Regulation of epicardial cell differentiation during development and disease

描述（由申请人提供）：心外膜是一个单细胞层间皮片，围绕心脏并含有多能祖细胞群。令人惊讶的是，心外膜向间质转化（EMT）导致心脏成纤维细胞和冠状血管形成的精确时刻，扩散无法为心脏提供燃料。心外膜胎儿基因程序在缺血性心脏病中被重新唤醒，并导致冠状动脉新生血管和纤维化。令人惊讶的是，目前已知的调节机制都无法解释EMT如何与生理需求完美同步发生。血清反应因子（SRF）是一种广泛表达的转录因子，其通过与组织特异性或信号响应性辅因子相互作用来控制基因表达程序。胚胎和成人心血管功能取决于SRF和心肌素之间的相互作用，心肌素在心肌细胞和平滑肌细胞中特异性表达，组成性核，并且是激活编码收缩蛋白的基因所必需的。相反，肌红蛋白相关转录因子（MRTF）-A和MRTF-B广泛表达，但在细胞质中保持休眠，直到生理信号导致其核积累。MRTF-A和-B促进间充质/肌成纤维细胞类型的分化，以响应越来越多的激动剂，包括Rho-Rho激酶、TGF-β 1和机械张力。我们最近发表的数据揭示了MRTF-A在心肌梗死后肌成纤维细胞分化和瘢痕形成中的关键作用。我们的初步研究表明，SRF，MRTF-A和MRTF-B在胚胎和成人心外膜中富集，并且是EMT所必需的。此外，MRTF/SRF活性由缺氧诱导，并与Wilms肿瘤1（WT 1）合作促进间充质表型，包括指导线索（Wnt信号传导）和细胞骨架组分的协调调节。基于我们的初步数据和其他人的工作，我们假设心外膜中的生理性缺氧促进了MRTF、SRF和WT 1之间的协同相互作用，从而在发育和疾病期间驱动EMT、冠状血管形成和心脏成纤维细胞产生。我们将测试这一假设与三个特定的目标，将定义的分子机制，在心外膜转录调控。目的1将确定如何MRTFs和SRF控制心外膜细胞功能在发育过程中使用这些因素的条件删除和谱系示踪实验小鼠。目的2将确定的转录机制，心外膜细胞的命运和功能，通过定义的表达签名合作调节SRF，MRTFs，和WT 1在心外膜，并确定调节这个基因调控轴的生理线索。目的3将明确MRTF-SRF在心肌梗死后心外膜衍生细胞分化和心功能中的作用。这些研究将测试一个范式转变假说，解释了心外膜细胞迁移和分化的生理线索的协调调节，并揭示了缺血性心脏病治疗的新的治疗靶点。

DESCRIPTION (provided by applicant): The epicardium is a single cell-layer mesothelial sheet that surrounds the heart and harbors a multi-potent progenitor cell population. Amazingly, epicardial-to-mesenchymal transition (EMT) leads to cardiac fibroblast and coronary vessel formation at the precise moment that diffusion fails to fuel the heart. The epicardial fetal gene program is re-awakened in ischemic heart disease and contributes to coronary neoangiogenesis and fibrosis. Surprisingly, none of the currently known regulatory mechanisms explain how EMT occurs in perfect synchrony with physiological demand. Serum response factor (SRF) is a widely expressed transcription factor that controls gene expression programs through interactions with tissue specific or signal responsive co- factors. Embryonic and adult cardiovascular function depends upon interactions between SRF and myocardin, which is specifically expressed in cardiomyocytes and smooth muscle cells, constitutively nuclear, and required for activation of genes encoding contractile proteins. Conversely, myocardin-related transcription factor (MRTF)-A and MRTF-B are broadly expressed, but held dormant in the cytoplasm until physiological signals lead to their nuclear accumulation. MRTF-A and -B promote differentiation of a mesenchymal / myofibroblast cell type in response to a growing list of agonists, including Rho-Rho kinase, TGF-¿1, and mechanical tension. Our recently published data reveal a critical role for MRTF-A in myofibroblast differentiation and scar formation following myocardial infarction. Our preliminary studies reveal SRF, MRTF-A and MRTF-B are enriched in the embryonic and adult epicardium and are required for EMT. Further, MRTF/SRF activity is induced by hypoxia and promotes a mesenchymal phenotype in cooperation with Wilms tumor 1 (WT1), including the coordinated regulation of guidance cues (Wnt signaling) and cytoskeletal components. Based on our preliminary data and the work of others, we hypothesize that physiological hypoxia in the epicardium promotes the synergistic interaction between MRTFs, SRF, and WT1 that drives EMT, coronary vessel formation and cardiac fibroblast production during development and disease. We will test this hypothesis with three Specific Aims that will define the molecular mechanisms underlying transcriptional regulation in the epicardium. Aim 1 will determine how MRTFs and SRF control epicardial cell function during development using conditional deletion of these factors and lineage tracing experiments in mice. Aim 2 will determine the transcriptional mechanism governing epicardial cell fate and function by defining the expression signature cooperatively regulated by SRF, MRTFs, and WT1 in the epicardium, and identify the physiological cues that modulate this gene regulatory axis. Aim 3 will define the role of MRTF-SRF in epicardial derived cell differentiation and cardiac function following myocardial infarction. These studies will test a paradigm-shifting hypothesis that explains the coordinated regulation of epicardial cell migration and differentiation by physiological cues and reveal novel therapeutic targets for the treatment of ischemic heart disease.