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Title: From cell to muscle : development of new ways to study inherited cardiomyopathies based on engineered heart tissue
Author: Owen, Thomas
ISNI:       0000 0004 7658 1702
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2018
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The ACTC E99K mutation is associated with hypertrophic cardiomyopathy (HCM- predominantly apical) and left ventricular non-compaction in a large group of patients from Northwest Spain. The aim of this thesis is to set up a new way of characterising patient phenotypes combining the technologies of engineered heart tissue and iPSCs. Punch biopsies were taken from 18 individuals (14 mutation carriers and 4 related non-carriers), fibroblasts were expanded, and 6 samples (2 mutation carriers and one healthy relative each from two different families) were taken forward for Sendai virus reprogramming. Five fibroblast lines were successfully reprogrammed, which was confirmed by staining for pluripotency markers. To make isogenic pairs of cells CRISPR-Cas9 PiggyBac gene editing was used to correct the E99K mutation in two disease carrying lines, and the mutation was knocked into one wild type line. We also analysed sudden cardiac death in the ACTC1 E99K mouse model. Higher rates of SCD were seen in enriched CBA/Ca mice, but SCD was almost absent in pure black6 mice. Using two photon microscopy, pico-sirius red staining, and gene expression we showed up regulation of fibrosis in young SCD mice. In addition, pure black6 mice were also found to have the same number of calcium release events as wild type. Pluripotency was confirmed after targeting of iPSCs, and differentiation into cardiomyocytes was achieved in all six lines: Donor I E99K, Donor I WT, Donor II E99K, Donor II WT, Donor III WT, Donor III E99K. The presence of the mutation in iPSC-CM was confirmed with an antibody specific to the ACTC E99K mutation. Donor I WT line had significantly fewer cardiomyocytes which affected the subsequent experiments. Under stimulation Donor II E99K and Donor III E99K had significantly longer relaxation times than wild type isogenic pairs. Isogenic pairs were treated with increasing concentrations of calcium Donor II E99K and Donor I E99K 3 were hypercontractile compared to Donor III WT, however, this hypercontractilty was not restored in the Donor II line when the mutation was corrected to wild type. Moreover, hypercontractility was not induced in the Donor III line with the E99K mutation knocked in compared to its wild type isogenic line. Lastly, under stimulation we also found a higher rate of abnormal arrhythmogenic like contractions in Donor II E99K and Donor III E99K compared to their wild type isogenic pairs. iPSC-CM with the E99K mutation are a useful model to understand HCM and have been shown to recapitulate some aspects of the human disease. Data suggest that poor relaxation and arrhythmic events are directly related to the mutation, while hypercontractility may be related to a co-inherited genotype or epigenetic change.
Supervisor: Marston, Steve ; Harding, Sian Sponsor: British Heart Foundation
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral