Effects Of Icariin On Directional Differentiation Of Mouse Embryonic Stem Cells Into Cardiomyocytes And Relative Modulating Mechanisms In Vitro

Embryonic stem (ES) cells are totipotent cells derived from the inner cell mass of the pre-implantation blastocyst or the morula cells. These cells can be induced to differentiate into multiple cell types, including spontaneously beating cardiomyocytes, under certain conditions in vitro. Exploitation of ES cell-derived cardiomyocytes has facilitated the analysis of early cardiac development and has permitted in vitro "gain-of-function" or "loss-of-function" genetic studies. In the most recently developed test, the embryonic stem cell test is used to assess the embryotoxic potential of test chemicals. This differentiation system also offers the chance to develop new treatment strategies as the applications for cardiac repair and cardiac tissue regeneration. Up to now, there have been few reports on the inducible effects of the traditional Chinese medicine involved in ES cells on their directional differentiation in vitro. The processes by which ES cells differentiate into cardiomyocytes involve molecular biological activities and signal transduction, therefore it could provide the opportunity for exploring the effects of cardioactive drugs on differentiation. Drugs could interfere the expressions of cardiac developmental-dependent genes, sarcomreic proteins, ion channels, or signal transduction during differentiation in vitro, which closely recapitulates the action of drugs in vivo. This differentiation system of ES cells is in favor of detecting the specific or unique targets of cardioactive drug action. A new treating method is explored by combining cardioactive drug with stem cells transplantation, which ispossible to promote the success of implantation therapy and increase clinical effects.Icariin (ICA) is one of the constituents of Epimedium, a traditional Chinese herbal medicine. It possesses many kinds of biological actions, particularly in cardiovascular function improvement, hormone regulation, immunological function modulation, anti-tumor and anti-virus activity. It has been reported that ICA is a major active component of the tablets of "Xin-shen-ning" used for the treatment of heart disease (cardiac angina and myocardial ischemia). Our former work mainly focused on the metabolization of ICA by human intestinal bacteria in vitro and the estrogenic-like activity of the ICA series compounds related to the metabolisms. However, the pharmacological effects and mechanisms of action of ICA on cardiovascular system are not yet known.In this paper, the specific properties of ES cells and the classic methods of cultures were used to investigate the inducible effects of ICA and its two metabolites on the directional differentiation of ES cells into cardiomyocytes in vitro, with a view to clarify the modulating mechanisms of ICA involved in the differentiation and to obtain the the specific or unique targets of action of ICA. This research also provides the experimental basis and a new method for clinical application of combining ICA with stem cells transplantation for treating the infarction myocardium by differentiating into myocardial cells.1. Inducible effects of ICA and its two metabolites on the directional differentiation of ES cells into cardiomyocytesCultures of differentiating ES cells were established by the formation of embryoid bodies (EBs) in hanging drop cultures. When EBs were plated separately onto gelatin-coated 24-well culture plates, ICA, icaritin (ICT), or desmethylicaritin (DICT) was added to the differentiation medium. The final concentrations of testdrugs were designated according to the preliminary test results as follows: 10"7, 10'8, 10"9 mol-L"1 ICA, 10"7 mol-L"1 ICT, and 10"7 mol-L"1 DICT. ES cells treated with 10"8 mol-L"1 retinoic acid (RA) or with 0.1% DMSO solvent were used as positive or negative controls. Differentiation cultures were monitored every day with light microscopy to record the morphology and the number of the spontaneously beating EBs. The rhythmically beating EBs were considered to be spontaneously beating cardiomyocytes, which was defined as a marker of successful differentiation. According to the percentage of the beating EBs, the inducing effects of ICA, ICT, or DICT on the directional differentiation of ES cells into cardiomyocytes were evaluated by means of the concentration-effect and time-effect relationship curves.ICA at 10"7 mol-L'1 possessed the best inducible effects on the differentiation of ES cells into cardiomyocytes. During the course of differentiation, the total percentage of beating EBs treated with 10"7 mol-L"1 ICA, ICT, and DICT was 91% CPO.001), 59% CP<0.01), and 49%, respectively. ES cells were remarkably induced into the rhythmically beating EBs with ICA in a concentration- and time- dependent manner. The results of concentration-effect relationship manifested that the percentage of beating EBs treated with different concentrations of 10"7, 10"8, 10"9 mol-L"1 ICA was 91% (P < 0.001), 76% (P < 0.001) and 56%, respectively. Although only 51 % of the EBs in control cultures contained beating clusters, 75 % of the EBs treated with 10"8 mol-L"1 RA differentiated into beating cardiac clusters (PO.001).The results of time-effect relationship revealed that the percentage of differentiation cultures containing contracting EBs with 10"7 mol-L"1 ICA reached a peak level of 84 % on d 7+9. The potential of EBs with 10"7 mol-L"1 or 10"8 mol-L"1 ICA to undergo cardiac differentiation was significantly enhanced in comparison with those in control cultures over a period from d 7+5 (P < 0.05). It suggested ICA could accelerate and increase the directional differentiation of ES cells intocardiomyocytes.2. ICA involving the molecular biological activities during the differentiation of ES cells into cardiomyocytesBased on the above results, the aim of this section was to confirm the cardiomyocytes derived from ES cells and to investigate the possible mechanisms of inducible action of ICA involved in the differentiation of ES cells into cardiomyocytes. For making sure it, the expressions of cardiac developmental-dependent genes were detected by RT-PCR, the expressions of cardiac-specific sarcomeric proteins were verified by immunocytochemistry, as well as the chronotropic responses of ES cell-derived cardiomyocytes to (3-AR agonist or antagonist were observed by measuring the beating frequency.RT-PCR analysis manifested that in the early differentiation stage before the formation of EBs, when ES cells were treated with ICA at lO^mol-L"1 for 24 h, the mRNA level of BMP2 was remarkedly increased. lO^mol-L'1 ICA also resulted in a significantly accelerated and increased mRNA levels of trascription factors GATA4 and Nkx2.5, cardiac-specific genes a-MHC, MLC-2v and |3-AR on early cardiac developmental stage in a time-dependent manner. It suggested the promoting effect of ICA on cardiac differentiation was related to advancing and increasing mRNA levels of the relative cardiac developmental-dependent genes.The differentiated beating cardiac cells stained positively with anti-a-actinin mAb and anti-troponin T mAb. ICA at lO^mol-L'1 accelerated the differentiation of ES cells into cardiomyocytes expressing cardiac-specific proteins for sarcomeric structures. On d 7+5, about 48% of the cells stained positively for sarcomeric a-actinin in the control, and no positive troponin T-protein-labelled cells were observed. While about 64% of the cardiomyocytes treated with 10"7 mol-L'1 ICA stained positively for a-actinin, and 5% of the cells contained positive troponinT-protein-labelled sarcomeres, which was significantly enhanced in comparison with those in control (48%, 0%) (PO.001). With continued differentiation (d 7+11), the proportion of the cells contained sarcomeric a-actinin staining was barely changed, yet the proportion of cardiomyocytes induced by 10"7 mol-L'1 ICA stained for troponin T-protein-labelled sarcomeres was significantly increased by about 16% in contrast with the case of 6% in the control (.PO.05). It indicated that ICA involved accelerating the sarcomerogenesis for a-actinin and cardiac troponin T during the cardiac differentiation.The positive chronotropic responses of the ES-derived cardiomyocytes were observed after administration of isoprenaline (ISO, p-AR agonist). The effect of ISO on cardiomyocytes was different between early and late developmental stage. On d 7+7 and d 7+11, the beating frequency could be significantly increased in the present of ISO in a concentration-dependent manner. The cardiomyocytes derived from ES cells induced by ICA were more sensitive to ISO than those in control (PO.05). Furthermore, the positive chronotropic effect was interrupted by propranolol (PRO, P-AR antagonist). It suggested that ICA could increase the expression of p-AR genes and promote its maturation, which resulted in higher sensitivity of cardiomyocytes to ISO.3. ICA-mediated early modulation during cardiomyocytes differentiation in ES cellsThe purpose of this section focused on the early modulating mechanisms of inducible effects of ICA during the differentiation stage before a shift to the cardiomyocyte phenotype. To prove the partial mechanisms, flow cytometry was used to detect the modulation of cell cycle and apoptosis. p53 expression in the early differentiation phase was analyzed by RT-PCR and Western blot methods. The concentrations of cAMP and cGMP in ES cells were mensurated by 125I-cAMP and125I-cGMP double antibody radioimmune methods. On the other hand, endogenous nitric oxide (NO) concentration in ES cells during differentiation was evaluated by Griess reaction. Aminoguanidine (AG) was used to confirm the influence of NO on the process which ICA induced the differentiationof ES cells into cardiomyocytes.Flow cytometry revealed that 10"7 mol-L"1 ICA treatment for 48 h could evoke the accumulation of cells in G0/G1 (.P<0.01), reduce the proportion of cells in S phase, and accelerate apoptosis of the cell population (P<0.05). ICA resulted in a significantly increased expressions of p53 mRNA and protein for 24-48 h treatment by RT-PCR and Western blot analysis. It indicated that p53 may play a direct role in apoptosis on early differentiation of ES cells mediated by ICA. During day 7+0 and 7+9 cardiac developmental stage, 10"7 mol-L"1 ICA increased the level of p53 mRNA, but caused a parallel decrease in the level of p53 protein. The decline in p53 protein was associated with the increase in the level of mdm2 protein after ICA treatment. It also suggested that a decline in p53 protein due to the increasing expression of mdm2 protein for degradation would favor the production of newly formed cardiomyocytes.The result of radioimmunoassay manifasted the levels of cAMP and cGMP in ES cells were 0.233 mmol-L"1 and 0.060 mmol-L"1, cAMP/cGMP ratio was 3.875. When ES cells were treated with ICA at 10"7 mol-L"1 for 24 h, the levels of cAMP and cGMP significantly increased to 0.660 mmol-L"1 and 0.083 mmol-L"1 (P<0.05). Meanwhile, cAMP/cGMP ratio also increased to 7.870. Treatment of ES cells with ICA for 48 h resulted in keeping up elevating the level of cAMP and the ratio of cAMP/cGMP (PO.01), but the level of cGMP hardly increased. It hinted that the partial mechanisms of inducible effect of ICA were associated with elevating the cAMP/cGMP ratio in ES cells on early differentiation events before a shift to the cardiomyocyte phenotype.In the control, supernatant NO production was low. When ES cells treated withICA for 24 and 48 h, supernatant NO production was elevated (P<0.05). In particular, the NO-cGMP pathway was involved in the early differentiation of ES cells treated with ICA for 24 h. During the course of differentiation (from d 7 to d 7+9), ICA facilitated the directional differentiation of ES cells into cardiomyocytes and the supernatant NO generation increased. In the present of AG, the NO generation during the differentiation was decreased significantly. In addition, AG could significantly delay and decrease the incidence of contracting EBs compared with control cells. The beating EBs were not observed until d 7+2, and the percentage of them was only about 35% on d 7+9. However, in ICA together with AG culture, though AG resulted in a decrease in the percentage of beating EBs treated with ICA to about 75% on d 7+9, the potential of EBs to undergo cardiac differentiation was significantly enhanced in comparison with those in control with AG cultures over a period from day 7+1 to d 7+9 (PO.05). It implied that another mechanism of inducible effect of ICA involved promoting the pathway of NO generation by the up-regulation of iNOS expression during cardiac differentiation stage.Conclusion1. Among threee compounds, ICA had the best inducible effects on the directional differentiation of ES cells into cardiomyocytes at 10"7 mol-L*1.2. The promoting effect of ICA on cardiac differentiation was related to advancing and increasing mRNA levels of BMP2, trascription factors GATA4 and Nkx2.5, cardiac-specific a-MHC, MLC-2v and P-AR genes on early cardiac developmental stage. ICA involved accelerating the sarcomerogenesis for a-actinin at early differentiation stage and cardiac troponin T during late cardiac differentiation. The cardiomyocytes derived from ES cells induced by ICA were more sensitive to ISO.3. The partial mechanisms of inducible effects of ICA were associated with regulating the cell cycle, inducing apoptosis, as well as the modulating of p53 during early differentiation and cardiac development. Meanwhile, it was related to elevating the cAMP/cGMP ratio in ES cells on early differentiation events before a shift to the cardiomyocyte phenotype. In addition, ICA involved promoting the pathway of NO generation by the up-regulation of iNOS expression during differentiation stage.