Assessing The Impact Of Drug Treatment On Cardiomyocyte Function
Application NoteCell AnalysisAssessing the Impact of DrugTreatment on Cardiomyocyte FunctionThrough combined analysis of contractility, metabolicflux, and cellular oxygenationiPS Cardiomyocyte Contractility:Cells cultured on RTCA E-Plate Cardio 96Measured on xCELLigence RTCA CardioAllows interrogation of ContractilityCell Metabolism:Cells cultured on E-Plate Cardio 96Measured on TRF Fluorescence Plate ReaderAgilent assays monitor mitochondrial function(MitoXpress Xtra), glycolytic flux (pH-Xtra) andcellular oxygenation (MitoXpress Intra).Workflow Integration:Allows measurement on E-Plates such thatmetabolism and contractility can be measuredsequentially on the same test plate.AuthorsRyan McGarrigle, Conn Carey,and James HynesAgilent Technologies, Inc.AbstractIn this application note, we demonstrate the feasibility of combiningmicroelectrode-based iPS cardiomyocyte contractility measurements with amicroplate-based bioenergetics assessment to better characterize cellularresponses to drug treatment. Contractility was assessed on 96-well E-PlateCardio 96 using the Agilent xCELLigence RTCA Cardio system while cell metabolismwas measured on the same E-plate using a multiplexed fluorometric measurementof O2 consumption with Agilent MitoXpress Xtra, glycolytic flux with Agilent pH Xtra,and cellular oxygenation using Agilent MitoXpress Intra.
IntroductionCardiotoxicity and related cardiac impairment remain oneof the main reasons for both drug withdrawal1 and FDAblack box warning2 and are a significant cause of compoundattrition in preclinical development. In vitro assays are capableof better characterizing cardiac response to drug treatmentsand are therefore of significant importance to better predictsuch adverse effects in vivo.Cardiac tissue requires an uninterrupted supply of respiratorysubstrates to meet the very high ATP demand imposed bycontinuous beating. Over 95% of this ATP is generated byoxidative phosphorylation (OXPHOS) with the necessarymitochondrial network taking up approximately one-thirdof cardiomyocyte cell volume. Energy starvation andmitochondrial dysfunction are therefore significant factorsin the progression of cardiotoxicity and so detection of suchmetabolic dysfunction is an important aspect of cardiotoxicityscreening. This detection is best achieved by monitoring thetwo main ATP generating processes, OXPHOS and glycolysis.In vivo, the most important respiratory substrates forATP production are pyruvate and fatty acyl CoA, however,cardiomyocyte metabolism is particularly adaptableand substrates such as amino acids, lactate, and ketonebodies can also be used. Examples of this adaptabilityinclude hypoxia inducible factor (HIF) mediated metabolicresponses to hypoxia and ischemia and the shift from fattyacid oxidation (FAO) to glucose metabolism that occursin hypertrophic cardiac tissue. These adaptions highlightthe importance of information on substrate preferenceand oxygenation when designing and interpreting in vitrocardiomyocyte analyses.As cardiac contraction is the main ATP consumer, thecoupling of contractility to ATP production, and by extension,mitochondrial activity, is critically important to normalcardiomyocyte function, particularly as the mitochondrialreticulum also regulates intracellular calcium homeostasisand a multitude of critical signally pathways. The ability torelate cardiomyocyte beating to alter metabolic activity wouldtherefore be of significant utility.Figure 1. A simplified schematic of the inter-relationship between cardiomyocyte metabolism and beating activity. OXPHOS produces most of the ATP needed,with pyruvate and Acyl CoA being the main respiratory substrates. By measuring beating, OXPHOS (via O2 consumption), glycolytic flux (via extracellularacidification), and cellular oxygenation a more complete picture of cardiomyocyte function can be established.2
Mitochondrial dysfunction and contractilityacidification as a result of energy production. Both reagentscan be measured using dual-read TR-F (time resolvedfluorescence) detection.3 This allows measurement onE-Plates such that, if necessary, metabolism and contractilitycan be measured sequentially on the same test plate.Furthermore, cellular oxygenation measurements with theAgilent MitoXpress Intra intracellular oxygen assay canbe conducted between xCELLigence RTCA time points (inparallel but on plate reader platform) if desired.Contractility is measured by culturing iPS cardiomyocyteson E-Plate Cardio 96 and measuring them on the AgilentxCELLigence RTCA Cardio system in real time. The E-Platehas interdigitated impedance (IMP) microelectrode arrayson the bottom of each well. IMP electrodes measurecellular impedance, which is affected by the number of cellscovering the electrode, the morphology of the cells, andthe degree of the cell attachment. The fast sampling rateof IMP measurement (12.9 ms/77 Hz) allows capturingtemporal rhythmic changes in cell morphology and degreeof cell attachment to the plate associated with contractionof cardiomyocytes. Therefore, the Cardio system is used topredict drug-induced proarrhythmia, contractile liability, andchronic toxicity of drugs under development.Results and discussioniPS cardiomyocytes maintain beat rates in the presence ofmitochondrial inhibitors.To assess the effects of metabolism on beat rate,cardiomyocytes were treated with mitochondrial modulatorson an E-Plate. Beat rates were assessed 0.5 and 24 hourspost-treatment (Figure 2A). Interestingly 1 µM FCCPinfluenced the beat rate at both time points suggesting thatcardiomyocytes cannot recover following mitochondrialuncoupling (Figure 2A). Lower concentrations did not reducethe beat rate.Cell metabolism is measured using the Agilent MitoXpressXtra oxygen consumption assay to assess mitochondrialfunction and the Agilent pH-Xtra glycolysis assay, whichuses extracellular acidification (ECA) to assess glycolyticfunction. Soluble metabolic sensor reagents show a changein fluorescence signal in response to changes in oxygen orMetabolism Testing0.5 h post treatment30s30sO 2 ConsumptionGlycolytic Flux (ECA)ATP50024 h post-treatmentVehicleFCCP600BContractility Testing% EffectA1 µM4003002001000.1 µM00.01 µMDMSO(1 µM)Antimycin A(1 µM)Rotenone(1 µM)FCCP(2.5 µM)10 µM0.1 µMVehicleRotenoneC1 µM1 µM100 nM10 nM350900O2 Consumption300 GlycolyticFlux (ECA)Baseline800600200500150400300100200500O2 ConsumptionGlycolytic Flux (ECA)Baseline700250% EffectAntimycinVehicle10000.5Antimycin (µM)100510FCCP (µM)Figure 2. The impact of mitochondrial impairment on cardiomyocyte beating. Beating is maintained in the presence of mitochondrial inhibitors through increasedglycolytic ATP supply. 30 s xCELLigence traces at 0.5 and 24 hours post-treatment (A). O2 consumption, extracellular acidification, and ATP were measured atfixed concentrations (B). O2 consumption, extracellular acidification dose responses for antimycin (C) and FCCP (D). Data presented relative to untreated control.3
Measuring oxygen consumption rates using MitoXpressXtra confirmed that antimycin A and rotenone decreasemitochondrial respiration as oxygen consumption decreasesacutely upon treatment (Figure 2B). FCCP was shown toincrease oxygen consumption but as mitochondria areuncoupled, they are unable to generate ATP (Figure 2B).Analysis of the extracellular acidification using pH-Xtraglycolysis assay shows that when mitochondria are inhibitedor uncoupled, glycolysis is increased (Figure 2B). There isa clear concentration-dependent increase in acidification(Figure 2C) suggesting that ATP depletion is amelioratedthrough increased glycolysis in cardiomyocytes.Together, this suggests that increased glycolysis suppliesthe cells with enough ATP to facilitate cardiomyocytebeating despite the lack of mitochondrial ATP fromoxidative phosphorylation. This is consistent with previousobservations on specific cell lines.4Cell metabolism is tightly coupled to contractile activityThe β-adrenoreceptor agonist, isoproterenol is used for thetreatment of bradycardia (slow heart rate). Figure 3A showsbeat rate traces of cardiomyocytes using the xCELLigenceRTCA, treatment with isoproterenol increased the beat rateby 45% compared to control 30 minutes post drug addition(Figure 3A). Isoproterenol also caused a similar increase inoxygen consumption (Figure 3B).These data suggest that when the beat rate is elevated,the increased ATP demand is met by increasing aerobicATP production through mitochondrial respiration(Figure 3B). An antimycin A control was included to measurenon-mitochondrial oxygen consumption. Acidification ratesdid not increase (data not shown) suggesting that OXPHOSrather than glycolysis is supplying the additional ATP required.Changes in cellular oxygenation were measured usingMitoXpress Intra. Figure 4 demonstrates that untreatedcardiomyocytes under these conditions experience 14%oxygen, 7% less than ambient oxygen due to respirationand other non-mitochondrial background oxygen-consumingprocesses. When cells are treated with antimycin A,experienced oxygen increases to around ambient levels( 21%) as aerobic ATP production has been inhibited.4Conversely, cells treated with isoproterenol were shownto have an increased beat rate, and therefore oxygenconsumption experience as low as 6% oxygen as a result ofthe increased oxygen consumption. This causes a significantbut temporary reduction in oxygen availability with values of 6% observed for 15 minutes despite cells being culturedand measured at 21% O2.AUntreatedIsoproterenol (1µM)BIsoproterenol (1µM)34MitoXpress Xtra (µs)Inhibitory concentrations of antimycin A and rotenone (1 µM)did not have a significant impact on beat rates at both timepoints (Figure 2A). This suggests that cardiomyocytes canstill generate ATP. High concentrations of antimycin A didreduce beat rates after 24 hours.323028Untreated2624Antimycin A222030405070608090Time (min)Figure 3. Impact of isoproterenol on cardiomyocyte beat rate measuredon an Agilent xCELLigence RTCA Cardio system (A) and cardiomyocytemetabolism (B) measured on an advanced TR-F detection compatiblefluorescence plate reader. Increased oxygen consumption caused morerapid oxygen depletion.Compound treatment% O220Reduction inintracellular O2driven byrespirationAntimycin (1µM)1816Untreated14Further O2depletioncaused byIsoproterenoltreatment121086Isoproterenol (1µM)102030405060TimeFigure 4. Impact of isoproterenol on cardiomyocyte oxygenationmeasured using advanced TR-F detection fluorescence plate reader withatmospheric control.
Contractility can be perturbed using several compoundssuch as nifedipine or E-4031. Nifedipine is used to treatand manage angina, high blood pressure, and several otherconditions, it acts as an L-type Ca2 channel antagonist.Figure 5A demonstrates the dose-dependent effects ofnifedipine on contractile force, while Figure 5B illustrates adose-dependent decrease in cardiomyocyte O2 consumption.Extracellular acidification was also reduced (data not shown).The hERG channel inhibitor E-4031 causes an irregular beatrate pattern (Figure 6A), which also causes a decrease inoxygen consumption and a minor decrease in acidificationrates (Figure 6B). Suggesting that with a decrease inATP demand the cell responds by decreasing both ATPgenerating pathways.A20sBNifedipineVehicle10 nM25 nM100 nM0.25 µM604000.011100[Nifedipine] (nM)Figure 5. The impact of nifedipine on the beat rate (A) and metabolism(B). Beating was measured 30 minutes post-treatment. A range ofconcentrations from 10 nM to 1 µM were assayed. Metabolism datapresented as oxygen consumption rate as a percentage of untreated control.B1.0 µM10080% EffectVehicleFresh media containing the MitoXpress Xtra reagent,150 µL/well was added before measurement. Compoundswere added directly, then all wells were sealed withprewarmed HS oil. Plates were measured kinetically for2.5 to 3.0 hours at 37 C (Ex 380 nm, Em 650 nm, andAdvanced dual-read TR-F plate reader detection)The sample plate is placed in CO2 free incubator 3 hoursbefore measurement, to remove CO2. Samples were washedthree times using respiration buffer (1 mM phosphate)prepared using the buffer tablet provided. 150 µL ofrespiration buffer containing the pH-Xtra reagent was addedto sample wells. Compounds were added directly, and theplate was measured kinetically for 2.5 hours at 37 C (Ex380 nm, Em 615 nm, and Advanced dual-read TR-F platereader detection).20E-4031Oxygen consumption assayGlycolysis assay1.0 µM40sInduced pluripotent stem cells cardiomyocytes were suppliedby NCARDIA. Cells were plated onto fibronectin-coatedE-Plate Cardio 96 and placed in culture for 2 to 3 days,performing media changes as per the manufacturer’sinstructions. Cells were plated at 4 to 5 104 cells/well forpH-Xtra and MitoXpress-Xtra assays.1000.5 µMACell culture12080% Effect50 nMMaterials and methods6040200O 2 Consumption Glycolytic Flux(ECA)Figure 6. The impact of E-4031 on the beat rate (A) and metabolism (B).Beating was measured 30 minutes post-treatment. A single concentration1 µM of E-4031 was used. Metabolism data presented as oxygenconsumption rate and ECA as a percentage of untreated control.Cellular oxygenation assayCells were loaded with MitoXpress-Intra reagentovernight (14 hours) in a E-Plate Cardio 96 the day beforemeasurement. Cells were washed twice and 150 µL of freshmedia was added. The plate was measured kinetically at37 C. (Ex 380 nm, Em 650 nm, and Advanced dual-read TR-Fplate reader detection).Contractile assayiPS-cardiomyocytes were plated on 96 well E-Plates andimpedance measurements were recorded at selected timepoints (60 seconds sweep at a sampling rate of 77 Hz). Drugtreatment was initiated once the culture showed 40 to 60synchronic beats/min. The data were normalized to baseline.5
ConclusionThe combination of Agilent MitoXpress Xtra, MitoXpress Intra,and pH-Xtra metabolic assays with the xCELLigence RTCACardio system and E-Plate Cardio 96 enabled the sequentialmeasurement of metabolism and contractility from thesame sample using the same plate. Using the dual-read TR-Fmeasurement approach on conventional TR-F plate readersinforms on oxygen consumption and ECA. The combineduse of microplate-based contractility and metabolismmeasurements has been demonstrated to generate amore complete picture of cardiomyocyte response to drugtreatment and allows the delineation of inter-relationshipsbetween cardiomyocyte beating and the underlyingbioenergetic processes. This multiparametric workflow helpsto improve data density per well of sample.Complete impairment of OXPHOS through treatment withelectron transport inhibitors did not immediately impaircardiomyocyte beating. Increased ECA suggests that ATPsupply is maintained through increased glycolytic fluxallowing beating to continue for 24 hours post-treatment.The β-adrenoreceptor agonist isoproterenol increased beatrate and caused a significant increase in O2 consumptionbut little change in ECA. This suggests that increased ATPdemand is being met through OXPHOS rather than glycolysis.The L-type Ca2 channel antagonist nifedipine reducedcontractile force and caused a dose-dependent reductionin both oxygen consumption and ECA, indicative of reducedOXPHOS and glycolytic activity in response to treatment.This combined analysis of critical cardiomyocyte functionstherefore delivers a more holistic and informative in vitrocardiotoxicity screen in that it related cellular function tothe metabolic activity driving that function. In so doing,it provides additional mechanistic information as to thecause of observed alterations in cardiomyocyte metabolismor contractility.www.agilent.com/chemFor Research Use Only. Not for use in diagnostic procedures.DE44202.2461458333This information is subject to change without notice. Agilent Technologies, Inc. 2021Printed in the USA, February 1, 20215994-2987ENThese highly informative workflows allow users to interrogatemetabolic modulators of cardiomyocyte function. As betterin vitro cardiac models are developed, knowing the metabolicphenotype is essential to ensure that assays appropriatelyreflect mature cardiomyocyte biology. Reliance on glycolysisor OXPHOS shapes how these cells will respond to drugsand how they will survive in environments that they can beexposed to such as nutrient deprivation or hypoxia. Theseworkflows allow for assessment of contractility followed bymetabolic interrogation with the same biomaterial withouthaving to re-plate or potentially differentiate additionalcardiomyocytes for parallel measurements. This saves oncell consumption while improving data density and deliveringmultiparameter outputs from single samples. The flexibility ofthese workflows makes them well-positioned to characterizeboth metabolism and cardiomyocyte function under a rangeof conditions including drug screening, nutrient deprivation,hypoxia, and ischemia/reperfusion. Integrating these AgilentCell Analysis technologies offers a complete solution forassessing cardio-metabolism.References1. Lawrence, C. L. et al. In Vitro Models of Proarrhythmia. Br.J. Pharmacol. 2008 Aug, 154(7), 1516–22. Dykens J. A.; Will Y. The Significance of MitochondrialToxicity Testing in Drug Development. Drug Discov. Today2007 Sep, 12(17–18), 777–85.3. Hynes J. et al. A High-Throughput Dual Parameter Assayfor Assessing Drug-Induced Mitochondrial DysfunctionProvides Additional Predictivity Over Two EstablishedMitochondrial Toxicity Assays. Toxicol. In Vitro 2013 Mar,27(2), 560–9.4. Marroquin L. D. et al. Circumventing the Crabtree Effect:Replacing Media Glucose with Galactose IncreasesSusceptibility of Hepg2 Cells to Mitochondrial Toxicants.Toxicol. Sci. 2007 Jun, 97(2), 539–47.
E-Plate Cardio 96 and placed in culture for 2 to 3 days, performing media changes as per the manufacturer's instructions. Cells were plated at 4 to 5 104 cells/well for pH-Xtra and MitoXpress-Xtra assays. Oxygen consumption assay Fresh media containing the MitoXpress Xtra reagent, 150 µL/well was added before measurement. Compounds
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