Valdebenito, Gabriel Esteban; (2024) Modelling the impact of the m.3243A»G mutation on mitochondrial bioenergetics and calcium homeostasis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Mutations in the mitochondrial DNA (mtDNA) give rise to rare, debilitating disorders with limited treatment options. Disease modelling is crucial for understanding pathogenic mechanisms and to enable drug screening. This thesis focuses on the mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome, which is most frequently caused by the m.3243A>G mutation in the mtDNA. This mutation causes a multisystem disorder that includes muscle weakness, the focus of the work in this thesis.  To explore the impact of the mutation, I characterised mitochondrial function and calcium homeostasis in various patient derived cell types expressing the m.3243A>G mutation. In order to study muscle pathophysiology and metabolism, I applied a novel protocol to generate skeletal myofibers from human-induced pluripotent stem cells (hiPSCs) carrying the m3243A>G mutation and expressing different mutant loads. Chapter three demonstrates the effects of m.3243A>G on calcium handling in cybrid cells and patient-derived fibroblasts. These effects are manifested by changes in ER and cytosolic calcium concentration, and an increase in mitochondrial calcium uptake despite the decreased mitochondrial membrane potential of mutant cells. Chapter four characterises hiPSCs showing stable levels of the m.3243A>G mutation, without affecting the pluripotency or cellular bioenergetics. I report the successful differentiation of hiPSCs into myofibers with the mutation, alongside an isogenic control line, using a small molecule protocol. Through proteomic analysis, muscle progenitors are compared to myofibers, revealing that myofibers express the contractile machinery despite the m.3243A>G mutation. Chapter five explores altered cell signaling and bioenergetics in mutant hiPSC-derived myofibers, unveiling a decrease in mitochondrial function, an increase in glucose metabolism, an altered NAD+/NADH redox state, possibly increased utilization of NADH shuttles, and the activation of the PI3K/AKT/mTOR pathway to a different degree than seen in fibroblasts and cybrid cells. Chapter six investigates changes in cytosolic calcium signaling in myofibers, revealing that myofibers exhibit an elevated resting cytosolic calcium concentration and that they fatigue more quickly in response to repetitive electrical stimulation. The findings presented in this thesis demonstrate that: (I) cytosolic and mitochondrial calcium homeostasis are altered in cybrid cells, patient fibroblasts, and myofibres derived from hiPSCs, playing a crucial role in shaping the phenotype of the disease, and (II) hiPSC-derived myogenic cells serve as a valuable in vitro model for exploring the consequences of the m.3243A>G mutation, recapitulating critical aspects of mitochondrial myopathy observed in patients carrying this mitochondrial mutation, and could be used as a platform for improving drug discovery and testing.

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