Advanced mitochondrial development in induced pluripotent stem cell-derived cardiomyocytes through integration of metabolic medium, nanopatterning and electrostimulation

The generation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offers an unlimited source of patient-specific human CMs. However, the application of iPSC-CM in vitro-models to guide the selection of drugs for individual patients in the clinic remains a vision. A significant obstacle is the immature phenotype of iPSC-CMs, which alters their sensitivity to physiological and pathophysiological stimuli as well as to proarrhythmic and toxic agents, compared to adult CMs. The metabolism of CMs plays a critical role in overcoming this limitation, as particularly the transition from glycolysis to β-oxidation of fatty acids is essential for their maturation into a more adult-like phenotype.

In this study, we demonstrate advanced mitochondrial development in iPSC-CMs by integrating the use of lipid-supplemented maturation medium (MM) with cell alignment on nanopatterned surfaces (NP) and the induction of increased contractile workload through electrostimulation (ES).

Through systematic and parallel testing of these three stimuli, we identified ES as the pivotal factor for enhancing mitochondrial development and metabolic maturation. Morphological analysis revealed that NP induced the alignment of iPSC-CMs, enhanced Connexin-43 membrane expression as well as α-actinin/ryanodine receptor 2 colocalisation, and increased sarcomere length in comparison to cells in MM. The combination with ES further improved the function of iPSC-CMs, demonstrated by their ability to adapt to a high pacing frequency of 2 Hz. These changes were accompanied by a gradual metabolic maturation of iPSC-CMs achieved through combination of MM with NP and NP+ES.

RNA-seqencing and pathway enrichment analysis of genes upregulated in the MM+NP+ES group vs. MM group revealed significant enrichment of genes involved in electron transport chain, tricarboxylic acid cycle, mitochondrial biogenesis, glucose metabolism, and fatty acid oxidation. In contrast, these gene clusters showed only slight upregulation in the MM+NP group compared to the MM. Real-time PCR studies confirmed an increased expression of markers associated with mitochondrial development, including PPARGC1α, PPARα, OPA1, and MFN2, in iPSC-CMs of the MM+NP+ES group. Furthermore, we found an increased Tom20 staining intensity in iPSC-CMs matured under MM+NP+ES as well as MM+ES compared to MM, showing that ES is most important for mitochondrial development, while NP has a minor effect. Subsequent analysis against the transcription factor target database identified that the genes upregulated by MM+NP+ES in comparison to MM were enriched in TFAM (mitochondrial transcription factor A)- and HMCES (5-hydroxymethylcytosine binding, embryonic stem cell-specific)-related target gene sets. TFAM is crucial for the transcription, replication, and packaging of mitochondrial DNA (mtDNA), while HMCES helps to maintain genomic and mtDNA integrity during oxidative stress.

In summary, our findings highlight the importance of enhanced contractile activity in promoting advanced mitochondrial development, as well as metabolic and functional maturation of iPSC-CMs. Furthermore, our results strongly suggest that TFAM and HMCES play significant roles in the endogenous mechanisms underlying mitochondrial development. In future studies, we plan to utilize this experimental model to investigate  mitochondrial cardiomyopathies using patient-specific iPSC-CMs.