Downregulation of SRF-signaling drives polyploidy development of human induced pluripotent stem cell-derived cardiomyocytes in response to contractile activity

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an unlimited source of human cardiomyocytes for medical research and clinical application. Nevertheless, the immature phenotype of iPSC-CMs in comparison to adult CMs represents a major limitation. A variety of approaches, such as lipid-enriched media, hormone treatment or biophysical cues, were shown to improve the structural properties and the contractile as well as electrophysiological function of iPSC-CMs.

However, the development of polyploidy, representing a central aspect of CM maturation as approximately 70% of adult CMs are polyploid, was only investigated in a few studies and the mechanisms underlying polyploidy development in CMs are still unclear.

In this study, we demonstrate that cultivation of iPSC-CMs under the combined influence of lipid-enriched maturation medium (MM), nanopatterning (NP), and electrical stimulation (ES, 2 Hz) promotes the development of polyploidy. Analysis of the DNA content in iPSC-CMs using flow cytometry revealed a significant increase of the polyploid (≥ 4n) population. We identified ES as the pivotal stimulus for polyploidy development, as the DNA content of iPSC-CMs cultured under the influence of MM or the combination of MM+NP was unaltered compared to cells in B27 medium. Co-staining of DNA and the cell cycle marker Ki‑67 confirmed an increased cell cycle activity of the ES-stimulated iPSC-CMs. Importantly, we observed an increased proportion of polyploid Ki67^- iPSC-CMs under the influence of MM+NP+ES compared to cells in B27-medium, MM or MM+NP, confirming the presence of polyploid, cell-cycle inactive iPSC-CMs. To gain insight into the molecular mechanisms of how ES promotes polyploidy, we performed RNA-sequencing and identified that SRF target genes were strongly downregulated in iPSC-CMs cultured under ES. In agreement with this finding, SRF protein levels were decreased in CMs from the MM+NP+ES group compared to the other groups. As the SRF target gene cluster included many genes involved in cell cycle regulation and cell proliferation, we specifically evaluated the expression of genes that regulate cell cycle progression. We found that activators of G2/M checkpoints (CCNB1-3, CDK1) and cytokinesis (ANLN, SEPTIN7/2) were downregulated, whereas CDK inhibitors (CDKN1A, CDC20) were upregulated in response to ES. In contrast, we did not observe any significant changes in the gene sets controlling the G1- and S-phase progression or the G1/S checkpoint. Therefore, our data support that ES induces a cell cycle arrest after the S-phase, before exit from M-phase, which may lead to bi-nucleation or nuclear polyploidy.

Taken together, our data provide strong evidence that downregulation of SRF-signaling in response to an increased contractile activity represents as an endogenous maturation mechanism of iPSC-CMs leading to the development of polyploidy.