title: Shox2 regulates the pacemaker gene program in embryoid bodies.
abstract: The pacemaker tissues of the heart are a complex set of specialized cells that initiate the rhythmic heartbeat. The sinoatrial node (SAN) serves as the primary pacemaker, whereas the atrioventricular node can serve as a subsidiary pacemaker in cases of SAN failure or block. The elucidation of genetic networks regulating the development of these tissues is crucial for understanding the mechanisms underlying arrhythmias and for the design of targeted therapies. Here we report temporal and spatial self-organized formation of the pacemaker and contracting tissues in three-dimensional aggregate cultures of mouse embryonic stem cells termed embryoid bodies (EBs). Using genetic marker expression and electrophysiological analyses we demonstrate that in EBs the pacemaker potential originates from a localized population of cells and propagates into the adjacent contracting region forming a functional syncytium. When Shox2, a major determinant of the SAN genetic pathway, was ablated we observed substantial slowing of spontaneous contraction rates and an altered gene expression pattern including downregulation of HCN4, Cx45, Tbx2, Tbx3, and bone morphogenetic protein 4 (BMP4); and upregulation of Cx40, Cx43, Nkx2.5, and Tbx5. This phenotype could be rescued by adding BMP4 to Shox2 knockout EBs in culture from days 6 to 16 of differentiation. When wild-type EBs were treated with Noggin, a potent BMP4 inhibitor, we observed a phenotype consistent with the Shox2 knockout EB. Altogether, we have generated a reproducible in vitro model that will be an invaluable tool for studying the molecular pathways regulating the development of cardiac pacemaker tissues.
authors.affiliation: 1 Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center , New Orleans, Louisiana.
authors.forename: Sherin I
authors.initials: SI
authors.lastname: Hashem
authors.forename: May L
authors.initials: ML
authors.lastname: Lam
authors.forename: Shirley S
authors.initials: SS
authors.lastname: Mihardja
authors.forename: Steven M
authors.initials: SM
authors.lastname: White
authors.forename: Randall J
authors.initials: RJ
authors.lastname: Lee
authors.forename: William C
authors.initials: WC
authors.lastname: Claycomb
mesh_terms: D000818:Animals; D055415:Bone Morphogenetic Protein 4 / Q000235:genetics / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D002478:Cells, Cultured; D018920:Coculture Techniques; D017630:Connexins / Q000235:genetics / Q000378:metabolism; D058732:Embryoid Bodies / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D004742:Enhancer Elements, Genetic / Q000235:genetics; D050993:GATA6 Transcription Factor / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D051379:Mice; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055748:SOXB1 Transcription Factors / Q000235:genetics* / Q000378:metabolism; D012849:Sinoatrial Node / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D020825:T-Box Domain Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C525810:Bmp4 protein, mouse; D055415:Bone Morphogenetic Protein 4; D017630:Connexins; D050993:GATA6 Transcription Factor; C578706:Hcn4 protein, mouse; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse; C097348:T-Box Domain Protein 2; D020825:T-Box Domain Proteins