Elucidating the Role of Microenvironment Mechanics in Regulating Cardiac Myofibroblast Plasticity

项目总结 纤维化是大量心脏病理疾病的基础，从遗传性心肌病到 缺血性心力衰竭。尽管在识别触发信号的分子信号方面取得了实质性进展 静止心脏成纤维细胞(CFs)的特征激活及其转分化为 肌成纤维细胞(MyoFBs)，人们对其长期命运的机制知之甚少 持续性，这是开发有效的抗纤维化疗法的主要障碍。 此K99/R00应用程序描述了一个五年研究培训计划，该计划建议利用(I)人 诱导多能干细胞来源的心脏成纤维细胞(IPSC-CFs)，(Ii)可调生物工程材料 机械特性，以及(Ii)单细胞多组学平台，以研究支配 MyoFB的命运和可塑性。鉴于CFs和MyoFBs对细胞外基质(ECM)的敏感性 僵硬，申请者赵相坤博士将测试ECM介导的机械调制的假说 信号通过与已知调节的可溶性因子协同作用加强MyoFBs的去分化 纤维化形成的主要途径。在目标1(K99)中，赵博士将使用报告的IPSC线路(带有荧光标记 典型的MyoFB‘标记’基因，如CFP-TAGLN)和一种新的动态软化水凝胶体系 实时表征机械卸载对MyoFB命运的影响。在《目标2》(K99)中，赵博士将 研究细胞外基质软化和转化生长因子-β途径在调节MyoFB状态中的协同作用，(I)通过 检测机械敏感转录因子之间的刚性依赖的蛋白质相互作用(例如，是- 相关蛋白1(YAP))，以及(Ii)通过鉴定ECM刚性下游的表观遗传调节因子。 转座酶转座酶可及染色质的细胞分析(scatac-seq)。在《目标3》(R00)中，赵博士将确定 沿着细胞的机械感觉装置的潜在的可药物靶标，并在 工程心脏组织和压力超负荷诱导的肥大和心力衰竭的小鼠模型。 拟议的研究建立在Pi Sangkyun Cho博士在生物材料、蛋白质组学、 和ECM机械生物学，同时在(一)记者IPSC-CFS提供新的培训机会，(二)单细胞 多组学平台，以及(Iii)动物模型。导师约瑟夫·吴博士是IPSCs和心血管领域的先驱 共同导师萨拉·海尔肖恩博士是生物材料和再生医学方面的领先专家， 他的指导与吴博士的指导相辅相成。咨询委员会成员Jeffery Molkentin博士(心脏 纤维化)、约瑟夫·希尔(心力衰竭模型)和Michal Snyder(单细胞基因组学)提供了更多的专业知识 和指导。总而言之，精心设计的研究培训计划、卓越的指导团队和 斯坦福大学杰出的环境预计将有助于推动赵博士实现他的长期目标 在生物工程和心血管疾病的交叉点建立一个独立的研究项目 基质生物学。

PROJECT SUMMARY Fibrosis underlies a vast number of cardiac pathological conditions, ranging from genetic cardiomyopathies to ischemic heart failure. Although substantial progress has been made in identifying molecular signals that trigger the characteristic activation of quiescent cardiac fibroblasts (CFs) and their transdifferentiation into myofibroblasts (MyoFBs), far less is known about the mechanisms that govern their long-term fate and persistence, which presents major obstacles to the development of effective anti-fibrotic therapies. This K99/R00 application describes a five-year research training plan that proposes to leverage (i) human induced pluripotent stem cell-derived cardiac fibroblasts (iPSC-CFs), (ii) engineered biomaterials with tunable mechanical properties, and (ii) single-cell multiomics platforms to investigate molecular mechanisms that govern MyoFB fate and plasticity. Given the well-established sensitivity of CFs and MyoFBs to extracellular matrix (ECM) stiffness, the applicant Dr. Sangkyun Cho will test the hypothesis that modulation of ECM-mediated mechanical signaling potentiates the de-differentiation of MyoFBs, by synergizing with soluble factors known to regulate major pathways in fibrogenesis. In Aim 1 (K99), Dr. Cho will use reporter iPSC lines (with fluorescently tagged canonical MyoFB ‘marker’ genes, e.g., CFP-TAGLN) and a novel dynamically softening hydrogel system to characterize in real-time the effects of mechanical unloading on MyoFB fate. In Aim 2 (K99), Dr. Cho will investigate the synergy between ECM softening and the TGF-beta pathway in regulating MyoFB states, (i) by examining stiffness-dependent protein interactions among mechanosensitive transcription factors (e.g., yes- associated protein 1 (YAP)), and (ii) by identifying epigenetic regulators downstream of ECM stiffness with single- cell assay for transposase transposase-accessible chromatin (scATAC-seq). In Aim 3 (R00), Dr. Cho will identify potential druggable targets along the cell’s mechanosensory apparatus, and test candidate compounds in engineered heart tissues and a mouse model of pressure-overload induced hypertrophy and heart failure. The proposed studies build upon PI Dr. Sangkyun Cho’s well-suited prior training in biomaterials, proteomics, and ECM mechanobiology, while providing new training opportunities in (i) reporter iPSC-CFs, (ii) single-cell multiomics platforms, and (iii) animal models. Mentor Dr. Joseph Wu is a pioneer in iPSCs and cardiovascular biology, and co-mentor Dr. Sarah Heilshorn is a leading expert in biomaterials and regenerative medicine, whose mentorship complements that of Dr. Wu. Advisory Committee members Drs. Jeffery Molkentin (cardiac fibrosis), Joseph Hill (heart failure models), and Michal Snyder (single-cell genomics) provide additional expertise and guidance. In Summary, the well-tailored research training plan, exceptional mentoring team, and an outstanding Environment at Stanford University are anticipated to help propel Dr. Cho toward his long-term goal of establishing an independent research program at the intersection of bioengineering and cardiovascular stromal biology.