| Background and objectiveRecent research demonstrated that ischemic heart disease and acute myocardial infraction remain the leading cause of mortality in developing countries. The myocardial ischemia caused by coronary circulatory disturbance may lead to the decreased of cardiomyocytes, even heart failure when it is seriously. The critical problem is searching the approach which can increase the number of cardiomyocytes to cure ischemic heart disease. Recently, stem cell therapy is used as a tool in myocardial regeneration. However, previous studies focus on using embryonic stem cells or mesenchymal stem cells in stem cell transplantation therapy. But the rate of migration and differentiation of these transplantations was low and immunological rejection may happen. Recent researches discovered there is a reservoir of multipotent stem cells. It contradicted with traditional concept, which regard the mammalian heart as a terminally differentiated organ. These myocyte precursors undergoing mitosis and owning the potential of differentiation were called cardiac stem cells (CSCs).Recent studies revealed that CSCs expressing some stem cell markers (c-kit, MDR-1, and Sca-1). CSCs can be divided into a variety of cell types:sca-1~+ CSCs, c-kit+ CSCs, SP cells, Isl1+ CSCs and Cardiospheres and cardiosphere-derived cells (CDCs). These stem cells can differentiate into three cardiac lineages:the myocardial cells, vascular endothelial cells and vascular smooth muscle cells. Due to the long growth cycle of SP cells, it is not suitable to study in vitro. It is difficult to culture c-kit+ CSCs after separation. Isl1+ cells exist only in embryonic or postnatal young heart. As a result, raw materials are not easy to acquire. The cells in CDCs express different surface antigen, so the stem cell lines are adulterated. However, the sca-1~+ CSCs were abundant in heart. In addition, this type stem cells can be isolated easily by magnetic cell separation system. After extracting, the cultivation of sca-1~+ CSCs is easier than other types of stem cells. Furthermore, the expression of sca-1 may play a crucial role in cardioprotection. Therefore, sca-1~+ CSCs as an obtained material can guarantee the experiment proceeding successfully.Sphingosylphosphorylcholine (SPC), a naturally occurring bioactive lipid, originate from lipid metabolism. SPC is the major component of high-density lipoproteins (HDL) in blood plasma. The endogenic character endows it less toxicity, and high efficiency to be uptake by cells, then, activate many cellular signaling pathways more easily. The bioactive lipid regulated some physiological processes, for example:proliferation, cell migration, angiogenesis, anti-apoptotic, anti-tumor and wound healing. Previous studies in our laboratory revealed that SPC can protect endothelial and myocardial cells against apoptosis through promoting autophagy. But the effect on cell differentiation is less known. Previous studies have shown that SPC promote mesenchymal stem cell differentiate into vascular smooth muscle cells through activating Rho-associated protein kinases, remaining the unknown effect on myocardial cells.To answer these scientific questions mentioned above, we investigated the function of SPC in differentiate of sca-1~+ CSCs to cardiomyocytes and the underling molecular mechanisms. Here, we reported that SPC as a more effective tool to inducing stem cell differentiation, which might be a reason for SPC protecting the heart from infarction damage. The clarification of the underlying mechanism will support new strategies for heart regeneration.Results1. Extraction of endogenous sca-1~+ cardiac stem cells of adult mice.We performed flow cytometry analysis to confirm the purity of CSCs during culture in vitro after isolating through using magnetic cell separation system. CSCs were treated with SPC or vehicle for 2 to 3 weeks, then changes of the morphology were observed through invert microscope.2. The differentiation of mouse heart-derived sca-1~+ cells into myocardial cell was induced by SPC.The cells were treated with different concentrations for various time. The result of qPCR showed that SPC promoted transcription of GATA4, Nkx2.5, cTnt and Mef2c at 2 and 3 weeks. Furthermore, increased expression of cTnt was verified by immunofluorescence. Our result of GATA4 immunofluorescence assay showed that SPC promoted translocation of GATA4 to nucleus. We detected the role of SPC on the proliferation of Sca-1+ cells and embryonic rat heart-derived H9c2 cells by EdU assay. Our data suggested that the upregulation of cardiac markers were not because SPC enhanced the proliferation of pre-existing cardiac marker-positive cells. All the results verified the function of SPC in inducing CSC differentiation to cardiomyocytes.3. JNK is involved in SPC-stimulated CSC differentiation to cardiomyocytes.JNK participated in stem cell differentiation to cardiomyocytes and SPC-stimulated apoptosis. Here, we reported that SPC activated JNK at 1 and 2 weeks by western blot. Furthermore,the expression of ROCK1 can be activated by SPC, while ROCK2 was not. Then we used the specific inhibitor of JNK to analyze their functions in SPC-induced differentiation. JNK inhibitor SP600125 weakened SPC promoted the expression of myocardial marker and nucleus translocation of GATA4.4. SPC may induce the differentiation of sca-1~+ stem cells through JNK/STAT3.STAT3 is vital to stem cell differentiation, and might be regulated by JNK. Western blot assay showed that SPC promoted phosphorylation of STAT3 Ser727 and Tyr705. At the same time, we demonstrated that SPC can promote the translocation of STAT3 into nuclear. Next, we sought to verify the role of STAT3 and its relationship with JNK in SPC-induced myogenic transition. JNK inhibitor SP600125 inhibited phosphorylation of STAT3 at 2 weeks, suggested JNK located at the upstream of STAT3. STAT3 inhibitor stattic reduced the expression myocardial marker and changed GATA4 nucleus location, which suggested that STAT3 is involved in SPC-induced sca-1~+ cell differentiation as JNK, and revealed a JNK/STAT3 pathway in this process.5. The differentiation of sca-1~+ stem cells induced by SPC was also through the GSK3p modulated β-catenin signaling pathway.Inhibition of P-catenin might be beneficial for myogenic differentiation. Our western blot assays showed that phosphorylation of P-catenin was increased by SPC at 1 h, and maintained at a high level till 1 and 2 weeks. The phosphorylation of GSK3β experienced a short decrease at 1 h, then increased at 1 and 2 week. LiCl and SB216763, inhibitors of GSK3β, were used combined with SPC. As a result, LiCl and SB216763 were both increased β-catenin and inhibited SPC-stimulated cardiomyocyte differentiation. The immunofluorescence assay showed that these inhibitors also inhibited SPC-induced cTnt expression and GATA4 nucleus translocation. We used XAV-939, one of inhibitors of β-catenin, to inhibit the effect of beta-catenin. As the results, XAV-939 were obviously increased SPC-stimulated transcription of GATA4, Nkx2.5, cTnt and Mef2c and GTAT4 nucleus translocation, which suggested that inhibiting the function of P-catenin could further enhance the differentiation induced by SPC. As conclusion, SPC induced CSC differentiation to cardiomyocytes through β-catenin pathway.6. There is no crosstalk between JNK/STAT3 and P-catenin signaling pathways.To clarify the relationship between JNK/STAT3 and P-catenin signaling pathways, we used a series of inhibitors. As a result, JNK and STAT3 inhibitors did not change the activities of GSK3β. Meanwhile, there no changes of JNK and STAT3 were observed when we treated cells with SPC associated with GSK3β inhibitors, which suggested that there is no crosstalk between JNK/STAT3 and P-catenin signaling pathways.7. Lipid raft is a modulator to activation of JNK/STAT3 signaling pathway.Lipid rafts are cholesterol-rich microdomains of cell membranes that have an important roles in receptor-mediated signal transduction. We used P-cyclodextrin to destroy the lipid raft firstly, western blot revealed, SPC-stimulated phosphorylation of JNK and STAT3 were both inhibited when lipid raft was disrupted, but SPC-inhibited activity of P-catenin was not changed, which suggested that lipid raft is a modulator to JNK/STAT3 signaling pathway. We detected the changes of lipid raft associated protein, Rho-associated protein kinases (ROCKs), in SPC treated cells. The results showed that ROCK1 could be inhibited by SPC at the early stage, which might explain the molecular mechanisms of the activation of JNK and STAT3. In conclusion, SPC promotes the differentiation of resident sca-1 positive cardiac stem cells to cardiomyocytes through lipid raft/JNK/STAT3 and β-catenin signaling pathways. |
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