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 developmen...