Puerarin Promotes Differentiation Of Working Cardiomyocytes Derived From Murine Embryonic Stem Cells And Its Underlying Mechanism

Aims:Currently CMs derived from stem (ES) cells represent the most promising CMs for cardiac regenerative medicine.lt is important to screen and identify chemical compounds that induce high efficiency cardiac differentiation.Phytoestrogen puerarin is a traditional Chinese herbal medicine to treat many cardiac diseases. The aim of this study was to investigate the possible inducible effects of puerarin on embryonic stem cells differentiation and regulatory mechanism in vitro.Methods:Effect of puerarin on proliferation of murine ES cells were analyzed via MTT assay, then were differentiated using The classic "hanging drop-Suspension-adherent" three step differentiation protocol with or without 1?mol/L?10?mol/L?100?mol/L puerarin. Differentiating ES cells were analyzed via morphological analysis for diameter of embryonic bodys (EBs) and percentage of beating EBs, immunofluorescence staining for?-sarcomeric-actinin, RT-PCR for specific genes of different mesodern, western blot for serine-threonine kinase (Akt) and phosphorylation of the serine-threonine kinase (P-Akt).Results:Puerarin (100?mol/L) resulted in significantly increased number of cardiac cells and percentage of beating EBs,100?mol/L puerarin was the optimal concentration Puerarin resulted in a significantly increased the number of?-sarcomeric-actinin positive cells on differentiation day 12. RT-PCR results showed expressions of endoderm, cardiac (mesoderm) and endothelial cells specific gene and decreased expressions of neuronal cells (ectoderm) specific gene at early differentiation stages. Furthermore, we confirmed that embryoid body (EB)-size increased by puerarin might affect the direction of differentiation. Puerarin also facilitated phosphorylation of the serine-threonine kinase (Akt). Conclusions:Taken together, our data suggest that PI3K/Akt pathway might mediate effects of puerarin on proliferation and differentiation of mES cells. Aims:Ventricular cells are obviously the best candidate for reconstructing injured ventricles. However, Cardiomyocytes derived from embryonic stem cells(ES cells) are the mixture of pacemaker-, ventricular-, atrial-like cells, which can be distinguished based on their electrophysiological properties. Therefore, it is important to screen and identify chemical compounds that induce high efficiency cardiac differentiation and specialization of ESCs. Phytoestrogen puerarin has protective effect against myocardial reperfusion injury, has been used clinically for treatment of myocardial infarction. In the present work we aimed to investigate the effects of puerarin on cardiac differentiation of ESCs and its ventricular specialization.Methods:Murine ES cells were differentiated using standard embryoid body-based differentiation protocol with or without 100?mol/L puerarin. Flow cytometry, semi-quantitative RT-PCR and patch clamp were employed to assess differentiating ES cells.Results:Puerarin resulted in a significantly increased percentage of ES cells-derived cardiomyocytes (ES-CMs), the up-regulated transcript levels of transforming growth factor-beta (TGF-?)1,2,3, and Wnt11, but not Wnt3a, bone morphogenetic protein (BMP)2 and 4. The expressions of T-box5, myocyte enhancer factor 2c, and a-myosin heavy chain are significantly increased in early differentiation stage. In advanced differentiation stage, puerarin enhanced expressions of ventricular-specific myosin light chain 2 ventricular transcrip. Patch-clamp analysis confirmed that puerarin doubled percentage of ventricular-like cells.Conclusions:Our results suggest that puerarin promotes cardiac differentiation and enhances specialization of mES cells into ventricular-like cells through regulating expression of multiple genes involved in cardiac development and ventricle differentiation.