Project Summary/Abstract Hypertrophy Cardiomyopathy (HCM) is the most prevalent hereditary cardiovascular disease – affecting 1 in 500 individuals. Advanced forms of the disease clinically present with hypercontractility, hypertrophy (enlargement of the organ and individual cardiomyocytes) and fibrosis. Several single-point mutations in b- myosin heavy chain (MYH7), Myosin Binding Protein C (MYBPC3), and Troponin (cTn) have been associated with HCM and increased contractility at the organ level. However, the kinetics at the molecular level remain unclear, as different sarcomeric protein mutations can result in increased, decreased, or unchanged force production. A knowledge gap persist in understanding how these altered kinetics at the molecular level lead to the more advanced hypertrophic phenotype of HCM at the cellular level. Interestingly, the Hippo Pathway has been demonstrated to be activated during developmental growth, quiescent during cardiac homeostasis, and reactivated in pathological growth (i.e. HCM). However its involvement in the disease, in particular the initiation of the hypertrophic phenotype, is poorly understood. Here, we aim to understand whether homeostatic mechanical signaling through the canonical growth regulator, Hippo-YAP, is altered 1) by changes in the biomechanics of single HCM mutant cardiomyocytes and 2) by alterations in the mechanical environment. We propose to use human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) genetically edited to harbor point mutations associated with HCM, as a reduce ordered model to study the relationship between mechanical signaling and hypertrophic growth. We will modulate mechanical stresses (i.e. diseased conditions) in healthy and diseased cardiomyocytes by treatment with inotropic drugs and culture in fibrotic-like stiff conditions and track the resulting signaling events by fluorescently labeling the key regulatory protein of the Hippo pathway (YAP). To further elucidate the mechanism by which YAP is contributing to the phenotypes of HCM we have developed a novel optogenetic tool, termed OptoYAP, which provides full temporal and spatial control of the Hippo pathway. Lastly we aim to understand the mechanism behind the reactivation of YAP in pathological conditions by perturbing the mechanical signaling by the nucleus. We hypothesize that 1) changes in force production alter the homeostatic mechano-signaling of the Hippo pathway to initiate cellular hypertrophy and 2) subsequent changes to the extracellular environment (stiffening) compounds this effect leading to a feedforward signal progressing the disease phenotypes. 3) pathological YAP signaling is driven by excessive force transmission by the cytoskeleton resulting in nuclear deformation. Our results will provide insights into HCM progression and provide a testbed for therapeutic options in treating HCM.