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FULL PAPERToxicology

Study of time-dependent pharmacokinetics of mouse embryonic stem cell-derived cardiomyocytes for drug screeningZhen Qi1, Misato KaMoshida2, Miho TaMai2,3, Masamichi hirose4 and Yoh-ichi Tagawa1,2*1 School of Life Science and Technology, Tokyo Institute of Technology, 4259 B51, Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8501,

Japan2 Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 B51,

Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8501, Japan3 Faculty of Dental Medicine, Hokkaido University, Hokkaido, Kita 13-jo, Nishi 7-chome, Kita-ku, Sapporo 060-8586, Japan4 Department of Pathophysiology and Pharmacology, School of Pharmaceutical Sciences, Iwate Medical University, 1-1-1 Yahaba, Shiwa-gun, Iwate

028-3694, Japan

Abstract. Core clock proteins play a significant role in maintaining physiological functions, including the metabolism in organisms in a circadian pattern. Metabolism is critical for drug efficiency and pharmacokinetics, which depend on circadian rhythm in animals and humans. Although alternative results are expected in animal experiments, there are limited reports on the influence of circadian rhythm on drug metabolism in culture. We observed the circadian rhythm in mouse embryonic stem (ES) cell-derived cardiomyocytes, as well as in the animal, after forskolin stimulation. The clock-synchronized mouse ES cell-derived cardiomyocytes exhibited time-dependent drug responses. This synchronized circadian rhythm could be maintained for up to three days by forskolin stimulation after every 24 hr. The beating rates of mouse ES cell-derived cardiomyocytes followed a circadian pattern. In conclusion, we established a mouse ES cell-derived cardiomyocyte culture model that exhibited a circadian beating pattern and time-dependent drug responses for up to three days. This model would serve as a valuable tool for chronotherapeutic research in culture.Key words: embryonic stem cell, cardiomyocyte, circadian rhythm, drug screening, chronotherapy

highlights

1. Mouse ES cell-derived cardiomyocytes exhibited gene expression and drug responses similar to that of atrial cardiomyocytes in RT-PCR, beat counting, and electrocardiograph analyses.

2. Circadian rhythms of beating rate change and drug responses could be initiated into the mouse ES cell-derived cardiomyocytes through forskolin stimulation. The forskolin-induced circadian rhythm could be maintained every 24 hr for at least three days.

3. This culture model would be a useful tool for drug screening.

Introduction

Novel drug development is a cost- and time-intensive process, with an average development period of 8.5 years and an average cost of approximately US $897 million (in 2000 US dollars) [1]. Preclinical drug development involves drug screening in culture and in animal experiments. Owing to the inaccuracy of drug evaluation in culture, approximately 93% of drugs are not pursued after animal experiments [2]. Moreover, drugs that successfully enter the market are withdrawn as well [3]. Therefore, it is necessary

to establish a reliable culture model that is comparable to the animal system.

Most organisms follow a 24 hr day and night cycle. This cycle significantly affects physiological functions in animals [4]. Therefore, experimental animals are maintained under the fixed lighting cycle condition in a day. Each circadian cycle in the body is synchronized by the suprachiasmatic nucleus (SCN) located in the hypothalamus in mammals [5]. Light is a dominant factor that influences the circadian rhythm [6] and is detected by the retina and transmitted to SCN through the retino-hypothalamic tract [7]. SCN relays this signal to the entire body, ultimately inducing the synchronization of peripheral tissue and organ patterns [8]. A transcriptional-translational feedback loop is responsible for the circadian rhythm. The CLOCK/BMAL1 heterodi-

*Correspondence to: Tagawa, Y.: ytagawa@bio.titech.ac.jp (Supplementary material: refer to J-STAGE https://www.jstage.jst.go.jp/browse/trs/)

Received: Dec. 13, 2019; Accepted: Jan. 24, 2020

Translat Regulat Sci. 2(1): 7–13, 2020; doi: 10.33611/trs.2_7

©2020 Catalyst Unit

This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives (by-nc-nd) License. (CC-BY-NC-ND 4.0: https://creativecommons.org/licenses/by-nc-nd/4.0/)

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mer initiates the expression of clock-controlled genes [9], including Per1, Per2, Per3, Cry1, and Cry2; conversely, the products of the clock-controlled genes block the CLOCK/BMAL1 activity [10]. Although nearly all cell types follow the circadian rhythm at the unicellular level, they cannot sense light and exhibit random circadian rhythm as a whole in culture. Circadian rhythm is crucial for metabolism in animals and leads to time-dependent drug responses [11–13]. However, culture models with circadian rhythm have not been reported. It is necessary to establish a culture model with circadian rhythm to evaluate drug effects and toxicity in a setting comparable to physiological conditions.

The beating rate changes significantly during the day [14], leading to time-dependent drug responses [15]. A variety of cardiovascular diseases are caused by circadian rhythm disruption [16]. The preparation of primary cardiomyocytes is technique-dependent and time-intensive. For large-scale drug screening, a large number of mice are required for primary cardiomyocyte preparation. Considering issues associated with animal rights, a replacement method is necessary. Ascorbic acid can enhance mouse ES cell dif-ferentiation into cardiomyocytes by increasing collagen synthesis [17]. Cardiomyocytes could be differentiated from mouse ES cells through embryoid body (EB) formation as well. These cardiomyocytes exhibit considerable adapt-ability after ectopic transplantation into the mouse heart [18]. Drug responses in these cardiomyocytes are yet to be evaluated. Primary cardiomyocytes cells would completely deviate from the circadian rhythm after several days in culture [19]. Primary cardiomyocytes without circadian rhythm could not be used for time-dependent drug effects and toxicity study in culture. We endeavored to establish a cardiac model with circadian rhythm in culture.

Materials and Methods

drugs for studying cardiomyocyte functionIsoproterenol (Sigma-Aldrich, Tokyo, Japan,), proprano-

lol hydrochloride (Enzo Life Sciences, Inc., Tokyo, Japan), (R)-(−)-phenylephrine hydrochloride (Sigma-Aldrich), and prazosin hydrochloride (Sigma-Aldrich) were diluted in the mouse ES cell differentiation medium at a final concentration of 10 µM. One millimolar acetylcholine was prepared by diluting acetylcholine chloride in the mouse ES cell differentiation medium at a final concentration of 1 µM. Atropine sulfate salt monohydrate (Sigma-Aldrich) was diluted in the ES cell differentiation medium at a final concentration of 100 µM. Forskolin (LC Laboratories, Wo-burn, MA, USA) contained in DMSO (Sigma-Aldrich) was added to the mouse ES cell differentiation medium at a final concentration of 10 µM. After 2 hr of forskolin stimulation, cells were washed with PBS, and the medium was changed to forskolin-free mouse ES cell differentiation medium. The bottom of cardiomyocyte culture dishes was marked for microscopic observation (Olympus, Tokyo, Japan). The beats were counted every minute. The action potential of mouse ES cell-derived cardiomyocytes was recorded us-ing a microelectrode array system MED 64 (Alpha MED Scientific Inc., Osaka, Japan).

Cells and animalsMouse ES cell line ST1 was established from the BALB/

cA mouse [20]. Mouse embryonic fibroblasts (MEFs) were collected from limbs, legs, and tail of 12.5-day-old BALB/cA mouse embryo. Total RNA from atrial and ventricular tissues were collected from 2-, 7-, and 28-day-old BALB/cA mice. Primary atrial cardiomyocytes were collected from the atrium of 28-day-old mice. The hearts were col-lected from these mice, and the atrium and ventricles were removed under microscopic observation. Both MEF cells and primary atrial cardiomyocytes were cultured with 10% Fetal Bovine Serum (FBS, Thermo Fisher Scientific, Tokyo, Japan) in Dulbecco’s modified Eagle’s medium (DMEM, high glucose, Thermo Fisher Scientific). The BALB/cA mice (CLEA Japan, Inc., Tokyo, Japan) were maintained in an air-conditioned room (22–24°C) under a 12 hr light cycle and allowed free access to food and water. All mice were maintained at the animal facility at the Tokyo Institute of Technology. All animal protocols were approved by the Animal Experimentation Committees of the Tokyo Institute of Technology.

Culture and differentiation of murine ES cellsThe mouse ES cells were cultured as following ways. The

mouse ES cells were cultured in a mitomycin C (Kyowa Hakko Kirin, Tokyo, Japan)-treated mouse embryonic fibroblast feeder layer and the medium (15% FBS (Thermo Fisher Scientific), DMEM, high glucose) with 1 mM sodium pyruvate (Thermo Fisher Scientific), 0.1 mM nonessential amino acid (Thermo Fisher Scientific), 2,000 U/ml mouse leukemia inhibitory factor (LIF, Chemicon International, Temecula, CA, USA), and 0.1 mM 2-mercaptoethanol (Sigma-Aldrich) was changed every day. The feeder-free mouse ES cells were cultured in a medium (PD0325901, CHIR99021, and LIF was added into ESGRO Complete Basal medium (Chemicon) at a final concentration of 100 Unit/ml.) that was changed every day. The mouse ES cells were passaged every 3–5 days.

For differentiation, mouse ES cells were dissociated us-ing trypsin and re-suspended in a differentiation medium (Iscove’s modified Dulbecco’s medium; Invitrogen, Tokyo, Japan) containing 15% FBS, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 0.1 mM 2-mercaptoethanol). The suspension was then used to create a hanging drop at a concentration of 200 cells/µl cells were cultured under the following conditions: 5% CO2, 37°C for five days [21]. Under these conditions, mouse ES cells could form EBs. EBs were then plated in gelatin coated dishes; the day of plating was denoted as day 0 (A0). The differentiation medium was changed every two days. After 3–5 days of culture, cardiomyocyte beating could be observed.

rNa extraction and reverse transcription-polymerase chain reaction analysis

The total RNA was extracted from mouse heart tissues using the Acid Guanidium Thiocyanate-Phenol Chloroform method [22]. Total RNA extraction from cultured cells was conducted following instructions provided in the Quick-Gene RNA cultured cell kit S RC-S (KURABO, Tokyo, Japan). Reverse transcription was conducted following

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instructions from Super Script II Reverse transcriptase kit (Thermo Fisher Scientific). PCR was performed using Ex Taq polymerase (Takara bio, Kusatsu, Japan). The LAS-4000 system (Fujifilm, Tokyo, Japan) and Multi Gauge program version 3.1 (Fujifilm) were used for PCR product analysis. All primers used in this study were purchased from Euro-fins Genomics (Supplementary Table 1).

real time PCrReal time PCR was performed using the Step One Plus

(Applied Biosystems, Tokyo, Japan) system; the Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) was used for detection. The ∆∆Ct method was used for relative quantification of the data during analyses.

statistical analysisData were compared using Student’s t-test and one-way

ANOVA. The error bar represented as mean ± SEM from three independent samples. Differences were considered significant for P<0.05 [*], P<0.01 [**], P<0.005 [***].

Results

Mouse es cell-derived cardiomyocytes and primary atrial cardiomyocytes exhibited similar pharmacological responses

Identifying the properties of mouse ES cell-derived car-diomyocytes is necessary. Total RNA was extracted from ES cells after three days of culturing and from EBs at 0, 2, 5, 10, and 12 days in culture. Total RNA was prepared from atrial and ventricular tissues collected from 2-, 7-, and 28-day-old mouse hearts. The expression of genes of mouse ES cell-derived cardiomyocytes and atrial and ventricular tissues from the mouse heart was compared. The mouse ES cell-derived cardiomyocytes at five days cultured EBs expressing Anp, Mlc-2α, β-Mhc, and α-Mhc. The marker- Mlc-2v for ventricle cardiomyocytes was not expressed in mouse ES cell-derived cardiomyocytes even after 12 days in culture (Fig. 1A). The gene expression verified that mouse ES cell-derived cardiomyocytes were similar to atrial cardiomyocytes. For evaluating mouse ES cell-derived and primary atrial cardiomyocytes, α/β-adrenaline, muscarine receptor agonists isoproterenol, phenylephrine, and acetyl-choline, and antagonists propranolol, prazosin, and atropine were added into the medium, respectively. Both mouse ES cell-derived and primary atrial cardiomyocytes exhibited increased beating rate after stimulation by isoproterenol and phenylephrine, and a decreased beating rate after stimulation by propranolol, prazosin, and atropine (Fig. 1B–D). Although the beating rate of mouse ES cell-derived cardiomyocytes decreased directly after acetylcholine stimulation, it returned to the baseline after 15 min of stimu-lation. The beating rate of primary atrial cardiomyocytes underwent no significant changes immediately after ace-tylcholine stimulation, while it increased significantly after 15 min of stimulation. The cardiomyocyte action potential for mouse ES cell-derived cardiomyocytes was recorded after stimulation by isoproterenol and propranolol. The beating rates increased visibly after isoproterenol stimula-tion and decreased after propranolol stimulation (Fig. 1E).

This indicated that mouse ES cell-derived and primary atrial cardiomyocytes exhibited similar responses against β-adrenaline stimulation.

Circadian rhythm-synchronized mouse es cell-derived cardiomyocytes exhibited significant time-dependent drug effects

For identifying circadian rhythm in the mouse heart, total RNA was extracted from BALB/cA mouse heart at zeitgeber time (ZT) 2 and ZT 14. The expression of clock gene Per1 was upregulated at ZT 14 compared to that at ZT 2, while Bmal1 expression was upregulated at ZT 2 compared to that at ZT 14 (Fig. 2A). The clock genes, Per1 and Bmal1, exhibited circadian and antiphase expression patterns. It remains unknown whether circadian rhythm could be synchronized for mouse ES cell-derived cardiomyocytes. Initiation of cir-cadian rhythm synchronization was induced using forskolin in mouse ES cell-derived cardiomyocytes. The clock gene Per1 expression increased directly after forskolin stimula-tion, stabilizing at approximately ZT 6. Conversely, Bmal1 expression peaked at ZT 12 and gradually decreased until ZT 24 (Fig. 2B). Based on the results, 24 hr oscillating and antiphase expression was observed for clock genes Per1 and Bmal1. For verifying time-dependent drug effects in mouse ES cell-derived cardiomyocytes, these cardiomyocytes were treated with β-adrenaline receptor agonist-isoproterenol for 1 hr at ZT 0 and ZT 18. The clock-synchronized mouse ES cell-derived cardiomyocytes had significantly higher beating rates after isoproterenol stimulation at ZT 18 than at ZT 0 (Fig. 2C). The time-dependent drug responses in circadian rhythm-synchronized mouse ES cell-derived cardiomyocytes were significant.

The mouse es cell-derived cardiomyocytes maintained circadian rhythm for a length of three days

The circadian rhythm was synchronized by light each day in animals, and it was maintained in culture as well. The mouse ES cell-derived cardiomyocytes were stimulated thrice by forskolin every 24 hr, and the total RNA was extracted. In these cardiomyocytes, the clock genes Per1 and Bmal1 exhibited oscillating and antiphase expression every 24 hr for three days (Fig. 3A, 3B). For confirming the circadian rhythm in the beating rate, the beating rate of these cardiomyocytes was evaluated. The beating rates increased directly after the first forskolin stimulation. For the second and third forskolin stimulation, the mouse ES cell-derived cardiomyocytes displayed decreasing beating rates immediately after forskolin stimulation. Thereafter, the beating rates changed in a circadian pattern every 24 hr for a length of three days (Fig. 3C). The circadian rhythm became a dominant factor that controlled beating rates after the second forskolin stimulation.

Discussion

Acetylcholine stimulation activates the potassium cur-rent, thereby hyperpolarizing cardiomyocyte resting action potential [23]. This explains why both primary atrial and mouse ES cell-derived cardiomyocytes did not respond to acetylcholine directly after stimulation and their beating rate

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Translational and Regulatory Sciences

Fig. 1. The mouse embryonic stem (ES) cell-derived cardiomyocytes undergo gene expression and drug responses similar to atrial cardiomyocytes. (A) The mouse ES cell-derived cardiomyocytes (the left graph) were compared with atrial and ventricular tissues from mice (the right graph; the number represents days after birth). The gene Anp has an atrial function, Mlc-2v has a ventricular function, and Mlc-2a is an atrial marker. β-Mhc and α-Mhc represent immature and mature cardiomyocyte markers, respectively. Hprt is a housekeeping gene. (B) The mouse ES cell-derived cardiomyocytes (the upper graph) and primary atrial cardiomyocytes (the lower graph) were administered both β-adrenaline receptor agonist-isoproterenol (dashed arrow) and antagonist-propranolol (solid arrow) at two different time points (n=3). (C) ES cell-derived cardiomyocytes (the upper graph) and to primary atrial cardiomyocytes (the lower graph) were administered the α-adrenaline receptor agonist-phenylephrine (dashed arrow) and antagonist-prazosin (solid arrow) (n=3). (D) Mouse ES cell-derived cardiomyocytes (the upper graph) and primary atrial cardiomyocytes (the lower graph) were administered the muscarine receptor agonist-acetylcholine (dashed arrow) and antagonist-atropine (solid arrow) (n=3). (E) The cardiomyocyte action potential indicated the response of mouse ES cell-derived cardiomyocytes to β-adrenaline receptor agonist-isoproterenol and antagonist-propranolol. Error bars represent mean ± SEM. *P<0.05, **P<0.01. EB, embryoid body; ns, not significant.

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increased between 5 and 15 min. The mouse ES cell-derived cardiomyocytes following circadian rhythm responded to isoproterenol (β-adrenergic agonist) in a time-dependent manner. This model could predict the time-dependent drug effects in culture. Drug toxicity depends on time as well. This model could contribute to drug screening as well as to time-dependent therapeutic study in culture by avoiding toxicity and the magnification of drugs.

Moreover, the circadian rhythm in this model could be maintained for at least three days by forskolin stimulation every 24 hr. The beating rate of mouse ES cell-derived cardiomyocytes increased after the first forskolin stimula-

tion, which may have been induced by the upregulated cAMP levels [24, 25]. Mice are nocturnal animals, and their heart rate would increase at night, which is antiphase to Per1 expression [26]. After the second and third forskolin stimulation, the beating rate decreased in the as well as in culture animal. Circadian rhythm, and not cAMP, becomes the predominant factor controlling the beating rate. This mechanism needs to be elucidated in future studies. The circadian rhythm was introduced in mouse ES cell-derived cardiomyocytes after the second forskolin stimulation. The circadian pattern was observed in gene expression as well as in the physiological beating of cardiomyocytes. It could be

Fig. 2. Mouse embryonic stem (ES) cell-derived cardiomyocytes followed synchronized circadian rhythm. (A) Core clock genes, Bmal1 (white column) and Per1 (black column) exhibited oscillating and antiphase expression pattern in mouse heart RNA samples (n=3). (B) The mouse ES cell-derived cardiomyocytes exhibited circadian rhythmic expression of clock genes such as Per1 (the upper graph) and Bmal1 (the lower graph) after forskolin stimulation (the dashed arrow) (n=3), ***P<0.005, determined by one-way ANOVA. The triangle connected with a dashed line and the point connected with a solid line represent the forskolin-stimulated and non-stimulated groups, respectively. (C) Clock-synchronized mouse ES cell-derived cardiomyocytes exhibited time-dependent drug effects (black column) compared to the unsynchronized control group (white column). The response of mouse ES cell-derived cardiomyocytes to isoproterenol was much more efficient at zeitgeber time (ZT) 18 than at ZT 0 (ZT 0 corresponds to the time point directly after forskolin stimulation); no significant difference was observed in unsynchronized groups (white column) (n=3). Error bars represent mean ±SEM. *P<0.05, **P<0.005. The “ns” represents not significant.

Fig. 3. Circadian rhythm in mouse embryonic stem (ES) cell-derived cardiomyocytes was maintained by forskolin stimulation after every 24 hr. (A) After forskolin stimulation every 24 hr (the dashed arrow), the expression of the clock gene Per1 exhibited a daily oscillating pattern that was maintained for three days (n=3). (B) The oscillating expression of the clock gene-Bmal1, which is antiphase to the clock gene-Per1, was maintained by forskolin stimulation induced every 24 hr (the dashed arrow) for three days (n=3). (C) The beating rate of mouse ES cell-derived cardiomyocytes exhibited a time-dependent oscillating pattern that was maintained by forskolin stimulation (the dashed arrow) for three days (n=3). Error bars represent mean ± SEM. The dashed arrow represents forskolin stimulation. *P<0.05, **P<0.01, ***P<0.005, one-way ANOVA. ZT, zeitgeber time.

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considered that this model could simulate circadian rhythm in animals. Since mouse ES cell-derived cardiomyocyte culture model with circadian rhythm was established suc-cessfully, a human iPS cell-based culture model following a circadian pattern in physiological functions would be a logical step for further studies on drug screening.

Conclusion

The mouse ES cell-derived cardiomyocytes have gene expression and drug responses similar to atrial cardio-myocytes. Circadian rhythm-synchronized mouse ES cell-derived cardiomyocytes exhibit significant time-dependent drug effects compared to unsynchronized cells. Circadian rhythm could be maintained in mouse ES cell-derived cardiomyocytes for least three days by forskolin stimulation every 24 hr. This culture model used the circadian rhythm to establish a novel method of chronotherapeutic research in culture.

FundingThis study was supported by a Grant-in-Aid for Scientific

Research (A) [No. 25242040], for Challenging Exploratory Research [No. 18K19905], for Early-Career Scientists [No. 19K20655] from the Japan Society for the Promotion of Science (JSPS) and a Grant-in-Aid for Scientific Research on Innovative Areas [No. 23119003] from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan and by Building of Consortia for the Development of Human Resources in Science and Technology, Ministry of Education, Culture, Sports, Science and Technology, Japan.

acknowledgementWe would like to thank Ishibe Keiko in Shimadzu

Techno-Research who purified mineral oil to high quality for the cardiomyocyte differentiation.

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