Generation of Human Cardiomyocytes With Adult-Like Metabolism by a Chemically Defined Medium for Mitochondrial Disease Modeling

BACKGROUND: Human induced pluripotent stem cell (iPSC)–derived cardiomyocytes (iPSC cardiomyocytes) as a model for cardiac disease have become increasingly important, bolstered by the Food and Drug Administration’s initiative to promote novel alternative methods for biomedical research. A key limitation of metabolically immature iPSC cardiomyocytes is their limited ability to accurately recapitulate mitochondria-dependent pathologies, such as Friedreich ataxia–associated cardiomyopathy. Adult cardiomyocytes are characterized by a high density of interfibrillar mitochondria, abundant respiratory complexes (I–V) that form supercomplexes, and efficient utilization of fatty acids to drive mitochondrial respiration. These unique metabolic structures and functions are largely missing in iPSC cardiomyocytes. Further, whether iPSC cardiomyocytes can efficiently utilize fatty acids for respiration in the presence of glucose, a unique adult feature, remains unclear. METHODS: We developed a chemically defined, 11-component maturation medium (MM11) designed using a bioinformatics-guided strategy that identified high-fidelity transcriptional markers of adult cardiac metabolic maturity. Human iPSC cardiomyocytes were treated with MM11 for 2 weeks and evaluated at the molecular, structural, and functional levels, with an emphasis on mitochondrial maturity. We benchmarked our results with isolated primary adult human cardiomyocytes. Last, we tested whether this method could enable us to more accurately model Friedreich ataxia cardiomyopathy. RESULTS: MM11 treatment increased expression of metabolic pathways, including mitochondrial respiration and fatty acid metabolism, at the transcript and protein levels. Ultrastructural analyses revealed the formation of interfibrillar mitochondria and mitochondrial respiratory supercomplexes, a feature never shown in previous reports. Functionally, MM11-treated iPSC cardiomyocytes exhibited substantially increased oxidative phosphorylation and a metabolic preference for fatty acid utilization in the presence of physiologic substrates, as determined by oxygen consumption assays and 13 C isotope tracing. CONCLUSIONS: MM11 promotes robust metabolic maturation of human iPSC cardiomyocytes, enabling sophisticated mitochondrial architecture and substrate utilization. These metabolically matured cardiomyocytes more faithfully model mitochondria-dependent cardiac pathologies, including ischemia-reperfusion injury and Friedreich ataxia cardiomyopathy, improving the use of iPSC cardiomyocytes for mechanistic studies of human heart disease.