Tpm1 Promotes Differentiation Of Rat Cardiac Fibroblasts Into Cardiomyocyte-like Cells Through The PI3K-Akt Pathway

As the most abundant cells in the adult mammalian myocardium(70%),cardiac fibroblasts not only provide physical support for cardiomyocyte-like cells,but also have a strong ability of proliferation and metabolism,which can provide nutritional support for cardiomyocyte-like cells by autocrine and paracrine.CFs have strong ability of differentiation for many kinds of cells(osteoblasts,adipocytesand cardiomyocytes)after treated with various inducers both in vivo and in vitro.In the case of myocardial injury,part of CFs has the ability to spontaneously migrate to the infarct area and transforms into myocardial cells.CFs are an attractive source for cardiac regeneration and myocardial repair if they could be reprogrammed into cardiomyocytes.Relevant studies have shown that CFs can transform into myocardial cellsin vitro,express the specific protein of cardiomyocytes,and possess the unique electrophysiological characteristics of myocardium.However,the introduction of exogenous genes still has the problems of low efficiency and poor safety,which limits its clinical application.In addition to this method,many studies in recent yearshave shown that small molecular compounds can completely replace transcription factors and promote cell reprogramming,which undoubtedly provides a new method and idea for people to realize the transformation of CFs into cardiomyocyte-like cells.Small molecule compounds have different combination,in which the key chemicals such as SB431542 can induce fibroblast differentiation by inhibiting the TGF-? signaling pathway.As an adenylate cyclase AC activator,forskolin can induce the phosphorylation of PKA-mediated connexin and promote the expression of myocardial genes.Dorsomorphin,as an inhibitor of the BMP signaling pathway,can induce smad activation and promote the production of cardiomyocyte-like cells.Due to the different signaling pathways of various small molecules,they are respectively involved in the activation of different specific genes,and the activated transcription factors of these genes can activate the downstream target genes and initiate the differentiation of CFs into cardiomyocyte-like cells.However,the key genes that regulate the final differentiation of cardiomyocytes under their combined effects remain unknown.Therefore,it is necessary to further analyze the expression profile to observe which genes play a key regulatory role in the differentiation process of cardiomyocyte-like cells and what pathways they use to regulate the transformation of CFs into cardiomyocyte-like cells.Clarifying these problems will provide experimental basis and theoretical support for revealing the efficient transformation mechanism of CFs into cardiomyocyte-like cells,mobilizing the potential of cardiac repair of CFs,and making efficient use of CFs to replace damaged cardiomyocytes in vivo.In order to reveal the molecular mechanism of CFs to cardiomyocyte differentiation,this study intends to use small molecule compounds combination inducing CFs in vitro,observing the differentiation efficiency and the expression rules of specific proteins and genes of the myocardium during differentiation.In addition,RNA-seq technology was used to screen the genes with significant changes in the process of CFs transformation into cardiomyocyte-like cells.After that,the purpose genes were transfected into the CFs,and their regulating function of myocardial cell differentiation were detected,and the mechanism of key gene regulating CFs to cardiomyocyte-like cells was discussed preliminarily to lay the cytological foundation and theoretical basis for further mobilizing the cardiac endogenous myocardial regeneration and improving the myocardial repair effect.1 The combination of small molecule compounds CFDSV can effectively induce the differentiation of cardiac fibroblasts into cardiomyocyte-like cells CFs were isolated and cultured by differential adherent method to detect its purity and identify its multidirectional differentiation potential.Using the combination of small molecule compounds(5?mol/L CHIR99021,10?mol/L forskolin,1?mol/L dorsomorphin,5?mol/L SB431542 ? 200nmol/L valproiz acid,CFDSV)induced CFs to myocardial cell in vitro and detected the cell toxicity through the cell proliferation and apoptosis assay.By detecting the expression changes of myocardial specific proteins and genes at different time periods(3d,8d,12 d and 16d)after induction,it was to determine the induction ability of CFs to myocardial differentiation in vitro and the genes involved in differentiation.After myocardial induction,the cell body significantly became smaller,and the shape gradually became round from the long spindle,which remained stable until 16 days after induction.Cell proliferation was significantly inhibited.cTnT appeared on the 3rd day after induction and was strongly positively expressed in the cytoplasm,and the number of cTnT positive cells increased from less(0.035%)to more(42.6%).Cx43,?-actin,Gata4,Tbx5,Mef2 c were strongly positively expressed after induction.?-actin appeared at 12 days and dispersed in the cytoplasm,while Gata4 and Mef2 c were mainly expressed in the nucleus.Cx43 were spot-expressed at the junction between myocardial cells,and increased with the induction time,reaching the maximum on the 16 th day.Tbx5 appeared at 3 days after induction,distributed around the nucleus,reached the peak at 8 days,and then gradually disappeared,which showing complementary expression with cTnT.In addition,Sox2 decreased gradually with induction.The results showed that the mixture of small molecule compounds CFDSV could induce cardiac fibroblasts to gradually lose their pluripotency and differentiate into cardiomyocyte-like cells with high induction efficiency.2 Tpm1 is an important gene that positively regulates the differentiation of CFs into cardiomyocyte-like cells To identify the genes that changed during induction,the total RNA from the CFDSV induction group and the control group were extracted for transcriptome sequence analysis.Gene Ontology enrichment analysis and KEGG pathway analysis were conducted on the genes expressing differences based on the sequencing results.Then m RNA content of the genes with significant differences were detected by RT-PCR to further determine the data reliability and preliminarily determine the genes with significant differences.By RNA-seq analysis,we obtained a total of 2441 differentially expressed genes in the CFDSV induction group.Through comparative analysis,1141 genes were up-regulated and 1300 genes were down-regulated.Go enrichment analysis showed that up-regulated genes were most closely related to cardiac development,contractile fiber and muscle tissue development.The downregulated genes were most closely related to the biological development process.KEGG analysis revealed that upregulated genes were mainly enriched in cell adhesion,PI3K-Akt signaling pathway and hypertrophic cardiomyopathy,while down-regulated genes were enriched in fibrosis occurs,Wnt pathway,TGF and other signaling pathways.RT-PCR verified that the up-regulated expressions of myocardial related genes Tnnt2,Actc1,Myh6,Mef2 c,Pdlim3,Myh11,Tpm1,Nppa,Tpm2 and down-regulated expressions of Wnt2 b,Tgfbi,Col7a1,Sox2 and Fbln1 were basically consistent with the sequencing results.The expression levels of Tpm1 and Tpm2 were significantly increased,and the over-expressed plasmids of Tpm1 and Tpm2 were constructed to transfect CFs.Comprehensive analysis,Tpm1 was significantly increased the expression of myocardial related genes,and was selected as candidate gene for further identification.To further investigate the role of Tpm1 in myocardial differentiation,the overexpression and interference plasmid of Tpm1 were transfected into the CFs.The expression of myocardial related proteins and genes(cTnT,Cx43 and ?-actin)were detected to observe the effect of Tpm1 on the differentiation of CFs into cardiomyocyte-like cells.The results showed that the expression of Tpm1,cTnT,Cx43 and ?-actin in CFs were significantly higher in Tpm1 overexpression group than that of the control group.On the contrary,myocardial related proteins and genes in CFs were still negatively expressed in the Tpm1 interference group although pretreated with CFDSV.These results suggest that Tpm1 can positively regulate the differentiation of CFs into cardiomyocyte-like cells.3 Tpm1 promotes the differentiation of CFs into cardiomyocyte-like cells by activating the PI3K-Akt pathway By comparing the gene enrichment pathway of cells in the CFDSV induction group and the control group,more genes were found to be enriched on the PI3K-Akt pathway in the CFDSV induction group.In order to prove the PI3K-Akt pathway was involved in CFs to myocardial differentiation process,western blotting was used to test the downstream molecules of the PI3K-Akt pathway after induction,The detection results of downstream pathway molecules showed that induced CFs activated the downstream Akt protein phosphorylation of PI3K-Akt signaling pathway after myocardial induction by CFDSV,and PI3 K inhibitor LY294002 pretreatment has an inhibitory effect on this process,thereby inhibiting the downstream Akt phosphorylation and reducing the expression of cTnT.Further analysis of the role of PI3K-Akt pathway in promoting myocardial differentiation showed that the expression of P-Akt and cTnT increased after transfection with Tpm1 overexpressed virus.After pretreatment with PI3 K specific inhibitor Ly294002,the expression level of P-Akt level and cTnT were significantly reduced,indicating that Tpm1 may be involved in activating the PI3K-Akt signaling pathway and promoting the differentiation of CFs into cardiomyocyte-like cells by up-regulating the expression of myocardial specific genes.Conclusions: The combination of small molecule compounds CFDSV can promote the myocardial differentiation of CFs;Multiple genes are involved in the process of CFs differentiation into myocardium,among which Tpm1 is the key gene that positively regulates the differentiation of CFs into cardiomyocyte-like cells;Tpm1 activates myocardial related proteins through the PI3K-Akt pathway,and promotes the differentiation of CFs into cardiomyocyte-like cells.