Contractility assessment using aligned human iPSC-derived cardiomyocytes.

INTRODUCTION: Cardiac safety assessment, such as lethal arrhythmias and contractility dysfunction, is critical during drug development. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been shown to be useful in predicting drug-induced proarrhythmic risk through international validation studies. Although cardiac contractility is another key function, fit-for-purpose hiPSC-CMs in evaluating drug-induced contractile dysfunction remain poorly understood. In this study, we investigated whether alignment of hiPSC-CMs on nanopatterned culture plates can assess drug-induced contractile changes more efficiently than non-aligned monolayer culture. METHODS: Aligned hiPSC-CMs were obtained by culturing on 96-well culture plates with a ridge-groove-ridge nanopattern on the bottom surface, while non-aligned hiPSC-CMs were cultured on regular 96-well plates. Next-generation sequencing and qPCR experiments were performed for gene expression analysis. Contractility of the hiPSC-CMs was assessed using an imaging-based motion analysis system. RESULTS: When cultured on nanopatterned plates, hiPSC-CMs exhibited an aligned morphology and enhanced expression of genes encoding proteins that regulate contractility, including myosin heavy chain, calcium channel, and ryanodine receptor. Compared to cultures on regular plates, the aligned hiPSC-CMs also showed both enhanced contraction and relaxation velocity. In addition, the aligned hiPSC-CMs showed a more physiological response to positive and negative inotropic agents, such as isoproterenol and verapamil. DISCUSSION: Taken together, the aligned hiPSC-CMs exhibited enhanced structural and functional properties, leading to an improved capacity for contractility assessment compared to the non-aligned cells. These findings suggest that the aligned hiPSC-CMs can be used to evaluate drug-induced cardiac contractile changes.

[Cardiotoxicity risk assessment of anti-cancer drugs and future perspectives].

Cardiotoxicity is a serious adverse effect of anti-cancer drugs. Anti-cancer drug-induced cardiotoxicity are arrhythmia, cardiac contractile dysfunction, coronary artery disease, and hypertension, which affect to the quality of life in patients with cancer. In particular, cardiac contractile dysfunction is a life-threatening symptom leading to heart failure, suggesting that it is very important to predict the risk of developing the contractile dysfunction by anti-cancer drugs. Recently, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can be used to assess the risk of drug-induced arrhythmias. This prompts us to evaluate other cardiotoxic effects such as contractility dysfunction and structural toxicity with hiPSC-CMs. Since anti-cancer drug-induced contractility dysfunction are considered to be induced by chronic exposure, we have developed a method to assess chronic contractility dysfunction by imaging analysis of hiPSC-CMs. BMS-986094, which failed in clinical trials due to the occurrence of heart failure, was used as a positive compound. We found that chronic exposure to BMS-986094 decreased the contraction and relaxation velocity in hiPSC-CMs. Doxorubicin was observed to decrease cytotoxicity and both contraction and relaxation velocities in hiPSC-CMs. We are currently further evaluating other anti-cancer drugs with different mode-of-actions using hiPSC-CMs and assess the predictivity and utility of contractile assessment using hiPSC-CMs by comparing with real-world data. Here, we introduce our novel method to assess the chronic contractility of hiPSC-CMs by imaging analysis and discuss the future perspectives for assessing the anti-cancer drug-induced cardiotoxicity.

Contractility assessment of human iPSC-derived cardiomyocytes by using a motion vector system and measuring cell impedance.

Predicting drug-induced cardiotoxicity during the non-clinical stage is important to avoid severe consequences in the clinical trials of new drugs. Human iPSC-derived cardiomyocytes (hiPSC-CMs) hold great promise for cardiac safety assessments in drug development. To date, multi-electrode array system (MEA) has been a widely used as a tool for the assessment of proarrhythmic risk with hiPSC-CMs. Recently, new methodologies have been proposed to assess in vitro contractility, such as the force and velocity of cell contraction, using hiPSC-CMs. Herein, we focused on an imaging-based motion vector system (MV) and an electric cell-substrate impedance sensing system (IMP). We compared the output signals of hiPSC-CMs from MV and IMP in detail and observed a clear correlation between the parameters. In addition, we assessed the effects of isoproterenol and verapamil on hiPSC-CM contraction and identified a correlation in the contractile change of parameters obtained with MV and IMP. These results suggest that both assay systems could be used to monitor hiPSC-CM contraction dynamics.

Proarrhythmia Risk Assessment Using Electro-Mechanical Window in Human iPS Cell-Derived Cardiomyocytes.

Evaluation of drug-induced cardiotoxicity is still challenging to avoid adverse effects, such as torsade de pointes (TdP), in non-clinical and clinical studies. Numerous studies have suggested that human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a useful platform for detecting drug-induced TdP risks. Comprehensive in vitro Proarrhythmia Assay (CiPA) validation study suggested that hiPSC-CMs can assess clinical TdP risk more accurately than the human ether-a-go-go-related assay and QT interval prolongation. However, there were still some outliers, such as bepridil, mexiletine, and ranolazine, among the CiPA 28 compounds in the CiPA international multi-site study using hiPSC-CMs. In this study, we assessed the effects of the positive compound dofetilide, the negative compound aspirin, and several CiPA compounds (bepridil, mexiletine, and ranolazine) on the electromechanical window (E-M window), which were evaluated using multi-electrode array assay and motion analysis, in hiPSC-CMs. Similar to previous in vivo studies, dofetilide, which has a high TdP risk, decreased the E-M window in hiPSC-CMs, whereas aspirin, which has a low TdP risk, had little effect. Bepridil, classified in the high TdP-risk group in CiPA, decreased the E-M window in hiPSC-CMs, whereas ranolazine and mexiletine, which are classified in the low TdP-risk group in CiPA, slightly decreased or had little effect on the E-M window of hiPSC-CMs. Thus, the E-M window in hiPSC-CMs can be used to classify drugs into high and low TdP risk.

Chronic cardiotoxicity assessment of BMS-986094, a guanosine nucleotide analogue, using human iPS cell-derived cardiomyocytes.

Predicting drug-induced side effects in the cardiovascular system is very important because it can lead to the discontinuation of new drugs/candidates or the withdrawal of marketed drugs. Although chronic assessment of cardiac contractility is an important issue in safety pharmacology, an in vitro evaluation system has not been fully developed. We previously developed an imaging-based contractility assay system to detect acute cardiotoxicity using human iPS cell-derived cardiomyocytes (hiPSC-CMs). To extend the system to chronic toxicity assessment, we examined the effects of the anti-hepatitis C virus (HCV) drug candidate BMS-986094, a guanosine nucleotide analogue, which was withdrawn from phase 2 clinical trials because of unexpected contractility toxicities. Additionally, we examined sofosbuvir, another nucleotide analogue inhibitor of HCV that has been approved as an anti-HCV drug. Motion imaging analysis revealed the difference in cardiotoxicity between the cardiotoxic BMS-986094 and the less toxic sofosbuvir in hiPSC-CMs, with a minimum of 4 days of treatment. In addition, we found that BMS-986094-induced contractility impairment was mediated by a decrease in calcium transient. These data suggest that chronic treatment improves the predictive power for the cardiotoxicity of anti-HCV drugs. Thus, hiPSC-CMs can be a useful tool to assess drug-induced chronic cardiotoxicity in non-clinical settings.

Assessment of Contractility in Human iPS Cell-Derived Cardiomyocytes Using Motion Vector Analysis.

Human-induced pluripotent stem cell (iPSC) technology paves the way for next-generation drug-safety assessment. In particular, human iPSC-derived cardiomyocytes, which exhibit electrical activity, are useful as a human cell model for assessing QT-interval prolongation and the risk of the lethal arrhythmia Torsade de Pointes (TdP). In addition to proarrhythmia assay, contractile behavior has received increased attention in drug development. In this study, we developed a novel high-throughput in vitro assay system using motion vectors to evaluate the contractile activity of iPSC-derived cardiomyocytes as a physiologically relevant human platform. The methods presented here highlight the use of commercially available iPSC-derived cardiomyocytes, iCell cardiomyocytes, for contractility evaluation recorded by the motion vector system.

Comprehensive Cardiotoxicity Assessment of COVID-19 Treatments Using Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Coronavirus disease 2019 (COVID-19) continues to spread across the globe, with numerous clinical trials underway seeking to develop and test effective COVID-19 therapies, including remdesivir. Several ongoing studies have reported hydroxychloroquine-induced cardiotoxicity, including development of torsade de pointes (TdP). Meanwhile, human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are expected to serve as a tool for assessing drug-induced cardiotoxicity, such as TdP and contraction impairment. However, the cardiotoxicity of COVID-19 treatments has not been fully assessed using hiPSC-CMs. In this study, we focused on drug repurposing with various modes of actions and examined the TdP risk associated with COVID-19 treatments using field potential using multi-electrode array system and motion analysis with hiPSC-CMs. Hydroxychloroquine induced early after depolarization, while remdesivir, favipiravir, camostat, and ivermectin had little effect on field potentials. We then analyzed electromechanical window, which is defined as the difference between field potential and contraction-relaxation durations. Hydroxychloroquine decreased electromechanical window of hiPSC-CMs in a concentration-dependent manner. In contrast, other drugs had little effect. Our data suggest that hydroxychloroquine has proarrhythmic risk and other drugs have low proarrhythmic risk. Thus, hiPSC-CMs represent a useful tool for assessing the comprehensive cardiotoxicity caused by COVID-19 treatments in nonclinical settings.

[Development of a new assessment for cardio-oncology and its international trend].

Cardiac safety assessments play a key role in drug development. New non-clinical cardiac safety risk assessments, such as the use of iPSC-derived cardiomyocytes, have been validated by several consortiums both in Japan and abroad. The emerging multidisciplinary field of cardio-oncology has been recognized more important. The success of new cancer therapies has improved life expectancy of cancer patients, hence more attention has been paid to cardiotoxicities associated with existing and new anti-cancer therapies, such as cardiomyocyte injury and heart failure, vascular injury and hypertension or thrombosis, which accelerated coronary artery disease. In addition to the well-studied proarrhythmia risk, some cardiotoxicities, such as contractility impairment, are expected to be evaluated by iPSC-derived cardiomyocytes. Here we developed a novel imaging-based in vitro contractility assay using iPSC-derived cardiomyocytes. In the review, we would like to discuss the current status and future perspectives in the assessment of cardiac contractile function by anti-cancer agents.

Cardiotoxicity Assessment of Drugs Using Human iPS Cell-Derived Cardiomyocytes: Toward Proarrhythmic Risk and Cardio-Oncology.

Growing evidence suggests that Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs) can be used as a new human cell-based platform to assess cardiac toxicity/safety during drug development. Cardiotoxicity assessment is highly challenging due to species differences and various toxicities, such as electrophysiological and contractile toxicities, which can result in proarrhythmia and heart failure. To explore proarrhythmic risk, the Multi-Electrode Array (MEA) platform is widely used to assess QT-interval prolongation and the proarrhythmic potential of drug candidates using hiPSC-CMs. Several consortiums, including the Comprehensive in vitro Proarrhythmia Assay (CiPA) and the Japanese iPS Cardiac Safety Assessment (JiCSA), have demonstrated the applicability of hiPSC-CMs/MEA for assessing the torsadogenic potential of drug candidates. Additionally, contractility is a key safety issue in drug development, and efforts have been undertaken to measure contractility by a variety of imaging-based methods using iPS-CMs. Therefore, hiPSC-CMs might represent a standard testing tool for evaluating the proarrhythmic and contractile potentials. This review provides new insights into the practical application of hiPSC-CMs in early or late-stage nonclinical testing during drug development.

Development of torsadogenic risk assessment using human induced pluripotent stem cell-derived cardiomyocytes: Japan iPS Cardiac Safety Assessment (JiCSA) update.

Cardiac safety assessment is challenging because a better understanding of torsadogenic mechanisms beyond hERG blockade and QT interval prolongation is necessary for patient safety. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide a new human cell-based platform to assess cardiac safety in non-clinical testing during drug development. The multi-electrode array (MEA) platform is a promising electrophysiological technology to assess QT interval prolongation and proarrhythmic potential of drug candidates using hiPSC-CMs. The Japan iPS Cardiac Safety Assessment (JiCSA) has established an MEA protocol to evaluate the applicability of hiPSC-CMs for assessing the torsadogenic potential of compounds and completed a large-scale validation study using 60 compounds. During our study, an international multi-site study of hiPSC-CMs was performed by the Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative using 28 compounds. We have comparatively analyzed our JiCSA datasets with those of CiPA using the CiPA logistical and ordinal linear regression model. Regardless of the protocol differences, the evaluation results of the 28 compounds were very similar and highly predictable for torsadogenic risks. Thus, an MEA-based approach using hiPSC-CMs would be a standard testing method to evaluate proarrhythmic potentials. This review paper would provide new insights into the hiPSC-CMs/MEA method required for its regulatory use.

International Multisite Study of Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Drug Proarrhythmic Potential Assessment.

To assess the utility of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as an in vitro proarrhythmia model, we evaluated the concentration dependence and sources of variability of electrophysiologic responses to 28 drugs linked to low, intermediate, and high torsades de pointes (TdP) risk categories using two commercial cell lines and standardized protocols in a blinded multisite study using multielectrode array or voltage-sensing optical approaches. Logistical and ordinal linear regression models were constructed using drug responses as predictors and TdP risk categories as outcomes. Three of seven predictors (drug-induced arrhythmia-like events and prolongation of repolarization at either maximum tested or maximal clinical exposures) categorized drugs with reasonable accuracy (area under the curve values of receiver operator curves ∼0.8). hiPSC-CM line, test site, and platform had minimal influence on drug categorization. These results demonstrate the utility of hiPSC-CMs to detect drug-induced proarrhythmic effects as part of the evolving Comprehensive In Vitro Proarrhythmia Assay paradigm.

Overexpression of KCNJ2 in induced pluripotent stem cell-derived cardiomyocytes for the assessment of QT-prolonging drugs.

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes hold great potentials to predict pro-arrhythmic risks in preclinical cardiac safety screening, although the hiPSC cardiomyocytes exhibit rather immature functional and structural characteristics, including spontaneous activity. Our physiological characterization and mathematical simulation showed that low expression of the inward-rectifier potassium (IK1) channel is a determinant of spontaneous activity. To understand impact of the low IK1 expression on the pharmacological properties, we tested if transduction of hiPSC-derived cardiomyocytes with KCNJ2, which encodes the IK1 channel, alters pharmacological response to cardiac repolarization processes. The transduction of KCNJ2 resulted in quiescent hiPSC-derived cardiomyocytes, which need pacing to elicit action potentials. Significant prolongation of paced action potential duration in KCNJ2-transduced hiPSC-derived cardiomyocytes was stably measured at 0.1 µM E-4031, although the same concentration of E-4031 ablated firing of non-treated hiPSC-derived cardiomyocytes. These results in single cells were confirmed by mathematical simulations. Using the hiPSC-derived cardiac sheets with KCNJ2-transduction, we also investigated effects of a range of drugs on field potential duration recorded at 1 Hz. The KCNJ2 overexpression in hiPSC-derived cardiomyocytes may contribute to evaluate a part of QT-prolonging drugs at toxicological concentrations with high accuracy.

A new paradigm for drug-induced torsadogenic risk assessment using human iPS cell-derived cardiomyocytes.

INTRODUCTION: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are anticipated to be a useful tool for conducting proarrhythmia risk assessments of drug candidates. However, a torsadogenic risk prediction paradigm using hiPSC-CMs has not yet been fully established. METHODS: Extracellular field potentials (FPs) were recorded from hiPSC-CMs using the multi-electrode array (MEA) system. The effects on FPs were evaluated with 60 drugs, including 57 with various clinical torsadogenic risks. Actual drug concentrations in medium were measured using the equilibrium dialysis method with a Rapid Equilibrium Dialysis device. Relative torsade de pointes (TdP) scores were determined for each drug according to the degree of FP duration prolongation and early afterdepolarization occurrence. The margins were calculated from the free concentration in medium and free effective therapeutic plasma concentration. Each drug's results were plotted on a two-dimensional map of relative TdP risk scores versus margins. RESULTS: Each drug was categorised as high, intermediate, or low risk based on its location within predefined areas of the two-dimensional map. We categorised 19 drugs as high risk; 18 as intermediate risk; and 17 as low risk. We examined the concordance between our categorisation of high and low risk drugs against the torsadogenic risk categorisation in CredibleMeds®. Our system demonstrated high concordance, as reflected in a sensitivity of 81%, specificity of 87%, and accuracy of 83%. DISCUSSION: These results indicate that our torsadogenic risk assessment is reliable and has a potential to replace the hERG assay for torsadogenic risk prediction, however, this system needs to be improved for the accurate of prediction of clinical TdP risk. Here, we propose a novel drug induced torsadogenic risk categorising system using hiPSC-CMs and the MEA system.

Electrophysiological Characteristics of Human iPSC-Derived Cardiomyocytes for the Assessment of Drug-Induced Proarrhythmic Potential.

The aims of this study were to (1) characterize basic electrophysiological elements of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) that correspond to clinical properties such as QT-RR relationship, (2) determine the applicability of QT correction and analysis methods, and (3) determine if and how these in-vitro parameters could be used in risk assessment for adverse drug-induced effects such as Torsades de pointes (TdP). Field potential recordings were obtained from commercially available hiPSC-CMs using multi-electrode array (MEA) platform with and without ion channel antagonists in the recording solution. Under control conditions, MEA-measured interspike interval and field potential duration (FPD) ranged widely from 1049 to 1635 ms and from 334 to 527 ms, respectively and provided positive linear regression coefficients similar to native QT-RR plots obtained from human electrocardiogram (ECG) analyses in the ongoing cardiovascular-based Framingham Heart Study. Similar to minimizing the effect of heart rate on the QT interval, Fridericia's and Bazett's corrections reduced the influence of beat rate on hiPSC-CM FPD. In the presence of E-4031 and cisapride, inhibitors of the rapid delayed rectifier potassium current, hiPSC-CMs showed reverse use-dependent FPD prolongation. Categorical analysis, which is usually applied to clinical QT studies, was applicable to hiPSC-CMs for evaluating torsadogenic risks with FPD and/or corrected FPD. Together, this results of this study links hiPSC-CM electrophysiological endpoints to native ECG endpoints, demonstrates the appropriateness of clinical analytical practices as applied to hiPSC-CMs, and suggests that hiPSC-CMs are a reliable models for assessing the arrhythmogenic potential of drug candidates in human.

Assessment of testing methods for drug-induced repolarization delay and arrhythmias in an iPS cell-derived cardiomyocyte sheet: multi-site validation study.

A prospective comparison study across 3 independent research laboratories of a pure IKr blocker E-4031 was conducted by using the same batch of human iPS cell-derived cardiomyocytes in order to verify the utility and reliability of our original standard protocol. Field potential waveforms were recorded with a multi-electrode array system to measure the inter-spike interval and field potential duration. The effects of E-4031 at concentrations of 1 to 100 nM were sequentially examined every 10 min. In each facility, E-4031 significantly prolonged the field potential duration corrected by Fridericia's formula and caused early afterdepolarizations occasionally resulting in triggered activities, whereas it tended to decrease the rate of spontaneous contraction. These results were qualitatively and quantitatively consistent with previous non-clinical in vitro and in vivo studies as well as clinical reports. There were inter-facility differences in some absolute values of the results, which were not observed when the values were normalized as percentage change. Information described in this paper may serve as a guide when predicting the drug-induced repolarization delay and arrhythmias with this new technology of stem cells.