Document Type

Dissertation - Open Access

Award Date

2016

Degree Name

Doctor of Philosophy (PhD)

Department / School

Chemistry and Biochemistry

First Advisor

Adam Hoppe

Keywords

actin, cadiac muscle, FRET, myosin, tropomyosin, troponin

Abstract

Heart disease is the leading cause of death in the United States. Understanding heart function at the molecular level is critical for developing of more effective treatments. In the cardiac muscle, the thin filament is composed by troponin (Tn), tropomyosin (Tm), and F-actin. It provides Ca2+-dependent regulation of contraction by modulating myosin attachment and force generation in a cooperative scheme. However, this mechanism remains unclear. To understand thin filament activation, we studied the binding and functional properties of Tn and Tm to F-actin at single particle resolution by employing fluorescence image colocalization, in vitro motility assays, and Förster resonance energy transfer based on fluorescence lifetime imaging (FLIM-FRET). Our results suggest that under physiologically relevant conditions, Tn and Tm binding to Factin is not cooperative and it is not affected by Ca2+. This suggests that one single type of interaction is involved in fully regulated thin filaments. Thin filament activation has been confirmed by in vitro motility assays, where phosphorylation of serine 23 and 24 in TnI, truncation of TnT C-terminal region, and incorporation of a Ca2+ desensitizer altered the Ca2+ response to filament sliding. FLIM-FRET measurements revealed an allosteric dependence on thin filament activation as a function of myosin and Ca2+. Our results provide evidence of multiple allosteric elements within thin filaments responsible for the molecular modulation of cardiac muscle activation.

Library of Congress Subject Headings

Myocardium.

Heart -- Molecular aspects.

Muscles -- Molecular aspects.

Cytoplasmic filaments.

Description

Includes bibliographical references (pages 124-137)

Format

application/pdf

Number of Pages

191

Publisher

South Dakota State University

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Rights Statement

In Copyright