For the past decade, cell-based therapies have been the focus of research to investigate their potential to treat ischemic heart disease. The translation to human clinical studies depends on the demonstration of therapeutic efficacy and safety, particularly when transplanted in the subacute and chronic post-MI phase. A number of studies were identified that reported the effect of hiPSC-CMs on cardiac outcomes when transplanted at least 7 days post-myocardial infarction. The mean sample size of the published studies was 30 (±17) animals with a mean follow-up duration of 51 (±37) days. hiPSC-CM transplantation enhanced systolic function through augmented myocardial contractility, decreased infarct size, attenuated ventricular remodeling, and enhanced angiogenesis in the infarct and border zones in both small and large animal models. This effect was enhanced by co-transplantation with cells of vascular or adipose origin and is associated with high expression of VEGF in most studies. Despite this effect, transplanted hiPSC-CMs were structurally immature with limited survival at the endpoint. Epicardial delivery was associated with better efficacy outcomes and lower rates of arrhythmia. No study reported teratoma formation or immune rejection. From the current literature, there appears to be no consensus on the extent to which hiPSC-CMs improved systolic function, nor the degree to which this arises directly from integration of the new myocardium or from a paracrine-mediated mechanism. The nature of this paracrine mechanism and ways to improve the maturity and survival of implanted cardiomyocytes are issues that have yet to be resolved. In summary, while therapeutic benefit from cell therapy is clear, further research is required to establish whether the key mechanisms require a cellular component.

Optical clearing combined with light-sheet microscopy enables high-resolution imaging of extended tissue at scale. However, standard mesoSPIM systems are optimised for intact organs and are not suited to thin tissue slices. We present an oblique compensation scanning method using obliquely mounted samples held between refractive-index-matched slides in a 3D-printed frame. This enables mechanical de-skewing during acquisition, minimising post-processing requirements. We demonstrate feasibility in fluorescent bead phantoms and rabbit heart tissue, achieving a 4.8 × reduction in processing time and a 1.5 × improvement in axial resolution ((13.15±1.36) μm to (8.72±1.80) μm) compared to conventional z scan. The oblique compensation acquisition method extends mesoSPIM's utility to fragile, laterally extended tissue sections.

Mathematical models of cardiac cell electrical activity include numerous parameters, making calibration to experimental data and individual-specific modeling challenging. This study applies Sobol sensitivity analysis, a global variance-decomposition method, to identify the most influential parameters in the Shannon model of rabbit ventricular myocyte action potential (AP). The analysis highlights the background chloride current (IClb) as the dominant determinant of AP variability. Additionally, the inward rectifier potassium current (IK1), fast/slow delayed rectifier potassium currents (IKr, IKs), sodium-calcium exchanger current (INaCa), the slow component of the transient outward potassium current (Itos), and L-type calcium current (ICaL) significantly affect AP biomarkers, including duration, plateau potential, and resting potential. Exploiting these results, a hierarchical reduction of the model is performed and demonstrates that retaining only six key parameters can capture sufficiently well individual biomarkers, with a coefficient of determination exceeding 0.9 for selected cases. These findings improve the utility of the Shannon model for personalized simulations, aiding applications like digital twins and drug response predictions in biomedical research.

Despite advances in the treatment of ST-elevation myocardial infarction (MI) with primary percutaneous coronary intervention, the risk of remote ventricular arrhythmias (VA) and sudden death remains significant. Reperfused MI may support faster ventricular tachycardia (VT)1–3, potentially due to smaller isthmuses. While cardiac MRI has an expanding role in risk assessment and planning VT ablation, the link between scar characteristics and VT cycle length is poorly defined. This study compared VT inducibility and cycle length in vivo in rabbits with MI or ischemia-reperfusion (IR) to controls, with structural and functional characterisation using high-resolution ex vivo imaging. Adult male New Zealand White rabbits underwent either permanent coronary ligation (MI, n=16), temporary ligation for 60 minutes (IR, n=8), or no intervention (controls, n=7). In vivo PES was perfomed on a subset (n=20). Langendorff-perfused hearts were imaged using optical mapping (OM) with RH237 dye to identify late activating sites in sinus rhythm, and conduction discontinuities quantified by maximal phase difference (PDmax) in activation time4. Sustained VT was induced ex vivo through burst pacing, with flecainide (1µM) if required. Fixed MI hearts were scanned with 7T MRI for high-resolution T1-weighted imaging (125µm3) and histology with Masson’s Trichrome. OM and histology data were mapped onto 3D MRI-rendered structures. Scar volume, surface area and transmurality were calculated. Sustained VT (>30 seconds) in vivo was induced in 29% (n=2/7) of MI hearts, 50% (n=3/6) of I-R hearts and 0/7 control hearts (p=0.11). MI scars were more transmural (84±4% vs 44±13%, p<0.0001) and extensive (38±17% vs 24±11%, p=0.043) than IR, which had a preserved epicardial rim. Scar volume, surface area or transmurality did not impact VA inducibility. VT cycle length was positively correlated with scar transmurality (R2=0.97, p=0.003), while no correlation was found with scar volume or surface area. IR hearts often had late LV apical activation in sinus rhythm (6/8 vs. 2/16 MI hearts, p=0.0047 and 0/7 controls, p=0.0005). Apical conduction times were longer in MI/IR hearts with evidence of an epicardial rim (n=9/24: 16ms±2 vs. 6ms±1, p <0.0001). Late activation was not associated with VA inducibility, whereas conduction discontinuity (PDmax) was increased in hearts with sustained VA (2.6ms ± 0.9 vs 1.4ms ± 0.7; p=0.028). Sustained VT was inducible ex vivo in 16/16 MI, 7/8 IR hearts and 1/5 of control hearts (MI/IR vs. controls, p<0.0001). During VT, lines of conduction block aligned with regions of late activation in sinus rhythm or discontinuous conduction. Scar transmurality strongly correlates with VT cycle length, with less transmural scars linked to faster VT. Critical VT substrate in hearts with subendocardial scars may be associated with an epicardial rim, thus standard endocardial ablation may not prevent VT recurrence.

A major challenge in cardiac research is the limited translatability of drug screening and toxicity assays due to the use of in vitro models that poorly mimic the native cardiac environment. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer a promising route forward, but conventional 2D culture on rigid substrates hinders their functional maturation and predictive accuracy. This study addresses this problem by investigating the effect of hybrid fibronectin-based hydrogels with tunable stiffness on the mechanical and electrical properties of hiPSC-CMs. We engineered hydrogels with stiffness mimicking the lowest range of neonatal heart tissue stiffness (2-4 kPa) and compared hiPSC-CM behavior on these substrates to that on standard fibronectin-coated glass. Our results demonstrate that hydrogel culture promotes more uniform and stable cardiomyocyte contractions, as evidenced by increased single peak percentages and altered contraction duration. Electrophysiological analysis revealed that hydrogel stiffness influences action potential duration and signal amplitude. Furthermore, hiPSC-CMs on hydrogels exhibited enhanced cell-matrix and cell-cell adhesion, indicating improved structural and functional connectivity. Drug testing with known cardioactive compounds, including isoproterenol and nifedipine, revealed distinct differences in drug responses between hydrogel and glass cultures, suggesting that hydrogels provide a more physiologically relevant platform for assessing drug effects. This work highlights the potential of engineered hydrogel substrates to enhance the functional maturity and predictive accuracy of hiPSC-CMs for cardiac research and drug development.

AIMS:Electrical integration of human-induced pluripotent stem-cell-derived cardiomyocyte (hiPSC-CM)-based tissue with the host myocardium is a requirement of successful regeneration therapy. This study was designed to identify electrical coupling in the acute phase (1-2 h) post-grafting using an ex vivo model. METHODS AND RESULTS:Small, engineered heart tissues (mini-EHTs), consisting of ∼50 000 hiPSC-CMs on a hydrogel (spontaneous rate 0.34 ± 0.05 Hz), were loaded with Cal520-AM. EHTs were implanted sub-epicardially into a Langendorff-perfused rabbit heart after blebbistatin treatment. For up to 100 min, a continuous pseudo-electrocardiogram was recorded during sinus rhythm (rate 2.0-3.5 Hz). At 25 min intervals, EHT calcium transients (CaTs) were recorded for 10-20 s (no contraction group). To study the influence of mechanical activity, blebbistatin was washed off after implantation (contraction recovery group). Periodic entrainment of EHTs with the myocardium was detected less often (P = 0.011) in the no contraction group (1/9 hearts) than in the contraction recovery group (5/6 hearts). The average coupling delay (QRS-CaT) and the difference in consecutive delays (Δdelay) were 89 ± 50 and 10 ± 3 ms, respectively (n = 12 traces; N = 6 hearts). Coupling ratios (QRS:CaT) varied from 2:1 to 4:1. These coupling parameters were not significantly different in the two experimental groups. Modelling of hiPSC-CM tissue separated by a 25 μm saline gap from the myocardium demonstrated field-effect coupling with similarly variable activation delays. Importantly, coupling failed with a gap of 100 μm. CONCLUSION:EHT entrainment is possible immediately after grafting and has features compatible with field-effect coupling. Sensitivity to the gap dimensions may explain why entrainment is more common in actively contracting myocardium.

The complexity of the cellular proteome is massively expanded by a repertoire of chemically distinct reversible post-translational modifications (PTMs) that control protein localisation, interactions, and function. The temporal and spatial control of these PTMs is central to organism physiology, and mis-regulation of PTMs is a hallmark of many diseases. Here we present an approach to manipulate PTMs on target proteins using nanobodies fused to enzymes that control these PTMs. Anti-GFP nanobodies fused to thioesterases (which depalmitoylate protein cysteines) depalmitoylate GFP tagged substrates. A chemogenetic approach to enhance nanobody affinity for its target enables temporal control of target depalmitoylation. Using a thioesterase fused to a nanobody directed against the Ca(v)1.2 beta subunit we reduce palmitoylation of the Ca(v)1.2 alpha subunit, modifying the channel's voltage dependence and arrhythmia susceptibility in stem cell derived cardiac myocytes. We conclude that nanobody enzyme chimeras represent an approach to specifically manipulate PTMs, with applications in both the laboratory and the clinic.

Conversion and prevention of atrial fibrillation (AF) are essential requirements for its treatment. Abnormal leakage of calcium from the sarcoplasmic reticulum via the ryanodine receptor 2 (RyR2) during diastole is considered a major cause of AF. Although catheter ablation has made it possible to stop persistent AF, it does not address recurrence. In this context, we describe the additional pharmacological and antiarrhythmic effects of the RyR2 inhibitor M201-A, including the results of a phase I study. M201-A inhibited G-protein-gated atrial K+ channel at an IC50 value of 0.35 μM, which was associated with a 38% increase in the atrial effective refractory period (ERP) and high atrial selectivity with an atrial ERP/ventricular ERP of 5.7. M201-A inhibited rapid component of the delayed rectifier potassium current (IC50 value of 0.43 μM) but induced only a limited prolongation in the action potential duration in rabbit ventricular cells and human induced pluripotent stem cell-derived cardiomyocytes. M201-A is an R-enantiomer of the active metabolite M201-R of K201. After orally administering 540 mg K201 to patients with AF, the unbound drug concentration of M201-R was 30-fold higher (approximately 15-fold for M201-A) than that of K201, and AF was converted in 6 of the 12 patients (50%) during the first 2 hours following administration. M201-A was the main contributor with a stronger bioactive metabolite than M201-B (S-enantiomer) and M201-R. Furthermore, M201-A was 5-fold more biologically active than K201 in humans due to protein binding rates. These pharmacological properties hold promise as a new therapeutic strategy for AF, particularly the prevention of recurrence after catheter ablation. SIGNIFICANCE STATEMENT: Abnormal leakage of calcium from the sarcoplasmic reticulum via the ryanodine receptor 2 (RyR2) is considered a major cause of atrial fibrillation (AF). M201-A had inhibitory effects on RyR2 and G-protein-gated atrial K+ channel. It had high atrial selectivity with limited QT interval corrected by Fridericia's formula prolongation and proarrhythmic risk. It was the most active conversion metabolite when K201 was administered orally in patients with paroxysmal AF. These pharmacological effects of M201-A, combined with its inhibitory actions on RyR2, provide a promising therapeutic option for treating AF.

Previously, we introduced a microscope design that enabled rapid, random-access well plate imaging [ eLife, 10, e56426 (2021)10.7554/eLife.56426]. Here, we implement this design in a low-cost, compact, and portable prototype (the Exeter Multiscope) and apply it to the problem of capturing the contraction of cardiomyocyte monolayers, which have been plated into nine wells within a 96-well plate. Using a transmissive rather than reflective geometry, each well is sampled using 500 × 500 pixels across a 1.4 × 1.4 mm field of view, acquired in three colours at 3.7 Hz per well. The use of multiple illumination wavelengths provides post-hoc focus selection, further increasing the level of automation. The performance of the Exeter Multiscope is benchmarked against industry standard methods using a commercial microscope with a motorised stage and demonstrates that the Multiscope can acquire data almost 40 times faster. The data from both Multiscope and the commercial systems are processed by a 'pixel variance' algorithm that uses information from the pixel value variability over time to determine the timing and amplitude of tissue contraction. This algorithm is also benchmarked against an existing algorithm that employs an absolute difference measure of tissue contraction.

For the preclinical phase of the drug development, the ICH guideline S7B describes assays to assess cardiotoxicity focusing on potentially life threatening, acute effects causing delayed ventricular repolarization and an increase in proarrhythmic risk. However, there exist no guidelines to assess long-term cardiotoxicity although a variety of treatments e.g. with anti-cancer drugs like anthracyclines cause life threatening conditions including cardiomyopathy. HESI's Cardiac Safety Committee Stem Cell Work Group organized and executed a multi-site study with 12 blinded compounds (doxorubicin, erlotinib, sunitinib, pentamidine, arsenic trioxide, BMS-986094, milrinone, nilotinib, endothelin-1, vinblastine, vincristine, vinorelbine) of different known mechanism causing long-term cardiotoxicity in humans. We participated in HESI's multi-site study with the objective to reveal whether microelectrode array (MEA) recordings from hiPSC-derived cardiomyocytes are suitable for assessing drug-induced long-term cardiotoxicity. Cardiomyocytes were cultured on microelectrode arrays. On day of experiment, baseline recordings were taken before compounds were added as single dose in 4 concentrations and 5 replicates each. Recordings were taken at 1 h, 24 h, 48 h, 72 h, and 96 h post compound addition. On each MEA, doxorubicin was added at 300 nM (n = 4) and dofetilide at 3 nM (n = 4) as positive controls for long-term or acute cardiotoxic response, respectively. The MEA parameters FPD, FPDc, beat period, field potential amplitude and beating arrest were analyzed. After 1 h treatment, only Nilotinib prolonged FPD and FPDc and endothelin-1 decreased beat period and amplitude. Long-term dose and time dependent FPD/FPDc prolongations were observed with sunitinib, pentamidine, nilotinib, and endothelin-1. Vincristine and vinblastine slightly prolonged FPD/FPDc. Vinorelbine decreased the FP amplitude. Sunitib, pentamidine, vinorelbine, BMS-986094 and doxorubicin arrested beating at higher doses. As expected, the non-cardiotoxic drug erlotinib did not reveal any effect. As exception for the cardiotoxic drugs, only arsenic trioxide and milrinone did not show any effect. Overall, we assessed 9 of 11 compounds correctly with clear long-term cardiotoxic effects on hiPSC-CMs from 8 compounds and erlotinib with no effects as expected. Two compounds appeared to be false negative, namely milrinone and arsenic trioxide. Thus, MEA recordings from hiPSC-CM are a powerful tool to assess long-term cardiotoxicity.

The Health and Environmental Sciences Institute (HESI) is a nonprofit organization focused on resolving global health challenges through collaborative science. HESI achieves this by engaging scientists from around the world in the public, including health regulatory authorities, and private sectors and coalescing around a specific topic. The HESI Cardiac Safety Committee (CSC) officially formed in 2008 to improve public health by reducing unanticipated cardiovascular-related adverse effects from pharmaceuticals or chemicals. Since the publication by Pierson et al. (2013) outlining achievements, the HESI CSC has continued to champion cardiac safety, having a major impact on the field of cardiovascular safety assessments. This has been attained through numerous prospective studies, retrospective analyses, workshops, symposia and more than 24 peer review publications. These publications detail results of the work over the past decade and while reporting results is important, the lasting impacts on the field of CV safety show the real value of collaborative science. Examples of this work include details of the Comprehensive In vitro Proarrhythmia Assay (CiPA) initiative and subsequent updated ICH E14/S7B Q&As, as well as the related stem cell validation studies and best practices. Similarly, challenges and opportunities in both manual and automated patch clamp studies for proarrhythmic prediction and use in an in silico model. A better understanding of assay sensitivity and specificity can lead to better interpretation and implementation. HESI studies on in vivo and in vitro structure and functional assessments have contributed in these areas. Earlier detection of potential failure modes allow for better drug development and design. HESI team are investigating promising new biomarkers to detect hemostatic changes earlier in the process. Answering challenging questions as a collaborative team, sharing data for the benefit of the larger scientific community, and helping young scientists advance their careers are just a few of the outcomes of the last 10 years of the CSC. Future challenges ahead and the direction of the CSC will be offered for input.

iPSC-derived cardiomyocytes (iPSC-CMs) have an established role determining the effects of drugs after acute (<1 h) exposure. A host of toxic actions have been shown to develop during chronic (>12 h) drug exposure, including hERG trafficking issues; in vivo tests used to detect these are expensive, involve complex interpretation and may include species dependent differences. To assess the performance of an extended iPSC-CM assay to investigate longer term toxic hERG trafficking actions using serum-free solutions. Functional cellular assays were monitored over a prolonged (72 h) exposure time at 24 h intervals: (i) Electrophysiology using optical voltage measurements (ii) contractile kinetics and amplitude using a camera-based algorithm (iii) iPSC-CM monolayer integrity based on assessment of wide field images and (iv) extracellular lactate dehydrogenase (LDH) measurements that assess plasmalemma integrity. iCell2 (FujiFilm-CDI) iPSC-CMs were seeded on a 96well plate and incubated in a serum free media (FluoroBrite, ThermoFisher). Automated image and signal analysis was completed using Clyde Biosciences a proprietary analysis platform (CellOPTIQ®). 6 concentrations of compounds known to alter hERG trafficking were studied and chronic toxicity, electrophysiological, metabolic dysfunction were assessed. All drugs showed effects across the parameters studied with clear prolongation of APD90 after 24 h and prolonging further at 48 h. Functional data from the DMSO control wells were stable over 72 h, but small, significant loss of cell integrity and raised LDH signals were detected after 72 h. iPSC-CMs can be used to assess medium-long term actions of hERG trafficking drugs allowing mechanistic interpretation. This represents a highly specific system for chronic assessment of human cardiovascular toxicity risk such as hERG trafficking.

Collagen IV is a major constituent of basement membranes and mutations in the genes COL4A1 and COL4A2 present clinically as a variable, multi-system disorder called COL4A1 (Gould) syndrome. Evidence from case reports supports a cardiac component to this disease, but the phenotypic and functional implications affecting the heart, their progression and underlying mechanisms all remain poorly characterised. Indeed, the role of the basement membrane (BM) in adult cardiac disease remains underexplored. We set out to address these knowledge gaps by combining in-depth phenotypic and molecular analyses of a Col4a1 mutation on cardiac biology in a murine model (Col4a1+/svc) of Gould Syndrome. This revealed morphological cardiac defects including cardiomyocyte hypertrophy with myocardial and vascular fibrotic remodelling that impaired cardiac function. The Col4a1 mutation causes systolic and diastolic dysfunction with reduced left ventricular developed pressure. Mechanistically, we show these defects are due to secretion of mutant protein and BM defects rather than collagen misfolding and proteotoxic stress. The BM defects lead to a pro-fibrotic state with increased fibrillar collagen deposition, cardiac stiffness, and ECM compositional defects. These are accompanied by altered regulation of pathways involved in sarcomere formation, sarcolemma stability and cardiomyocyte metabolism, establishing a molecular signature of COL4A1-related cardiac disease. Intriguingly, aspects of this molecular signature including cardiac metabolic pathways, regulation of cardiac muscle contraction and BM component expression, are shared with common cardiomyopathies such as coronary micro-embolism, and dilated, ischemic and hypertrophic obstructive cardiomyopathies. By defining the molecular and phenotypic cardiac components of Gould syndrome these data show that the BM is essential for maintaining systolic and diastolic function and that alterations to the BM leads to a fibrotic response. These data increase insight into the role of the basement membrane and collagen IV in cardiac biology, and highlights mechanisms shared between Gould syndrome and common adult cardiac disease.

Recent high-throughput experiments unveil substantial electrophysiological diversity among uncoupled healthy myocytes under identical conditions. To quantify inter-cell variability, the values of a subset of the parameters in a well-regarded mathematical model of the action potential of rabbit ventricular myocytes are estimated from fluorescence voltage measurements of a large number of cells. Statistical inference yields a population of nearly 1200 cell-specific model variants that, on a population-level replicate experimentally measured biomarker ranges and distributions, and in contrast to earlier studies, also match experimental biomarker values on a cell-by-cell basis. This model population may be regarded as a random sample from the phenotype of healthy rabbit ventricular myocytes. Univariate and bivariate joint marginal distributions of the estimated parameters are presented, and the parameter dependencies of several commonly used electrophysiological biomarkers are determined. Parameter values are weakly correlated, while summary metrics such as the action potential duration are not strongly dependent on any single electrophysiological characteristic of the myocyte. Our results demonstrate the feasibility of accurately and efficiently fitting entire action potential waveforms at scale.

Surgical coronary ligation is the standard technique to induce myocardial infarction (MI) in rabbits but is associated with procedural trauma and the generation of thoracic adhesions. Percutaneous coronary occlusion avoids these shortcomings and is established in pigs but has only been applicable to large rabbits because of a mismatch between the equipment used and target vessel size. Here, we describe a new scalable approach to percutaneous MI induction that is safe and effective in 2.5–3.5-kg rabbits.

Systolic and diastolic functions are coordinated in the heart by myofilament proteins that influence force of contraction and calcium sensitivity. Fine control of these processes is afforded by a variety of post-translation modifications that occur on specific proteins at different times during each heartbeat. Cardiac myosin binding protein-C is a sarcomeric accessory protein whose function is to interact transiently with actin, tropomyosin and myosin. Previously many different types of post-translational modification have been shown to influence the action of myosin binding protein-C and we present the first report that the protein can be modified covalently by the small ubiquitin like modifier protein tag. Analysis by mass spectrometry suggests that there are multiple modification sites on myosin binding protein-C for this tag and single point mutations did not serve to abolish the covalent addition of the small ubiquitin like modifier protein. Functionally, our data from both model human embryonic kidney cells and transfected neonatal cardiac myocytes suggests that the modification reduces phosphorylation of the filament protein on serine 282. In cardiac myocytes, the hypo-phosphorylation coincided with a significantly slower relaxation response following isoprenaline induced contraction. We hypothesise that this novel modification of myosin binding protein-C represents a new level of control that acts to alter the relaxation kinetics of cardiac myocytes.

Background and PurposeThe ryanodine receptor 2 (RyR2) is present in both the heart and kidneys, and plays a crucial role in maintaining intracellular Ca2+ homeostasis in cells in these organs. This study aimed to investigate the impact of M201-A on RyR2, as well as studying its effects on cardiac and renal functions in preclinical and clinical studies.Experimental ApproachFollowing the administration of M201-A (1,4-benzothiazepine-1-oxide derivative), we monitored diastolic Ca2+ leak via RyR2 and intracellular Ca2+ concentration in isolated rat cardiomyocytes and in cardiac and renal function in animals. In a clinical study, M201-A was administered intravenously at doses of 0.2 and 0.4 mgkg-1 once daily for 20 min for four consecutive days in healthy males, with the assessment of haemodynamic responses.Key ResultsIn rat heart cells, M201-A effectively inhibited spontaneous diastolic Ca2+ leakage through RyR2 and exhibited positive lusi-inotropic effects on the rat heart. Additionally, it enhanced natriuresis and improved renal function in dogs. In human clinical studies, when administered intravenously, M201-A demonstrated an increase in natriuresis, glomerular filtration rate and creatinine clearance, while maintaining acceptable levels of drug safety and tolerability.Conclusions and ImplicationsThe novel drug M201-A inhibited diastolic Ca2+ leak via RyR2, improved cardiac lusi-inotropic effects in rats, and enhanced natriuresis and renal function in humans. These findings suggest that this drug may offer a potential new treatment option for chronic kidney disease and heart failure. image

We apply a novel microscope architecture, the Exeter Multiscope, to the problem of acquiring image data in rapid succession from nine wells of a 96 well plate. We demonstrate that the new microscope can detect contraction in cardiomyocyte monolayers which have been plated into these wells. Each well is sampled using 500 x 500 pixels across a 1.4 x 1.4 mm field of view, acquired in three colours at 3.7 Hz per well. The use of multiple illumination wavelengths provides post-hoc focus selection, further increasing the level of automation. The performance of the Exeter Multiscope is benchmarked against industry standard methods using a commercial microscope with a motorised stage and demonstrates that the Multiscope can acquire data almost 40 times faster. The data from both Multiscope and the commercial systems are processed by a ‘pixel variance’ algorithm that uses information from the pixel value variability over time to determine the timing and amplitude of tissue contraction. This algorithm is also benchmarked against an existing algorithm that employs an absolute difference measure of tissue contraction. ### Competing Interest Statement The authors have declared no competing interest.

The Health and Environmental Sciences Institute (HESI) is a nonprofit organization dedicated to resolving global health challenges through collaborative scientific efforts across academia, regulatory authorities and the private sector. Collaborative science across non-clinical disciplines offers an important keystone to accelerate the development of safer and more effective medicines. HESI works to address complex challenges by leveraging diverse subject-matter expertise across sectors offering access to resources, data and shared knowledge. In 2008, the HESI Cardiac Safety Committee (CSC) was established to improve public health by reducing unanticipated cardiovascular (CV)-related adverse effects from pharmaceuticals or chemicals. The committee continues to significantly impact the field of CV safety by bringing together experts from across sectors to address challenges of detecting and predicting adverse cardiac outcomes. Committee members have collaborated on the organization, management and publication of prospective studies, retrospective analyses, workshops, and symposia resulting in 38 peer reviewed manuscripts. Without this collaboration these manuscripts would not have been published. Through their work, the CSC is actively addressing challenges and opportunities in detecting potential cardiac failure modes using in vivo, in vitro and in silico models, with the aim of facilitating drug development and improving study design. By examining past successes and future prospects of the CSC, this manuscript sheds light on how the consortium's multifaceted approach not only addresses current challenges in detecting potential cardiac failure modes but also paves the way for enhanced drug development and study design methodologies. Further, exploring future opportunities and challenges will focus on improving the translational predictability of nonclinical evaluations and reducing reliance on animal research in CV safety assessments.