The generation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offers an unlimited source of patient-specific human cardiomyocytes. However, the use of iPSC-CM in vitro-models to guide the clinic selection of particular drugs for individual patients remains a vision. A major limitation represents the immature phenotype of iPSC-CMs, which alters their sensitivity towards physiological and pathophysiological stimuli, cardioactive drugs or toxic substances, in comparison to adult cardiomyocytes (CMs). Recently, our group established a fatty acid-supplemented maturation medium (MM), which significantly enhanced the structural, metabolic and functional maturation of iPSC-CMs.

In this study, we aim to generate iPSC-CMs with an advanced maturation state by combining the use of MM with the alignment of the cells on nanopatterned surfaces (NP) and induction of an increased contractile workload by electrical stimulation (ES).

Under the combined influence of MM, NP and ES, iPSC-CMs develop a more complex cellular structure, as demonstrated by the increased expression ratio of TNNI3/TNNI1 and the increased cell size (FSC), granularity (SSC) as well as cTNT-intensity in flow cytometry measurements. Determination of the cellular DNA-content further demonstrated a significantly increased population of polyploid (4n) CMs, specifically induced by ES, but not by MM or MM with NP. Examination of the cell cycle marker Ki-67 in this population confirmed a higher proportion of Ki-67-negative polyploid iPSC-CMs, a key hallmark of adult CMs.

Analysis of the mitochondrial development as a critical aspect of the metabolic maturation showed that MM alone strongly increased the expression of markers for mitochondrial development including PPARGC1α and PPARα, which was further enhanced under the combined influence of MM, NP and ES. This was accompanied by an increased cellular mitochondrial content, determined by an increased Tom20-intensity in flow cytometry experiments. Interestingly, the combination of MM with NP alone in the absence of ES did not promote mitochondrial development, indicating the importance of the increased workload through ES for these adaptations.

The electrophysiological properties of iPSC-CMs were investigated by measuring sodium channel I_Na and hERG channel current I_Kr using automated and manual patch-clamp. Our data demonstrated a strong increase of I_Na through cultivation in MM, which is further increased by combination with NP and ES but not by NP alone. In contrast, I_Kr was synergistically enhanced by cultivation on NP and application of ES.

Taken together, our results demonstrate the synergistic effect of MM, NP and ES to induce advanced maturation of iPSC-CMs. Interestingly, the comparison of the effects of NP alone and NP combined with ES reveals the distinct effect of these stimuli on ploidy, metabolism and electrophysiological properties of iPSC-CMs. Following studies aim to decipher the pathways underlying the diverse and synergistic effects of the maturation stimuli and to evaluate the value of the advanced maturation state for the risk assignment of cardioactive drugs.