PROJECT SUMMARY This comprehensive training program described in this five-year proposal is designed to prepare Dr. Staudt to transition to a career as a productive independent investigator focused on deciphering the molecular mechanisms underlying diastolic heart disease. Over the course of the described studies, Dr. Staudt will expand his skills in induced pluripotent stem cell-derived cardiomyocyte phenotyping at the single-cell level, as well as acquire new skills in biophysical analysis of pathogenic myosin mutations and high-throughput assay design and analysis. Further, he will obtain substantial experience and training in mentorship, scientific presentation, and grant writing. These skills will be crucial for his successful transition to independence. This proposal assembles an impressive team of world-renowned experts in cardiovascular biology to guide him, including his co-mentors, Drs. Mark Mercola and Euan Ashley, as well as the members of his mentorship committee, Drs. Joseph Wu, Marlene Rabinovich, and Don Bers. Their support will provide the resources and mentorship that Dr. Staudt needs to succeed in an independent academic career at the conclusion of the K08 award. The scientific aim of this project is to uncover mechanisms leading to diastolic dysfunction, an abnormal stiffness of the ventricular muscle that contributes to nearly half of heart failure cases. Specifically, this proposal focuses on Pediatric Restrictive Cardiomyopathy (RCM), a severe genetic disorder characterized by isolated, profound diastolic dysfunction. Patients with this disease have few treatment options, stemming from a relative lack of understanding of the molecular mechanisms underlying this disease. This proposal leverages novel stem cell models of RCM combined with high-throughput measurement of diastolic function to probe these mechanisms. In Aim 1, multiple cellular models of RCM will be characterized and compared to determine whether different classes of RCM mutations act via similar or divergent mechanisms. Aim 2 uses a functional genomics approach to determine whether different mutations evoke distinct pathogenic mechanisms that converge on a similar clinical presentation. Aim 3 focuses on a unique line from a patient with severe, pediatric onset RCM caused by a de novo mutation in cardiac myosin. In this aim, a novel multi-scale approach links the biophysical effects of this RCM mutation on myosin molecules to the physiologic changes in whole cells, and compares this to a comparable, previously characterized myosin mutation that causes Hypertrophic Cardiomyopathy, a more common but generally less severe disease.