Modulation of cardiac muscle contractility by phosphorylation, HCM and DCM causing mutations and small molecules
File(s)
Author(s)
Sheehan, Alice Mary
Type
Thesis
Abstract
Mutations in sarcomeric proteins that cause familial hypertrophic cardiomyopathy and dilated cardiomyopathy have been shown to abolish the coupled relationship between troponin I phosphorylation and myofilament Ca2+-sensitivity, a phenomenon referred to as uncoupling. In normal heart, PKA phosphorylation of troponin I Ser22 and 23 leads to a 2-fold decrease in Ca2+-sensitivity and a corresponding increase in the rate of Ca2+ release from TnC and is essential for the lusitropic response to adrenergic stimulation. Therefore, uncoupling results in a blunted response to β1-adrenergic activation and has been demonstrated in animal models with hypertrophic cardiomyopathy and dilated cardiomyopathy mutations at a cellular, tissue and whole animal level. However, the molecular mechanisms and physiological relevance of uncoupling as a common phenomenon in cardiomyopathy-associated sarcomeric mutations are not well-understood. In this study, I have employed a multidisciplinary approach to probe for the presence of troponin uncoupling in mutation-containing cardiomyopathy models at an atomistic, molecular and cellular level. I have employed molecular dynamics simulation to elucidate how the structure and dynamics of troponin can give rise to physiological properties of cardiomyopathy. Additionally, I have investigated small molecules analogues of EGCG and silybin for recoupling properties that can restore the abolished relationship between troponin I phosphorylation and myofilament Ca2+-sensitivity in vitro, identifying a number of promising recoupling agents via in vitro motility assay. Moreover, I have demonstrated uncoupling at the cellular level as a blunting of the time to relaxation in intact cardiomyocytes following β-adrenergic stimulation and have developed a methodology that is capable of distinguishing cellularly-active recoupling molecules from candidates that are toxic. I have identified two promising recoupling agents, silybin B and resveratrol, using the contractile study presented herein, demonstrated by a reversal of the blunted phenotype. My investigation has demonstrated the feasibility of small molecules as recoupling agents and their therapeutic potential.
Version
Open Access
Date Issued
2019-09
Date Awarded
2020-02
Copyright Statement
Creative Commons Attribution NonCommercial Licence
Advisor
Marston, Steven
Gould, Ian
Sponsor
British Heart Foundation
Publisher Department
National Heart & Lung Institute
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)