Collaborative science in action: A 20 year perspective from the Health and Environmental Sciences Institute (HESI) Cardiac Safety Committee.

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.

Challenging the status quo: a framework for mechanistic and human-relevant cardiovascular safety screening.

Traditional approaches to preclinical drug safety assessment have generally protected human patients from unintended adverse effects. However, these assessments typically occur too late to make changes in the formulation or in phase 1 and beyond, are highly dependent on animal studies and have the potential to lead to the termination of useful drugs due to liabilities in animals that are not applicable in patients. Collectively, these elements come at great detriment to both patients and the drug development sector. This phenomenon is particularly problematic in the area of cardiovascular safety assessment where preclinical attrition is high. We believe that a more efficient and translational approach can be defined. A multi-tiered assessment that leverages our understanding of human cardiovascular biology, applies human cell-based in vitro characterizations of cardiovascular responses to insult, and incorporates computational models of pharmacokinetic relationships would enable earlier and more translational identification of human-relevant liabilities. While this will take time to develop, the ultimate goal would be to implement such assays both in the lead selection phase as well as through regulatory phases.

Development of a pharmaceutical database as an aid to the nonclinical detection of drug-induced cardiac toxicity.

The Health and Environmental Sciences Institute (HESI) Cardiac Safety Committee designed and created a publicly accessible database with an initial set of 128 pharmacologically defined pharmaceutical agents, many with known cardiotoxic properties. The database includes specific information about each compound that could be useful in evaluating hypotheses around mechanisms of drug-induced cardiac toxicity or for development of novel cardiovascular safety assays. Data on each of the compounds was obtained from published literature and online sources (e.g., DrugBank.ca and International Union of Basic and Clinical Pharmacology (IUPHAR) / British Pharmacological Society (BPS) Guide to PHARMACOLOGY) and was curated by 10 subject matter experts. The database includes information such as compound name, pharmacological mode of action, characterized cardiac mode of action, type of cardiac toxicity, known clinical cardiac toxicity profile, animal models used to evaluate the cardiotoxicity profile, routes of administration, and toxicokinetic parameters (i.e., Cmax). Data from both nonclinical and clinical studies are included for each compound. The user-friendly web interface allows for multiple approaches to search the database and is also intended to provide a means for the submission of new data/compounds from relevant users. This will ensure that the database is constantly updated and remains current. Such a data repository will not only aid the HESI working groups in defining drugs for use in any future studies, but safety scientists can also use the database as a vehicle of support for broader cardiovascular safety studies or exploring mechanisms of toxicity associated with certain pharmacological modes of action.

Analyzing captive breeding outcomes to inform reintroduction practice: lessons from the pookila (Pseudomys novaehollandiae).

Captive breeding is often used to produce individuals for reintroduction programs in order to reestablish a species in an area where it has become locally extinct. To maximize the likelihood of establishing a self-sustaining population in the wild, an analysis of data from captive breeding programs is commonly undertaken to (1) increase the quantity of individuals and rate at which they can be released, and (2) maintain or improve the genetic and phenotypic quality of individuals. Here we demonstrate how the knowledge gained from these analyses can also be applied to decision-making during the design of subsequent reintroductions to further advance a reintroduction program toward success. We conducted an analysis of data from a captive breeding program for the threatened pookila (Pseudomys novaehollandiae, New Holland mouse) spanning 6 years. We found evidence for relationships between the reproductive output of pookila and behavioral, demographic, experiential, health, and physiological predictors. Based on a biological interpretation of these results, and with reference to a checklist of all known translocation tactics, we recommend 11 specific design elements to maximize the probability of pookila reproduction postrelease (thereby improving the likelihood of reintroduction success). These recommendations should be interpreted as hypotheses to be evaluated and refined in future reintroduction trials for the pookila. The uncertainty around the postrelease survival and reproduction of a species that is common in reintroduction practice warrants the creative use of existing data to inform adaptive management. Indeed, there is a wealth information in well-kept captive breeding records that is currently underused by reintroduction practitioners. The direct integration of knowledge derived from captive breeding (where available) with decision-making for reintroductions, as described here, will help navigate these uncertainties, which would benefit the conservation of both understudied and well-known species around the world.

Genetic mixing in conservation translocations increases diversity of a keystone threatened species, Bettongia lesueur.

Translocation programmes are increasingly being informed by genetic data to monitor and enhance conservation outcomes for both natural and established populations. These data provide a window into contemporary patterns of genetic diversity, structure and relatedness that can guide managers in how to best source animals for their translocation programmes. The inclusion of historical samples, where possible, strengthens monitoring by allowing assessment of changes in genetic diversity over time and by providing a benchmark for future improvements in diversity via management practices. Here, we used reduced representation sequencing (ddRADseq) data to report on the current genetic health of three remnant and seven translocated boodie (Bettongia lesueur) populations, now extinct on the Australian mainland. In addition, we used exon capture data from seven historical mainland specimens and a subset of contemporary samples to compare pre-decline and current diversity. Both data sets showed the significant impact of population founder source (whether multiple or single) on the genetic diversity of translocated populations. Populations founded by animals from multiple sources showed significantly higher genetic diversity than the natural remnant and single-source translocation populations, and we show that by mixing the most divergent populations, exon capture heterozygosity was restored to levels close to that observed in pre-decline mainland samples. Relatedness estimates were surprisingly low across all contemporary populations and there was limited evidence of inbreeding. Our results show that a strategy of genetic mixing has led to successful conservation outcomes for the species in terms of increasing genetic diversity and provides strong rationale for mixing as a management strategy.

The incidence of spontaneous arrhythmias in telemetered beagle dogs, Göttingen Minipigs and Cynomolgus non-human primates: A HESI consortium retrospective analysis.

INTRODUCTION: Characterization of the incidence of spontaneous arrhythmias to identify possible drug-related effects is often an important part of the analysis in safety pharmacology studies using telemetry. METHODS: A retrospective analysis in non-clinical species with and without telemetry transmitters was conducted. Electrocardiograms (24 h) from male and female beagle dogs (n = 131), Göttingen minipigs (n = 108) and cynomolgus non-human primates (NHP; n = 78) were analyzed. RESULTS: Ventricular tachycardia (VT) was observed in 3% of the dogs but was absent in minipigs and NHPs. Ventricular fibrillation (VF) was not observed in the 3 species. Ventricular premature beats (VPBs) were more frequent during daytime and atrioventricular blocks (AVBs) were more frequent at night in all species. A limited number of animals exhibited a high arrhythmia frequency and there was no correlation between animals with higher frequency of an arrhythmia type and the frequency of other arrythmias in the same animals. Clinical chemistry or hematology parameters were not different with or without telemetry devices. NHP with a transmural left ventricular pressure (LVP) catheter exhibited a greater incidence of VPBs and PJCs compared to telemetry animals without LVP. DISCUSSION: All species were similar with regards to the frequency of ventricular ectopic beats (26-46%) while the dog seemed to have more frequent junctional complexes and AVB compared to NHP and minipigs. Arrhythmia screening may be considered during pre-study evaluations, to exclude animals with abnormally high arrhythmia incidence.

Mini Safe Havens for population recovery and reintroductions 'beyond-the-fence'.

In response to the ongoing decline of fauna worldwide, there has been growing interest in the rewilding of whole ecosystems outside of fenced sanctuaries or offshore islands. This interest will inevitably result in attempts to restore species where eliminating threats from predators and competitors is extremely challenging or impossible, or reintroductions of predators that will increase predation risk for extant prey (i.e., coexistence conservation). We propose 'Mini Safe Havens' (MSHs) as a potential tool for managing these threats. Mini Safe Havens are refuges that are permanently permeable to the focal species; allowing the emigration of individuals while maintaining gene flow through the boundary. Crucial to the effectiveness of the approach is the ongoing maintenance and monitoring required to preserve a low-to-zero risk of key threats within the MSH; facilitating in-situ learning and adaptation by focal species to these threats, at a rate and intensity of exposure determined by the animals themselves. We trialled the MSH approach for a pilot reintroduction of the Australian native New Holland mouse (Pseudomys novaehollandiae), in the context of a trophic rewilding project to address potential naïveté to a reintroduced native mammalian predator. We found that mice released into a MSH maintained their weight and continued to use the release site beyond 17 months (525 days) post-release. In contrast, individuals in temporary soft-release enclosures tended to lose weight and became undetectable approximately 1-month post-release. We discuss the broad applicability of MSHs for population recovery and reintroductions 'beyond-the-fence' and recommend avenues for further refinement of the approach. Supplementary Information: The online version contains supplementary material available at 10.1007/s10531-022-02495-6.

The Challenges of Predicting Drug-Induced QTc Prolongation in Humans.

The content of this article derives from a Health and Environmental Sciences Institute (HESI) consortium with a focus to improve cardiac safety during drug development. A detailed literature review was conducted to evaluate the concordance between nonclinical repolarization assays and the clinical thorough QT (TQT) study. Food and Drug Administration and HESI developed a joint database of nonclinical and clinical data, and a retrospective analysis of 150 anonymized drug candidates was reviewed to compare the performance of 3 standard nonclinical assays with clinical TQT study findings as well as investigate mechanism(s) potentially responsible for apparent discrepancies identified. The nonclinical assays were functional (IKr) current block (Human ether-a-go-go related gene), action potential duration, and corrected QT interval in animals (in vivo corrected QT). Although these nonclinical assays demonstrated good specificity for predicting negative clinical QT prolongation, they had relatively poor sensitivity for predicting positive clinical QT prolongation. After review, 28 discordant TQT-positive drugs were identified. This article provides an overview of direct and indirect mechanisms responsible for QT prolongation and theoretical reasons for lack of concordance between clinical TQT studies and nonclinical assays. We examine 6 specific and discordant TQT-positive drugs as case examples. These were derived from the unique HESI/Food and Drug Administration database. We would like to emphasize some reasons for discordant data including, insufficient or inadequate nonclinical data, effects of the drug on other cardiac ion channels, and indirect and/or nonelectrophysiological effects of drugs, including altered heart rate. We also outline best practices that were developed based upon our evaluation.

Ongoing declines of woodland birds: Are restoration plantings making a difference?

Woodland birds are a species assemblage of conservation concern, and their persistence in fragmented agricultural landscapes is dependent on both the preservation of existing woodland remnants and the implementation of restoration plantings. However, little is known about the habitat-use and persistence of birds in fragmented agricultural landscapes. We present a detailed, population-oriented study of woodland birds in temperate eucalypt woodland restoration plantings and remnant woodland patches in the South-west Slopes bioregion of New South Wales, Australia. First, we undertook a 3-yr mark-recapture project to assess annual survival and site fidelity in restoration plantings and woodland remnants. We supplemented our recapture efforts with resightings of color-banded individuals. Second, we tracked individual birds of two species, Superb Fairywren (Malurus cyaneus) and Willie Wagtail (Rhipidura leucophrys), and documented snapshots of their home ranges and movement patterns during the breeding season. Annual survival in the woodland bird assemblage was lower than expected (51%). Home ranges of the Superb Fairywren were positively correlated with patch size, and were constrained by patch edges in linear sites. Superb Fairywrens and Willie Wagtails were more likely to travel longer distances between substrates while foraging in linear sites. Willie Wagtails engaged in significant gap-crossing (up to 400 m) between adjacent habitat patches. Our findings indicate that (1) patch isolation and certain patch configurations place resident birds at an energetic disadvantage, and (2) in our study area, woodland bird populations are continuing to decline. We recommend landscape-scale habitat restoration programs aim to address ongoing population declines. Studies such as ours conducted over longer time periods would provide a deeper understanding of habitat use and population processes of woodland birds in fragmented agricultural landscapes.

Can We Panelize Seizure?

Seizure liability remains a significant cause of attrition in drug discovery and development, leading to loss of competitiveness, delays, and increased costs. Current detection methods rely on observations made in in vivo studies intended to support clinical trials, such as tremors or other abnormal movements. These signs could be missed or misinterpreted; thus, definitive confirmation of drug-induced seizure requires a follow-up electroencephalogram study. There has been progress in in vivo detection of seizure using automated video systems that record and analyze animal movements. Nonetheless, it would be preferable to have earlier prediction of seizurogenic risk that could be used to eliminate liabilities early in discovery while there are options for medicinal chemists making potential new drugs. Attrition due to cardiac adverse events has benefited from routine early screening; could we reduce attrition due to seizure using a similar approach? Specifically, microelectrode arrays could be used to detect potential seizurogenic signals in stem-cell-derived neurons. In addition, there is clear evidence implicating neuronal voltage-gated and ligand-gated ion channels, GPCRs and transporters in seizure. Interactions with surrounding glial cells during states of stress or inflammation can also modulate ion channel function in neurons, adding to the challenge of seizure prediction. It is timely to evaluate the opportunity to develop an in vitro assessment of seizure linked to a panel of ion channel assays that predict seizure, with the aim of influencing structure-activity relationship at the design stage and eliminating compounds predicted to be associated with pro-seizurogenic state.

Use of the ZDF rat to model dietary fat induced hypercoagulability is limited by progressive and fatal nephropathy.

INTRODUCTION: Zucker diabetic fatty (ZDF) rats are used widely as an animal model of metabolic syndrome and insulin resistance. Our study focused on the effects of high versus low dietary fat on the development of Type 2 diabetes in obese male ZDF rats (fa/fa), including biomarkers to detect early signs of hypercoagulability and vascular injury in the absence of overt thrombosis. METHODS: In this study, male (5/group) 10-week-old CRL:ZDF370(obese) rats were fed low (LFD, 16.7% fat) or high fat (HFD, 60% fat) diet for 12 or 15 weeks. Cohorts of 5 rats within diet groups were scheduled for sample collection after weeks 12 and 15. RESULTS: HFD-fed ZDF rats had oily coats, lower rates of food consumption, more accelerated weight gain and increased serum cholesterol (+15%) and triglyceride concentrations (+75%) vs. LFD-fed ZDF rats. Urinary ketones were observed only in HFD-fed ZDF rats and greater urine glucose and protein concentrations in HFD-fed ZDF vs. LFD-fed ZDF rats were seen. Hemostasis testing showed ~2-fold greater fibrinogen concentration, increased von Willebrand factor concentration, and high thrombin generation in HFD-fed ZDF vs LFD-fed ZDF rats. Increased mortality in the HFD-fed ZDF rat was attributed to exacerbations of altered carbohydrate metabolism as evidenced by ketonuria and nephropathy leading to renal failure. DISCUSSION: This characterization shows that the ZDF rat at the age, sex and weight used in this study is highly sensitive to dietary fat content that can exacerbate prothrombotic, metabolic and renal disturbances and increase mortality.

Repolarization studies using human stem cell-derived cardiomyocytes: Validation studies and best practice recommendations.

Human stem cell-derived cardiomyocytes (hSC-CMs) hold great promise as in vitro models to study the electrophysiological effects of novel drug candidates on human ventricular repolarization. Two recent large validation studies have demonstrated the ability of hSC-CMs to detect drug-induced delayed repolarization and "cellrhythmias" (interrupted repolarization or irregular spontaneous beating of myocytes) linked to Torsade-de-Pointes proarrhythmic risk. These (and other) studies have also revealed variability of electrophysiological responses attributable to differences in experimental approaches and experimenter, protocols, technology platforms used, and pharmacologic sensitivity of different human-derived models. Thus, when evaluating drug-induced repolarization effects, there is a need to consider 1) the advantages and disadvantages of different approaches, 2) the need for robust functional characterization of hSC-CM preparations to define "fit for purpose" applications, and 3) adopting standardized best practices to guide future studies with evolving hSC-CM preparations. Examples provided and suggested best practices are instructional in defining consistent, reproducible, and interpretable "fit for purpose" hSC-CM-based applications. Implementation of best practices should enhance the clinical translation of hSC-CM-based cell and tissue preparations in drug safety evaluations and support their growing role in regulatory filings.

Publisher Correction: Cross-site and cross-platform variability of automated patch clamp assessments of drug effects on human cardiac currents in recombinant cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Echocardiographic and hemodynamic indices of myocardial contractility simultaneously evaluated in telemetered beagle dogs: A HESI-sponsored cross-company evaluation.

INTRODUCTION: Alterations in cardiac contractility can have significant clinical implications, highlighting the need for early detection of potential liabilities. Pre-clinical methods to assess contractility are typically invasive and their translation to human measures of cardiac function are not well defined. Clinically, cardiac function is most often measured non-invasively using echocardiography. The objective of these studies was to introduce echocardiography into standard large animal cardiovascular safety pharmacology studies and determine the feasibility of this combination. METHODS: A consortia of laboratories combined their data sets for evaluation. At each site, telemetered beagle dogs, in a 4 × 4 Latin square crossover study design (n = 4), were administered either pimobendan (positive inotrope) or atenolol (negative inotrope) orally at clinically relevant dose levels. Standard telemetry parameters were collected (heart rate, mean arterial blood pressure, etc.) continuously over 24 h, as well as derived contractility endpoints: QA interval and LV +dP/dtmax. At Tmax, echocardiography was performed in conscious dogs with minimal restraint to collect contractility parameters: ejection fraction (EF) and fractional shortening (FS). RESULTS: Correlations between telemetry and echo contractility endpoints showed that, in general, a change in LV +dP/dtmax of 1000 mmHg/s translates to a 5.2% change in EF and a 4.2% change in FS. Poor correlations were shown between QA interval derived simultaneously, to both EF and FS. DISCUSSION: Comparing data from telemetry-only groups to those that included echocardiography collections showed no effect in the ability to interpret test article-related effects, providing the foundation for the inclusion of echocardiography without compromising standard telemetry data quality.

Cross-site and cross-platform variability of automated patch clamp assessments of drug effects on human cardiac currents in recombinant cells.

Automated patch clamp (APC) instruments enable efficient evaluation of electrophysiologic effects of drugs on human cardiac currents in heterologous expression systems. Differences in experimental protocols, instruments, and dissimilar site procedures affect the variability of IC50 values characterizing drug block potency. This impacts the utility of APC platforms for assessing a drug's cardiac safety margin. We determined variability of APC data from multiple sites that measured blocking potency of 12 blinded drugs (with different levels of proarrhythmic risk) against four human cardiac currents (hERG [IKr], hCav1.2 [L-Type ICa], peak hNav1.5, [Peak INa], late hNav1.5 [Late INa]) with recommended protocols (to minimize variance) using five APC platforms across 17 sites. IC50 variability (25/75 percentiles) differed for drugs and currents (e.g., 10.4-fold for dofetilide block of hERG current and 4-fold for mexiletine block of hNav1.5 current). Within-platform variance predominated for 4 of 12 hERG blocking drugs and 4 of 6 hNav1.5 blocking drugs. hERG and hNav1.5 block. Bland-Altman plots depicted varying agreement across APC platforms. A follow-up survey suggested multiple sources of experimental variability that could be further minimized by stricter adherence to standard protocols. Adoption of best practices would ensure less variable APC datasets and improved safety margins and proarrhythmic risk assessments.

Revegetation and reproduction: do restoration plantings in agricultural landscapes support breeding populations of woodland birds?

Restoration plantings are frequently occupied by native wildlife, but little is known about how planting attributes influence breeding by, and persistence of, fauna populations. We monitored breeding success of woodland birds in restoration plantings in a fragmented agricultural landscape in south-eastern Australia. We documented nest fate and daily nest survival (DSR) in plantings and remnant woodland sites. We analysed the influence on breeding success of patch attributes (size, shape, type) compared to other potentially influential predictors such as nest-site and microhabitat variables. We found that, in general, patch attributes did not play a significant role in determining breeding success for woodland birds. However, we examined a subset of species of conservation concern, and found higher DSR for these species in restoration plantings than in similarly sized woodland remnants. We also found negative effects of patch size and linearity on DSR in species of conservation concern. The primary cause of nest failure was predation (91%). We used camera trap imagery to identify the most common nest predators in our study sites: native predatory bird species, and the introduced red fox (Vulpes vulpes). Our findings are further evidence of the value of restoration plantings and small habitat patches for bird populations in fragmented agricultural landscapes. We recommend controlling for foxes to maximise the likelihood that restoration plantings and other woodland patches in Australia support breeding populations of woodland birds. More broadly, our study highlights the importance of taking a detailed, population-oriented approach to understanding factors that influence habitat suitability for fauna of conservation concern.

Considerations for an In Vitro, Cell-Based Testing Platform for Detection of Drug-Induced Inotropic Effects in Early Drug Development. Part 2: Designing and Fabricating Microsystems for Assaying Cardiac Contractility With Physiological Relevance Using Human iPSC-Cardiomyocytes.

Contractility of the myocardium engines the pumping function of the heart and is enabled by the collective contractile activity of its muscle cells: cardiomyocytes. The effects of drugs on the contractility of human cardiomyocytes in vitro can provide mechanistic insight that can support the prediction of clinical cardiac drug effects early in drug development. Cardiomyocytes differentiated from human-induced pluripotent stem cells have high potential for overcoming the current limitations of contractility assays because they attach easily to extracellular materials and last long in culture, while having human- and patient-specific properties. Under these conditions, contractility measurements can be non-destructive and minimally invasive, which allow assaying sub-chronic effects of drugs. For this purpose, the function of cardiomyocytes in vitro must reflect physiological settings, which is not observed in cultured cardiomyocytes derived from induced pluripotent stem cells because of the fetal-like properties of their contractile machinery. Primary cardiomyocytes or tissues of human origin fully represent physiological cellular properties, but are not easily available, do not last long in culture, and do not attach easily to force sensors or mechanical actuators. Microengineered cellular systems with a more mature contractile function have been developed in the last 5 years to overcome this limitation of stem cell-derived cardiomyocytes, while simultaneously measuring contractile endpoints with integrated force sensors/actuators and image-based techniques. Known effects of engineered microenvironments on the maturity of cardiomyocyte contractility have also been discovered in the development of these systems. Based on these discoveries, we review here design criteria of microengineered platforms of cardiomyocytes derived from pluripotent stem cells for measuring contractility with higher physiological relevance. These criteria involve the use of electromechanical, chemical and morphological cues, co-culture of different cell types, and three-dimensional cellular microenvironments. We further discuss the use and the current challenges for developing and improving these novel technologies for predicting clinical effects of drugs based on contractility measurements with cardiomyocytes differentiated from induced pluripotent stem cells. Future research should establish contexts of use in drug development for novel contractility assays with stem cell-derived cardiomyocytes.

Considerations for an In Vitro, Cell-Based Testing Platform for Detection of Adverse Drug-Induced Inotropic Effects in Early Drug Development. Part 1: General Considerations for Development of Novel Testing Platforms.

Drug-induced effects on cardiac contractility can be assessed through the measurement of the maximal rate of pressure increase in the left ventricle (LVdP/dtmax) in conscious animals, and such studies are often conducted at the late stage of preclinical drug development. Detection of such effects earlier in drug research using simpler, in vitro test systems would be a valuable addition to our strategies for identifying the best possible drug development candidates. Thus, testing platforms with reasonably high throughput, and affordable costs would be helpful for early screening purposes. There may also be utility for testing platforms that provide mechanistic information about how a given drug affects cardiac contractility. Finally, there could be in vitro testing platforms that could ultimately contribute to the regulatory safety package of a new drug. The characteristics needed for a successful cell or tissue-based testing platform for cardiac contractility will be dictated by its intended use. In this article, general considerations are presented with the intent of guiding the development of new testing platforms that will find utility in drug research and development. In the following article (part 2), specific aspects of using human-induced stem cell-derived cardiomyocytes for this purpose are addressed.

Electrophysiological characterization of drug response in hSC-derived cardiomyocytes using voltage-sensitive optical platforms.

INTRODUCTION: Voltage-sensitive optical (VSO) sensors offer a minimally invasive method to study the time course of repolarization of the cardiac action potential (AP). This Comprehensive in vitro Proarrhythmia Assay (CiPA) cross-platform study investigates protocol design and measurement variability of VSO sensors for preclinical cardiac electrophysiology assays. METHODS: Three commercial and one academic laboratory completed a limited study of the effects of 8 blinded compounds on the electrophysiology of 2 commercial lines of human induced pluripotent stem-cell derived cardiomyocytes (hSC-CMs). Acquisition technologies included CMOS camera and photometry; fluorescent voltage sensors included di-4-ANEPPS, FluoVolt and genetically encoded QuasAr2. The experimental protocol was standardized with respect to cell lines, plating and maintenance media, blinded compounds, and action potential parameters measured. Serum-free media was used to study the action of drugs, but the exact composition and the protocols for cell preparation and drug additions varied among sites. RESULTS: Baseline AP waveforms differed across platforms and between cell types. Despite these differences, the relative responses to four selective ion channel blockers (E-4031, nifedipine, mexiletine, and JNJ 303 blocking IKr, ICaL, INa, and IKs, respectively) were similar across all platforms and cell lines although the absolute changes differed. Similarly, four mixed ion channel blockers (flecainide, moxifloxacin, quinidine, and ranolazine) had comparable effects in all platforms. Differences in repolarisation time course and response to drugs could be attributed to cell type and experimental method differences such as composition of the assay media, stimulated versus spontaneous activity, and single versus cumulative compound addition. DISCUSSION: In conclusion, VSOs represent a powerful and appropriate method to assess the electrophysiological effects of drugs on iPSC-CMs for the evaluation of proarrhythmic risk. Protocol considerations and recommendations are provided toward standardizing conditions to reduce variability of baseline AP waveform characteristics and drug responses.