Metabolic fatty acid substrates enhance the structural and mechanical performance of human-induced pluripotent stem cell-derived cardiomyocytes.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer a powerful platform for disease modeling, drug discovery, and regenerative therapies. However, their clinical and research utility remains limited by their immature, fetal-like phenotype. In the human heart, postnatal metabolic maturation involves a critical switch from glycolysis to fatty acid β-oxidation, enabling efficient ATP production via oxidative phosphorylation. In this study, we investigated whether mimicking this metabolic shift in vitro by culturing hiPSC-CMs in a fatty acid-based maturation medium (FAM) could enhance their structural and functional development compared to a conventional glucose-based medium (GLM). hiPSC-CMs cultured in FAM for two weeks exhibited significant improvements in morphological, metabolic, and mechanical maturation markers. Morphologically, FAM-cultured CMs exhibited enhanced sarcomeric organization, increased cellular alignment, and a more elongated and rod-like shape, which are characteristics typically associated with mature CMs. Ultrastructural analysis further confirmed improved maturation, revealing more organized sarcomeres and densely packed mitochondria compared to GLM-cultured CMs. Metabolically, FAM-cultured CMs demonstrated a clear shift from glycolytic to oxidative metabolism, as evidenced by higher mitochondrial membrane potential, increased oxidative phosphorylation capacity, elevated ATP production, and reduced glycolytic activity. These metabolic adaptations indicate a more adult-like energy profile, consistent with enhanced fatty acid β-oxidation. Mechanically, FAM-cultured cardiomyocytes exhibited enhanced functional maturity, as evidenced by faster calcium transients and greater contraction amplitude, indicating improvements in specific electrophysiological properties. In conclusion, fatty acid supplementation effectively promotes the structural, metabolic, and mechanical maturation of hiPSC-CMs, resulting in a more adult-like phenotype. This strategy provides a robust and straightforward approach to enhance the physiological relevance of hiPSC-CMs for preclinical applications in disease modeling, drug testing, and regenerative medicine.