Overexpression of KCNJ2 enhances maturation of human-induced pluripotent stem cell-derived cardiomyocytes

Jingjun Zhou; Baiping Cui; Xiaochen Wang; Hongkun Wang; Junnan Zheng; Fengfeng Guo; Yaxun Sun; Hangping Fan; Jiaxi Shen; Jun Su; Jue Wang; Haige Zhao; Yiquan Tang; Tingyu Gong; Ning Sun; Ping Liang

doi:10.1186/s13287-023-03312-9

Overexpression of KCNJ2 enhances maturation of human-induced pluripotent stem cell-derived cardiomyocytes

Stem Cell Res Ther. 2023 Apr 15;14(1):92. doi: 10.1186/s13287-023-03312-9.

Authors

Jingjun Zhou^#^{1

2}, Baiping Cui^#^{3

4}, Xiaochen Wang^#^{1

2}, Hongkun Wang^{1

2}, Junnan Zheng⁵, Fengfeng Guo^{1

2}, Yaxun Sun⁶, Hangping Fan^{1

2}, Jiaxi Shen^{1

2}, Jun Su^{1

2}, Jue Wang^{1

2}, Haige Zhao⁵, Yiquan Tang⁷, Tingyu Gong^{8

9}, Ning Sun¹⁰, Ping Liang^{11

12}

Affiliations

¹ Key Laboratory of Combined Multi-Organ Transplantation, Ministry of Public Health, The First Affiliated Hospital, Zhejiang University School of Medicine, 79 Qingchun Road, Hangzhou, 310003, Zhejiang, China.
² Institute of Translational Medicine, Zhejiang University, Hangzhou, 310029, Zhejiang, China.
³ Institute of Geriatrics (Shanghai University), Affiliated Nantong Hospital of Shanghai University (The Sixth People's Hospital of Nantong), School of Medicine, Shanghai University, Nantong, 226011, China.
⁴ Shanghai Engineering Research Center of Organ Repair, School of Medicine, Shanghai University, Shanghai, 200444, China.
⁵ Department of Cardiovascular Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310003, Zhejiang, China.
⁶ Department of Cardiology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, 310016, Zhejiang, China.
⁷ State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Institutes of Brain Science, Fudan University, Shanghai, 200032, China.
⁸ Key Laboratory of Combined Multi-Organ Transplantation, Ministry of Public Health, The First Affiliated Hospital, Zhejiang University School of Medicine, 79 Qingchun Road, Hangzhou, 310003, Zhejiang, China. 11918392@zju.edu.cn.
⁹ Institute of Translational Medicine, Zhejiang University, Hangzhou, 310029, Zhejiang, China. 11918392@zju.edu.cn.
¹⁰ Wuxi School of Medicine, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214028, Jiangsu, China. sunning@jiangnan.edu.cn.
¹¹ Key Laboratory of Combined Multi-Organ Transplantation, Ministry of Public Health, The First Affiliated Hospital, Zhejiang University School of Medicine, 79 Qingchun Road, Hangzhou, 310003, Zhejiang, China. pingliang@zju.edu.cn.
¹² Institute of Translational Medicine, Zhejiang University, Hangzhou, 310029, Zhejiang, China. pingliang@zju.edu.cn.

^# Contributed equally.

Abstract

Background: Although human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are a promising cell resource for cardiovascular research, these cells exhibit an immature phenotype that hampers their potential applications. The inwardly rectifying potassium channel K_ir2.1, encoded by the KCNJ2 gene, has been thought as an important target for promoting electrical maturation of iPSC-CMs. However, a comprehensive characterization of morphological and functional changes in iPSC-CMs overexpressing KCNJ2 (KCNJ2 OE) is still lacking.

Methods: iPSC-CMs were generated using a 2D in vitro monolayer differentiation protocol. Human KCNJ2 construct with green fluorescent protein (GFP) tag was created and overexpressed in iPSC-CMs via lentiviral transduction. The mixture of iPSC-CMs and mesenchymal cells was cocultured with decellularized natural heart matrix for generation of 3D human engineered heart tissues (EHTs).

Results: We showed that mRNA expression level of KCNJ2 in iPSC-CMs was dramatically lower than that in human left ventricular tissues. KCNJ2 OE iPSC-CMs yielded significantly increased protein expression of K_ir2.1 and current density of K_ir2.1-encoded I_K1. The larger I_K1 linked to a quiescent phenotype that required pacing to elicit action potentials in KCNJ2 OE iPSC-CMs, which can be reversed by I_K1 blocker BaCl₂. KCNJ2 OE also led to significantly hyperpolarized maximal diastolic potential (MDP), shortened action potential duration (APD) and increased maximal upstroke velocity. The enhanced electrophysiological maturation in KCNJ2 OE iPSC-CMs was accompanied by improvements in Ca²⁺ signaling, mitochondrial energy metabolism and transcriptomic profile. Notably, KCNJ2 OE iPSC-CMs exhibited enlarged cell size and more elongated and stretched shape, indicating a morphological phenotype toward structural maturation. Drug testing using hERG blocker E-4031 revealed that a more stable MDP in KCNJ2 OE iPSC-CMs allowed for obtaining significant drug response of APD prolongation in a concentration-dependent manner. Moreover, KCNJ2 OE iPSC-CMs formed more mature human EHTs with better tissue structure and cell junction.

Conclusions: Overexpression of KCNJ2 can robustly enhance maturation of iPSC-CMs in electrophysiology, Ca²⁺ signaling, metabolism, transcriptomic profile, cardiomyocyte structure and tissue engineering, thus providing more accurate cellular model for elucidating cellular and molecular mechanisms of cardiovascular diseases, screening drug-induced cardiotoxicity, and developing personalized and precision cardiovascular medicine.

Keywords: Electrophysiology; Human-engineered heart tissues; KCNJ2; Maturation; iPSC-CMs.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cardiotoxicity
Cell Differentiation / genetics
Coculture Techniques
Humans
Induced Pluripotent Stem Cells* / metabolism
Myocytes, Cardiac / metabolism
Potassium Channels, Inwardly Rectifying* / genetics
Potassium Channels, Inwardly Rectifying* / metabolism

Substances

KCNJ2 protein, human
Potassium Channels, Inwardly Rectifying