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