PDGFRalpha+ fibroblast-like cells contribute to cardiac excitability

项目摘要 最近的研究发现了一种新的“成纤维细胞样”细胞在几个器官表达受体酪氨酸 激酶，PDGFRa（血小板衍生生长因子受体-α）。使用小鼠品系（Pdgfratm 11（EGFP）Sor/J），其中 PDGFRα+细胞通过表达编码组蛋白2B-eGFP融合蛋白的转基因进行组成型标记 由于Pdgfra蛋白由Pdgfra的内源性细胞特异性启动子驱动，我们能够分离、纯化和研究Pdgfra蛋白。 包括心脏在内的多种器官的成纤维细胞样细胞的功能和表型。我们有 证明这些细胞是可兴奋的，并对激动剂作出反应，迅速激活大离子电流。 我们已经通过免疫检测进一步证实了eGFP报告基因是PDGFRa+细胞独有的，使用 针对PDGFRα的抗体，从而确定这些成纤维细胞样细胞为PDGFRα+细胞。在 针对这一建议的初步研究，我们发现PDGFRα+细胞广泛分布于心肌中， 特别是在窦房结（SAN）和心房组织中。PDGFRa+细胞的显著群体表达PDGFRa+细胞。 经典的成纤维细胞标志物波形蛋白，并且灵长类动物中相同的群体被PDGFR α抗体标记 这表明该群体对应于心脏的心脏成纤维细胞样细胞（CFC）。使用 eGFP报告基因后，我们通过FACS从SAN和心房组织中分离和纯化心脏PDGFRa+细胞。我们 随后对SAN中PDGFRa+细胞的表征揭示了这些细胞是可兴奋的，并且能够 通过激活非选择性阳离子通道（NSCC）产生自发起搏活动。 值得注意的是，我们发现这些细胞与相邻的肌细胞形成缝隙连接。进一步调查 PDGFRa+细胞的自发性和这些细胞表型的更广泛表征将是 有两个具体目的：1）确定心脏PDGFRα+细胞的基因转录特征， 决定这种细胞表型。2)确定NSCC在产生自发内向电流中的作用 在PDGFRα+ CFC和调节心脏AP波形中的作用。成功完成提供的新见解 这一研究计划的提出将有助于更好地了解PDGFRa+的自动性和表型 CFCs在SAN和心房组织中的分布，可能为起搏器新的治疗靶点的开发提供支持 缺陷

Project Summary Recent studies have identified a novel “fibroblast-like” cell in several organs that express the receptor tyrosine kinase, PDGFRa (platelet-derived growth factor receptor-a). Using a mouse strain (Pdgfratm11(EGFP)Sor/J) in which PDGFRα+ cells are constitutively labeled by expression of a transgene encoding a histone 2B-eGFP fusion protein driven by the endogenous, cell-specific promoter for Pdgfra, we are able to isolate, purify, and study the function and phenotype of fibroblast-like cells from a variety of organs, including the heart. We have demonstrated that these cells are excitable and respond to agonists with rapid activation of large ionic currents. We have further confirmed that the eGFP reporter is exclusive to PDGFRa+ cells by immunodetection using antibodies against PDGFRα, thus definitively establishing these fibroblast-like cells as PDGFRα+ cells. In preliminary studies for this proposal, we found that PDGFRα+ cells are widely distributed in cardiac muscles, especially in sinoatrial nodal (SAN) and atrial tissues. A significant population of PDGFRa+ cells expresses the classical fibroblast marker, vimentin, and the same population is labeled by PDGFRa antibodies in the primate heart, indicating that this population corresponds to the cardiac fibroblast-like cells (CFCs) of the heart. Using the eGFP reporter, we isolated and purified cardiac PDGFRa+ cells by FACS from SAN and atrial tissues. Our subsequent characterization of PDGFRa+ cells in the SAN revealed that these cells are excitable and are capable of generating spontaneous pacemaker activity through activation of a nonselective cation channel (NSCC). Notably, we found that these cells form gap junctions with neighboring myocytes. Further investigation of the automaticity of PDGFRa+ cells and more extensive characterization of the phenotypes of these cells will be pursued in two Specific Aims: 1) Determine the genetic transcript signature of cardiac PDGFRα+ cells that determine this cellular phenotype. 2) Determine the role(s) of NSCCs in generating spontaneous inward currents in PDGFRα+ CFCs and in modulating cardiac AP waveform. Novel insights provided by successful completion of this research proposal will facilitate a better understanding of the automaticity and phenotype of PDGFRa+ CFCs in SAN and atrial tissues and may support the development of novel therapeutic targets for pacemaker deficiencies.