Bone Morphogenetic Protein 4 Promoted The Differentiation Of Tbx18-positive Epicardial Progenitor Cells To Pacemaker Cells

Sinoatrial node?SAN?,located at the junction of superior vena cava and right atrium,is the natural pacemaker.As the population ages,incidence of cardiac syncope and sudden death caused by SAN lesions increased.It is of great significance to clarify regulatory network in the differention process of pacemaker cells and roles of transcription factors during embryogenesis for building biological pacemakers which adapting to physiological needs.Epicardial progenitor cells,majority of which express Tbx18 transcription factor,have potentials to differentiate to heart cells such as fibroblasts,vascular smooth muscle cells and pacemaker cells.Lack of Tbx18 leads to serious lack of sinoatrial node head structure.Hence,Tbx18⁺ epicardial progenitor cells?EPCs?may be one of the most worth studying seed cells for establishing biological pacemakers.In the exploration trial of genetic recombination directly which transformed embryonic fibroblasts into myocardial cells,increased bone morphogenetic protein 4?BMP4?could upregulate rates of conversion into myocardial cells with spontaneous pacemaker function by 150 times.Therefore,it was of important value to clarify the role of BMP4 in differentiation of Tbx18⁺ EPCs into pacemaker cells.This study aimed to investigate the role of BMP4 in differentiation of Tbx18⁺EPCs into pacemaker cells and its mechanisms by establishing lineage tracing Tbx18-Cre/Rosa26^REYFP models of in vitro.Part1 Breeding and identification of Tbx18-Cre and YFP transgenic miceObjectives: Tbx18-Cre female mice and conditioned Cre report Rosa26^REYFP male mice were bred and identified respectively for building double-heterogeneous genetic lineage tracing models based on genetically engineered mice,which aimed to provide animals for tracing differentiated fates of embryonic Tbx18⁺ cells.Methods:Transgenic male mice of Tbx18-Cre and Rosa26^REYFP were mated and bred with C57BL/6 female mice.The ethanol method was used for extracting genomic DNA and PCR were used for identifying offspring genotype.Results:Female mice carrying Tbx18-Cre and male mice carrying Rosa26^REYFP were obtained successfully.Conclusions:A simple and efficient screening technology platform was set up and Transgenic mice of Tbx18-Cre and Rosa26^REYFP were bred in order to provide animals for subsequent experiments.Part2 Establishment of Tbx18⁺ fate-tracing EPCs in vitroObjectives:To culture high purity,good growth state EPCs of E11.5 and build Tbx18⁺ fate-tracing EPCs.Methods:Tbx18-Cre female mice were mated with Rosa26^REYFP male mice and then E11.5 embryonic heart was taken out for adhensive culture while embryonic tissure was used for identifying genotype by PCR.Tbx18⁺EPCs were screened under fluorescence microscope after climbling out from edges of heart tissue.Results:Fluorescence screening method was simple and effective.More than 95% of the E11.5 EPCs were Tbx18 positive cells.Conclusions:With the Tbx18-Cre/Rosa26^REYFP double transgenic mouse models,Tbx18⁺EPCs were traced and differentiated fates of Tbx18⁺ EPCs were traced.Part3 BMP4 promoted the differentiation of Tbx18⁺EPCs to pacemaker cellsObjectives:To determine whether BMP4 promote the differentiation of Tbx18⁺ EPCs to pacemaker cells.Methods:Cells cultured were divided into BMP4,BMP4 + LDN193189 group,control group and LDN193189 group.HCN4,the marker of pacemaker cells,was detected by means of immunofluorescence and fluorescence quantitative PCR.Results : BMP4 promoted the expression of HCN4 in Tbx18⁺ EPCs.The longer the intervention time,the more the expression of HCN4.Conclusions:BMP4 promoted the differentiation of Tbx18⁺ EPCs to pacemaker cellsPart4 Mechanisms of differentiation of Tbx18⁺ EPCs to pacemaker cells promoted by BMP4Objectives:To explore the regulation network of differentiation of Tbx18⁺ EPCs to pacemaker cells promoted by BMP4.Methods: Cells cultured were divided into BMP4,BMP4 + LDN193189 group,control group and LDN193189 group.Method of fluorescence quantitative PCR was used to detect mRNA levels of BMP4?SHOX2 ? Tbx3 ? GATA4 ? Nkx2.5.BMP4 targets were selected,then immunofluorescence was used to detect the expression in protein levels.Afterwards,GATA4,the BMP4 downstream target,was inhibited by RNAi technology.Cells cultured were divided into BMP4,BMP4+GATA4-RNAi group,control group,negative control group,GATA4-RNAi group to detect GATA4,BMP4,HCN4,Nkx2.5 for illustrating the relationship between upstream and downstream.Results:Data from fluorescent quantitative PCR shown that BMP4 upregulated GATA4 mRNA expression,downregulated Nkx2.5 mRNA expression but could not influence the expression of SHOX2,Tbx3,BMP4.Data from immunofluorescence shown that BMP4 upregulated GATA4 mRNA expression,downregulated Nkx2.5 mRNA expression but could not influence the expression of BMP4.GATA4-RNAi effectively lowered expression of GATA4?HCN4,upregulated Nkx2.5 and had no effect on BMP4 by means of fluorescence quantitative PCR and immunofluorescence.Conclusions : BMP4 promoted differentiation of Tbx18⁺EPCs to pacemaker cells by upregulation of GATA4.Nkx2.5,located in the downstream of BMP4 and GATA4,was the negative regulatory factor of differentiation of Tbx18⁺EPCs to pacemaker cells.