Various environmental chemicals affect the beating parameters of iPSC-derived cardiomyocytes, ina concentration-response pattern with varied potencies

iPSC-derived cardiomyocytes generally recapitulate the relative risk classifications of compoundslisted under the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative

Similarity in bioactivity profiles within compound classes (e.g. carbamate pesticides) areobservable using the Toxicological Priortization Index (ToxPi)

This work demonstrates the feasibility of using an organotypic population-based human in vitro model to quantitatively assess the cardiotoxic potential of

chemicals for which little cardiotoxicity information is available.

An In Vitro Human Population Model for Screening Environmental Chemicals for the Cardiotoxicity Hazard

Sarah D. Burnett1, Alex Blanchette1, Fred Wright2, Weihsueh Chiu1, and Ivan Rusyn1

1Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX; and 2Bioinformatics Research Center, North Carolina State University, Raleigh, NC

INTRODUCTION

OBJECTIVESCONCLUSIONS

ACKNOWLEDGEMENTS

REFERENCES

RESULTS

Bioactivity Visualization with the Toxicological Prioritization Index (ToxPi)

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To characterize cardiophysiologic and phenotypic alterations in iPSC-derived cardiomyocytesfollowing exposure to environmental chemicals

To characterize variation in effects on cardiomyocytes related to chemical class and to visualizesimilarities in chemical bioactivities using the Toxicological Prioritization Index (ToxPi) software

Characterize the cardiotoxicity hazard of environmental chemicals

MATERIALS & METHODS

Ca2+ flux traces for two individuals reveal qualitative and quantitative differences in cardiophysiologic performance. First Column: Untreatedcardiomyocytes show a donor-specific regular beat pattern. Second Column: Positive inotropic effects, induced by the β-adrenergic receptor agonistisoproterenol, were observable for all individuals tested. Third Column: The in vitro phenotype associated with clinical QT-prolongation was observablewith varying characteristics across individuals following exposure to sotalol, a β-blocker/K+ channel antagonist. Fourth Column: Tetraoctylammoniumbromide (TAB) induced cell death, and no beating was observed in any individual post-treatment.

• Abdo N, Xia M, Brown CC, Kosyk O, Huang R, Sakamuru S, Zhou YH, Jack JR, Gallins P, Xia K, Li Y, Chiu WA,Motsinger-Reif A, Austin CP, Tice R, Rusyn I, Wright F. Population-based in vitro hazard and concentration-response assessment of chemicals: the 1000 genomes high-throughput screening study. Environmental HealthPerspectives, 2015.

• Ferri N, Siegl P, Corsini A, Herrmann J, Lerman A, Benghozi R. Drug attrition during pre-clinical and clinicaldevelopment: Understanding and managing drug-induced cardiotoxicity. Pharmacology & Therapeutics, 2013.

• Prüss-Ustün A, Wolf J, Corvalán C, Neville T, Bos R, Neira M. Diseases due to unhealthy environments: Anupdated estimate of the global burden of disease attributable to environmental determinants of health. Journalof Public Health, 2016.

• Sirenko O, Grimm F, Ryan K, Iwata Y, Chiu W, Parham F, Wignall J, Anson B, Cromwell E, Behl M, Rusyn I, Tice R.In vitro cardiotoxicity assessment of environmental chemicals using an organotypic human induced pluripotentstem cell-derived model. Toxicology and Applied Pharmacology, 2017.

• Burnett S, Blanchette A, Grimm F, House J, Reif D, Wright F, Chiu W, Rusyn I. Population-based toxicity screeningin human induced pluripotent stem cell-derived cardiomyocytes. Toxicology and Applied Pharmacology, 2019.

QR-CODE

This work was supported by EPA STAR grant #RD83516601, NIH T32 grant #T32-ES-026568-01, andinstitutional support from Texas A&M University.

FUTURE WORK Analyze population variability in effects on cardiomyocytes following exposure to test chemicals

Characterize and visualize chemical bioactivity profiles on a population level using ToxPi and usehierarchal clustering to determine similarities between chemicals based on their bioactivities

Observable variation in effects on the phenotype Decay To Rise Time Ratio by chemicals with different risk classifications for Torsades de pointes (TdP, aform of polymorphic ventricular tachycardia with long QT interval as a risk factor) under the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative.Plots show chemical-induced changes in normalized Decay To Rise Time Ratio with increasing dose and the corresponding POD value. More pronouncedeffects are indicated by lower POD. Similar concentration-response patterns were observed among donors. First Column: More pronounced increases inDecay To Rise Time Ratio following treatment with compounds classified as high risk under the CiPA initiative. Second Column: Less pronounced increasesor lack of effect in Decay To Rise Time Ratio following treatment with compounds classified as low risk under the CiPA initiative.

Ca2+ Flux is a Cardiophysiologic Indicator with Individual Specificity

Variation in Effects on Cardiomyocytes by CiPA Risk Classification

Observable chemical-to-chemical variation in potency for Peak Frequency (BPM). Potency is indicated by POD, with alower POD value representing higher potency. Each point represents the median POD value across 5 donors. Colorsrepresent different chemical classifications (CiPA pharmaceuticals are compounds listed under the Comprehensive in vitroProarrhythmia Assay initiative). Ibutilide, a high-risk CiPA compound, and isoproterenol, a β-adrenergic receptor agonist,induced effects on BPM at relatively low concentrations. Conversely, food constituents folic acid and clove leaf oilgenerally did not affect BPM. Various environmental chemicals affected BPM with varied potencies.

Variation in potency of effects on cardiomyocytes for Peak Frequency (BPM). Potency is indicated by POD, with a lowerPOD value representing higher potency. Box plots show the median and interquartile range of POD values across 5donors, and whiskers represent the 5th and 95th percentile confidence intervals.

Variation in Potency Ranked by Chemical Classification

A B

Cell Culture: iCell Cardiomyocytes from 5 “healthy”donors (without associated cardiovascular diseasephenotype) of both sexes and different ethnicitieswere engineered by Cellular Dynamics International(CDI) and cultured according to manufacturerprotocols using an established standard operatingprocedure for 2 weeks until synchronous beatingwas observed.

Chemical Library: Concentration-response datawere collected for a total of 1029 chemicalscomprising pharmaceuticals, pesticides, flameretardants, polycyclic aromatic hydrocarbons (PAHs),plasticizers, plastics, and food constituents, atconcentrations ranging from 0.1 to 100 μM.

In Vitro Cardiotoxicity Assay – Cardiophysiology:Following 90 minutes of chemical exposure, effects oncardiophysiology were assessed by monitoring theintracellular Ca2+ flux of synchronously contractingcardiomyocytes using the FLIPR Tetra system (MolecularDevices LLC). A data processing algorithm for improvedquantitative integration of kinetic calcium flux traces wasdeveloped in R Studio. Four parameters describingcardiophysiology were selected for further analysis: BPM,Decay To Rise Time Ratio, Peak Amplitude CV, and PeakSpacing CV.

In Vitro Cardiotoxicity Assay – High-Content Imaging:After the 90-minute cardiophysiology read, cytotoxicityscreening was performed using the ImageXPress MicroConfocal High-Content Imaging System (MolecularDevices) following staining with Hoechst 33342 andMitoTracker. Five parameters describing cytotoxicity wereselected for further analysis: Total Cells, Nuclei Intensity,Mitochondrial Area, Mitochondrial Intensity, andMitochondrial Area Divided By Total Cells.

Overview of the study design: >1000 compounds,comprising a diverse chemical space, were screened in iPSC-derived cardiomyocytes from 5 different human donors.Functional cardiophysiology measurements and high-content imaging-based cellular/mitochondrial toxicityphenotypes were used to measure observable variation incardiotoxicity hazard.

Screening Library:

>1000 Pharmaceutical and Environmental Chemicals

Concentration-response assessment

Dose-Response Profiling

60%Caucasian

Adverse effects on the cardiovascularsystem are a major liability in drugdevelopment and post-marketsurveillance, so the potential forcardiotoxicity is carefully assessed fordrug candidates

Epidemiologic data suggest that ~35% ofischaemic heart disease burden may beattributable to environmental risk

No in vivo or in vitro cardiotoxicity testsare required for non-pharmaceuticals, soenvironmental compounds are seldomtested for their cardiotoxic potential

The extent to which the chemicals incommerce and the environment maycontribute to human cardiovasculardisease morbidity is essentially unknown

Human induced pluripotent stem cell(iPSC)-derived cardiomyocytes constitutean organotypic in vitro model forcardiotoxicity testing that is clinically-relevant

iPSC-derived cardiomyocytes have beenderived from multiple individuals anddemonstrate biological relevance, highsensitivity and specificity, and consistentreproducibility of both baseline andtreatment-induced characteristics

We hypothesize that a population ofiPSC-derived cardiomyocytes from adiverse set of normal human donors canbe used to assess the potentialcardiotoxicity hazard of environmentalchemicals

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Vehicle (0.5% DMSO)

Positive Inotrope (10 µM Isoproterenol)

K+ Channel Antagonist (10 µM Sotalol)

Chemical-to-Chemical Variation in Potency

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Additional Cardiophysiology Endpoints: Peak Frequency (BPM) Decay:Rise Time Ratio

Cytotoxic Control(50 µM TAB)

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19.8 ± 0.6

Observable similarity in bioactivity profiles between 4 chemicals of the carbamate pesticide class on “standard donor”cardiomyocytes, visualized using the Toxicological Prioritization Index (ToxPi) Graphical User Interface (GUI). PODvalues were generated based on the dose-response for each of 9 phenotypes and converted into ToxPi scores, whichindicate relative bioactivity. ToxPi scores were then used to generate a pie chart for each tested chemical, with each ofthe 9 phenotypes represented by one slice. Larger slices (closer to 1) indicate higher relative bioactivity.

Chemical Class

Isoproterenol

Clove leaf oil

Folic acid

Ibutilide

Chlorpyrifos

Pyridaben

Diphenolic acid

Bisphenol A

High Risk CiPA Compounds Low Risk CiPA Compounds

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Log(Dose)

Log(Dose) Log(Dose)

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POD = 0.615 µM

POD = 0.254 µM

POD = 1.192 µM

POD = 100 µM

POD = 100 µM

POD = 8.574 µM

21.6 BPM 39.0 BPM 11.4 BPM 0 BPM

16.8 BPM 33.0 BPM 13.8 BPM 0 BPM

CarbarylAldoxycarb Indoxacarb Aldicarb oxime

BPM

Decay To RiseTime Ratio

Peak Amplitude CV

Peak SpacingCV

Total Cells

NucleiIntensity

Mitochondrial Area

Mitochondrial Intensity

Mitochondrial Area DividedBy Total Cells

0 0.5 1

ToxPi Score

Donor Pool5 individual, “healthy” donors

iPSC reprogramming and differentiation

Environmental chemical screening

Cytotoxicity Endpoints: Total Cells Nuclei Intensity Mitochondrial Area,

Mitochondrial Intensity Mitochondrial Area

Divided By Total Cells

Bioactivity Visualization

Carbamate ester: