Human iPS Cell-derived Cardiomyocytes Research Articles

Low-adhesion culture selection for human iPS cell-derived cardiomyocytes

Despite progress in generating cardiomyocytes from pluripotent stem cells, these populations often include non-contractile cells, necessitating cardiomyocyte selection for experimental purpose. This study explores a novel cardiomyocyte enrichment mechanism: low-adhesion culture selection. The cardiac cells derived from human induced pluripotent stem cells were subjected to a coating-free low-adhesion culture using bovine serum albumin and high molecular weight dextran sulfate. This approach effectively increased the population of cardiac troponin T-positive cardiomyocytes. Similar results were obtained with commercially available low-adhesion culture dishes. Subsequently, we accessed the practicality of selection of cardiomyocytes using this phenomenon by comparing it with established methods such as glucose-free culture and selection based on puromycin resistance genes. The cardiomyocytes enriched through low-adhesion culture selection maintained autonomous pulsation and responsiveness to beta-stimuli. Moreover, no significant differences were observed in the expression of genes related to subtype commitment and maturation when compared to other selection methods. In conclusion, cardiomyocytes derived from pluripotent stem cells were more low-adhesion culture resistant than their accompanying non-contractile cells, and low-adhesion culture is an alternative method for selection of pluripotent stem cell-derived cardiomyocytes.

Evaluation of Contractile Function Using Human iPS Cell-derived Cardiomyocytes

Cardiotoxicity induced by anti-cancer drugs is a significant concern for patients undergoing cancer treatment. Some anti-cancer drugs can damage cardiac muscle cells directly or indirectly, potentially leading to severe heart failure. Various risk factors, including the type and dosage of chemotherapy agents as well as patient background, contribute to the development of cardiotoxicity. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), which enable patient-specific toxicity prediction, hold great promise in this regard. However, the practical implementation of hiPSC-CMs-based prediction of anti-cancer drug-induced cardiotoxicity still faces hurdles. One major challenge involves establishing and optimizing experimental systems for evaluating contractile dysfunction, the ultimate output of heart failure, using hiPSC-CMs. Such efforts are currently underway globally, focusing on tailoring functional evaluation systems to the characteristics of hiPSC-CMs. In this paper, we provide an overview of the contraction mechanisms of cardiac cells and introduce a method of measuring contraction that we have developed, and discuss the current status of contractile function evaluation methods using hiPSC-CMs.

Abstract 13757: Suppression of Ischemia-Hypoxia Reperfusion Injury in Human iPS Cell-Derived Cardiomyocytes Through Downregulation of EGR1 Mediated by miR-124-3p

Background: Ischemic heart diseases are one of the leading causes of death globally. Although timely revascularization is performed, some cases may still result in heart failure due to ischemia-hypoxia reperfusion (IH/R) injury. Early growth response 1 (EGR1) was reported to involved in reperfusion injury, but its specific role in IH/R-induced apoptosis in human cardiomyocytes (CMs) remains unexplored. Hypothesis: EGR1 is involved in the exacerbation of the IH/R-mediated apoptosis in human CMs, and miR-124-3p acts as an antagonist of EGR1. Methods: Human induced pluripotent stem cell (hiPSC)-derived CMs were cultured in serum-free low-glucose medium with a hypoxic chamber. EGR1 expression and the extent of apoptosis were evaluated by qRT-PCR, Western blotting (WB), and immunofluorescent assay (IFA). RNA-seq was conducted to identify specific pathways. SiRNA targeting EGR1 and miR-124-3p mimic were used to downregulate EGR1 under IH/R. Results: qRT-PCR (Control vs IH/R: 1.000 ± 0.000 vs 5.749 ± 2.947; P<0.01) and WB (0.037 ±0.038 vs 0.953 ± 0.306; P<0.0001) analyses revealed higher expression of EGR1 in IH/R group. IFA demonstrated a higher ratio of cleaved Caspase-3-positive apoptotic cells in IH/R group (0.202 ±0.206 vs 0.377 ± 0.252; P<0.001). RNA-seq analysis indicated significant downregulation of pathways related to cell survival in IH/R. The siRNA downregulated EGR1 successfully, as confirmed by qRT-PCR, WB, and IFA (IH/R Water vs IH/R siRNA: 0.612 ±0.307 vs 0.257 ±0.122; P<0.01), which led to the suppression of apoptosis (0.288 ±0.143 vs 0.142 ±0.049; P<0.05). miR-124-3p mimic downregulated EGR1 under IH/R conditions, as shown by PCR (IH/R Water vs IH/R miRNA mimic: 3.731 ±1.126 vs 2.568 ±0.959; P<0.0001) and WB (0.947 ±0.438 vs 0.478 ±0.277; P<0.05). IFA proved the suppression of EGR1 by miR-124-3p mimic (0.414 ±0.101 vs 0.062 ±0.064; P<0.0001), which subsequently reduced CM apoptosis (0.234 ±0.127 vs 0.092 ±0.047; P<0.001). Conclusion: EGR1 participate in exacerbating apoptosis mediated by IH/R in human CMs. The downregulation of EGR1 suppresses IH/R-induced apoptosis. Treatment with miR-124-3p targeting EGR1 may potentially serve as a novel therapeutic approach for cardioprotection in the future.

Development of Self-Pulsatile hybrid cylindrical structure using human iPS cell-derived cardiomyocytes and polydimethylsiloxane

In this study, a self-pulsatile cylindrical structure was developed by hybridizing a sheet of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM) and a polydimethylsiloxane (PDMS) tube. The dynamic contractive deformation and the internal flow behavior generated by self-pulsation were characterized. Time-variation of the minimum principal strain was evaluated using the high speed imaging system and the digital image correlation method. It was found that the strain varied in the range of 0.08 – 0.1 and clearly synchronized with the self-pulsation. It was also confirmed that the theoretical model combining a viscoelastic and an active stress models well coincided with the experimental strain data. It was also found that the flow inside the tube had a rate of 3.48 nL/min, and was synchronized with its self-contraction.

Temperature Dependence of the Beating Frequency of hiPSC-CMs Using a MEMS Force Sensor.

It is expected that human iPS cell-derived cardiomyocytes (hiPSC-CMs) can be used to treat serious heart diseases. However, the properties and functions of human adult cardiomyocytes and hiPSC-CMs, including cell maturation, differ. In this study, we focused on the temperature dependence of hiPSC-CMs by integrating the temperature regulation system into our sensor platform, which can directly and quantitatively measure their mechanical motion. We measured the beating frequency of hiPSC-CMs at different environmental temperatures and found that the beating frequency increased as the temperature increased. Although the rate at which the beating frequency increased with temperature varied, the temperature at which the beating stopped was relatively stable at approximately 20 °C. The stopping of beating at this temperature was stable, even in immature hiPSC-CMs, and was considered to be a primitive property of cardiomyocytes.

Effects of cigarette smoke extracts (combustible and heated tobacco) on rat and human iPS cell-derived cardiomyocytes.

Smoking is a risk factor of cardiovascular diseases. However, direct effects on cardiomyocytes, especially on intracellular Ca2+ dynamics and on human cardiomyocytes, are not fully elucidated. In addition, the effects of heated cigarettes, which have been increasingly used in recent years, on human diseases have not been extensively studied. In this study, we compared the effects of cigarette smoke extracts (CSEs) from heated (Ploom X, IQOS) and combustible cigarettes (1R6F, Hi-Lite) on cell viability, contractile function and intracellular Ca2+ dynamics in rat and human iPS cell-derived cardiomyocytes. MTS assays indicated that all CSEs reduced cell viability of cultured myocytes, with toxicity in the order of combustible > heated CSEs. Functional analysis showed that 1% CSE significantly reduced the spontaneous beating rate of cultured cardiomyocytes only in the combustible cigarette group. In contrast, in freshly isolated cardiomyocytes, cell shortening was reduced in all CSE-treated groups. Intracellular Ca2+ measurement revealed that abnormal Ca2+ dynamics are induced by combustible CSEs. Kinetic analysis of Ca2+ transients suggests that CSE accelerates the rate of Ca2+ uptake into the sarcoplasmic reticulum (SR), but also causes excessive Ca2+ leakage from the SR, leading to diastolic Ca2+ overload. Similar contractile dysfunction (reduced spontaneous beating rate and contractility) was also observed in human iPS-derived cardiomyocytes. These results indicate that combustible cigarettes attenuate contractile function by altering intracellular Ca2+ dynamics. On the other hand, heated cigarettes were also found to exhibit myocardial toxic effects, albeit to a lesser degree than combustible cigarettes, suggesting the need for caution when smoking cigarettes.

Echinochrome Prevents Sulfide Catabolism-Associated Chronic Heart Failure after Myocardial Infarction in Mice.

Abnormal sulfide catabolism, especially the accumulation of hydrogen sulfide (H2S) during hypoxic or inflammatory stresses, is a major cause of redox imbalance-associated cardiac dysfunction. Polyhydroxynaphtoquinone echinochrome A (Ech-A), a natural pigment of marine origin found in the shells and needles of many species of sea urchins, is a potent antioxidant and inhibits acute myocardial ferroptosis after ischemia/reperfusion, but the chronic effect of Ech-A on heart failure is unknown. Reactive sulfur species (RSS), which include catenated sulfur atoms, have been revealed as true biomolecules with high redox reactivity required for intracellular energy metabolism and signal transduction. Here, we report that continuous intraperitoneal administration of Ech-A (2.0 mg/kg/day) prevents RSS catabolism-associated chronic heart failure after myocardial infarction (MI) in mice. Ech-A prevented left ventricular (LV) systolic dysfunction and structural remodeling after MI. Fluorescence imaging revealed that intracellular RSS level was reduced after MI, while H2S/HS- level was increased in LV myocardium, which was attenuated by Ech-A. This result indicates that Ech-A suppresses RSS catabolism to H2S/HS- in LV myocardium after MI. In addition, Ech-A reduced oxidative stress formation by MI. Ech-A suppressed RSS catabolism caused by hypoxia in neonatal rat cardiomyocytes and human iPS cell-derived cardiomyocytes. Ech-A also suppressed RSS catabolism caused by lipopolysaccharide stimulation in macrophages. Thus, Ech-A has the potential to improve chronic heart failure after MI, in part by preventing sulfide catabolism.

Cardiac cell sheet engineering for regenerative medicine and tissue modeling.

Stem cell biology and tissue engineering are essential techniques for cardiac tissue construction. We have succeeded in fabricating human cardiac tissue using the mass production technology of human iPS cell-derived cardiomyocytes and cell sheet engineering, and we are developing regenerative medicine and tissue models to apply this tissue to heart disease research. Cardiac tissue fabrication and tissue functional evaluation technologies for contractile and electrophysiological function are indispensable, which lead to the functional improvement of bioengineered human cardiac tissue.

TRPC3-Nox2 Protein Complex Formation Increases the Risk of SARS-CoV-2 Spike Protein-Induced Cardiomyocyte Dysfunction through ACE2 Upregulation.

Myocardial damage caused by the newly emerged coronavirus (SARS-CoV-2) infection is one of the key determinants of COVID-19 severity and mortality. SARS-CoV-2 entry to host cells is initiated by binding with its receptor, angiotensin-converting enzyme (ACE) 2, and the ACE2 abundance is thought to reflect the susceptibility to infection. Here, we report that ibudilast, which we previously identified as a potent inhibitor of protein complex between transient receptor potential canonical (TRPC) 3 and NADPH oxidase (Nox) 2, attenuates the SARS-CoV-2 spike glycoprotein pseudovirus-evoked contractile and metabolic dysfunctions of neonatal rat cardiomyocytes (NRCMs). Epidemiologically reported risk factors of severe COVID-19, including cigarette sidestream smoke (CSS) and anti-cancer drug treatment, commonly upregulate ACE2 expression level, and these were suppressed by inhibiting TRPC3-Nox2 complex formation. Exposure of NRCMs to SARS-CoV-2 pseudovirus, as well as CSS and doxorubicin (Dox), induces ATP release through pannexin-1 hemi-channels, and this ATP release potentiates pseudovirus entry to NRCMs and human iPS cell-derived cardiomyocytes (hiPS-CMs). As the pseudovirus entry followed by production of reactive oxygen species was attenuated by inhibiting TRPC3-Nox2 complex in hiPS-CMs, we suggest that TRPC3-Nox2 complex formation triggered by panexin1-mediated ATP release participates in exacerbation of myocardial damage by amplifying ACE2-dependent SARS-CoV-2 entry.

Abstract 14660: Truncating Variants in MYBPC3 Cause Ubiquitin Proteasome System Dysfunction Related to Hypertrophic Cardiomyopathy in Human Induced Pluripotent Stem Cell Model

Introduction: Hypertrophic cardiomyopathy (HCM) is the most common inherited monogenic cardiomyopathy. Cardiac myosin binding protein C ( MYBPC3 ) is the most frequently mutated gene in this disease, and most pathogenic variants in MYBPC3 are truncating variants including splice site variants. The ubiquitin proteasome system (UPS), a major intracellular protein degradation system, plays an important role in the heart. Although impairment of UPS is reportedly caused by truncated cMyBP-C, the underlying molecular mechanism causing HCM is largely unknown. Hypothesis: Truncating variants in MYBPC3 lead to impaired UPS activity in human iPS cell-derived cardiomyocytes (hiPSC-CMs) and are associated with HCM phenotypes. Methods: Patient derived iPSC clones were generated from HCM patients with MYBPC3 variants (IVS11-9G>A and IVS27+1G>A). Corrected isogenic control of IVS27+1G>A was generated by base editing. To evaluate UPS activity, we employed ubiquitin fusion degradation reporter, Ub-G76V-GFP, which provides fluorescence readout of UPS activity. BNP expression of hiPSC-CMs were evaluated by antibody labeling and fluorescence-activated cell sorting (FACS). Contractility and Ca 2+ transient were measured from single biopatterned hiPSC-CMs. Results: Both MYBPC3 variant lines exhibited UPS impairment as reflected by increased GFP fluorescence compared to isogenic and healthy controls (Fig. A). IVS27+1G>A showed higher expression of BNP (Fig. B and C). Enhanced contractility and Ca 2+ handling abnormalities were observed in MYBPC3 variant iPSC-CM lines as compared to control (Fig. D and E). Conclusions: Impairment of UPS activity may be associated with a marker of cellular hypertrophy, and contractility and Ca 2+ handling abnormalities in hiPSC-CMs with MYBPC3 truncating variants. These findings support prior reports suggesting that decreased UPS activity contribute to the pathophysiological mechanism of HCM caused by truncating MYBPC3 variants.

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.

Phenotypic screen identifies FOXO inhibitor to counteract maturation and promote expansion of human iPS cell-derived cardiomyocytes

Achieving pharmacological control over cardiomyocyte proliferation represents a prime goal in therapeutic cardiovascular research. Here, we identify a novel chemical tool compound for the expansion of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. The forkhead box O (FOXO) inhibitor AS1842856 was identified as a significant hit from an unbiased proliferation screen in early, immature hiPSC- cardiomyocytes (eCMs). The mitogenic effects of AS1842856 turned out to be robust, dose-dependent, sustained, and reversible. eCM numbers increased >30-fold as induced by AS1842856 over three passages. Phenotypically as well as by marker gene expression, the compound interestingly appeared to counteract cellular maturation both in immature hiPSC-CMs as well as in more advanced ones. Thus, FOXO inhibitor AS1842856 presents a novel proliferation inducer for the chemically defined, xeno-free expansion of hiPSC-derived CMs, while its de-differentiation effect might as well bear potential in regenerative medicine.

Cardiotoxicity assessment of EGFR-TKI using human iPS cell-derived cardiomyocytes

Cardiotoxicity assessment of EGFR-TKI using human iPS cell-derived cardiomyocytes

Protective effect of nobiletin against doxorubicin-induced cardiotoxicity in human iPS cell-derived cardiomyocytes.

Protective effect of nobiletin against doxorubicin-induced cardiotoxicity in human iPS cell-derived cardiomyocytes.

Modulation of muscle redox and protein aggregation rescues lethality caused by mutant lamins

Mutations in the human LMNA gene cause a collection of diseases called laminopathies, which includes muscular dystrophy and dilated cardiomyopathy. The LMNA gene encodes lamins, filamentous proteins that form a meshwork on the inner side of the nuclear envelope. How mutant lamins cause muscle disease is not well understood, and treatment options are currently limited. To understand the pathological functions of mutant lamins so that therapies can be developed, we generated new Drosophila models and human iPS cell-derived cardiomyocytes. In the Drosophila models, muscle-specific expression of the mutant lamins caused nuclear envelope defects, cytoplasmic protein aggregation, activation of the Nrf2/Keap1 redox pathway, and reductive stress. These defects reduced larval motility and caused death at the pupal stage. Patient-derived cardiomyocytes expressing mutant lamins showed nuclear envelope deformations. The Drosophila models allowed for genetic and pharmacological manipulations at the organismal level. Genetic interventions to increase autophagy, decrease Nrf2/Keap1 signaling, or lower reducing equivalents partially suppressed the lethality caused by mutant lamins. Moreover, treatment of flies with pamoic acid, a compound that inhibits the NADPH-producing malic enzyme, partially suppressed lethality. Taken together, these studies have identified multiple new factors as potential therapeutic targets for LMNA-associated muscular dystrophy.

Human iPS cell-derived cardiomyocytes aligning on flexible gelatin fiber network show contraction and pharmacological responses relevant to human heart

Human iPS cell-derived cardiomyocytes aligning on flexible gelatin fiber network show contraction and pharmacological responses relevant to human heart

High-Throughput Drug Screening System Based on Human Induced Pluripotent Stem Cell-Derived Atrial Myocytes ∼ A Novel Platform to Detect Cardiac Toxicity for Atrial Arrhythmias.

Evaluation of proarrhythmic properties is critical for drug discovery. In particular, QT prolongation in electrocardiograms has been utilized as a surrogate marker in many evaluation systems to assess the risk of torsade de pointes and lethal ventricular arrhythmia. Recently, new evaluation systems based on human iPS cell-derived cardiomyocytes have been established. On the other hand, in clinical situations, it has been reported that the incidence of atrial arrhythmias such as atrial fibrillation has been increasing every year, with the prediction of a persistent increase in the near future. As to the increased incidence of atrial arrhythmias, in addition to the increased population of geriatric patients, a wide variety of drug treatments may be related, as an experimental method to detect drug-induced atrial arrhythmia has not been established so far. In the present study, we characterized the atrial-like cardiomyocytes derived from human induced pluripotent stem cells and examined their potential for the evaluation of drug-induced atrial arrhythmia. Atrial-like cardiomyocytes were induced by adding retinoic acid (RA) during the process of myocardial differentiation, and their characteristics were compared to those of RA-free cardiomyocytes. Using gene expression and membrane potential analysis, it was confirmed that the cells with or without RA treatment have atrial or ventricular like cardiomyocytes, respectively. Using the ultra-rapid activating delayed rectifier potassium current (IKur) channel inhibitor, which is specific to atrial cardiomyocytes, Pulse width duration (PWD) 30cF prolongation was confirmed only in atrial-like cardiomyocytes. In addition, ventricular like cardiomyocytes exhibited an early after depolarization by treatment with rapidly activating delayed rectifier potassium current (IKr) channel inhibitor, which induces ventricular arrhythmia in clinical situations. Here, we have established a high-throughput drug evaluation system using human iPS cell-derived atrial-like cardiomyocytes. Based on the obtained data, the system might be a valuable platform to detect potential risks for drug-induced atrial arrhythmias.

Isometric contraction force measurement of hiPSC-CMs on a movable plate with a feedback-controlled MEMS cantilever probe

We propose a measurement method with a feedback system to evaluate the contraction forces of human iPS cell-derived cardiomyocytes (hiPSC-CMs) in a dynamic mechanical environment. The measurement of the hiPSC-CM contraction forces is important for regenerative medicine; however, conventional methods are not able to evaluate these forces when the cells are subjected to a dynamic load similar to that from the heart. The proposed measurement system is composed of a micromachined piezoresistive cantilever attached to a feedback-controlled piezo stage and a micromachined movable plate where cells are cultured. A high sampling rate (2 kHz) and real-time control of the cell length were realized via the feedback-controlled piezo stage, while the contraction forces were measured by the cantilever. We evaluated the contraction forces of hiPSC-CMs in conditions of isometric and auxotonic contractions. Due to feedback-controlled loading for isometric contraction, the cell shrinkage was controlled to be less than 200 nm. Auxotonic contraction forces of 3.9 μN were measured without feedback control, while the contraction forces of isometric contraction were 6.0 μN with feedback-controlled loading. The results showed that the method leads to a work-loop evaluation of the hiPSC-CM cardiac cycle.

Machine Learning Techniques to Classify Healthy and Diseased Cardiomyocytes by Contractility Profile.

Cardiomyocytes derived from human induced pluripotent stem (iPS) cells enable the study of cardiac physiology and the developmental testing of new therapeutic drugs in a human setting. In parallel, machine learning methods are being applied to biomedical science in unprecedented ways. Machine learning has been used to distinguish healthy from diseased cardiomyocytes using calcium (Ca2+) transient signals. Most Ca2+ transient signals are obtained via terminal assays that do not permit longitudinal studies, although some recently developed options can circumvent these concerns. Here, we describe the use of machine learning to identify healthy and diseased cardiomyocytes according to their contractility profiles, which are derived from brightfield videos. This noncontact, label-free approach allows for the continued cultivation of cells after they have been evaluated for use in other assays and can be readily extended to organs-on-chip. To demonstrate utility, we assessed contractility profiles of cardiomyocytes obtained from patients with Timothy Syndrome (TS), a long QT disease which can lead to fatal arrhythmias, and from healthy individuals. The videos were processed and classified using machine learning methods and their performance was evaluated according to several parameters. The trained algorithms were able to distinguish the TS cardiomyocytes from healthy controls and classify two different healthy controls. The proposed computational machine learning evaluation of human iPS cell-derived cardiomyocytes' contractility profiles has the potential to identify other genetic proarrhythmic events, screen therapeutic agents for inducing or suppressing long QT events, and predict drug-target interactions. The same approach could be readily extended to the evaluation of engineered cardiac tissues within single-tissue and multi-tissue organs-on-chip.

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.