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

总结 在正常发育和应激条件下，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，代谢活性氧（ROS）是重要的 可以促进心肌细胞分化的信号分子;然而细胞如何翻译氧化还原信号 转化为功能性细胞结果的机制还知之甚少。此外，对这一过程的错误监管 心脏相关疾病，包括先天性心脏缺陷（CHD）和心力衰竭（HF），使这个问题， 特别是临床相关性。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（Nox 4），一种提供代谢产生的主要来源的酶， H2 O2在心脏发育过程中作为肌节组装的中心枢纽。我们的建议是新颖的， 创新的原因有几个：1）我们将采用人类诱导多能干细胞衍生的心肌细胞 （hiCM）策略作为模型，使我们能够测试Nox 4在心脏谱系定型期间的作用，以及2）我们 已经采用了最先进的成像，遗传学和光遗传学方法，3）测试细胞代谢如何 与心肌的功能单位肌节组件相连。具体来说，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Nox 4肌节的作用， 组装以及在CM过程中需要Nox 4的催化活性和线粒体定位 分化（目的1），我们将测试的假设，即Nox 4启动肌节形成的调控局灶性 粘附激酶（FAK）在FA样结构称为protocostameres（目标2），我们将采用新的状态- 最先进的遗传学和光遗传学工具，以获得对Nox 4如何调节CM的定量见解 活细胞的分化和收缩性（Aim 3）。我们的研究结果将填补我们知识的一个主要空白， 细胞代谢通知CM成熟，它也将确定在这个过程中的关键球员。重要的是这 工作将在基于人类细胞的模型系统中进行，这可能会加快新的 用于治疗心脏病的疗法。 奖学金培训计划利用劳里博耶博士的专业知识，沿着进行了一些合作 包括艾德博伊登博士的实验室。博耶博士是了解 决定细胞命运的分子机制。我们最近还与脊椎动物专家合作， 心脏发育生物学，包括卡罗琳伯恩斯博士，以获得更深入的了解保守的 肌节结构调节的基础机制。因此，我将接受世界一流的培训， 不同领域，包括分子和细胞生物学以及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 以及我作为独立研究者的发展

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.