Effect Of Slingshot-1L Phosphatase On Cardiomyocytes Differentiation Of Human Bone Mesenchymal Stem Cells

Adult cardiomyocytes(CMs) belong to permanent cells with limited ability of regeneration. Therefore, people are trying to seach newer ways to make the infracted cardiomyocytes to regenerate and promote revascularization.Recently, stem cellular transplantation has the possibility to replace infarctial cardiomyocytes and to restore function. So it becomes one of the hottest reach fields in rencent years.Mesenchymal stem cells(MSCs)are the stem cells derived from mesoderm, possessing great multi-potent differentiation ability. MSCs can differentiate into many different kinds of cells under different environments.But the mechanisms of MSCs differentiated into cardiomyocytes are still not very clear . As reported, cytoskeleton can play an important role in the process of MSCs differentiation.Slingshot(SSH) is a specific cofilin phosphatase. It can activate cofilin, inhibit the polymerization of actin filament,as a result, it participates in the rearrangement of cytoskeleton. This indicate that SSH may educe important effect in the process of MSCs'differentiation. Therefore, the stable transfected hMSCs cell line was established to study the effect of SSH phosphatase on hMSCs differentiation into cardiomyocytes. It may provide helpful theory support and laboratory basis for further study. A series of experiments were designed to investigate the effect of SSH-1Lphosphatase on hMSCs differentiation into cardiomyocytes.Reslults:1,A standardized platform has been established for hMSCs and the hMSCs maintaining stable characteristics, providing an enough cell source for further study.2,We determined the proper inducing concentration of 5-Aza is 10μM by cell proliferation detection and observed the morphology of hMSCs differentiate into CMs.3,We hint that 5-Aza can successfully induce hMSCs differentiating into CMs.4,The retroviral vector pLNCX-SSH-1L was transfected into packaging cell PA317 by lipofectamine. The transfectants were selected and viral titers were 5×108 CFU /L,collect viral supernatant to infect hMSCs, abtain the monoclones overexpressing SSH-1L. 5,We carryed out a series of detections by Immunocytochemical staining, immuno- fluorescence, RT-PCR and Western blot to confirm that the stable transfected hMSCs cell line over-expression Slingshot mediated by retrovirus was established successfully, providing an enough cell source for further study on the effect of SSH phosphatase on hMSCs differentiation into CMs.6,Karyotype analysis showing that Cr group(hMSCs which were not transfected), Scr (hMSCs which were transfected with vacant vector) and SSH group(hMSCs which were transfected with vector encoding SSH-1L) all maintained stable and normal diploid nucleus type without transform.7,During hMSCs differentiate into CMs induced by 5–Aza for four weeks , their morphology change was observed .Compared to the Cr group and Scr group,SSH group hMSCs showed multiple branches after one week, connecting with adjoining cells forming myotube-like structures after 2~3weeks and the myotube-like structures increased strikingly after 3~4weeks.On the basis of these experiments,we conclude that SSH group hMSCs are more powerful in differentiating into CMs than the other two control groups.8,We detected the microfilament changes during hMSCs differentiate into CMs induced by 5– Aza after four weeks. Cr, Scr and SSH group hMSCs'microfilament are all like long straight fiber bundle along the cell's major axis with regular arrangement without induced.After induced by 5– Aza for one week, Cr group and Scr group hMSCs'microfilament basically along the cell's major axis but with irregular arrangement,while SSH group hMSCs'microfilament spread to all directions. After induced for 2~3weeks, Cr group and Scr group hMSCs'microfilament spread to all directions, while SSH group hMSCs'microfilament join to one another and the actin gather together forming like intercalated disk structures. After induced for 3~4weeks, Cr group and Scr group hMSCs'microfilament join to one another forming like myotube-like structures, while SSH group hMSCs'actin gather together in microfilament junctions and forming like intercalated disk structures increase dramatically. On the basis of these experiments,we draw the conclusion that SSH-1Lplay an important role in hMSCs differentiation into CMs through regulating the rearrangement of cytoskeleton.9,We then measured the hMSCs'action potential by current clamp and find that the hMSCs which were not induced by 5-Aza not exhibit action potential,while,those hMSCs after induced exhibit weak action potential and the action potential of SSH group is stronger than the other two control groups. 10,RT-PCR analysis of mRNA expression for cardiac-specific genes in hMSCs shows that the genes can't be detected in Cr, Scr and SSH group hMSCs without induced by 5-Aza. After induced by 5– Aza for one week, hMSCs express GATA4 and cTnT, which are early phase makers of cardimyogenic lineage.While SSH group hMSCs began to expressβ-MHC andα-sarcomeric actin which are late markers of cardiac lineage. After induced by 5– Aza for two weeks,Cr and Scr group hMSCs began to expressβ-MHC andα-sarcomeric actin.The mRNA expression level for cardiac-specific genes increased along with the elongation of induced time and the cardiac-specific gene expression level is much higher in SSH group hMSCs than in the other two control groups.11,Western blot analysis showed that the expression of cardiac-specific protein can't be detected in Cr, Scr and SSH group hMSCs without induced by 5-Aza. After induced by 5– Aza for one week, SSH group hMSCs began to express Desmin, known to be early markers of myogenic differentiation. After induced for two weeks, Cr and Scr group began to express Desmin and then SSH group hMSCs begin to express of cardiac-specific protein cTnⅠandα-sarcomeric actin. While,Cr and Scr group expressed cTnⅠandα-sarcomeric actin until induced for three weeks. The cardiac-specific protein expression increased with the elongation of induced time and the SSH group hMSCs protein expression level is much higher than the other two control groups.In short, this study systemically elucidate that 5-Aza can induce the differentiation of hMSCs into CMs in vitro. We confirmed that SSH-1L can efficiently promote MSCs'differentiation into CMs through regulating the rearrangement of cytoskeleton for the first time. Our work offers helpful theory support and laboratory basis for further investigate on the mechanisms of stem cells differentiate into CMs.