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Title: Developing a model of mitochondrial heteroplasmy in human pluripotent stem cell-derived cardiomyoctes
Author: Pinto Ricardo, Carolina
ISNI:       0000 0004 7427 7360
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2018
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Hypertrophic cardiomyopathy and arrhythmias are common in patients with heteroplasmic mtDNA mutations but their pathogenesis and how the levels of mutation change in patients over time remain poorly understood. Therefore, a robust in vitro model of heteroplasmy in cardiomyocytes (CMs) is currently needed. This thesis addressed the hypothesis that human induced pluripotent stem cell-cardiomyocytes (hiPSCs-CMs) with heteroplasmic mitochondrial dysfunction recapitulated cardiac features associated with mtDNA disease. We have applied maturation strategies to identify culture conditions where the maximal metabolic potential of healthy hiPSC-CMs could be achieved to later determine if that was compromised in models of heteroplasmy. Time in culture and T3 did not improve mitochondrial function in hiPSC-CMs and consequently were not further applied. Antimycin A and menadione impair the electron transport chain and increase the production of reactive oxygen species and were used to generate a drug-induced model of heteroplasmy using healthy hiPSC-CMs. We found that the cells’ mitochondrial function recovered from antimycin A and menadione damage, possibly due to a quick mitochondrial turnover and so this approach could not be pursued. Instead, CMs were generated by differentiating hiPSCs reprogrammed from somatic cells of a patient with the A3243G mtDNA mutation. The levels of heteroplasmy increased with hiPSC passaging and we obtained pure populations of A3243G hiPSC-CMs with heteroplasmy levels ranging from 23 to 45% and preserved structural properties. Most functional properties were also maintained but mutant hiPSC-CMs showed a delay in the initiation of the calcium transient, which could explain the episodes of bradycardias that occur in patients with mtDNA diseases. Here, we have demonstrated that hiPSC-CMs can be used to investigate the cardiac abnormalities associated with mtDNA disease but concluded that higher levels of heteroplasmy would be required to detect a disease phenotype in these cells. Future work on heteroplasmic drifts in hiPSC culture will provide crucial information about the pathogenesis of mtDNA disease and into the drift that might happen with age in children resulting from mitochondrial replacement therapy, where a small amount of mutant mtDNA is carried over.
Supervisor: Harding, Sian ; Terracciano, Cesare Sponsor: Imperial College London ; British Heart Foundation
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral