Abstract Skeletal muscle accounts for 40% of body mass and defines in significant ways who we are as human beings. From the essential underpinnings of breathing, to the basic day-to-day movements of sitting, standing, and walking, skeletal muscle function enables the fullness of the human condition. Numerous skeletal muscle diseases cause marked contractile dysfunction leading to significantly diminished overall wellbeing and lifespan in humans. Therefore, preventing or reversing muscle dysfunction has significant health relevance. This proposal focuses on the sarcomere - the functional unit of striated muscle – known to underlie multiple forms of contractile dysfunction. In Nemaline myopathy, severe muscle weakness arises from hypoactive sarcomeres, while the severe muscle contractures characteristic of Distal Arthrogryposis stem from hyperactive sarcomeres. Other disorders, including inherited Muscular dystrophies, also involve altered sarcomere function. These diseases establish the sarcomere as a crucial, yet highly underserved, target for therapeutic intervention. Skeletal muscle diseases involving defective sarcomeres have no cure or effective treatments. A major challenge to progress centers on the inherent complexities of sarcomere regulation. Recently, novel ON/OFF myosin cross-bridge activation states under mechano-sensing regulatory control have been proposed to interface with the troponin- tropomyosin system to regulate contraction. Working together, through dynamic inter-myofilament signaling, this new view of sarcomere regulation has significant implications for muscle health and disease. To date, the data supporting this model derives mainly from biophysical studies, with physiological relevance unclear and critical to elucidate. We developed and validated a novel FRET-based sarcomere activation biosensor integrated into the myofilaments of intact skeletal muscle. Preliminary data shows the biosensor detects conformational changes in troponin, serving as a signaling nexus for real time reporting load-dependent inter-myofilament signaling regulation of sarcomere activation during physiological contractions in live skeletal muscles. Guiding hypothesis: Healthy skeletal muscle function requires precise sarcomere activation accomplished by dynamic inter-myofilament signaling wherein thin filament regulation initiates and myosin sustains sarcomere activation during physiological contraction; consequently, defective inter-myofilament signaling causes disease. The Aims are to investigate physiological mechanisms of inter-myofilament signaling in regulating sarcomere activation during twitch contractions in intact skeletal muscles and to investigate the effects myosin binding protein C as a key mechano-sensor governing inter-myofilament signaling processes in regulating sarcomere activation during twitch contractions in intact skeletal muscles. Elucidating the mechanisms underlying physiologically relevant mechano-sensitive inter-myofilame...