The myofilament basis of striated muscle function: Experimental and computational models

Thesis (Ph.D.)--University of Washington, 2018For over a century, understanding the subcellular mechanisms that regulate striated muscle function has been of great interest to biophysicists and physiologists. During contraction-relaxation cycles, both skeletal and cardiac myocytes undergo tremendous structural and geometrical dynamics that are driven by complex sarcomere-level regulatory processes. Yet, despite much effort, many sarcomere-based mechanisms of the regulation of muscle function have remained unresolved. The body of work presented here is aimed at revealing such mechanisms by leveraging recent advances in structural, mechanical, and computational biology. Specifically, we integrate: (i) experimental techniques to study sarcomere mechanics in situ, (ii) high-resolution X-ray diffraction-based imaging modalities of myofilament and sarcomere structure, (iii) spatially explicit computational models of sarcomere mechanics and energetics, and (iv) genetic engineering of murine models of heart failure harboring cardiomyopathy-associated myofilament protein mutations. In doing so, we provide insight into the molecular mechanisms that govern the regulation (and dysregulation) of straited muscle function. Moreover, this work focusses on uncovering contributions to regulatory mechanisms of muscle function from each of the three main myofilaments of the sarcomere: the myosin-containing thick filament, the actin-containing thin filament, and the giant filamentous protein titin. First, we describe a spatially explicit computational model that explores how titin stiffness affects contractile mechanics and energetics. We find that increasing titin stiffness inhibits the ability of myosin motors to energetically compete with the added titin-based strain in the thick filament and are therefore less efficient in generating force compared with motors in series with compliant titin. Second, we measure the contributions of titin to the elasticity of the sarcomere in-situ in single intact skeletal muscle fibers throughout the duration of isometric tetanus. We show that titin the I-band region of titin has a dynamic stiffness that is tuned to the length of the sarcomere, likely mediated by load-dependent structural dynamics of the I-band-specific domains. Third, we present multiple studies that harness high-intensity synchrotron light to enable Angstrom-level X-ray diffraction-based measurements of the myofilament structure in cardiac muscle. Through this technique, we investigate the structural dynamics of myosin motors in resting and activated cardiac muscle and find that some positively inotropic interventions have no effect on resting sarcomere structure, whereas others perturb the resting state of the thick filament to potentiate the ensuing contraction. Lastly, we use a transgenic murine model harboring a loss-of-function tropomyosin mutation that causes dilated cardiomyopathy. We show that both sarcomere-level and tissue-level dysfunction can be prevented by rationally designing filament-specific interventions. To summarize, the work presented here highlights results from an integrative approach that aims to understand myofilament-specific mechanisms that govern regulation of both cardiac and skeletal muscle function. As a result, we answer questions that range from a basic understanding of molecular-level regulation of muscle function to developing potential therapeutics that may translate to the clinic for patients with specific types of congenital heart failure