The STK25 Signaling Pathway in Human Cardiac Cells

项目摘要 该提案描述了一个为期五年的指导计划，其具体目标是培养校长 心血管医学独立研究事业的研究者。该项目旨在传授技能， 和知识所需的申请人，以实现他的长期目标，利用组织工程研究 激酶信号通路。本项目的近期目标是：1）获得实验经验 从人iPS细胞开发工程化心脏微组织的必要性2）扩展申请人的知识 在干细胞生物学，生物医学工程和教师发展的适当课程基础3） 发展成为一名独立研究人员所需的必要行政技能4）建立一个机构， 这项工作将使资金作为一个独立的调查员。申请人将有资源实现 这些目标的指导下，他的导师教授Vunjak-Novakovic以及精心挑选的 咨询委员会，这将促进申请人从指导到独立研究的过渡。 项目描述 在初步工作中，人类心脏祖细胞被用作筛选重要的激酶组的工具。 心血管生理学中细胞存活的驱动因素。我们鉴定了丝氨酸/苏氨酸激酶STK 25， 心脏祖细胞存活的调节因子，并使用过表达和敲低研究表明， STK 25控制这些细胞中的5 '-AMP-活化蛋白激酶（AMPK）途径。这个受影响的细胞 增殖和代谢。STK 25还能够与AMPK的上游调节因子LKB 1结合。然后我们 在我们从诱导性多能干细胞（iPS）分化的心肌细胞中研究了这一途径 并发现使用CRISPR-Cas9系统缺失STK 25负调节AMPK途径。 此外，这被证明是有害的心肌细胞在简单应变的机械性能 测定。本提案的目标是调查STK 25如何调节AMPK背后的机制 并在基于细胞的测定中证明该调节的生理学意义。利用 利用我们在组织工程方面的专业知识，我们将从iPS中产生成熟的、成人样的人类心肌微组织。 细胞，并用它们来模拟这种信号通路的生理影响， STK 25和AMPK。这些数据将证明CPC作为心脏信号替代物的创新性 探索，STK 25影响心肌细胞功能的机制和 这条通路对心肌细胞性能的影响。

Project Summary This proposal describes a five-year mentored program with the specific goal of preparing the principal investigator for an independent research career in cardiovascular medicine. The project aims to impart the skills and knowledge required for the applicant to achieve his long-term goal of using tissue engineering to investigate kinase signaling pathways. The immediate goals of this project are to 1) gain the experimental experience necessary develop engineered cardiac microtissues from human iPS cells 2) expand the applicant's knowledge base with the appropriate coursework in stem cell biology, biomedical engineering and faculty development 3) develop the necessary administrative skills required to be an independent researcher 4) formulate a body of work that will enable funding as an independent investigator. The applicant will have the resources to achieve these goals under the guidance of his mentor Professor Vunjak-Novakovic as well as a carefully selected advisory board that will foster the transition of the applicant from mentored to independent research. Project Description In preliminary work, human cardiac progenitor cells were used as a tool for screening the kinome for important drivers of cell survival in cardiovascular physiology. We identified the serine/threonine kinase, STK25, as a regulator of cardiac progenitor cell survival and used both overexpression and knockdown studies to show that STK25 controls the 5'-AMP-activated protein kinase (AMPK) pathway in these cells. This impacted cell proliferation and metabolism. STK25 was also able to bind to LKB1, an upstream regulator of AMPK. We then investigated this pathway in cardiomyocytes which we differentiated from inducible pluripotent stem cells (iPS) and found that deletion of STK25 using a CRISPR-Cas9 system negatively regulated the AMPK pathway. Furthermore, this was shown to be deleterious to the mechanical performance of cardiomyocytes in simple strain assays. The goal of this proposal is to investigate the mechanism behind how STK25 regulates the AMPK pathway and to demonstrate the physiologic significance of that regulation in cellular based assays. Harnessing our expertise in tissue engineering, we will generate mature, adult-like human myocardial microtissues from iPS cells and use them to model the physiological impact of this signaling pathway with genetic manipulation of both STK25 and AMPK. This data will demonstrate the innovation of CPC's as a surrogate for cardiac signaling exploration, the mechanism by which STK25 impacts cardiomyocyte function and the therapeutic potential of this pathway on cardiomyocyte performance.