Investigating the role of NADPH oxidase 4 (Nox4) in cardiomyocyte maturation

摘要 在正常发育和应激状态下，新陈代谢在调节细胞命运和功能方面起着关键作用 所有组织和器官的状况。例如，代谢中的活性氧(ROS)很重要 可促进心肌细胞分化的信号分子；但细胞如何翻译氧化还原信号 对细胞功能结果的了解甚少。此外，对这一过程的错误监管也是基础 与心脏相关的疾病，包括先天性心脏病(CHD)和心力衰竭(HF)，使这一问题 具有特殊的临床相关性。基于我们的初步数据和广泛的文献分析，我们假设 NADPH氧化酶4(NOX4)，一种提供新陈代谢产生的 在心脏发育过程中，过氧化氢是肌节组装的中心枢纽。我们的建议很新颖，而且 创新有几个原因：1)我们将使用人类诱导的多能干细胞来源的心肌细胞 (HiCms)策略作为一个模型，允许我们测试NOX4在心脏谱系承诺中的作用，以及2)我们 采用了最先进的成像、遗传和光遗传学方法，3)测试细胞的新陈代谢 与肌节组装相连，肌节组装是心肌的功能单位。具体地说，通过结合基因 微扰研究和纳米分辨率成像方法，我们将测试NOX4肌节的作用 组装以及在CM中对NOX4‘S催化活性和线粒体定位的要求 分化(目标1)，我们将检验NOX4通过调节焦点启动肌节形成的假设 黏附激酶(FAK)在FA样结构称为原雄蕊(Aim 2)，我们将使用新的状态- 最先进的遗传和光遗传学工具，可定量了解NOX4如何调节CM 活细胞的分化和收缩能力(目标3)。我们的结果将填补我们在了解如何 细胞代谢决定着CM的成熟，它也将确定这一过程中的关键角色。重要的是，这 工作将在基于人体细胞的模型系统中进行，这可能会加快新技术的开发 心脏疾病的治疗方法。 该奖学金培训计划利用了Laurie Boyer博士的专业知识以及一些合作 麻省理工学院社区，包括埃德·博伊登博士的实验室。博耶博士是理解 决定细胞命运的分子机制。我们最近还与脊椎动物专家进行了合作 包括Caroline Burns博士在内的心脏发育生物学对保守的 支持肌节结构调节的机制。因此，我将接受世界级的培训 不同的领域，包括分子和细胞生物学以及在体外和体内心脏的遗传学 模型系统。麻省理工学院的环境和资源将极大地促进拟议目标的成功 以及我作为一名独立研究员的发展。

SUMMARY Metabolism plays key roles in regulating cell fate and function during normal development and under stress conditions in all tissues and organs. For example, metabolic reactive oxygen species (ROS) are important signaling molecules that can promote cardiomyocyte differentiation; yet how the cell translates redox signals into functional cellular outcomes is poorly understood. Moreover, faulty regulation of this process underpins cardiac-related illness including congenital heart defects (CHD) and heart failure (HF), making this question of particular clinical relevance. Based on our preliminary data and extensive literature analysis, we hypothesize that NADPH oxidase 4 (Nox4), an enzyme that provides the major source of metabolically generated H2O2 acts as a central hub for sarcomere assembly during heart development. Our proposal is novel and innovative for several reasons: 1) we will employ human induced pluripotent stem cell-derived cardiomyocytes (hiCMs) strategy as a model allowing us to test the role of Nox4 during cardiac lineage commitment, and 2) we have adapted state-of-the-art imaging, genetic, and optogenetic approaches, 3) to test how cellular metabolism is coupled to sarcomere assembly, the functional unit of the cardiac muscle. Specifically, by combining genetic perturbation studies and nanoscale resolution imaging approaches, we will test the role of Nox4 sarcomere assembly as well as the requirement of Nox4’s catalytic activity and mitochondrial localization during CM differentiation (Aim 1), we will test the hypothesis that Nox4 initiates sarcomere formation by regulation of focal adhesion kinase (FAK) at FA-like structures called protocostameres (Aim 2), and we will employ novel state- of-the art genetic and optogenetic tools to derive quantitative insights into how Nox4 regulates CM differentiation and contractility in live cells (Aim 3). Our results will fill a major gap in our knowledge of how cellular metabolism informs CM maturation and it will also identify key players in this process. Importantly, this work will be performed in a human cell-based model system, which may expedite the development of new therapies for the treatment of cardiac disease. The fellowship training plan leverages the expertise of Dr. Laurie Boyer along with a number of collaborations within the MIT community, including the lab of Dr. Ed Boyden. Dr. Boyer is a pioneer in understanding the molecular mechanisms that drive cell fate decisions. We have also recently worked with experts in vertebrate cardiac developmental biology including Dr. Caroline Burns to gain a deeper understanding of the conserved mechanisms underpinning regulation of sarcomere structure. Therefore, I will receive world class training in diverse areas including molecular and cellular biology as well as genetics in both in vitro and in vivo cardiac model systems. The environment and resources at MIT will greatly facilitate the success of the proposed aims and my development as an independent researcher.