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Metabolic remodeling in hiPSC-derived myofibers carrying the m.3243A>G mutation
Gabriel E Valdebenito1, Anitta R Chacko1, Chih-Yao Chung1
1Department of Cell and Developmental Biology, UCL, Gower Street, London WC1E 6BT, UK; Consortium for Mitochondrial Research, UCL, Gower Street, London WC1E 6BT, UK.
Abstract:
Mutations in mitochondrial DNA cause severe multisystem disease frequently associated with muscle weakness. The m.3243A>G mutation is the major cause of mitochondrial encephalomyopathy lactic acidosis and stroke-like episodes (MELAS). Experimental models that recapitulate the disease phenotype in vitro for disease modeling or drug screening are very limited. We have therefore generated hiPSC-derived muscle fibers with variable heteroplasmic mtDNA mutation load without significantly affecting muscle differentiation potential. The cells exhibit physiological characteristics of muscle fibers and show a well-organized myofibrillar structure. In cells carrying the m.3243A>G mutation, the mitochondrial membrane potential and oxygen consumption were reduced in relation to the mutant load. We have shown through proteomic, phosphoproteomic, and metabolomic analyses that the m.3243A>G mutation variably affects the cell phenotype in relation to the mutant load. This variation is reflected by an increase in the NADH/NAD+ ratio, which in turn influences key nutrient-sensing pathways in the myofibers. This model enables a detailed study of the impact of the mutation on cellular bioenergetics and on muscle physiology with the potential to provide a platform for drug screening.
Insights
Researchers developed a new cellular model for mitochondrial encephalomyopathy lactic acidosis and stroke-like episodes (MELAS) using patient-derived muscle fibers. This model accurately reflects disease impact on cellular energy production and muscle function, aiding drug discovery.
Area of Science:
- Cell Biology
- Genetics
- Biochemistry
Background:
- Mitochondrial DNA mutations cause severe multisystem diseases, often presenting as muscle weakness.
- The m.3243A>G mutation is a primary cause of mitochondrial encephalomyopathy lactic acidosis and stroke-like episodes (MELAS).
- Existing in vitro models for MELAS are limited for disease modeling and drug screening.
Purpose of the Study:
- To generate a novel in vitro model of MELAS using human induced pluripotent stem cell (hiPSC)-derived muscle fibers.
- To investigate the impact of variable heteroplasmic mtDNA mutation loads on muscle cell phenotype and function.
- To establish a platform for drug screening for MELAS.
Main Methods:
- Generation of hiPSC-derived muscle fibers with controlled m.3243A>G mutation loads.
- Assessment of muscle fiber differentiation, myofibrillar structure, mitochondrial membrane potential, and oxygen consumption.
- Comprehensive proteomic, phosphoproteomic, and metabolomic analyses to characterize cellular phenotype variations.
Main Results:
- hiPSC-derived muscle fibers maintained differentiation potential and myofibrillar organization.
- Cells with the m.3243A>G mutation showed reduced mitochondrial membrane potential and oxygen consumption, correlating with mutation load.
- Metabolomic analysis revealed an increased NADH/NAD+ ratio and altered nutrient-sensing pathways in mutant cells.
Conclusions:
- The developed hiPSC-derived muscle fiber model effectively recapitulates key aspects of MELAS pathophysiology.
- This model allows for detailed investigation of mutation load-dependent effects on cellular bioenergetics and muscle physiology.
- The model serves as a valuable platform for screening potential therapeutic compounds for MELAS.
