells in the experimental group were observed at 16th day after induced by 5-aza.Results:1 The construction and identification of eukaryotic expression vector PEGFP-N1-Nkx2-5Digestesd by XbaI and HindⅢ, a gene fragment of about 1500 bp was produced from pEFSA-HA-Nkx2-5 plasmid, which was the same size as the full-length of human Nkx2-5 gene (1585 bp). The results confirmed that plasmid pEFSA-HA-Nkx2-5 contains full-length of human Nkx2-5 gene. Using plasmid pEFSA-HA-Nkx2-5 as a template, a gene fragment of about 1500 bp was amplified, which was the same size as the full-length of human Nkx2-5 gene. With the full-length gene as a template, a gene fragment of about 1000 bp was amplified, which was the same size as the human Nkx2-5 Coding sequence (974bp). Nkx2-5 Coding sequence and vector pEGFP-N1 were digested with BamH I and Xho I respectively. The gene fragment (about 1000 bp) and vector (4.7 kb) both with BamH I and Xho I restriction enzyme sites were obtained. The DNA bands were recovered from the agarose gel respectively. After ligation and transformation, a gene fragment of about 1000 bp was amplified by bacteria colony PCR from the recombinant plamid. After digesting with BamH I and Xho I, the recombinant plasmid producted two gene fragments of about 1000 bp and 4.7 kb. With CMV promoter primer for sequencing, the sequence result was 98% agreed with Gene Bank for the human Nkx2-5 gene coding sequence. In the gene start area, there was an intact Kozak sequence. Terminator of gene was mutated and the reading frame was correctly identified. In this study, recombinant plasmid pEGFP-N1-Nkx2-5 was constructed successfully.2 Exogenous expression of Nkx2-5 gene induced the aggregated P19 cells to differentiate toward the cardiomyocytes The experiment confirmed that the appropriate cell seeding density for transfection was 2×105/ml. the best transfection system was the plasmid DNA(μg) and Lipofectamine2000 (μl)at a ratio of 1:3. And the stable selection concentration of G418 was 700μg/ml. In the transfected group, some cells expressed the green Nkx2-5-EGFP fusion protein 24 hours later after transfection with plasmid pEGFP-N1-Nkx2-5. 14 days later after being Selected with 700μg/ml G418, most cells expressed green fluorescent protein. These cells are P19 cells with exogenous expression of Nkx2-5 gene. The cells of transfected group and non-transfected group were aggregated for 4 days and formed some aggregates. After adhesion of the aggregates, some cells grew out and formed outgrowth around the aggregates. The cells in the outgrowth became larger and longer with extended processes and linked to each other. In transfected group, immunocytochemistry results showed that the three antibodies were not expressed at 4th day, but at 8th day, 12th day and 16th day, were all expressed, and the expression was gradually increased. There was obvious brown filament-like structure in the cytoplasm of the cells at 12th day and 16th day. The expression of Desmin at 16th day and 12th day was significantly different from that at 8th day (P <0.01). The expression ofα-sarcomeric actin at 16th day was significantly higher than that at 8th day (P<0.01). The expression of cTnT at 16th day was significantly higher than that at 8th day (P <0.01). The results of immunofluorescence showed that the co-expression ofα-sarcomeric actin and cTnT was detected at 8th day, 12th day, and 16th day after adhere culture, and the expression gradually increased. The overlapped expression of the two proteins was detected at the same position of the same cell. RT-PCR analysis showed that, in transfected group, GATA-4 expressed at 4th day after adherent culture. At 8th day and 12th day, the expression of GATA-4 was gradually increased but decreased at 16th day. ANP andα-MHC expressed at 8th day, 12th and 16th day, and the expression was gradually increased. The results showed that the expression of GATA-4 at 8th day, 12th day and 16th day was significantly different from that at 4th day (P<0.05 or P<0.01). The expression ofα-MHC at 12th day and 16th day was significantly different from that at 8th day (P <0.05 or P <0.01). The expression of ANP at 16th day was increased significantly compared with that at 8th day and 12th day (P <0.01). Western Blot results showed that, in transfected group,α-sarcomeric actin and cTnT were expressed at 8th day, and gradually increased at 12th day and 16th day. The expression ofα-sarcomeric actin at 12th day and 16th day was significantly increased compared with that at 8th day (P<0.01). The expression ofα-sarcomeric actin at 16th day was significantly increased compared with that at 12th day (P <0.05). The expression of cTnT at 16th day and 12th day was significantly increased compared with that at 8th day (P<0.01). The expression of cTnT at 16th day was also significantly increased compared with that at 12th day (P<0.01). The observation of ultrastructure revealed that some cells of transfected group after aggregation and adherent culture for 16 days became elongated cylindrical and there appeared cell junction between the adjacent cells. Beside the cell junction, some microfilament-like structure appeared. In cytoplasm of some cells, there was some dense myofilament-like structure arranged in parallel way,but sarcomere-like structure was not founed. No beating cells were found during the experiment. Results of immunocytochemistry, immunofluorescence, RT-PCR, and Western blot in non-transfected group were all negative. The ultrastructural result showed that no differentiated cell was found in non-transfected group.3 5-azacytidine induced monolayer cultured P19 cells with exogenous expression of Nkx2-5 gene to differentiate toward cardiomyocytesWith 5-aza inducing monolayer culture, the cells of experimental group and control group all grew slowly compared with the P19 cell without inductor. A number of cells turned round, dead and floated up every day. As the cells increased in number, they gathered and formed a state similar to adherent aggregates-like structure. The cells in the center of the aggregates were small and dense, while the surrounding cells became longer, changed into spindle-like shape with extended processes and arranged in radial way. When the aggregates-like structure was formed, the number of dead cells decreased. In the process of culture, beating cells were not found in either of the two groups. In experimental group,α-sarcomeric actin and cTnT expression was detected at 8th day after induced by 5-aza. At 12th day and 16th day, the expression of the two proteins was gradually increased. There was obvious brown filament-like structure in the cytoplasm of some cells. Statistics showed that, the expression ofα-sarcomeric actin at 16th day was significantly different from that at 12th day and 8th day (P <0.01). The expression of cTnT at 16th day was significantly different (P <0.01) compared with that at 8th day. In control group, the expression ofα-sarcomeric actin and cTnT was not detected at 4th day, 8th day,12th day and 16th day after induced by 5-aza. The results of immunofluorescence showed that,in experimental group, the co-expression ofα-sarcomeric actin and cTnT was detected at 8th day, 12th day, and 16th day after induced by 5-aza, and the expression gradually increased. The overlapped expression of the two proteins was detected at the same position of the same cell. In control group, the co-expression ofα-sarcomeric actin and cTnT was not detected. RT-PCR results showed that in experimental group, the expression of GATA-4 at 4th day, 8th day, 12th day and 16th day increased gradually. Alpha-MHC and ANP expressed at 8th day, 12th day and 16th day after induction and the expression also increased gradually. Statistics showed that the expression of GATA-4 at 12th day and 16th day was significantly different compared with that at 4th day (P<0.01). The expression of GATA-4 at 16th day was significantly different from that at 12th day and 8th day (P <0.01, P<0.05). The expression ofα-MHC at 16th day and 12th day was significantly increased compared with that at 8th day (P <0.01). The expression ofα-MHC at 16th day was significantly higher than that at 12th day too (P <0.01). The expression of ANP at 16th day and 12th day was significant increased compared with that at 8th day (P <0.01). The expression of ANP at 16th day was also significant higher than that at 12th day too (P <0.05). In control group, there was no ANP expression. GATA-4 expressed at 8th day, 12th day and 16th day, and the expression was increased gradually. The expression ofα-MHC appeared at 12th day and 16th day. Statistics showed that the expression of GATA-4 at 16th day had a significant difference with that at 8th day (P <0.05). There was no significantly different between the expression ofα-MHC at 12th and that at 16th day. RT-PCR results showed there was a difference between the two groups. The expression of GATA-4 andα-MHC in experimental group was earlier than that in control group. And at each time point, the expression of GATA-4 andα-MHC in experimental group was significantly increased compared with that in control group(P<0.01 or P<0.05), except theα-MHC expression at 12th day. The ANP was expressed in experimental group but not expressed in control group. Western Blot showed that, in experimental group,α-sarcomeric actin and cTnT were expressed at 8th day, 12th day and 16th day and the expression was increased gradually. Statistics showed that in experimental group, the expression ofα-sarcomeric actin at 16th day and 12th day was significantly increased compared with that at 8th day (P<0.01). The expression ofα-sarcomeric actin at 16th day was significantly higher than that at 12th day (P <0.01). The expression of cTnT at 16th day and 12th day was significantly higher than that at 8th day (P <0.01). The two proteins were not detected in control group. Observation of the ultrastructure showed that cell junction appeared between adjacent cells in experimental group. In the cytoplasm beside the cell junction there was some microfilament-like structure. Some of the adjacent cells extended mutual chimeric processes which were similar to the early myocardial intercalated disk-like structure. In the cytoplasm of some cells, some dense myofilament-like structure arranged in parallel way was found. Some area of the myofilament-like structure was dispersed with high electron density area, which was similar to the dark and light band structure of earlier stage sarcomere or the dense body and dense patch of smooth muscle.Conclusion:1 The human Nkx2-5 gene coding sequence was successfully amplified. The eukaryotic expression vector pEGFP-N1-Nkx2-5 was successfully constructed, which would lay a foundation for the further studies on the role of Nkx2-5 in heart development and cardiac differentiation. 2 Eukaryotic expression vector pEGFP-N1-Nkx2-5 can be expressed in P19 cells and can be used for the transfection and selection of eukaryotic cells.3 Exogenous expression of Nkx2-5 gene induced P19 cells only with aggregates formation and without inducing agents to differentiate toward cardiomyocytes. Exogenous expression of Nkx2-5 gene in P19 cells induced the expression of cardiac-specific markers and striated muscle markers and the expression of the markers increased gradually with prolongation of culture time. Exogenous expression of Nkx2-5 gene can induce myocardial differentiation from P19 cells.4 Cardiac differentiation can be induced from monolayer cultured P19 cells by 5-aza. And the cells expressed early cardiac-specific transcription factor and cardiac gene, but no protein expression was detected.5 P19 cells with exogenous expression of Nkx2-5 gene can be differentiated toward cardiomyocytes by 5-azacytidine induction under monolayer cultured condition, and expressed cardiac-specific transcription factor, skeletal muscle-specific and cardiac-specific genes in both the gene level and protein level. The expression of the markers was also increased gradually with prolongation of culture time.6 With monolayer culture and induced by 5-aza, the myocardial differentiation of P19 cells with exogenous expression of Nkx2-5 gene was better than that of non-transfecred P19 cells. It may suggest that Nkx2-5 gene can promote P19 cells to differentiate toward myocardial cells under monolayer culture condition.