The Study Of Mechanism On 4-phenylbutyric Acid Affecting The Differentiation Of Stem Cells Into Cardiomyocytes

Cardiac differentiation in vitro is a complex stepwise process that is tightly regulated by the chronological integration of intrinsic transcriptional program and extrinsic signaling pathways.The effect of exogenous signaling pathways is dependent on the cell context and the differentiation stage during cardiomyogenesis,exhibiting remarkable stage-specific and dose-dependent characteristics.There are several signaling pathways participating in the stepwise process of cardiac differentiation,such as TGF-?,Wnt,Notch,and Hedgehog.These signaling pathways exert distinct effect and govern the determination of cell fate during the critical steps of cardiac differentiation.Manipulating signaling pathways can direct the committed differentiation of stem cells to the cardiac lineages,thereby efficiently obtaining homogeneous and sufficient number of cardiomyocytes.Recently,the effect of some small molecule compounds on Cardiac differentiation is one of the hotspots.As a small molecule compound,4-PBA is not only endoplasmic reticulum stress inhibitor,but also histone deacetylase inhibitor.The differentiation of stem cell into cardiomyocytes requires cells to synthesize enzymes,antibodies,serum proteins and extracellular matrix and other components,and there will be a large number of new synthetic protein in endoplasmic reticulum.The differentiation of stem cells in cardiomyocytes cells in the process of 1-5 days need suspension culture to form embryoid bodies.The embryoid body is a three-dimensional spherical structure.And at the late stage cells need to adherent to culture and rapidly form multi-layer structure,there exist oxygen deficiency and lacking of energy,and these may cause endoplasmic reticulum stress.Epigenetic regulation of chromatin structure is the basis for activating or inhibiting of genes during embryonic development,which plays an important role in cardiac differentiation in vitro.Our research aims to study the effects and mechanisms of a small molecule compound 4-PBA on cardiac differentiation,and assess whether 4-PBA acts by reducing the endoplasmic reticulum stress or enhancing acetylation to affect differentiation.Our studies will provide a method for obtaining a large number of pure and functional cardiomyocytes from stem cells.The research contentTo investigate the effects of 4-PBA on differentiation of stem cells into cardiomyocytes,and further analyze the potential molecular mechanism.The research methods1.We used mouse embryonic stem cell R1 as the experimental material and 10-4 M VC as an inducer to establish cardiac differentiation model.The percentage of beating embryoid bodies was observed and counted.Immunofluorescence staining was used to detect the expression of a-actinin.Real-time PCR were performed to evaluate the cardiac specific expression of transcription factors to determine whether the differentiation model was established successfully.2.The expression of endoplasmic reticulum stress and acetylation gene was detected by real-time PCR in different stages of cardiac differentiation.3.Cells were treated with 4-PBA,TM,NaB and the solvent dimethyl sulfoxide(DMSO)was used as a control at different stages of cardiac differentiation.The effects of 4-PBA,TM,NaB on myocardial differentiation was determined by counting the number of beating embryoid bodies and real-time PCR and western blotting to analyzing cardiac-specific genes and proteins.4.Overexpression pRK-ATF4,which is endoplasmic reticulum stress marker.Real-time PCR detected cardiac-specific markers in the early stem cell into cardiac differentiation.The results of the study1.Establishment of myocardial differentiation model.With 10-4 M VC as the inducer,1-5 days in the bacterial dish suspension culture.we can see the formation of embryoid bodies in the third day,which the edge were smooth and the central were black.We could see the embryoid bodies beating spontaneous 7 days later,and on the 12th day of the number of the beating embryoid bodies was in the peak.Choose the 12th day cells trypsin digestion,immunofluorescence staining,and could be find a-actinin fibronectin expression,and there was a clear sarcomere structure.The differentiation of 0,3,5,7,9,12 days collected cells and extracted RNA,real-time PCR to detect Gata4 and Nkx2.5 on the fifth day of differentiation,and began to rise.The expression of Myl2,Myl7 and Tnnt2 in the cardiac differentiation was significantly up-regulated on the 9th day(p<0.01).It showed that the cardiac differentiation model was established successfully.2.The expression of endoplasmic reticulum stress and acetylation related geneCollecting cells and extracting RNA in the differentiation of 0,3,5,7,9,12 days.The expression of endoplasmic reticulum stress gene profiles were detected by real-time PCR.The results showed that the Bip,ATF4,Xbpl first decrease and then increase again and then decrease,and a significant rise at the time of 12th days,and the expression of CHOP first reduce and then rise,with the induction time increasing.From this we concluded that the expression of endoplasmic reticulum stress genes existed obvious stage effect,and endoplasmic reticulum stress was involved in cardiac differentiation.The expression of Hadc 2,4,6,7 and Cbp/P300 also has time phase significantly.3.The effects of 4-PBA on cardiac differentiationBecause the expression of endoplasmic reticulum stress genes exist obvious time period effect,and the differentiation was divided into the early(1-5 d),the intermediate(6-9 d),and the late(10-12 d)stages.Refine the time period to determine the best dosing time.Cells was treated with 4-PBA(1M)and the solvent dimethyl sulfoxide(DMSO)was used as a control at different stages of cardiac differentiation.The effects of 4-PBA on myocardial differentiation was determined by counting the number of beating embryoid bodies.The results showed that 4-PBA could promote cardiac differentiation at early stage,inhibit at mid-term,and promote at late stage.4-PBA was added at different point 1-2 days,3-5 days,1-5 days,and collected cells and extracted of RNA.The expression of cardiac structural genes Nkx2.5 and Gata4 were detected by real-time PCR.We found that compared with the other groups in 1-2 days Nkx2.5 and Gata4 expression increased most significantly(p<0.01).4-PBA was added at different point 6-7 days,8-9 days,6-9 days,10-11 days,11-12 days,10-12 days,and collected cells and extracted of RNA.The expression of cardiac structural genes Myl2 and Myl7 were detected by real-time PCR.We found that compared with the other groups in 6-7 days Myl2 and Myl7 expression decreased most significantly(p<0.01),and in 11-12 days increased most significantly(p<0.01).4.The potential molecular mechanism of 4-PBA effects the differentiation of stem cells into cardiomyocytes(1)As an inhibitor of endoplasmic reticulum stress,the effects of 4-PBA on cardiac differentiation has three stages.Cells were treated with endoplasmic reticulum stress of agonist tunicamycin(0.025?g/ml)TM,Sodium butyrate NaB(1M)and solvent dimethyl sulfoxide(DMSO),which was used as a control,at different stages of cardiac differentiation.Then counted the number of beating embryoid bodies.The expressions of cardiac-specific genes(Nkx2.5,Gata4,Myl2 and Myl7)were detected by real-time PCR.The results showed that the effect of TM on cardiac differentiation has an obvious promoting action.But there was no time phase effect.The effect of NaB on cardiac differentiation was similar to 4-PBA.(2)In the early stage of cardiac differentiation,transfected the pRK-ATF4,which was endoplasmic reticulum stress marker.We found that it has a positive effect on the expression of Nkx2.5 and Gata4,but the effect was not significant.We speculated that the effects of 4-PBA on myocardial differentiation may be independent of endoplasmic reticulum stress,but endoplasmic reticulum stress is involved in cardiac differentiation.ConclusionThe effects of 4-phenylbutyric acid(4-PBA)on embryonic stem cells into cardiomyocytes has three stages that promotes early,mid-term inhibit,and promotes late.We considered that the effect was not caused by endoplasmic reticulum stress,and the effect of NaB on cardiac differentiation was similar to that of 4-PBA but whether promoting cardiac differentiation through acetylation way also needs to be further explored.