Summary Mutations in genes that encode the proteins of the cardiac sarcomere are a primary cause of hypertrophic cardiomyopathy (HCM). HCM represents the most common genetic cardiac disorder and is characterized by a complex and progressive clinical course exhibiting broad phenotypic variability and, for patients carrying mutations in sarcomeric genes in particular, significant morbidity and mortality. Recent longitudinal studies have begun to define the natural history of HCM and revealed a “preclinical” stage in genotype-positive cohorts. Before the availability of genetic testing the majority of patients presented with significant symptoms and late stage cardiac remodeling, thus limiting their response to treatment. The ability to identify patients before the onset of irreversible HCM opens a therapeutic window whereby genotype- positive, phenotype-negative patients can be treated before the onset of pathogenic remodeling and thus perhaps change the natural history of this lifelong disorder. This goal will require the ability to precisely identify primary disease mechanisms at the level of the complex and dynamic cardiac sarcomere. An enduring clinical challenge in HCM remains the treatment of patients with diastolic dysfunction (impaired left ventricular relaxation) which manifests as shortness of breath, often with minimal exertion and is one of the most common manifestations in this otherwise diverse disorder, thus representing a significant unmet need. To date, our group has focused on mutations in the regulatory thin filament and developed a program that combines structural biophysics with in vitro experiment, in vivo animal models and an experimentally validated all-atom computational model of the cardiac thin filament (cTF). As the thin filament is the primary arbiter of cardiac relaxation at the molecular level we now propose to apply our integrated approach to first, in Aim 1, fully characterize (at the atomic level) three specific functional domains of