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Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Kinesiology

Year Degree Awarded

2018

Month Degree Awarded

February

First Advisor

Edward P. Debold

Subject Categories

Biophysics | Exercise Science

Abstract

Muscle contracts after calcium (Ca++) is released into the muscle cell, resulting from a cascade of events which result in myosin, the molecular motor of muscle, to produce force and motion. Myosin cyclically binds to a regulated thin filament, using the chemical energy of ATP to produce force and motion. Perturbations in muscle, such as a build-up of metabolic by-products or point mutations in key contractile proteins, can inhibit these functions in both skeletal and cardiac muscle either acutely or chronically. Despite the many years we have studied skeletal and cardiac muscle, we still do not have a clear picture of the effect of these perturbations at the molecular level. Indeed, we do not even have a clear picture of how muscle is activated at the molecular level. Such an understanding would provide a foundation for future work, and aid our understanding of perturbations such as muscle fatigue or point mutations. Recent advances in biophysical techniques have allowed us to directly observe both single myosin and small ensembles of myosin interacting with an actin filament. We build off this previous work by examining 1) a single myosin molecule interacting with a regulated actin thin filament, and 2) small ensembles of myosin working together to pull on a regulated thin filament. We set out to directly determine how Ca++ and myosin activate the thin filament and thus muscle. Our approach to directly observe these molecular phenomena across physiological Ca++ levels as well as high ATP concentrations is novel. The first goal of this work was to examine how 1) a single myosin interacts with a regulated thin filament and 2) how a small ensemble of myosins work together to generate force and motion in a Ca++ dependent manner as they interact with a regulated thin filament. The findings build on previous work using similar techniques, but are rich in data and provide a more physiological viewpoint. The second goal of this work was to examine a particular mutation that leads to hypertrophic cardiomyopathy. The current evidence suggests that this mutation alters both thin filament dynamics and myosin kinetics. This mutation was thus chosen not only to add to our understanding of this particular mutation in hopes of developing a therapeutic, but also to provide insight into the role of thin filament activation and myosin dynamics in hopes of better understanding muscle contraction overall. With our experiments, supplemented with a mathematical model, we have de-convolved the role of Ca++ and myosin in thin filament activation, and developed a model of muscle activation from the single molecule up to the scale of a cell.

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