Regulation of epicardial cell differentiation during development and disease

描述（由申请人提供）：心外膜是包围心脏的单层间皮层，含有多能祖细胞群。令人惊讶的是，心外膜到间充质转化（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，并有助于冠状动脉新生血管生成和纤维化。令人惊讶的是，目前已知的调节机制都无法解释EMT是如何与生理需求完美同步发生的。血清反应因子（SRF）是一种广泛表达的转录因子，通过与组织特异性或信号应答辅助因子的相互作用来控制基因表达程序。胚胎和成人的心血管功能取决于SRF和心肌蛋白之间的相互作用，而心肌蛋白在心肌细胞和平滑肌细胞中特异性表达，构成核，是激活编码收缩蛋白的基因所必需的。相反，心肌素相关转录因子(MRTF)-A和MRTF- b广泛表达，但在细胞质中处于休眠状态，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-¿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疤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，是EMT所必需的。此外，MRTF/SRF活性由缺氧诱导，并与Wilms tumor 1 （WT1）合作促进间充质表型，包括指导信号（Wnt信号）和细胞骨架成分的协调调节。根据我们的初步数据和其他人的工作，我们假设心外膜的生理性缺氧促进了mrtf、SRF和WT1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的产生。我们将用三个特定的目标来验证这一假设，这将定义心外膜转录调控的分子机制。目的1将通过条件删除这些因子和小鼠谱系追踪实验，确定mrtf和SRF在发育过程中如何控制心外膜细胞功能。目的2将通过定义心外膜中SRF、mrtf和WT1共同调控的表达特征，确定控制心外膜细胞命运和功能的转录机制，并确定调节该基因调控轴的生理线索。目的3将确定MRTF-SRF在心肌梗死后心外膜源性细胞分化和心功能中的作用。这些研究将验证一种范式转移假说，该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.