PDGFRalpha+ fibroblast-like cells contribute to cardiac excitability

项目摘要 最近的研究在几个器官中发现了一种新型的“成纤维细胞样”细胞，它表达酪氨酸受体 激酶，PDGFRA(血小板衍生生长因子受体-a)。使用小鼠品系(Pdgfratm11(EGFP)Sor/J)，其中 PDGFRα+细胞通过表达编码组蛋白2B-绿色荧光蛋白融合的转基因进行结构性标记 由PDGFRA的内源性细胞特异性启动子驱动的蛋白质，我们能够分离、纯化和研究 来自包括心脏在内的各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，并通过大离子电流的快速激活对激动剂做出反应。 我们通过免疫检测进一步证实了EGFP报告基因是PDGFRA+细胞所特有的。 抗血小板衍生生长因子受体α的抗体，从而确定这些成纤维细胞样细胞为血小板衍生生长因子受体α+细胞。在……里面 对这一提议的初步研究，我们发现PDGFRα+细胞广泛分布于心肌中， 尤其是在窦房结和心房组织中。大量的PDGFRA+细胞表达 经典的成纤维细胞标志物，波形蛋白，以及灵长类中的PDGFRA抗体标记的同一群体 心脏，表明这一群体对应于心脏的成纤维细胞样细胞(CFCs)。vbl.使用 以绿色荧光蛋白为报告材料，用流式细胞仪从窦房结和心房组织中分离纯化了PDGFRA+细胞。我们的 随后对窦房结中PDGFRA+细胞的特征表明，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)产生自发的起搏器活动。 值得注意的是，我们发现这些细胞与邻近的肌细胞形成缝隙连接。对事件的进一步调查 PDGFRA+细胞的自动化和对这些细胞表型的更广泛的表征将是 追求两个特定的目标：1)确定心脏PDGFRα+细胞的基因转录特征 确定这种细胞表型。2)确定NSCCS在产生自发内向电流中的作用(S) 在PDGFRCFCs和α+CFCs中以及在心脏AP波的调制中。成功完成提供了新的见解 这一研究建议的提出将有助于更好地理解PDGFRA+的自动机和表型 在窦房结和心房组织中的CFCs，并可能支持起搏器新治疗靶点的开发 不足之处。

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.