Zhu Xingyu, Soh Yee Kiat, Wan Mingxin, Pang Jeremy Kah Sheng, Leow Wei Liang, Tian Chong, Soh Boon Seng, Chan Eric Chun Yong
Department of Pharmacy and Pharmaceutical Sciences, Faculty of Science, National University of Singapore, Singapore, Singapore.
Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore, Singapore.
Drug Metab Dispos. 2025 Apr;53(4):100053. doi: 10.1016/j.dmd.2025.100053. Epub 2025 Mar 3.
Cardiac cytochrome P450 2J2 (CYP2J2) plays a significant role in cardiovascular homeostasis due to its dual functions in drug metabolism and the epoxidation of polyunsaturated fatty acids. Additionally, the inhibition of CYP2J2 by xenobiotics has been linked to drug-induced cardiotoxicity, warranting further investigation of this critical enzyme in cardiac systems. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) are physiologically relevant in vitro models that recapitulate relevant phenotypes important for cardiovascular research. However, no studies have so far characterized CYP2J2 expression and activities in these models. Here, we developed and validated H7 hESC-CMs as suitable in vitro models for investigating CYP2J2 in drug metabolism and cardiotoxicity. We first performed the genotyping and confirmed the presence of wild-type CYP2J2∗1/∗1 alleles in wild-type hESCs. Our optimized cardiomyocyte differentiation protocols yielded virtually pure (93.3% ± 6.8%) hESC-CMs, which exhibited P450 epoxygenase mRNA-expression profiles consistent with human cardiomyocytes, with CYP2J2 as the dominant isozyme and minor contributions from CYP2C8 and CYP2C9. By employing a CYP2J2-selective fluorescent substrate, ER-BnXPI, and astemizole as probe substrates, CYP2J2-mediated demethylation of both substrates exhibited typical Michaelis-Menten kinetics, which confirms functional CYP2J2 activities in vitro. Additionally, we demonstrated the capacity of CYP2J2 for arachidonic acid epoxidation, validating its ability to metabolize polyunsaturated fatty acid substrates. Finally, CYP2J2 activity in hESC-CMs was significantly inhibited by danazol and dronedarone, which are established CYP2J2 inhibitors known to cause cardiotoxicity. Ultimately, our study sheds novel insights on hESC-CMs as a suitable model for investigating CYP2J2-mediated metabolism and its inhibition in vitro. SIGNIFICANCE STATEMENT: H7 human embryonic stem cell-derived cardiomyocytes (hESC-CMs) were developed and validated as an in vitro model for investigating CYP2J2-mediated drug metabolism and its inhibition. By characterizing CYP2J2 transcriptional expression, catalytic activity, and inhibition response to established CYP2J2 inhibitors, our study confirmed functional CYP2J2 in hESC-CMs and ascertained that the model recapitulates the physiology of primary cardiomyocytes. This pioneering research highlights the potential of hESC-CMs in advancing our understanding of CYP2J2-mediated metabolism, its inhibition, and implications in drug-induced cardiotoxicity.
心脏细胞色素P450 2J2（CYP2J2）在心血管稳态中发挥着重要作用，因为它在药物代谢和多不饱和脂肪酸环氧化中具有双重功能。此外，异源生物对CYP2J2的抑制作用与药物诱导的心脏毒性有关，因此有必要进一步研究心脏系统中的这种关键酶。人胚胎干细胞衍生的心肌细胞（hESC-CMs）是体外生理相关模型，可概括对心血管研究重要的相关表型。然而，迄今为止尚无研究对这些模型中CYP2J2的表达和活性进行表征。在此，我们开发并验证了H7 hESC-CMs作为研究CYP2J2在药物代谢和心脏毒性中的合适体外模型。我们首先进行了基因分型，并确认野生型hESCs中存在野生型CYP2J2∗1/∗1等位基因。我们优化的心肌细胞分化方案产生了几乎纯的（93.3%±6.8%）hESC-CMs，其显示出与人心肌细胞一致的P450环氧化酶mRNA表达谱，以CYP2J2为主导同工酶，CYP2C8和CYP2C9的贡献较小。通过使用CYP2J2选择性荧光底物ER-BnXPI和阿司咪唑作为探针底物，两种底物的CYP2J2介导的去甲基化均表现出典型的米氏动力学，这证实了体外功能性CYP2J2活性。此外，我们证明了CYP对花生四烯酸环氧化的能力，验证了其代谢多不饱和脂肪酸底物的能力。最后，达那唑和决奈达隆显著抑制了hESC-CMs中的CYP2J2活性，这两种药物是已知会导致心脏毒性的CYP2J2抑制剂。最终，我们的研究为hESC-CMs作为研究CYP2J2介导的代谢及其体外抑制作用的合适模型提供了新的见解。意义声明：H7人胚胎干细胞衍生的心肌细胞（hESC-CMs）被开发并验证为研究CYP2J2介导的药物代谢及其抑制作用的体外模型。通过表征CYP2J2的转录表达、催化活性以及对已确定的CYP2J2抑制剂的抑制反应，我们的研究证实了hESC-CMs中功能性CYP2J2的存在，并确定该模型概括了原代心肌细胞的生理学特征。这项开创性研究突出了hESC-CMs在推进我们对CYP2J2介导的代谢、其抑制作用以及在药物诱导的心脏毒性中的意义的理解方面的潜力。
