KR102590512B1 - Dual-Cardiotoxicity Evaluation Method Based on human iPSC-derived cardiomyocytes and Multielectrode Assay - Google Patents

Abstract

The present invention relates to a dual-cardiotoxicity evaluation method including electrophysiological signal measurement and contractile force measurement based on multi-electrode analysis (MEA) using human induced pluripotent stem cell (iPSC)-derived cardiomyocytes.
The present invention detects electrophysiological changes due to drug treatment, etc. by analyzing changes in delayed field potential period (FPD), beat cycle, and amplitude, and measures contractile force based on impedance to confirm changes in body function, thereby detecting the two above. It was experimentally confirmed that the effect of drugs on the heart in vitro can be accurately and comprehensively analyzed through duplication of measurements. Therefore, the present invention can provide a competitive cardiotoxicity screening platform for candidate drug compounds.

Description

Dual-Cardiotoxicity Evaluation Method Based on human iPSC-derived cardiomyocytes and Multielectrode Assay}

The present invention relates to a dual-cardiotoxicity evaluation method including electrophysiological signal measurement and contractile force measurement based on multi-electrode analysis (MEA) using human induced pluripotent stem cell (iPSC)-derived cardiomyocytes.

The process of identifying adverse drug reactions (ADRs) is essential for new drug development, and many potential drug candidates fail due to side effects. Cardiac toxicity, which includes symptoms such as ventricular repolarization disorders, QT interval prolongation, arrhythmias, bradycardia, and tachycardia, is one of the major causes of drug withdrawal in the market. These symptoms are not limited to cardiovascular drugs and require cardiotoxicity safety testing for a wide range of drugs. In fact, more than 2,000 post-marketing drugs, including anticancer drugs and antibiotics, were found to have cardiac ADRs, and of the 462 drugs withdrawn from the market between 1953 and 2013, 14% caused serious cardiovascular side effects. In particular, not only can it have an acute effect with an early onset immediately after drug administration, but in the case of cardiotoxicity, a chronic effect with a delayed onset can occur. Therefore, even after preclinical screening, toxicities can be identified in Phase 2 (shortened follow-up period) and Phase 3 (extended follow-up period) trials or after market launch. Therefore, assessing potential cardiotoxicity is an important parameter in the pharmaceutical market and various prediction models have been proposed to prevent enormous socioeconomic costs.

In this context, the International Conference for Harmonization (ICH) presented Guidelines S7B and E14 in 2005 for non-clinical and clinical trials to assess cardiovascular risk for new drugs. ICH S7B, a guideline for the evaluation of nonclinical cardiotoxicity, recommends ventricular repolarization (QT interval prolongation) in cell lines transfected with human Ether-a-go-go-Related Gene (hERG) or cardiomyocytes (CM) isolated from animals or humans. It is based on The clinical trial guideline ICH E1T analyzes thorough QT/QTc studies of subjects to evaluate the proarrhythmic effects of non-antiarrhythmic drugs. By applying the above two guidelines and conducting preliminary screening, the number of drugs withdrawn from the market due to risk of ventricular arrhythmia was significantly reduced. However, a) hERG is not the only ion channel that controls ventricular repolarization, and b) prolongation of the QT interval alone is insufficient to be used as an indicator of clinical arrhythmia risk. Therefore, the narrow evaluation criteria resulted in false positives and led to the discontinuation of drugs with potential for treating arrhythmias. Therefore, the development of improved guidelines is needed to avoid these unintended consequences.

To overcome the limitations of the ICH S7B/E14 approach with high sensitivity and low specificity, the FDA launched the Comprehensive in vitro Proarrhythmia Assay (CiPA) project in 2013. The CiPA project will a) analyze drug effects on seven ion channels that complexly regulate ventricular repolarization, b) integrate information about the seven ion channels to build an in silico human ventricular tissue model, and c) electrical activity. and focused on arrhythmia risk assessment of drugs in an integrated biological system using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). In particular, hiPSC-CM has the advantage of being able to confirm changes in action potentials and repolarization regardless of mechanisms that directly affect ion channels or mechanisms that are difficult to predict from in silico reconstruction models. However, since hiPSC-CMs contain a mixed phenotype of cardiac cells (atria, nodules, and ventricles) and have an immature phenotype compared to adult ventricular cardiac myocytes, this is a limitation that needs to be overcome. In addition, the current platform, the Multielectrode Assay (MEA) measurement method or voltage-sensing optical platform, is limited to measuring electrical signals such as field potential (FP) and action potential of cardiomyocytes to detect arrhythmogenic cardiotoxicity. Therefore, measures of non-proarrhythmic cardiotoxicity, such as loss of contractility, should be added to summarize the actual cardiac response to the drug.

Non-patent Document 1: Bang, J. S. et al. Optimization of episomal reprogramming for generation of human induced pluripotent stem cells from fibroblasts. Animal cells and systems 22, 132-139, doi:10.1080/19768354.2018.1451367(2018). Non-patent Document 2: Ando, H. et al. A new paradigm for drug-induced torsadogenic risk assessment using human iPS cellderived cardiomyocytes. Journal of pharmacological and toxicological methods 84, 111-127, doi:10.1016/j.vascn.2016.12.003(2017).

Accordingly, the present inventors sought to construct, evaluate, and verify a modified human stem cell-based in vitro arrhythmia analysis system that can analyze changes in electrophysiological and physical functions according to CiPA, an internationally popular cardiotoxicity evaluation for new drug development. did.

Therefore, the purpose of the present invention is to provide a dual-cardiotoxicity evaluation method including electrophysiological signal measurement and contractile force measurement based on multi-electrode analysis (MEA) using human induced pluripotent stem cell (iPSC)-derived cardiomyocytes. .

The present invention includes the steps of i) measuring electrophysiological signals after treating myocardial cells with a test substance using a multi-electrode system; and

ii) measuring the contractile force using multi-electrode analysis after measuring the electrophysiological signal;

Provides a dual-cardiotoxicity evaluation method including.

According to a preferred embodiment of the present invention, the cardiomyocytes of step i) may be cardiomyocytes differentiated from hi-PSCs derived from human foreskin fibroblasts.

According to a preferred embodiment of the present invention, the cardiomyocytes of step i) are,

a) reprogramming human foreskin fibroblasts into hiPSCs;

b) Differentiating the hiPSCs into cardiomyocytes for 10 to 14 days;

c) purifying the differentiated cardiomyocytes for 4 to 8 days; and

d) maturing the purified cardiomyocytes for 10 to 14 days;

It may be manufactured including.

According to a preferred embodiment of the present invention, the cardiomyocytes of step i) may be highly purified, mature cardiomyocytes.

According to a preferred embodiment of the present invention, the electrophysiological signal measurement in step i) is selected from the group consisting of field potential duration (FPD), corrected field potential duration (FPDc), beat period, and spike amplitude. It may be measuring one or more of the following.

According to a preferred embodiment of the present invention, the electrophysiological signal measurement in step i) may be measured for 1 to 10 minutes 20 to 30 minutes after processing the test material.

According to a preferred embodiment of the present invention, the contractile force measurement in step ii) may be to measure the beat amplitude.

According to a preferred embodiment of the present invention, the contractile force in step ii) may be measured for 1 to 10 minutes after measuring the electrophysiological signal.

According to a preferred embodiment of the present invention, the electrophysiological signal measured in step i); and the contractile force measured in step ii); If all of these differ by more than 5% to 15% compared to the normal control group, it may be judged that the test substance exhibits cardiotoxicity.

According to a preferred embodiment of the present invention, the cardiotoxicity may be drug-induced cardiotoxicity.

According to a preferred embodiment of the present invention, the cardiotoxicity may mean arrhythmia.

The present invention detects electrophysiological changes due to drug treatment by analyzing changes in delayed field potential period (FPD), beat cycle, and amplitude, and measures contractility based on impedance to confirm changes in body function, thereby measuring two things. It was experimentally confirmed that the effect of drugs on the heart can be accurately and comprehensively analyzed in vitro through duplication of . Therefore, the present invention can provide a competitive cardiotoxicity screening platform for candidate drug compounds.

Figure 1 presents characterization of hiPSC-CMs and definition of key parameters for MEA-based FP activity and contractility. (A) Protocol for differentiating and purifying hiPSCs into CMs; (B) Cardiomyocyte-specific protein expression stained with α-actinin (green, FITC), cTnT (red, TRITC), and DAPI (blue); (C) FACS analysis of cTnT + cells in CM; represents.
Figure 2 presents characterization of hiPSC-CMs and definition of key parameters for MEA-based FP activity and contractility. (D) Experimental plan for recording before (baseline) and after drug treatment in MEA; (E) Morphology of 50,000 hiPSC-CMs seeded in 24-well MEA plates; represents.
Figure 3 presents characterization of hiPSC-CMs and definition of key parameters for MEA-based FP activity and contractility. (F) Four key parameters of FP activity: beat period, depolarizing spike amplitude, field potential period (FPD), and rate-corrected field potential period (FPDc); (G) Parameters of contractile beat amplitude; represents.
Figure 4 presents changes in four key parameters of FP activity during clinical torsadogenic drug treatment. (A) Nifedipine (0.3 μM); (B) Quinidine (1 μM). From the right, % change in FPD, FPDc, beat period and spike amplitude are shown, quantified across replicates across evaluation sites (N = 3 replicates).
Figure 5 presents changes in four key parameters of FP activity during clinical torsadogenic drug treatment. (C) E-4031 (0.03 μM); (D) Dofetilide (0.003 μM). From the right, % change in FPD, FPDc, beat period and spike amplitude are shown, quantified across replicates across evaluation sites (N = 3 replicates).
Figure 6 presents changes in four key parameters of FP activity during clinical torsadogenic drug treatment. (E) Terfenadine (0.1 μM); (F) Mexiletine (3 μM). From the right, % change in FPD, FPDc, beat period and spike amplitude are shown, quantified across replicates across evaluation sites (N = 3 replicates).
Figure 7 shows changes in beat amplitude, a key parameter of contractility, during clinical torsadogenic drug treatment. The left shows contractile force waveforms before (black) and after (red) administration, and the right shows the % change in beat amplitude. (A) Nifedipine (0.1 μM); (B) Quinidine (1 μM). The % change in beat amplitude was quantified across replicates across evaluation sites (N = 3 replicates).
Figure 8 shows changes in beat amplitude, a key parameter of contractility, during clinical torsadogenic drug treatment. The left shows contractile force waveforms before (black) and after (red) administration, and the right shows the % change in beat amplitude. (C) E-4031 (0.1 μM), (D) Dofetilide (0.01 μM). The % change in beat amplitude was quantified across replicates across evaluation sites (N = 3 replicates).
Figure 9 shows changes in beat amplitude, a key parameter of contractility, during clinical torsadogenic drug treatment. The left shows contractile force waveforms before (black) and after (red) administration, and the right shows the % change in beat amplitude. (E) Terfenadine (0.3 μM); (F) Mexiletine (3 μM). The % change in beat amplitude was quantified across replicates across evaluation sites (N = 3 replicates).
Figure 10 shows the results of monitoring changes in FP activity and contractile force according to Nifedipine administration treatment in hiPSC-CM. A merged image (right) showing the FP activity waveform (left, black when merged) and contractility (middle, red when merged) could be seen.

Hereinafter, the present invention will be described in more detail.

In the present invention, we developed a cardiotoxicity evaluation platform by applying our own high-purity human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) to multielectrode analysis (MEA) and confirmed its applicability in cardiotoxic drug evaluation through clinical torsadogenic drug treatment. . Due to species differences and local channel analysis, the existing cardiotoxicity assessment methods, Langendorff and hERG channel analysis, have limitations in the overall assessment of cardiac dysfunction. Additionally, since evaluation of cardiac contractility is an important indicator of cardiac toxicity, the present invention can provide a standard platform for evaluating cardiotoxicity by stimulation-contraction coupling based on multiple probe impedances.

To perform the cardiotoxicity assessment proposed by CiPA, it is important to produce hiPSC-derived highly functional and highly purified CM. Therefore, we produced a cell source that can be used in a cardiotoxicity assessment platform through a small molecule-based differentiation protocol and metabolic selection (Figure 1). It is difficult to mimic cardiac function in vivo solely through the expression of CM-specific markers cTnT and α-actinin and the formation of cardiac-specific sarcomeric structures, and maturation must be achieved. Therefore, it is essential to observe ionic currents and electrical conduction, which are key features of functional cardiac cells. Accordingly, the present inventors seeded hiPSC-CMs cultured for up to 30 days on MEA plates and underwent a stabilization process, and confirmed that field potential (FP) was well observed in mature cardiomyocytes (Figure 3 G). We then compared and evaluated electrophysiological changes in the platforms by treating them with previously known ion channel blockers.

In addition, we sought to more accurately evaluate the drug response of CM by measuring contractile force based on impedance along with changes in FP. Drug-induced electrocardiographic changes currently routinely used to assess cardiac toxicity do not reflect changes in body function or structural abnormalities due to morphological damage. Therefore, for a more accurate assessment of cardiotoxicity, contractility must be additionally evaluated to detect changes other than arrhythmia. Traditionally, optical image analysis methods, such as quantifying contraction velocity and sarcomere shortening of adult CMs, utilizing computational platforms to analyze microscopy videos of hiPSC-CMs, and tracking the translocation of intracellular Ca2+ using fluorescent dyes. This was done. Furthermore, MEA-based impedance measurements have improved throughput compared to optical analysis. Sensitivity, predictability, and specificity are similar, and structural cardiac toxicity and arrhythmia risk can be measured on the same platform. Therefore, it has the advantage of providing mechanistic insight into the cardiotoxicity of specific drugs.

We treated our platform with nifedipine, a calcium channel blocker, and found that FPD, FPDc, and beat duration decreased in a concentration-dependent manner (Figure 4 A) and beat amplitude, indicative of contractility, decreased (Figure 7 A); Additionally, it was confirmed that these two results were correlated (Figure 10). Nifedipine is an L-type calcium channel blocker used to treat patients with high blood pressure and heart failure. However, high doses are known to increase the risk of out-of-hospital cardiac arrest because they shorten action potentials and cause cardiac arrhythmias. In a previous study, exposure to nifedipine decreased Ca2+ wave amplitude and FPDc in hiPSC-CMs and reduced contractility in guinea pig hearts. That is, the cardiotoxicity evaluation platform of the present invention can evaluate changes in electrophysiology and contractility related to Ca2+ channels.

In contrast, when quinidine, E-4031, dofetilide, mexiletine, and terfenadine were administered, FPD and FPDc increased, spike amplitude decreased, and there was a significant change in beat amplitude. There was none (Figures 4 to 6). These drugs, except terfenadine (an antihistamine), are used as antiarrhythmic drugs and are known to inhibit hERG channels or multiple channels. When a heart cell is depolarized, K+ inside the cell moves out through the hERG channel and repolarizes the membrane potential. Dysfunction of hERG channels inhibits leakage of K+ ions and alters the transmembrane potential, leading to an increase in the action potential interval, a prolongation of the FPDc, and a decrease in spike amplitude. In terms of contractility, a previous study reported that hERG blocker could increase Ca2+ transients and change contractility in rat muscle cells in hiPSC-CMs, but contractility was not significantly affected even though activities such as delayed FPD and premature depolarization occurred. reported that it did not. Additionally, when hiPSC-CMs were treated with pentamidine, another hERG channel blocker, there was no significant change in FPD or beat amplitude in the short term. Taking the above results together, including the results for nifedipine, our data are consistent with the mechanisms by which each drug acts on ion channels and contractility.

In conclusion, we developed a hiPSC-CM/MEA platform that can screen for cardiotoxicity of new drug candidates through electrophysiology and impedance-based contractility, and evaluated it using six reference drugs with confirmed cardiotoxicity mechanisms. , it was confirmed that stimulation-contraction coupling can be comprehensively analyzed simultaneously. Using the present invention, it is possible to 1) identify the effects of drugs on cardiac structure or cytotoxic damage that may be missed through existing electrophysiological analysis, and 2) identify arrhythmia mechanisms that may be caused by complex ion channels. .

Therefore, the present invention includes the steps of i) measuring electrophysiological signals after treating cardiomyocytes with a test substance using a multi-electrode system; and

ii) measuring the contractile force using multi-electrode analysis after measuring the electrophysiological signal;

A dual-cardiotoxicity evaluation method including: can be provided.

According to a preferred embodiment of the present invention, the cardiomyocytes of step i) may be cardiomyocytes differentiated from hi-PSCs derived from human foreskin fibroblasts.

According to a preferred embodiment of the present invention, the cardiomyocytes of step i) are,

a) reprogramming human foreskin fibroblasts into hiPSCs; b) Differentiating the hiPSCs into cardiomyocytes for 10 to 14 days; c) purifying the differentiated cardiomyocytes for 4 to 8 days; and d) maturing the purified cardiomyocytes for 10 to 14 days; It may be manufactured including.

Preferably, the cardiomyocytes of step i) are,

a) reprogramming human foreskin fibroblasts into hiPSCs; b) Differentiating the hiPSCs into cardiomyocytes for 11 to 13 days; c) purifying the differentiated cardiomyocytes for 5 to 7 days; and d) maturing the purified cardiomyocytes for 11 to 13 days; It may be manufactured including.

More preferably, the cardiomyocytes of step i) are,

a) reprogramming human foreskin fibroblasts into hiPSCs; b) Differentiating the hiPSCs into cardiomyocytes for 12 days; c) purifying the differentiated cardiomyocytes for 6 days; and d) maturing the purified cardiomyocytes for 12 days; It may be manufactured including.

According to a preferred embodiment of the present invention, the cardiomyocytes of step i) may be highly purified, mature cardiomyocytes. The high purity may mean that the expression level of the cardiomyocyte specific marker cTnT is 90% to 100% upon FACS analysis, and more preferably, it may mean that the expression level of cTnT is 94% to 100%. The matured cardiomyocytes may refer to cardiomyocytes in which the sarcomere structure is confirmed, cTnT (cardiac troponin T) protein expression is confirmed, and ionic current and electrical conduction are well observed (Example 1) . Alternatively, the matured cardiomyocytes may mean cardiomyocytes with a baseline beating rate of 20-90 beats/min (i.e., 0.3-1.5 Hz) and a baseline spike amplitude of 0.3 mV or more for MEA measurement.

According to a preferred embodiment of the present invention, 10,000 to 100,000 cardiomyocytes in step i) may be seeded on the MEA plate.

The test substance in step i) may be used without limitation as long as it is a substance that needs to be confirmed as to whether it causes toxicity to the heart, but is preferably a toxic substance or a drug candidate. Additionally, the test substance in step i) may be treated at a concentration of 0.0001 to 30 μM, and preferably may be treated at a concentration of 0.001 to 3 μM.

According to a preferred embodiment of the present invention, the electrophysiological signal measurement in step i) consists of field potential duration (FPD), corrected field potential duration (FPDc), beat period, and spike amplitude as FP indicators. It may be measuring one or more items selected from the group.

According to a preferred embodiment of the present invention, the electrophysiological signal measurement in step i) may be to measure the FP index of cardiomyocytes that changes after treatment with the test substance. That is, the measurement of the electrophysiological signal in step i) may be performed for 1 to 10 minutes 20 to 30 minutes after processing the test material. Preferably, the electrophysiological signal measurement in step i) may be measured for 3 to 7 minutes 23 to 27 minutes after processing the test material, and more preferably, after processing the test material. This may be measured for 5 minutes after 25 minutes.

According to a preferred embodiment of the present invention, the contractile force measurement in step ii) may be to measure the beat amplitude.

According to a preferred embodiment of the present invention, the contractile force of step iii) may be measured by measuring the changing beat amplitude after measuring the electrophysiological signal. That is, the contractile force measurement in step ii) may be measured for 1 to 10 minutes after measuring the electrophysiological signal. Preferably, the contractile force in step ii) may be measured for 3 to 7 minutes after measuring the electrophysiological signal, and more preferably for 5 minutes after measuring the electrophysiological signal. It may be.

According to a preferred embodiment of the present invention, the electrophysiological signal measured in step i); and the contractile force measured in step ii); If all of these differ by more than 5% to 15% compared to the normal control group, it may be judged that the test substance exhibits cardiotoxicity. More preferably, the electrophysiological signal measured in step i); and the contractile force measured in step ii); If all of these differ by more than 10% compared to the normal control group, it may be judged that the test substance exhibits cardiotoxicity.

The electrophysiological signal may be any one or more selected from the group consisting of field potential duration (FPD), corrected field potential duration (FPDc), beat period, and spike amplitude as an FP indicator, and is preferably field potential duration. It may be any one or more selected from the group consisting of period (FPD), corrected field potential duration (FPDc), and beat period. The contractile force may be a change in beat amplitude.

According to a preferred embodiment of the present invention, the cardiotoxicity may be drug-induced cardiotoxicity.

According to a preferred embodiment of the present invention, the cardiotoxicity may mean arrhythmia.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it is obvious to those skilled in the art that the scope of the present invention should not be construed as limited by these examples. All chemical reagents used in the present invention were purchased from Tocris and Sigma-Aldrich. 1000-fold chemical stock solutions were prepared in DMSO and stored at -20 °C, and serial dilutions (1000-fold) of chemicals were further prepared in DMSO immediately prior to compound addition. A 10-fold final dilution of chemicals was prepared as disposable culture medium. The final concentration of DMSO in treated wells was 0.1%.

Generation and characterization of hiPSC-CMs for cardiotoxicity testing

<1-1> hiPSC-CM generation

CiPA is a revised cardiovascular pharmacology test targeting hiPSC-CMs for risk assessment in a system similar to the human environment. CM was produced in-house according to the direction suggested by CiPA and applied to MEA. hiPSCs were chemically differentiated using a defined protocol to generate beating CMs, and highly purified CMs were obtained through metabolic selection using sodium lactate (Figure 1 A).

Specifically, human foreskin fibroblasts (BJ-fibroblasts, ATCC; VA, USA) were reprogrammed using a reprogramming episomal vector (Non-patent Document 1). Afterwards, hiPSCs were seeded on Matrigel (Corning Inc.; Corning, NY, USA) coated cell culture dishes (Eppendorf; Hamburg, Germany) for differentiation into the cardiac lineage. When seeding hiPSCs, 5 μM Y-27632 (Tocris Bioscience; Bristol, UK) was added to mTeSRTM1 (STEMCELL Technologies Inc; Vancouver, BC, Canada) medium, and the medium was replaced 24 h later. Thereafter, the medium was changed daily for 3-4 days until confluency reached 90%. On day 0, cells were treated with CHIR99021 (Tocris) 6 μM in RPMI 1640 (Gibco) + B27 supplement (B27(-); Gibco). After 48 h, the medium was replaced with RPMI1640 + B27(-) supplemented with 2 μM C59 (Selleckchem; Houston, TX, USA) for an additional 48 h. On day 4, the medium was replaced with RPMI1640 + B27 supplement (B27(+); Gibco) and refreshed every 2 days until contraction occurred. When shrinking cells appeared on days 8 to 10, cells were isolated using TrypeLE (Gibco) and subcultured on Matrigel-coated dishes. From days 12 to 17, hiPSC-CMs were metabolically selected by replacing the medium with glucose-free RPMI1640 + B27(+) containing sodium L-lactate (Simgma-Aldrich; St. Louis, MO, USA). Purified. After purification, the medium was replaced with RPMI1640 + B27(+) every 2 days until day 30 to allow hiPSC-CMs to mature.

<1-2> hiPSC-CM characteristic analysis

To confirm the characteristics of the generated CM, we attempted to perform immunofluorescence staining and FACS analysis using a CM-specific marker antibody.

Specifically, for flow cytometry (FACS), hiPSC-CMs were fixed in 4% paraformaldehyde solution (PFA) for 20 min and resuspended in FACS solution after washing. Then, fixed hiPSC-CMs were stained using 1:200 PE-conjugated human anti-Cardiac Troponin T antibody (cTnT; Thermo Fisher Scientific; Waltham, MA, USA) for 30 min at 4 °C. hiPSC-CMs were analyzed using FACSCalibur and Cell Quest software (BD Biosciences; San Jose, CA, USA).

For immunofluorescence staining, hiPSC-CMs were plated on Matrigel-coated dishes and cultured for 5 days. Cells were then fixed with 4% PFA for 20 min at 4 °C, permeabilized with 0.03% Triton X-100 for 10 min at room temperature, and incubated with 3% normal goat serum in 0.03% Triton Blocked at room temperature for 30 min. Then, cells were incubated with 1:200 cTnT (Abcam; Cambridge, MA, USA) and α-sarcomeric actinin (α-actinin; Sigma-Aldrich; St Louis, MO, USA) in 0.03% Triton X-100 for one day at 4°C. dyed with On a shaker, cells were washed three times for 10 min with 0.03% Triton Together, they were incubated in the dark at room temperature for 2 hours. Cells were washed three times before nuclei were stained with DAPI (Thermo Fisher Scientific). All images were analyzed using a fluorescence microscope Nikon TE2000-U (Nikon; Japan).

As a result of staining for α-actinin and cTnT, the sarcomere structure, a CM-specific structure, was confirmed (Figure 1 B). Additionally, FACS analysis confirmed that 94.51% cTnT was expressed (Figure 1 C). These results show that the produced CM is a suitable source for MEA-based cardiotoxicity testing.

Defining two MEA-based analysis methods using hiPSC-CMs

Based on reported references for cardiotoxicity testing using MEA and CiPA, hiPSC-CMs were seeded and then stabilized to record baseline and FP activity and contractility after drug administration (Figure 2 D). FP of CM is mainly measured using MEA, but the Maestro Edge version of MEA can evaluate not only FP but also contractile force.

Specifically, to seed hiPSC-CMs in a 24-well MEA plate, 50 μg/ml fibronectin (Sigma-Aldrich) was coated on the center of the well at the recording site containing the electrode for 1 hour. After removing fibronectin, 6 ul of medium droplets were injected into 50,000 cells and seeded on the MEA plate in the form of droplets, and 500 µl of medium was added 1 hour later. The medium was changed every 2 days until electrophysiological signals from CM appeared, and the experiment was performed after about 6 days. On the day of the experiment, MEA plates seeded with hiPSC-CMs were transferred directly from the incubator to the MEA device, Maestro Edge version (Axion BioSystems Inc; Atlanta, GA, USA). Environmental controls (37 °C and 5% CO2) were used to maintain temperature and pH. To minimize the error margin of the electrical signal, baseline recordings were made after 25 min on the MEA, with FP measured for 5 min and contractility measured for an additional 5 min. Drugs were freshly prepared at 10 times the final concentration, 10% of the medium volume was removed from each well and an equal volume of prepared drug was added. Dosing was performed while the plate remained in the MEA device to minimize mechanical and temperature perturbations. FP and contractility were recorded for 5 min every 25 min after drug injection in the same manner as baseline recordings. In the experiment, one dose was injected per well and changes in electrophysiological signals of CM were measured in more than 3 wells for each drug concentration. Raw data of FP and contraction values were recorded in AxIS software and then analyzed using Cardiac Analysis Tool and AxIS Metric Plotting Tool (Axion BioSystems Inc). All data were expressed as mean ± SD.

As a result, it was confirmed that 50,000 cells of hiPSC-CM were well seeded on the electrode of the MEA plate (Figure 2 E). The heart undergoes depolarization and repolarization according to the inflow and outflow of ions, and blood circulates through the pumping action of contraction and relaxation. Ventricular CM, the constituent cells of the heart, reflect the function of the heart. Therefore, the complex waveforms derived from hiPSC-CMs appearing on MEA reflect variations in baseline parameters (Figure 3). FP consists of four main parameters: (1) FPD, (2) FPD corrected (FPDc), (3) beat period, and (4) spike amplitude (Figure 3 F). FPD is defined as the interval between the depolarizing spike and the peak of the repolarizing function and closely resembles the QT interval in clinical ECG. FPD measurements were rate corrected using the Fridericia correction (FPDc), the most commonly used rate correction formula in in vitro cardiotoxicity testing. The percent change between drug treatment and baseline conditions was calculated for each well. Beat duration was defined as the interval between two consecutive depolarizing spikes, and spike amplitude was an indirect measure of the effect of the drug on the rise of propagating action potentials. The main parameter of contractility is the beat amplitude ( Figure 3 G). The beat amplitude of the waveform based on the impedance signal indicates the degree of contraction.

Analysis of FP activity following clinical torsadogenic drug exposure

MEA-based cardiovascular pharmacology tests in CiPA and JiCSA (Japan iPS Cardiac Safety Assessment) were evaluated using a variety of drugs with clinical torsadogenic information. These drugs include cardiovascular drugs (anti-arrhythmia, anti-angina and anti-hypertensive drugs), quality-of-life drugs (anti-allergy, digestive, urinary and anti-platelet drugs), psychotropic drugs and anti-disease drugs (antibiotics, anti-malarial and anti-cancer drugs). . Using torsade de pointes (TdP) risk drugs that affect ion channels in CMs, we identified changes in four key parameters of FP in hiPSC-CMs (Figures 4 to 6, Tables 1 and 2). The dosage of the drug was determined according to reference literature (Non-patent Document 2).

1) Nifedipine - Nifedipine is an L-type Ca2+ channel inhibitor that belongs to the TdP risk category of drugs with isolated TdP case reports in humans. Treatment of hiPSC-CMs with nifedipine resulted in a dose-dependent decrease in FPD, FPDc, and beat duration. However, spike amplitude did not change significantly with dose.

2) Quinidine - Quinidine is an antiarrhythmic/class-Ia compound, a multi-channel blocker for Na+ and hERG and belongs to the repolarization-prolonging antiarrhythmic drugs in the TdP risk category. When hiPSC-CMs were treated with quinidine, FPD and FPDc increased, and beat cycle slightly increased in a dose-dependent manner. Spike amplitude also decreased slightly dose-dependently.

3) E-4031 - E-4301 is an antiarrhythmic/class III compound and known as hERG channel blocker, it belongs to class III antiarrhythmic drugs not marketed in TdP risk category. Treatment with E-4031 increased FPD and FPDc in a dose-dependent manner, with FPD increasing to 36.10 ± 6.63% at 0.03 μM. At 0.003 μM and 0.01 μM the beating period was unchanged, but at 0.03 μM the beating period increased to 11.89 ± 6.23% compared to the control. Spike amplitude decreased significantly with increasing concentrations of E-4031.

4) Dofetilide - Dofetilide is an antiarrhythmic/class III known hERG channel blocker and belongs to the repolarizing-prolonging antiarrhythmic drugs in the TdP risk category. When hiPSC-CMs were treated with dofetilide, FPD and FPDc increased in a dose-dependent manner. However, the beat cycle did not change regardless of concentration. Spike amplitude was consistent at 0.0003 μM and 0.001 μM but decreased at 0.003 μM.

5) Terfenadine - Terfenadine is an anti-allergy/histamine H1-receptor antagonist that inhibits hERG channels. Terfenadine is a drug that has been withdrawn or discontinued from the market due to an unacceptable TdP risk in at least one major regulatory area for the condition being treated in the TdP risk category. A slight increase in FPD and FPDc was observed upon treatment with terfenadine and was not detectable at 1 μM due to a rapid decrease in spike amplitude. Administration of terfenadine did not affect the beat cycle.

6) Mexiletine - Mexiletine is an antiarrhythmic/class Ib drug known to block the action of late Na+ channels and hERG channels, and it belongs to a group of drugs with isolated reports of TdP in humans in the TdP risk category. . Treatment of hiPSC-CMs with mexiletine increased FPD, FPDc, and beat duration in a dose-dependent manner. At a concentration of 30 μM, these parameters were not detectable due to a sharp decrease in spike amplitude.

drug Primary Ion current blocked TdP risk categories
(Redfern/Mirams) Experimental concentration (μM) One 2 3 4 Nifedipine Ca2 ⁺ 4 0.01 0.03 0.1 0.3 Qunidine Na ⁺ , hERG One 0.1 0.3 One - E-4031 hERG * 0.003 0.01 0.03 0.1 Dofetilide hERG One 0.0003 0.001 0.003 0.01 Terfenadine Ca ²⁺ , hERG 2 0.03 0.1 0.3 One Mexiletine Na ⁺ , hERG 4 One 3 10 30

Table 1 above shows the reference drugs included in the clinical torsadogenic drugs (TdP risk categories: 1, repolarization-prolonging antiarrhythmic drugs; 2, drugs acceptable in at least one major regulatory area for the condition being treated). Drugs withdrawn or discontinued from the market due to unknown TdP risk; 3, Drugs with a measurable incidence of TdP in humans or for which there are numerous case reports in the published literature; 4, Drugs with isolated reports of TdP in humans; 5, Drugs with isolated reports of TdP in humans Drugs for which no TdP reports have been published; *, unmarketed class III antiarrhythmia drugs; -, undisclosed drug).

drug Concentration (μM) FPD change (%) FPDc change (%) Beat cycle change (%) Spike amplitude change (%) Nifedipine 0.01 3.63±4.85 9.18±6.10 -14.34±4.43 0.95±0.44 0.03 -4.14±9.88 3.76±11.31 -20.95±4.82 -5.09±5.43 0.1 -28.88±3.54 -17.36±6.43 -35.83±6.17 5.59±3.41 0.3 -58.41±3.64 -45.81±5.16 -54.55±4.89 23.95±14.12 Qunidine 0.1 7.85±12.05 8.53±14.18 -1.07±7.43 1.18±17.08 0.3 19.39±9.16 17.85±13.40 5.90±16.44 -13.74±16.75 One 32.12±37.44 26.33±38.41 16.28±17.49 -31.22±12.46 E-4031 0.003 11.86±2.22 13.56±2.70 -4.38±2.12 -9.42±20.76 0.01 18.27±8.69 18.63±8.83 -0.90±1.12 -31.63±33.36 0.03 36.10±6.63 31.20±8.82 11.89±6.23 -92.48±7.24 Dofetilide 0.0003 4.93±5.82 6.05±6.84 -3.00±2.72 1.88±6.94 0.001 9.36±0.96 9.65±1.23 -0.76±1.90 -2.94±4.60 0.003 30.79±7.34 29.73±7.51 2.49±1.47 -49.67±33.28 Terfenadine 0.03 3.21±5.65 6.47±5.29 -8.89±3.96 -4.51±4.46 0.1 10.32±4.20 12.84±3.69 -6.48±5.39 -8.06±4.41 0.3 12.94±10.01 12.57±8.94 0.99±6.17 -67.00±17.96 One Not detected Not detected Not detected -100±0.00 Mexiletine One 1.92±7.06 2.16±7.40 -0.60±3.90 3.85±12.18 10 45.74±1.03 36.27±2.55 22.53±9.48 -82.94±8.33 30 Not detected Not detected Not detected -100±0.00

[Table 2] above shows the effect of drug concentration on the four main parameters of field potential activity.

Contractile force analysis following clinical torsadogenic drug exposure

TdP drugs are known to induce changes in the QT interval by blocking specific ion channels, which are mainly observed as electrophysiological changes (e.g. action potentials or FP activity) in the CM. However, little information is available on changes in myocardial contractility when TdP drugs are applied. Therefore, to more accurately predict cardiotoxicity, changes in beat amplitude, a key parameter of contraction, were confirmed after recording FP (Figures 7 to 9, Table 3). When nifedipine, a calcium channel blocker, was administered, beat amplitude decreased with increasing concentration, and after treatment with 0.3 μM nifedipine, beat amplitude decreased by 62.01 ± 9.00% (Figure 7 A). However, there was no significant change in the beat amplitude when various drugs that were hERG and multi-channel blockers were administered (Figures 7 to 9).

Interestingly, the waveform of contractile force was found to reflect not only beat amplitude but also parameters of FP such as FPD and beat period. In the case of nifedipine, the FPD and beat cycle decreased with increasing concentration, and the interval seen in one contraction decreased during nifedipine treatment, as can be seen from the waveform of the contractile force (Figure 7 A). Additionally, in the case of quinidine, E-4031, and Dofetilide, FPD increased as the concentration increased, and the contraction interval during administration slightly increased compared to base, as can be seen from the contraction force waveform (Figure 7, Figure 8). Contractile force analysis based on impedance values reflected some of the key parameters of FP activity and demonstrated the existence of new patterns that could not be found by FP analysis alone.

drug Concentration (μM) Beat amplitude change (%) Nifedipine 0.03 -9.32±12.16 0.1 -31.12±13.77 0.3 -62.01±9.00 Qunidine 0.1 2.63±10.29 0.3 -8.40±14.23 One -7.39±14.48 E-4031 0.01 -0.30±15.51 0.03 -6.36±26.17 0.1 -8.90±29.61 Dofetilde 0.001 2.73±8.79 0.003 3.28±12.17 0.01 0.15±14.12 Terfenadine 0.1 10.08±6.70 0.3 8.54±9.54 One Not detected Mexiletine 3 10.08±6.70 10 8.54±9.54 30 Not detected

Evaluation of FP/inotropic coupling for nifedipine

The FP/contractile waveforms of the nifedipine group with the greatest changes in FP key parameters and beat amplitude were combined (Figure 10). Beat durations in the FP and contractile waveforms were consistent before treatment, but a decrease in beat duration in the FP/contraction coupled waveform occurred after nifedipine exposure. In addition, it was confirmed that FPD and beat cycle capacity independently decreased with a decrease in beat amplitude. Treatments above 0.3 μM showed little contractility and decreased beat amplitude through the FP/contractile coupled waveform, but did not reduce the spike amplitude of FP. Above all, as a result of analyzing two indicators of FP and contractility simultaneously, the dual evaluation method of the present invention can complement each other to increase the predictability of cardiovascular toxicity screening.

Claims (11)

i) measuring electrophysiological signals after treating a test substance to highly purified, matured cardiomyocytes differentiated from human foreskin fibroblast-derived hi-PSCs using a multi-electrode system; and
ii) measuring the contractile force for 3 to 7 minutes using multielectrode analysis after measuring the electrophysiological signal;
As a dual-cardiotoxicity evaluation method comprising,
The cardiomyocytes of step i) are,
a) reprogramming human foreskin fibroblasts into hiPSCs;
b) Differentiating the hiPSCs into cardiomyocytes for 10 to 14 days;
c) purifying the differentiated cardiomyocytes for 4 to 8 days; and
d) maturing the purified cardiomyocytes for 10 to 14 days;
Dual-cardiotoxicity evaluation method, characterized in that it was produced including.
delete delete delete The method of claim 1, wherein the electrophysiological signal measurement in step i) is any one or more selected from the group consisting of field potential duration (FPD), corrected field potential duration (FPDc), beat period, and spike amplitude. Dual-cardiotoxicity evaluation method characterized by measuring.
The dual-cardiotoxicity evaluation method according to claim 1, wherein the electrophysiological signal measurement in step i) is measured for 1 to 10 minutes 20 to 30 minutes after treating the test substance.
The method of claim 1, wherein the contractile force in step ii) is measured by measuring the beat amplitude.
delete The method of claim 1, comprising: an electrophysiological signal measured in step i); and the contractile force measured in step ii); A dual-cardiotoxicity evaluation method characterized in that the test substance is judged to be cardiotoxic if all of these differ by more than 5% to 15% compared to the normal control group.
The method of claim 1, wherein the cardiotoxicity is drug-induced cardiotoxicity.
The method of claim 1, wherein the cardiotoxicity refers to arrhythmia.