MiR-126-Rac1/p67phox Signaling Takes A Critical Role In Icariin Induced Cardiac Differentiation Of Murine Embryonic Stem Cells

Embryonic stem (ES) cells can be cultured and maintained in undifferentiated state in vitro. Accumulated evidence suggested that cardiomyocyte differentiation of ES cells in vitro faithfully recapitulated cardiomyocyte differentiation in vivo. Thus cardiomyocyte differentiation of ES cells is considered as an efficiency model in vitro for cardiac developmental biology, cardiac pathology and new drug discovery. In our previous work, Icariin (ICA) facilitates the directional differentiation of ES cells into cardiomyocytes, and reactive oxygen species (ROS) signaling plays a vital role in the process. However, the mechanisms by which ROS are activated by ICA are still not understood.In the present studies, in order to reveal the underlying mechanisms of ICA in cardiomyocyte differentiation, the relationship between structure and inducing effect was investigated. Given that ICA is a prenylflavonid compound with a prenyl group at position8of ring A, we presume that protein prenylation may be responsible for its role in cardiomyocyte differentiation.Racl, which belongs to the small G protein Rho GTPases family, work as molecular switches to transduce intracellular signals from growth factors or G protein coupled receptors. As the cytosolic subunit of NADPH oxidase complex, Racl protein prenylation is essential to its migration from the cytosol to the plasma membrane where its attachment favors assembly of p67phox.This section demonstrated that Racl protein prenylation initiating ROS generation takes a critical role in ICA induced cardiomyocyte differentiation. Small interfering RNA (siRNA) targeting Racl (si-Racl), the plasmid with Racl CAAX box deletion (Racl△), the selective GGTase-I inhibitor GGTI-298, the selective FTase inhibitor FTI-277and the Racl GTP-binding inhibitor NSC23766were used to determine whether the ultimate activation of Racl is essential to ICA induced ROS generation.MicroRNAs (miRNAs) play an important role in regulating cardiomyocyte differentiation of ES cells by repressing the translation of selected mRNAs. With miRNAs arrays, we identified that miR-126increased markedly during ICA induced cardiomyocyte differentiation. miR-126mimics enhanced cardiomyocyte differentiation, while miR-126inhibitor abolished the differentiation. Accordingly, miR-126enhances the pro-angiogenic actions of VEGF (vascular endothelial growth factor).Based on the observations that VEGF elicits a burst of ROS that enhances angiogenesis, this section is aimed to investigate whether miR-126promotes Racl-p67phox membrane accumulation, thus activating ROS. Former studies have showed the role of Ca2+in cardiomyocyte differentiation, and Ca2+is the direct molecular target of ROS. The third section was aimed to explore whether miR-126-Racl/p67phox-ROS-Ca2+pathway involved in ICA induced cardiomyocyte differentiation.1. Racl protein prenylation initiating ROS generation takes a critical role in ICA induced cardiomyocyte differentiation of ES cellssi-Racl, Racl△, GGTI-298, FTI-277and NSC23766were used to determine the role of Racl protein prenylation in ICA induced cardiomyocyte differentiation. Intracellular ROS generation was measured using the fluorescent dye2’,7’-dichlorodihydrofluorescein diacetate (DCF-DA). The differential protein fractions (the membrane and cytosol fractions) of Racl were assessed by western blot. The sarcomeric structure of cardiomyocytes derived from ES cells was assessed by immunofluorescence with a-actinin antibody.The results demonstrated that ICA0.5,1,2,4h rapidly elevated Racl translocation to the membranes. si-Racl, Racl△, GGTI-298and NSC23766all could abolish the inducible effect of ICA, however, FTI-277couldn’t disrupt the effect. These results suggested that Racl protein prenylation was involved in ICA induced cardiomyocyte differentiation.2. miR-126/VEGF/Flk-1signaling is involved in ICA induced cardiomyocyte differentiation of ES cellsThe first section has explored the role of Racl in ICA induced cardiomyocyte differentiation, to further investigate the upstream target of ROS, miR-126, VEGF and Flk-1was analyzed. The expression of VEGF and Flk-1was assessed by western blot. The different expressions of miRNAs induced by ICA were assessed by miRNAs chips. The Flk-1inhibitor SU5416, the siRNAs targeting Flk-1(si-Flk-1) miR-126mimic and miR-126inhibitor was used to investigate the miR-126/VEGF/Flk-1signaling in ICA induced cardiomyocyte differentiation.The results demonstrated that VEGF is highly expressed in EBs on day5, when EBs initiated to differentiate into cardiomyocytes, VEGF showed a markedly decrease. Flk-1kept a relative steady expression during the differentiation. ICA slightly increased VEGF expression on day5+3. SU5416(5μM,2.5μM and1μM) all had no toxic effect on ES cells. SU5416couldn’t inhibit the spontaneous cardiomyocyte differentiation, as seen by the increased beating activity and a-actinin expression. SU5416was applied to ES cells from hanging drop cultures, it did inhibit the spontaneous cardiomyocyte differentiation. With microRNA arrays, we identified that miR-126increased markedly during ICA induced cardiomyocyte differentiation. Overexpression of miR-126in ES cells promoted cardiomyocyte differentiation by activating VEGF signaling, while knockdown of miR-126blocked cardiomyocyte differentiation by inhibiting VEGF signaling. ICA2h significantly enhanced the formation of Rac1-p67phox complex, which was disrupted by miR-126inhibitor.3. miR-126-Rac1/p67phox-ROS-Ca2+signaling participated in ICA induced cardiomyocyte differentiation of ES cellsBased on the first and second sections, in order to further verify the role of ICA in cardiomyocyte differentiation, this section is aimed to investigate the downstream target of ROS, the Ca2+. The co-locations of Ca2+handing proteins on endoplasmic reticulum (ryanodine receptors, RyRs; inositol1,4,5-triphosphate receptors, IP3Rs;(sarco) endoplasmic reticulum Ca2+-ATPase, SERCA) and α-actinin were assessed by fluorescence. The expressions of RyR2, IP3R2and SERCA2were assessed by western blot. The intracellular Ca2+transients were tested by Fluo4-AM staining.The results demonstrated that there was a correlation between α-actinin and RyR2, IP3R2or SERCA2during differentiation. The expressions of RyR2, IP3R2and SERCA2were increased in EBs treated with ICA. The concentration of intracellular Ca2+in EBs on day5was increased by ICA. The amplitude of the intracellular Ca2+transients was elevated in the cardiomyocytes derived from ES cells with ICA treatment. miR-126inhibitor could hinder the inducible effect of ICA.Conclusion:1. Rac1protein prenylation is catalysed by GGTase-I, addition of geranylgeranyl diphosphate to the cysteine residue in the CAAX sequence and subsequently migrates to the membrane where its attachment favors assembly of p67phox, thus activating NOXs and generating ROS. This process takes a critical role in ICA induced cardiomyocyte differentiation.2. At the early differentiation stage, up-regulation of miR-126induced by ICA promotes Racl-p67phox membrane accumulation may via VEGF and its receptor Flk-1, thus activating ROS.3. Ca2+signaling is regulated by ROS in ICA induced cardiomyocyte differentiation, miR-126inhibitor decreases ROS generation, thus down-regulation [Ca2+]i in day5EBs and RyR2, IP3R2, SERCA2proein expression in day5+7EBs and the amplitude of Ca2+transient in the cardiomyocytes derived from ES cells.