Regulation of epicardial cell differentiation during development and disease

描述(申请人提供)：心外膜是一个单细胞层间皮膜，包裹在心脏周围，含有多潜能的祖细胞群体。令人惊讶的是，心外膜向间充质转化(EMT)导致心脏成纤维细胞和冠状动脉血管的形成，正是在扩散无法为心脏提供燃料的时刻。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血管生成和纤维化。令人惊讶的是，目前已知的调控机制中没有一种解释EMT是如何与生理需求完全同步发生的。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反应性辅助因子相互作用来控制基因表达程序。胚胎和成人的心血管功能依赖于SRF和myocardin之间的相互作用，myocardin在心肌细胞和平滑肌细胞中特异表达，构成细胞核，是编码收缩蛋白的基因激活所必需的。相反，肌钙蛋白相关转录因子(MRTF)-A和MRTF-B广泛表达，但在细胞质中保持休眠状态，直到生理信号导致它们的核积累。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、转化生长因子-β1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丰富，是EMT所必需的。此外，MRTF/SRF活性在低氧条件下被诱导，并与Wilms Tumor 1(WT1)协同促进间充质表型，包括协调调节引导信号(Wnt信号)和细胞骨架成分。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低氧促进了MRTFS、SRF和WT1之间的协同作用，从而在发育和疾病过程中推动EMT、冠状动脉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检验这一假说，这三个目标将定义心外膜转录调控的分子机制。目的1通过在小鼠中有条件地删除这些因子和进行谱系追踪实验，确定MRTFS和SRF在发育过程中如何控制心外膜细胞的功能。目的2通过定义SRF、MRTF和WT1在心外膜中共同调控的表达信号，确定调控心外膜细胞命运和功能的转录机制，并确定调控该基因调控轴的生理信号。目的3明确MRTF-SRF在心肌梗死后心外膜来源细胞分化和心功能中的作用。这些研究将检验一种范式转换假说，该假说解释了生理线索对心外膜细胞迁移和分化的协调调节，并揭示了治疗缺血性心脏病的新靶点。

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.