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Title: Characterisation of dynamic cellular processes in human stem cell models of inherited cardiomyopathy using genetically encoded reporters
Author: Broyles, Connor Neil
ISNI:       0000 0004 8507 8527
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2020
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The demonstrated number 1 killer in the world is cardiovascular disease, with inherited cardiomyopathies the more dramatic scenario as children are forced to live with the condition from an early age. Small genetic changes to the myofilament proteins in cardiac myocytes can result in either hypertrophic cardiomyopathy where the heart becomes enlarged and fails to relax efficiently, or dilated cardiomyopathy where the chambers of the heart stretch and are unable to appropriately contract. Either case results in inefficient delivery of vital oxygen and nutrients to the body. However it is not simply that one gene causes hypertrophy or dilation, but rather that single mutations within the same gene can cause either disease. This complicates how the diseases are understood, and the search for treatments. To address this, individual mutations must be investigated and specific molecular pathologies known, in order to effectively target the true problem when managing the condition; forcing researchers to focus on individual families containing the mutation, resulting in personalised approaches to medical treatment. This thesis investigates two mutations found in different families that are known to result in hypertrophic and dilated cardiomyopathy: Troponin I arginine 145 → glycine (R145G) and Troponin T arginine 141 → tryptophan (R141W) respectively. This is performed in human stem cell derived cardiac myocytes, using novel genetically encoded reporters for detection of protein localisation and calcium ion sensing specifically at the myofilament. Because stem cell derived cardiac myocytes have foetal-like appearances and physiology, the outset of this work aimed to define if cells could be improved for greater myofilament alignment, sarcomere function, and calcium physiology by extending the time of culture prior to experimentation. In the later half of this thesis, the disease cases are investigated with wild- type cells after applying mutant versions of the genetic reporters used for characterisation of the dynamic functions, and novel patient derived cell lines; permitting characterisation of cells already containing the mutation. The first two chapters investigating cell structure, function and calcium physiology identified that maintaining the cells for 50 or more days beyond the initiation of the stem cell directed differentiation to cardiac myocytes was sufficient in evoking reliable and improved cellular appearances. These efforts also introduced the idea that troponin I and T have a certain degree of molecular mobility, observed via fluorescence recovery after photobleaching, that could potentially play a role in disease. Investigation of the hypertrophy causing mutation (R145G) revealed an increase in Troponin I mobility, and mutant cells with myofilament disarray and prolonged calcium transients that are expected of the disease. The increase in troponin I mobility with the mutation fits with the current knowledge of the sarcomere remaining active during relaxation; presenting a potential disease mechanism where the mutated protein is unable to remain attached to inhibit contraction. The investigation of the dilated mutation (R141W) revealed a decrease in Troponin T mobility, and an array of phenotypes for sarcomeric calcium handling that appears natural or contains a plateau in the decay. Both pathologies observed fit with the current understanding that the R141W mutation enhances Troponin T and Tropomyosin affinity to reduce the ability to reveal myosin binding sites for contraction initiation, forcing cells to require elevated calcium levels to initiate and sustain contraction. Overall, The work presented highlights the potential of genetically encoded reporters in the investigation of cardiac diseases on the single cell level.
Supervisor: Srinivas, Shankar ; Daniels, Matthew J. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Cardiovascular system--Diseases ; Troponin ; Troponin Mobility