Promotion of maturation in CDM3-induced embryonic stem cell-derived cardiomyocytes by palmitic acid.

Background: Myocardial infarction leads to myocardial necrosis, and cardiomyocytes are non-renewable. Fatty acid-containing cardiomyocyte maturation medium promotes maturation of stem cell-derived cardiomyocytes.

Objective: To study the effect palmitic acid on maturation of cardiomyocytes derived from human embryonic stem cells (hESCs) to optimize differentiation for potential treatment of myocardial infarction by hESCs.

Methods: hESCs were differentiated into cardiomyocytes using standard chemically defined medium 3 (CDM3). Up to day 20 of differentiation, 200 Mm palmitic acid were added, and then the culture was continued for another 8 days to mimic the environment in which human cardiomyocytes mainly use fatty acids as the main energy source. Light microscopy, transmission electron microscopy, immunofluorescence, reverse transcription-polymerase chain reaction, and cellular ATP assays, were carried out to analyze the expression of relevant cardiomyocyte-related genes, cell morphology, metabolism levels, and other indicators cardiomyocyte maturity.

Results: Cardiomyocytes derived from hESCs under exogenous palmitic acid had an elongated pike shape and a more regular arrangement. Sarcomere stripes were clear, and the cells color was clearly visible. The cell perimeter and elongation rate were also increased. Myogenic fibers were abundant, myofibrillar z-lines were regularly, the numbers of mitochondria and mitochondrial cristae were higher, more myofilaments were observed, and the structure of round-like discs was occasionally seen. Expression of mature cardiomyocyte-associated genes TNNT2, MYL2 and MYH6, and cardiomyocyte-associated genes KCNJ4, RYR2,and PPARα, was upregulated (p < 0.05). Expression of MYH7, MYL7, KCND2, KCND3, GJA1 and TNNI1 genes was unaffected (p > 0.05). Expression of mature cardiomyocyte-associated sarcomere protein MYL2 was significantly increased (p < 0.05), MYH7 protein expression was unaffected (p > 0.05). hESC-derived cardiomyocytes exposed to exogenous palmitic acid produced more ATP per unit time (p < 0.05).

Conclusion: Exogenous palmitic acid induced more mature hESC-CMs in terms of the cellular architecture, expression of cardiomyocyte maturation genes adnprotein, and metabolism.