| Embryonic stem (ES) cells are derived from the inner cell mass of the pre-implantation blastocyst or the morula cells, which can be cultured and maintained in undifferentiated state in vitro. Unlimited differentiation potential is one of the marked characters of ES cells, in other words, ES cells can differentiate into cell types of all three primary germ layers if cultured under the correct conditions. Accumulated evidence suggested that cardiomyocyte differentiation of ES cells in vitro faithfully recapitulated cardiomyocyte differentiation in vivo and cardiomyocytes derived from ES cells display properties similar to those observed in vivo or in primary cultures cultures: 1) Embryoid bodies (EBs) were developed before cardiomyocyte differentiaton, which was characterized with three primary germ layers; 2) Cardiac specific transcriptors or genes are expressed in a developmentally controlled manner; 3) Showing characteristic sarcomeric structures; 4) Possessing membrane-bound ion channels; 5) Its contractility could be triggered by cardiac-specific ion currents. For these reasons, cardiomyocyte differentiation of ES cells is considered as an efficiency model in vitro for cardiac developmental biology, cardiac pathology and cardiac disease therapy.As to pharmacology, "knock-in" technology in mouse ES cells is well-established and given the wide potential for its application in drug discovery. ES cells can be engineered to express reportor genes (usually GFP or luciferase) under control of a lineage-specific promoter, which offers the opportunity for high-throughput screening to identify leads, which induce differentiation towards the precutors of cardiomyocytes. Optimisation of the pharmacophore around the lead would generate low molecular weight compounds for control of differentiation to an early precursor cell. One can envisage later stages which would specify later precursor cells and ultimately fully differentiated cells. Fully differentiated cardiomyocyts would be valuable models for drug discovery. Compounds discovered at earlier stages may be useful drugs to stimulate regeneration of tissue in treatment of degenerative diseases, ischaemic heart disease or stroke. Moreover, ES-derived systems are of special importance for the investigation of embryotoxic properties of teratogenic agents. Chemicals could modulate the expressions of cardiac developmental-dependent genes, sarcomeric proteins, ion channels, or proteins involved in signal transduction way, which closely recapitulates the action of chemicals in vivo, when they are added to the differentiation processes.Although there have been few reports on the inducible effects of compounds on cardiomyocyte differentiation of ES cells, our previous work has demonstrated that icariin (ICA) could significantly stimulated cardiac differentiation of ES cells in vitro. ICA is an active ingredient of plant herba Epimedium, which possesses many kinds of biological actions, such as improving cardiovascular function, modulating hormone excretion and immunological functions, and displaying anti-tumor activities. Using a model system comprised of ES cells, we found that ICA could efficiently stimulated cardiac differentiation which is paralleled by an accelerated gene expression ofα-myosin heavy chain (α-MHC) and myosin light chain 2 (MLC-2v). But little is known about the mechanisms involved in ICA induced cardiomyocyte differentiation of ES cells.Cardiomyocyte differentiation can be divided in two processes: cardiogenesis and cardiac myofibrillogenesis. Early cardiogenesis is regulated by three families of transcription factors (i.e., Nkx2.5, MEF2C, and GATA4), which start to be fully expressed on d 5, while cardiac transcription factors (i.e.,α-MHC, MLC-2v) encoding contracting proteins have not appeared at that time. Thus, d 5 is a critical window at which the cardiac differentiation program becomes fully activated but no cardiac cells, identified by organized cardiac sarcomeric proteins or by functional automatic contractions, are yet present. The molecular pathways that control specification and differentiation of cardiomyocyte differentiation are differed in different processes. Any enviromental factors that affect cardiomyocyte differentiation are tranduced by these molecular pathways.In addition to the expression of cardiac specific transcription factors and genes, mitochondrial biogenesis is another critical event in differentiation processes. Mitochondrial biogenesis requires coordinated changes in the metabolic enzymes of oxidative phosphorylation, TCA cycle, and fatty acid oxidation. Expression of the hundreds of nuclear-encoded mitochondrial biogenesis related genes is co-regulated by a few nuclear transcription factors and co-activators. Any factors that interrupt mitochondrial biogenesis would prevent cardiomyocyte differentiation of ES cells.Our previous work has proved that ICA could induce cardiomyocyte differentiation of ES cells when it was added from d 5 and maintained from then on, which cover the two processes of differentiation. Therefore, it should be made clear that application at which term could ICA efficiently induced cardiomyocyte differentiation (Part I). Then, the possible mechanism involved in inducible effect of ICA was investigated (Part II). At last, the modulation of the most commonly factors (PPARαand PGC-1α) implicated in control of mitochondrial biogenesis by ICA during cardiomyocyte differentiation was addressed (Part III).1. Application of ICA from d 5 to d 8 could efficiently induced cardiomyocyte differentiation of ES cellsThe aim of this section is to confirm that application at which term could ICA most efficiently induce cardiomyocyte differentiation. Cultures of differentiating ES cells were established by the formation of EBs in hanging drop cultures. Differentiation cultures were monitored every other day with light microscopy to record the morphology and the number of the spontaneously beating EBs. EBs containing 2 or more beating foci were considered beating EBs, which was defined as a marker of successful differentiation. This scoring technique is based on the fact that an EB containing only a single beating cluster, is usually very small and not representative of a typical beating EB in which several beating clusters are usually visible. Immunofluorescence was adapted to identify cardiomyocytes derived from ES cells. Meatime, expresssion of Troponin-T andα-Actinin were evaluated on d 12 when EBs were treated with ICA at different term.Cardiomyocytes derived from ES cells were positive for Troponin-T andα-Actinin antibody and cross striations were observed at high magnification. During the course of differentiation, the total percentage of EBs containing beating areas was 58 %, 93 %, and 62 %, when EBs were treated with 10-7mol/L ICA at d 3-5, d 5-8, and d 8-12 respectively, indicating that application of ICA from d 5 to d 8 possessed the best inducible effect on cardiomyocyte differentiation of ES cells.In parallel, expression ofα-Actinin and Troponin-T was significantly improved when EBs were subjected to 10-7 mol/L ICA from d 5 to d 8. Meanwhile, the data also showed that protein level ofα-Actinin and Troponin-T in EBs were up-regulated by ICA in a dose-dependend manner.2. Involvement of p38MAPK and reactive oxygen species (ROS) in ICA induced cardiomyocyte differentiation of murine embryonic stem cells in vitroBased on the results of part I, this section was aimed to investigate the possible mechanisms of the inducible effect of ICA. Reactive oxygen species (ROS) generated by the N0X4 family of NADPH oxidases has been described to act as second messengers regulating cardiomyocyte differentiation of ES cells. Intracellular ROS level in undifferentiated ES cells or EBs was analyzed and expression of N0X4 was evaluated when EBs were treated with ICA. A time-course analysis was performed in order to determine MAPK activation throughout the ICA reatment period. To comfirm the impact of MAPK activity on ICA enhanced cardiomyocyte differentiation, EBs were treated with ICA alone or together with SB203580 (10μM), UO126 (10μM), and SP600125 (1μM) from d 6 to d 8. Subsequently, cardiomyocyte differentiation was assessed by either counting the number of spontaneously beating EBs on d 12 or determining expression of cardiac specific genes on d 10. To explore whether ICA stimulated generation of ROS triggered cardiogenesis via the activation of p38MAPK, the phosphorylation of p38MAPK and cardiomyocyte differentiation were evaluated when generation of ROS was inhibited. After 24 h (d 6), outgrown EBs were subjected to 10-7 mol/L ICA. When EBs were treated with ICA for 3 h, an increase in ROS generation was observed, which was inhibited in the presence of NADPH oxidase inhibitor DPI (10μM) and anti-oxidant Trolox (30μM). ROS levels remained elevated for several hours and decreased in 9 h as assessed by determination of DCF fluorescence.Outgrown EBs (d 6) were treated with 10-7 mol/L ICA for 24 and 48 h (i.e. d 7 and d 8 in differentiation) and phosphorylation of p38MAPK, ERK, and JNK were detected on d 7, d 8, and d 9(24 h after the withdrawl of ICA). As a result, in the absence of ICA treatment (control), p38MAPK activity peaked spontaneously on d 6 and followed with a significant decrease on d 8, while phosphorylation of p38MAPK was further activated and prolonged by ICA treatment. ICA stimulated activation of p38MAPK remained until d 8 (i.e. 48 h after the addition of ICA) and disappeared on d 9. These results implied that a high p38MAPK activity was associated with cardiogenesis and responsible for ICA induced cardiomyocyte differentiation. Activation of ERK showed a similar time course with JNK activity, although phosphorylation was strong on d 6 and remained elevated throughout the differentiation procedure, however, there was not any significantly change between control and the cells treated with ICA.It was shown that ICA stimulated cardiomyogenesis was abolished by SB203580, while SB203580 alone had slight effect on differentiation. Similarly, the increase in the expression ofα-MHC and MLC-2v following ICA treatment was inhibited in the presence of SB203580. On the contrary, co-cultured with UO126 or SP600125 could not blunt the inducible effect of ICA. Moreover, administration of UO126 or SP600125 alone had no effect on cardiomyocyte differentiation of ES cells.Co-treatment with NADPH oxidase inhibitor DPI abolished ICA stimulated p38MAPK phosphorylation in EBs. In parallel, when DPI was co-applied, inducible effect of ICA was completely abolished, while DPI alone did not affect differentiation, as evaluated by counting the percentage of spontaneously contracting EBs. 3. ICA promoted expression of PGC-1αand PPARαduring cardiomyocyte differentiaton of ES cellsThis section was aimed to analysis the effect of ICA on expression of PGC-1αand PPARα. First, Semi-quantitative RT-PCR and Western-blot was adapted to detect expression of PGC-1αand PPARα, and antagonist or agonist was adapted to evaluate the function of PPARαin differentiation process. Second, expression of PGC-1αand PPARαwere analyzed when EBs were treated with ICA.Both PPARαand its coactivatior PGC-1αwere increased during cardiomyocyte differentiation. A positive correlation between PPARαand Troponin-T expression was also observed by immunofluorescence in early differentiation. Application of PPARαantagonist GW6471 prevented cardiomyocyte differentiation as indicated by reduced expression of cardiac specific genes (α-MHC, MLC-2v) and cardiac sarcomeric proteins (α-Actinin, Troponin-T). However, gene expression of cardiac specific transcription factors (GATA4, Nkx2.5, and MEF2C) remained unchanged. Moreover, cardiomyocyte differentiation of EBs could be efficiently stimulated by WY14643 treatment, the specific agonist of PPARα. Taken together, these results suggested a facilitating role of PPARαin cardiomyocyte differentiation of murine ES cells in vitro.Expression of PPARαand PGC-1αincreased coincidently in early differentiation and the increase was dose-dependently up-regulated by ICA treatment. Phosphorylation of p38MAPK peaked on d 6 and decreased after d 8, and the activation was further enhanced and prolonged when EBs were subjected to ICA, which was concurrent with the elevation of PPARαand PGC-1α. Moreover, inhibition of p38MAPK pathway by SB203580 efficiently abolished ICA stimulated cardiomyocyte differentiation and resulted in capture of up-regulation of PPARαand PGC-1α. Summary1. Application of ICA from d 5 to d 8 possessed the best inducible effects on cardiomyocyte differentiation of ES cells followed by up-rugulated expression ofα-Actinin and Troponin-T2. ROS generation and the subsequent activation of p38MAPK are essential for the inducible function of ICA on cardiomyocyte differentiation of murine ES cells in vitro.3. ICA promoted expression of PGC-1αand PPARαduring cardiomyocyte differentiation of murine ES cells in vitro and the effect was partly responsible for activation of p38MAPK.
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