Myofilaments as regulators of heart function in disease

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Abstract Ischemic cardiomyopathy and heart failure are the leading causes of combined morbidity and mortality in humans. Herein, sarcomere dysfunction has a central role in disease pathogenesis. The sarcomere is the essential functional unit of cardiac muscle, directly responsible for the pumping action of the heart. The cardiac sarcomere is a multimeric contractile apparatus consisting of a thin myofilament-based allosteric regulatory complex together with the myosin-based thick myofilament that generates force. Interlacing myofilaments operate in synchrony to regulate and generate the forces necessary for heart performance. Beat-to-beat control of cardiac sarcomere activation refers to the status of the thin filament regulatory system in controlling the degree to which contraction is turned on and off during a twitch. Disruption in sarcomere function underlies the basis for numerous forms of acquired and inherited heart diseases affecting millions of people in this country. Thus, focus here on mechanistic insights into sarcomere regulation underscores the major health relevance of this proposal. Recently, emerging results have come to the fore positing synergistic inter-myofilament regulatory signaling mechanisms, including a new role of myosin cross-bridge ON/OFF states in controlling muscle contraction. Building on our sarcomere activation innovations featuring single unloaded cardiac myocytes, we have made a breakthrough methodological advance, permitting real-time recordings of sarcomere activation in intact cardiac muscle under physiological load. This system is capable of detecting, by intramolecular FRET, multiple myofilament activating ligands during the physiological time course of a single twitch contraction in intact cardiac muscle under load. Guiding hypothesis: During physiologically relevant twitch contractions under load, thin filament activation is controlled dynamically by multiple synergistic inter-myofilament regulatory inputs, including TnC bound Ca2+, TnI switch domain-TnC interaction, OFF to ON state myosin cross-bridges, and MyBP-C in live cardiac muscle. This proposal aims to investigate inter-myofilament signaling by altering the TnI molecular switch mechanism during the physiological time-course of a single cardiac twitch in live cardiac muscles under load; to investigate the mechanism of inter-myofilament signaling during the cardiac twitch contraction in live intact cardiac muscles by modification in myosin cross-bridges; and to investigate the role MyBP-C in inter-myofilament signaling during physiological twitch contractions in intact cardiac muscles. Enabled by our innovative approach, the new insights into inter-myofilament signaling mechanisms gained here will significantly impact our understanding of cardiac function. In turn, this provides the essential foundation to guide new therapeutic discovery for the diseased heart by leveraging the sarcomere as an excellent target for developing new treatments and therapies for the diseased heart.