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

Abstract

Yafei Zhou,1,2 Rui Zhou,1,2 Wenjun Huang,1,2 Jie Wang,1,2 Congshan Jiang,1,2 Anmao Li,1,2 Christopher LH Huang,3 Yanmin Zhang1,2,4 1Key Laboratory of Precision Medicine to Pediatric Diseases of Shaanxi Province, Affiliated Children’s Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi, People’s Republic of China; 2Shaanxi Institute for Pediatric Diseases, Affiliate Children’s Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi, People’s Republic of China; 3Physiological Laboratory, University of Cambridge, Cambridge, UK; 4Department of Cardiology, Xi’an Children’s Hospital, Affiliated Children’s Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi, People’s Republic of ChinaCorrespondence: Yanmin Zhang, Key Laboratory of Precision Medicine to Pediatric Diseases of Shaanxi Province, Affiliate Children’s Hospital of Xi’an Jiaotong University, No. 69 Xi Ju Yuan Xiang, Xi’an, Shaanxi, 710000, People’s Republic of China, Tel [phone], Fax [phone], Email [email protected] 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. Keywords: cardiomyocytes, induced pluripotent stem cells, dynamic development, gene expression, morphology, action potential

Keywords

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