Vetter, Anthony2020-02-262020-02-262017-12https://hdl.handle.net/11299/211813University of Minnesota Ph.D. dissertation. December 2017. Major: Integrative Biology and Physiology. Advisor: Joseph Metzger. 1 computer file (PDF); 189 pages.The cardiac sarcomere is the functional unit of force production in the heart. The sarcomere is a highly ordered near-liquid crystalline arrays of thin and thick filament proteins held in stoichiometric balance that work in a concerted fashion to orchestrate the heart’s pump function. Regulation of cardiac output is mediated by a combination of myocyte cell-intrinsic and cell-extrinsic factors that fine-tune the contractile machinery. Central to the regulation of the sarcomere is the heterotrimeric cardiac troponin complex (cTn) whose subunits include: cardiac troponin T (cTnT), an adaptor protein that tethers cTn into the ultrastructure of the thin filament via tropomyosin; cardiac troponin C (cTnC), a calmodulin-like EF-hand protein that confers calcium sensitivity to the thin filament; and cardiac troponin I (cTnI), a molecular switch that toggles between the actin filament and N-terminal lobe of cTnC. When associated with actin, cTnI prevents the azimuthal rotation of tropomyosin along the filament thereby concealing strong myosin binding sites along the thin filament. Therefore, cTn may be thought of as a binary switch regulated by calcium, toggling cTnI between the actin-associated inhibitory state and the cTnC-associated active state. The penultimate outcome of this dynamic structural process is changing the steric accessibility of myosin binding sites to cycling force producing myosin cross-bridges. Alterations in the sarcomeric structure-function relationship by both cell-intrinsic and cell-extrinsic factors underlie the pathology of numerous acquired and inherited cardiomyopathies. As such, gaining a greater understanding of the activation, inactivation, and molecular interactions within the sarcomere underpins and enables our ability to redress heart failure. For decades, tools have long been available to quantitatively detect and monitor the sarcolemmal depolarization and calcium transient that initiate contraction. Furthermore, the final output of the contractile machinery can be monitored by a variety of tools that measure the force production of the sarcomere. However, to this date, there has been a “black box” around the intervening processes which govern the sarcomere activation under physiological conditions. A preponderance of evidence derived from studies of isolated proteins, electron microscopy, and permeabilized steady-state muscle fibers has supported the theory that strong myosin binding is requisite to activate the sarcomere. However informative these studies are, they are limited by their non-physiological experimental conditions. Here, ex vivo cellular physiology enabled by a novel live-cell biosensor and in silico molecular dynamics simulations were used in tandem to investigate the activation of the sarcomere and specifically how the cTnC:cTnI interface is a critical juncture for determining contractility under physiological conditions. Evidence suggests that in juxtaposition to the long-standing three-state model wherein myosin binding to actin is necessary to shift the equilibrium of cTn complexes from the closed to the open state, calcium and cTnI switch peptide binding are sufficient to activate the sarcomere and permit force production by cycling cross-bridges under the live cell conditions described herein. Furthermore, when cTnI binds to the N-terminal lobe of cTnC, the interaction between helix 4 of cTnI and the A helix of cTnC is a primary determinant of contractility independent of changes in the calcium-transient. Collectively, this work enhances our knowledge of how the cardiac sarcomere is regulated and establishes it as a potent therapeutic target to improve heart pump function in ailing myocardium.enCardiacFRETMyofilamentSarcomereTroponinThesis or Dissertation