論文申請者氏名
論文審査担当者
主査：田中 光一
副査：木村 彰方
副査：野村 典正
李 敏
論 文 題 目
Overexpression of cardiac Kir2.1 channel corrects the response of human iPS-derived cardiomyocytes to QT-prolonging drugs
（論文内容の要旨）
＜ Abstract ＞
Drug-induced development of life threatening arrhythmias, Torsade de pointe, with QT prolongation is the side effect of a wide range of cardiac and non-cardiac medications. Currently, pre-clinical prediction of drug-induced proarrhythmia is performed with in vivo non-human animals, isolated non-human animal’s tissues, or non-cardiomyocytes heterologously overexpressing hERG channels.
Human induced pluripotent stem cell-derived cardiomyocytes (hiPS-cardiomyocytes) hold great promise to address cardiac safety issue. However, electrophysiological study revealed that hiPS-cardiomyocytes exhibited spontaneous beating activity, which represented relatively immature cardiomyocytes compared with adult human ventricular cardiomyocytes. Moreover, a QT prolonging drug E4031 depolarized maximum diastolic potential, degraded action potential configuration, and stopped action potential firing, resulting in failure to evaluate drug effects on cardiac repolarization processes. Pharmacological experiments suggested the low expression of the inward rectifier potassium channel Kir2.1 as the main cause of electrophysiologically immature phenotypes in hiPS-cardiomyocytes. Therefore, human KCNJ2 encoding Kir2.1 was introduced into hiPS-cardiomyocytes. QT prolonging drug did not affect maximum diastolic potential, and lengthened action potential duration and field potential duration in a dose dependent manner in KCNJ2 overexpressed cells. These results suggest that hiPS-cardiomyocytes overexpressing KCNJ2 would be a potential model for evaluation of drug-induced QT prolongation in human cardiomyocytes.
＜
Introduction
＞
Drug-induced cardiac arrhythmia characterized by QT prolongation, or Torsade de Pointe has been a major reason for withdrawal of developmental products at late stage clinical trials. In cardiac safety screening, great efforts are paid either to exclude hERG channel blockade using heterologous expressed single hERG channel referred as hERG assay or to exclude QT-prolonging effects in vivo non-human animals referred as QT-prolongation assay.
The progress in hiPS cell technology has made it possible to use human cardiomyocytes for cardiac safety screening.
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However, hiPS-cardiomyocytes resemble fetal like, immature cardiomyocytes in many aspects including circular shaped morphology, low expression of some major cardiac genes, spontaneous beating activity, slow upstroke velocity and depolarized maximum diastolic potential in action potentials.
Many studies provide evidences of immaturity of hiPS-cardiomyocytes compared with adult counterparts. Before adoption of hiPS-cardiomyocytes for cardiac drug testing, it is critical to understand how their immature phenotypes affect drug response. To our knowledge, there has been no systematical research focusing on this matter.
Responses of hiPS-cardiomyocytes to an established selective hERG blocker, E4031 were tested.
In single cell patch-clamp experiments, E4031 depolarized maximum diastolic potential, degraded action potential configuration, and stopped action potential firings; in multiple cellular cardiac sheet experiments, E4031 degraded field potential waveform and arrested sheet contraction. Therefore, using current immature hiPS-cardiomyocytes, it is difficult to evaluate drug effects on cardiac repolarization process.
To optimize hiPS-cardiomyocytes for cardiac safety testing, I sought to mimic electrical maturation by manipulating ion channel gene expression towards adult pattern. It is found that the lack of I
K1
is the major contributor to the immature electrophysiological characteristics of hiPS-cardiomyocytes.
Therefore, human KCNJ2 gene, encoding the inward rectifier K + channel, was introduced into hiPS-cardiomyocytes. Resultant cells acquired some properties similar to adult ventricular cardiomyocytes. Action potential configuration and field potential configuration were not affected by
E4031 and action potential duration and field potential duration were prolonged by QT prolonging drugs E4031 and chromanol 293B as in adult ventricular cardiomyocytes.
＜
Methods
＞ hiPS-CMs (201B7) were differentiated using the 3-dimontional embryoid body method. The purified hiPS-cardiomyocytes, named iCell-cardiomyocytes were purchased from iPS Academia Japan Inc.
Adenoviral infection was achieved by applying Ad-EGFP or Ad-KCNJ2-EGFP at 100 MOI for 24 h.
Spontaneous and paced field potentials were recorded from hiPS-CMs sheets with a MED64 multi-electrode array system.
Action potentials and membrane currents were recorded with the perforated configuration of the patch-clamp technique with an Axopatch 200B amplifier.
＜
Results
＞
Patch-clamp experiments revealed that hiPS-cardiomyocytes automatically fired action potentials with diverse configurations.
This expression pattern of I f
and I
K1 implies that either robust I f
or lack of I
K1
contributes to spontaneous contraction of hiPS-cardiomyocytes. When I f
current was blocked with 5 mM CsCl, spontaneous activity was not stopped and beating rate was not affected, indicating that I f current is not the main contributor to spontaneous activity. Thus, it is hypothesized that absence of functional I
K1
is the major contributor to spontaneous activity in hiPS-cardiomyocytes.
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Spontaneous activity was stopped by overexpression of KCNJ2. Therefore, the lack of I
K1
is the major contributor to spontaneous contraction of hiPS-cardiomyocytes.
A selective hERG channel blocker, E4031 was applied to GFP-expressed control cells.
Consequently, action potential configuration was degraded, and action potential duration was abbreviated rather than prolonged. In contrast, in iCell-cardiomyocytes with overexpressed KCNJ2, maximum diastolic potential was stabilized to a relatively hyperpolarized level and action potential duration was prolonged in a dose dependent manner.
＜
Discussion
＞
Recently, hiPS cells can be differentiated into cardiomyocytes indefinitely, and thus can provide us an access to a human cardiac model. However, one of the difficulties for their successful employment is their immature nature.
In this study, it was demonstrated that the lack of functional I
K1
current is the main mechanism for immature electrophysiological phenotypes of hiPS-cardiomyocytes. When hiPS-cardiomyocytes were exposed to E4031, action potential configuration was degraded resulting in failure to evaluate drug induced action potential prolongation.
By overexpressing Kir2.1, I generated electrophysiologically matured cardiomyocytes evidenced by trigger-induced excitability and ventricular like action potential configurations. The sensitivity and specificity of KCNJ2 overexpressed hiPS-cardiomyocytes were confirmed with an established hERG blocker, E4031. Action potential duration was prolonged in a dose dependent manner without affecting resting membrane potential.
＜
Conclusion
＞
These results suggest that overexpression of cardiac channel gene KCNJ2 restores responses of hiPS-cardiomyocytes to QT-prolonging drugs observed in mature cardiomyocytes.
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論文提出者氏名 李 敏 （甲 第 4829 号）
主査： 田中 光一
論文審査担当者 副査： 木村 彰方
副査： 野村 典正
論 文 題 目
Overexpression of cardiac Kir2.1 channel corrects the response of human iPS-derived cardiomyocytes to QT-prolonging drugs.
（論文審査の要旨）
新薬の開発を遅らせる大きな原因として、不整脈などの心循環器系の副作用がある。従来、不整脈・
心不全といった副作用を引き起こすリスクは、動物由来細胞や実験動物を用いて評価されてきた。し
かし、生命倫理や種差などの観点から、ヒト細胞を用いた副作用の正確な評価系の確立が期待されて
いる。不整脈のリスクを評価する系として、ヒト iPS 細胞由来の心筋細胞を用いる系が研究されてい
る。しかし、従来のヒト iPS 細胞由来心筋細胞は、心筋細胞への分化が不十分であり、ヒトで致死的
不整脈リスクが示されている化合物を評価することができない。申請者は、ヒト iPS 細胞由来心筋細
胞の電気生理学的特性を解析し、分化した心筋細胞に比べ、ヒト iPS 細胞由来心筋細胞では KCNJ2 チ
ャネルの発現が低下しいていることを見つけた。そこで、KCNJ2 チャネルをアデノウイルスベクターを
用い過剰発現させたヒト iPS 細胞由来心筋細胞を作製し、不整脈を起こす化合物のリスク評価系とし
て妥当かどうか検討した。KCNJ2 過剰発現ヒト iPS 細胞由来心筋細胞は、単一細胞でもシート状にした
場合でも、分化した心筋細胞と同じ特性を有し、既知の不整脈誘発化合物 (E4031 および 293B)を評価
することができた。
申請者の作製した KCNJ2 過剰発現ヒト iPS 細胞由来心筋細胞は、新しい医薬品候補化合部物の心臓
毒性評価系として期待される。