Cardiovascular diseases are the leading cause of death in western countries but limited knowledge about the underlying mechanisms hinders the progress of understanding and treating these diseases. Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) pose tremendous potential for studying the physiology and pathophysiology of human heart cells. The application of iPSC-CMs is limited by their immature phenotype when compared to adult ventricular CMs. 

In our group, we recently established an advanced culture medium (MM) to enhance the maturation state of iPSC-CMs. The medium was supplemented with a complex mixture of fatty acids and levels of glucose, lactate and insulin close to the physiological concentration range. In previous studies, we demonstrated that iPSC-CMs cultured in MM have higher metabolic activity, increased mitochondrial membrane potential and improved contractile activity. 

In this study, we performed a comprehensive characterization of iPSC-CMs cultured in MM for 3 weeks in comparison to the widely used RPMI/B27 medium. Analysis of the metabolic activity revealed a strong increase in cellular oxygen consumption rate, ATP production and spare capacity of the iPSC-CMs cultured in MM. In line with these findings, immunostainings of the mitochondrial protein Tom20 and single-cell analysis of cellular mitochondrial networks confirmed an increased mitochondrial area in iPSC-CMs maintained in MM, compared to RPMI/B27. Furthermore, we found an upregulation of PPARα and PPARGC1α, important transcription factors involved in mitochondrial development. 

Cultivation in MM further led to strong hypertrophic growth, an increased expression of structural genes associated with adult ventricular CMs, such as the cardiac troponin I (TNNI3) and predominant expression of the ventricular isoform of myosin light chain 2 (MYL7, MLC2V) in relation to the atrial isoform (MYL2, MLC2A). These findings were confirmed on protein level based on co-immunostaining of MLC2A and MLC2V and quantification of iPSC-CMs populations expressing predominantly MLC2V or MLC2A, or both MLC isoforms. The proportion of cells predominantly expressing MLC2V was significantly increased for iPSC-CMs cultured in MM.

Contractile activity and electrophysiological properties were measured to evaluate the effect of MM on the functionality of iPSC-CMs. Quantification of beating properties was done by video-based motion analysis. CMs cultured in MM had a reduced spontaneous beating frequency and were able to adapt to higher beating rates (2 Hz). In contrast, most of the iPSC-CMs cultured in RPMI/B27 failed to follow 2 Hz pacing frequency. In addition, MEA recordings were performed to examine electrophysiological function of iPSC-CMs maintained in MM versus RPMI/B27. Here, we observed an increased conduction velocity and sodium spike amplitude as well as prolonged field potential duration (FPDc) of the iPSC-CMs matured in MM. 

Our study emphasizes that the presence of fatty acids along with physiological metabolite concentration levels in the recently established MM strongly enhance the development of more mature iPSC-CMs in terms of cellular structure, metabolism and functionality. These findings are highly relevant for the understanding of disease mechanisms, like cardiac mitochondriopathy or sensitivity to hypoxia, for which structural and functional development is essential.