Gene Expression, Morphology, and Electrophysiology During the Dynamic Development of Human Induced Pluripotent Stem Cell-Derived Atrial- and Ventricular-Like Cardiomyocytes

Abstract

Background and Objectives

Gene expression, morphology, and electrophysiological combination are essential for assessing the dynamic development of human induced pluripotent stem cell-derived atrial- and ventricular-like cardiomyocytes (iPS-AM and iPS-VM, respectively).

Methods

For iPS-AM/VM differentiation, we performed the small molecule-based temporal modulation of the retinoic acid and bone morphogenetic protein signaling pathways. We investigated the gene expression and morphology using immunofluorescence, quantitative real-time polymerase chain reaction, flow cytometry, and transmission electron microscopy as well as registered electrophysiological functions using a whole-cell patch clamp on days 20, 30, and 60 post-differentiations.

Results

Pan-cardiomyocyte marker, including troponin T2 (TNNT2) and alpha-actinin-2 (ACTN2), expressions increased both in iPS-AMs and iPS-VMs. Similarly, the mRNA expression of both iPS-AM-specific markers, ie, natriuretic peptide A (NPPA), myosin light chain 7 (MYL7), and K+ channel Kir3.4 (KCNJ5), and iPS-VM-specific markers, ie, gap junction α-1 (GJA1), myosin light chain 2 (MYL2), and alpha-1-subunit of a voltage-dependent L-type calcium channel (CACNA1C), increased from 0 to 20 days, and then decreased from 30 to 60 days. Concerning morphology, cardiac troponin-T (cTnT) arrangement was progressively organized and developed from a disorderly myofibrillar distribution to an organized sarcomere pattern both in iPS-AMs and iPS-VMs. Mitochondrial numbers gradually increased and those of lipid droplets decreased during dynamic development. Regarding physiological function, the resting and action potential amplitudes remained statistically indifferent in both cell types, and the action potential duration was prolonged during the development.

Conclusion

IPS-AMs/VMs displayed dynamic development concerning their gene expression, morphology, and electrophysiological function. The discoveries of this study could provide novel insights into heart development and encourage further research.

Graphical Abstract

Keywords:

• cardiomyocytes
• induced pluripotent stem cells
• dynamic development
• gene expression
• morphology
• action potential

Data Sharing Statement

The data underlying this article will be shared upon reasonable request by the corresponding author.

Acknowledgments

Graphical abstract created with BioRender.

Disclosure

The authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

Additional information

Funding

This work was supported in part by the Natural Science Foundation of China [No. 82211530115 and 81974014, No. 82200399], Xi’an Talent Program [XAYC200023], Natural Science Foundation of Shaanxi Province [2022JQ-968, 2023-JC-YB-747, 2023-JC-YB-752, 2021JQ-024, 2022JQ-877], Ministry of Science and Technology [G2022040006L and DL2022040004L], Shaanxi Provincial International Science and Technology Cooperation Program Key Project [2024GH-ZDXM-38], Natural Science Foundation of Xi’an Health Commission [No.2022qn10], and Natural Science Foundation of Xi’an Children’s Hospital [No. 2021H02].