O'Callaghan, Benjamin John; (2021) Development of an in vitro Model to Explore the Impact of mtDNA Mutations on the Metabolism and Epigenome of Myogenic Cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The work presented in this thesis describes the successful establishment of an in vitro model of mitochondrial disease using hiPSC technology and targeted differentiation towards disease-relevant skeletal muscle cell types, which was then utilised to explore the impact of mtDNA disease pathogenic variants on histone acetylation modifications. hiPSC lines harbouring a number of different disease-associated mitochondrial DNA (mtDNA) pathogenic variants were first established, through reprogramming of mitochondrial disease patient fibroblasts. By taking advantage of the random segregation of mutant mtDNA in patient fibroblast populations, hIPSC lines with high heteroplasmy for disease modelling were successfully established, alongside isogenic control lines with low/undetectable levels. Whilst the m.8344A>G pathogenic variant is detrimental to cell reprogramming and pluripotency maintenance, the m.3243A>G does not overtly impact the reprogramming process. A targeted differentiation protocol was then optimised in order to differentiate selected mtDNA disease hIPSC lines into disease-relevant myogenic cell types including terminally differentiated myotubes which show characteristic deficits in mitochondrial function. Whilst mid-range m.8344A>G mutation loads do not overtly impact the myogenic differentiation process, impairments in both myogenic differentiation and subsequent maturation of myotubes are observed in lines harbouring mid-range and higher m.3243A>G mutation loads. Assessments of metabolically sensitive histone acetylation modifications at early and later stages of the myogenic differentiation process revealed cell lines harbouring the m.3243A>G pathogenic variant display widespread deficits in a histone acetylation deposition and/or maintenance. Together, the data presented in this thesis show that hiPSC-derived myogenic cell types represent a useful in vitro model for exploring early pathomechanisms contributing to myopathic phenotypes in mitochondrial disease states. These data show for the first time that mitochondrial deficits caused by mtDNA pathogenic variants can negatively impact metabolic fluxes contributing to histone acetylation deposition and associated physiologically relevant myogenic cell-identity transitions. Modulators of histone acetylation modifications might therefore represent a novel target for therapeutic intervention in human mitochondrial diseases.

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