Electrophysiology of Muscle Skeletal & Cardiac excitation & contraction Mike Clark, M.D.
IN VITRO ASSESSMENT OF EXCITATION-CONTRACTION … · Excitation-contraction coupling was then...
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• Axol hiPSC-vCMs show specific compound-relevant responses to known cardiotoxic compounds. • Impedance and EFP pharmacology shown here confirms the presence of I CaV and I Kr channels in Axol hiPSC-vCMs. • The use of a dual reading technology that is enabled on the CardioExcyte 96 system, allows for the detection of compound effects on both the contractility and electrophysiological properties of a beating network of hiPSC-vCMs. • We have shown that the CardioExcyte 96 (Nanion Technologies) system used in combination with Human iPSC-Derived Ventricular Cardiomyocytes (Axol Bioscience) is an effective system for assessing cardiac pro-arrhythmia using compounds from the CiPA validation toolbox. Introduction Human induced pluripotent stem cell-derived ventricular cardiomyocytes (hiPSC-vCMs) (Axol Bioscience) offer a physiologically relevant model for predictive toxicology screening in vitro. The CardioExcyte 96 (Nanion Technologies) is a hybrid screening instrument that simultaneously records cell contractility (impedance) and the extracellular electrical field potential (EFP) in a 96-well plate. Used in combination these tools could help predict the risk of human clinical pro-arrhythmias more accurately. Here we present data on the optimisation of hiPSC-vCMs on the CardioExcyte 96. We determined seeding parameters and identified the optimal time point for analysis. Excitation-contraction coupling was then assessed in response to three standard reference compounds from the Comprehensive in vitro Pro- arrythmia Assay (CiPA) guidelines. The three compounds tested were verapamil, a mixed ion channel blocker acting upon both L-type calcium channels (I CaV ) and potassium channels (I Kr ); nifedipine, a selective calcium channel (I CaV ) blocker; and dofetilide, a selective ion channel blocker for I Kr. Both verapamil and nifedipine exhibit low pro-arrhythmic risk whereas dofetilide is classified as a high risk pro-arrhythmic compound by the Cardiac Safety Consortium. The addition of each of these compounds altered contractility and electrical excitation in the hiPSC-vCMs. Here we have demonstrated that the CardioExcyte 96, a non-invasive, label- free, high temporal resolution tool may be used in conjunction with Axol hiPSC-vCMs to predict pro-arrythmic risk in vitro. IN VITRO ASSESSMENT OF EXCITATION-CONTRACTION COUPLING FOR PREDICTING PRO-ARRHYTHMIC RISK IN IPSC-DERIVED VENTRICULAR CARDIOMYOCYTES Priyanka Dutta-Passecker 1 , George Gibbons 1 , Krisztina Juhasz 2 , Zoe Allen 1 , Ying Shao 1 , Sonja Stölzle-Feix 2 , Corina Bot 4 , Ulrich Thomas 2 Leo Doerr 2 , Matthias Beckler 2 , Niels Fertig 2 , Yichen Shi 1 1 Axol Bioscience Ltd., Cambridge, UK, [email protected]; 2 Nanion Technologies, Munich, Germany, 3 Technical University of Munich, Munich, Germany, 4 Nanion Technologies Inc., Livingston, NJ, USA www.axolbio.com www.nanion.de Figure 3. The cardiotoxic effects of cardiac ion channel modulators on Axol hiPSC-vCMs Axol hiPSC-vCMs showed compound-relevant responses (light blue) to known cytotoxic compounds. A; DMSO control, showed no change in EFP but an increase in amplitude. B; verapamil and C; nifedipine, both reduced the impedance amplitude, increased the beat rate and reduced the EFP. D; dofetilide, a selective ion channel blocker for the rapidly activating delayed rectifier potassium channel (I Kr ), resulted in arrhythmic events typical of I Kr blockers. The CardioExcyte 96 (CE 96) is a hybrid system combining impedance and EFP recordings from the same monolayer of cells. • Recordings are performed at physiological temperature in an incubator, or using the environmental chamber. • The CardioExcyte 96 can be used in cardiac safety screening on a variety of hiPSC-CMs to assess effects of CiPA- relevant compounds. • The CardioExcyte 96 provides complementary data to other assays such as automated patch clamping. Features CE 96 Number of channels 96 Impedance and EFP measurements Pacing Cell adhesion and spreading assays Operation in standard incubator Environmental control (for use outside incubator) Temporal resolution (Imp/EFP) 1ms / 0.1ms Dedicated analysis and recording software Integration with CYBERNANO analysis Figure 1: CardioExcyte 96 sensor technology The CardioExcyte 96 combines impedance and Extracellular Field Potential (EFP) recordings to measure contractility and ion channel activity. A; The 96-well sensor plate contains gold electrodes embedded in each well. B; Manual overlay of impedance (upper trace) and EFP (lower trace) trace data recorded from an autonomously beating cardiac preparation. stimulation impedance culture chamber reference electrode sensing electrode Figure 2: Molecular and physiological characterisation of Axol hiPSC-vCMs A; Top Immunocytochemistry data showed the expression of ventricular cardiomyocyte markers (cardiac troponin-I; 93.5% expression). Data from Dr Christian Zuppinger, University of Bern and Prof Matt Daniels, University of Oxford. Bottom Western blot data confirmed that Axol hiPSC-vCMs express more cardiac troponin-T (cTnT) and α-Actinin than human skin fibroblasts (hSFs). Data from Abigail Robertson, University of Manchester. B; 87 % of Axol hiPSC-vCMs have a ventricular phenotype determined by MLC2v expression, compared to 13 % expressing atrial MLC2a (n=1). Data from Dr Christian Zuppinger, University of Bern. A. Protein marker expression B. Atrial vs. ventricular phenotype quantification Characterisation of Axol ventricular iPSC-CMs MLC2v (87%) vs MLC2a (13%) Nodal population not included A. Sensor Plate Impedance EFP B. Trace Data D. Dofetilide (I Kr ) A. DMSO control C. Nifedipine (I CaV ) B. Verapamil (I CaV & I Kr ) -5 0 5 10 15 20 0 5 10 15 Impedance / Ω Time / s -25 -20 -15 -10 -5 0 5 10 15 0.0 0.5 1.0 1.5 2.0 EFP / μV Time / s -5 0 5 10 15 20 25 0 5 10 15 Impedance / Ω Time / s -20 -10 0 10 20 30 0.0 0.5 1.0 1.5 2.0 EFP / μV Time / s -20 -15 -10 -5 0 5 10 15 20 25 30 0.0 0.5 1.0 1.5 2.0 EFP / μV Time / s -5 0 5 10 15 20 25 0 5 10 15 Impedance / Ω Time / s -5 0 5 10 15 20 25 0 5 10 15 Impedance / Ω Time / s -30 -20 -10 0 10 20 30 40 0.0 0.5 1.0 1.5 2.0 EFP/ μV Time / s Compound assessment CardioExcyte 96 technology Conclusions Materials and methods Cardiomyocyte culture: Human iPSC-Derived Ventricular Cardiomyocytes (ax2505, Axol Bioscience) were thawed on Fibronectin (ax0049)-coated plates according to the manufacturer’s protocol, and cultured for the first 24 hours in Cardiomyocyte Maintenance Medium (ax2530) + 10% FBS. The next day cells were switched to serum-free Cardiomyocyte Maintenance Medium. Thereafter, the medium was changed every 2 days. Plating and recording: After 4 days, cells were dissociated with Axol Unlock (ax0044) and re-plated at 30,000 cells per well on the CardioExcyte 96 Sensor Plate. After one week the following drug compounds were applied: dofetilide (10nM), nifedipine (100nM) and verapamil (200nM). Immunocytochemistry: Cells were fixed in 3 % PFA, permeabilised with 0.2 % Triton X-100 and blocked with BSA. Primary antibody was incubated overnight 4 ˚C, and secondary antibody coupled to Alexa Fluor ® dyes (Invitrogen) applied for 2 hours. Western blot: 30 μg protein run on 10 % SDS-PAGE gel for 70 minutes at 130 V and transferred to PVDF membrane. Membranes incubated with primary antibody overnight at 4 ˚C, washed and incubated with secondary antibody for 1 hour.
Transcript of IN VITRO ASSESSMENT OF EXCITATION-CONTRACTION … · Excitation-contraction coupling was then...
• Axol hiPSC-vCMs show specific compound-relevant responses to knowncardiotoxic compounds.
• Impedance and EFP pharmacology shown here confirms the presence of ICaVand IKr channels in Axol hiPSC-vCMs.
• The use of a dual reading technology that is enabled on the CardioExcyte 96system, allows for the detection of compound effects on both the contractilityand electrophysiological properties of a beating network of hiPSC-vCMs.
• We have shown that the CardioExcyte 96 (Nanion Technologies) systemused in combination with Human iPSC-Derived VentricularCardiomyocytes (Axol Bioscience) is an effective system for assessingcardiac pro-arrhythmia using compounds from the CiPA validation toolbox.
IntroductionHuman induced pluripotent stem cell-derived ventricular cardiomyocytes(hiPSC-vCMs) (Axol Bioscience) offer a physiologically relevant model forpredictive toxicology screening in vitro. The CardioExcyte 96 (NanionTechnologies) is a hybrid screening instrument that simultaneously recordscell contractility (impedance) and the extracellular electrical field potential(EFP) in a 96-well plate. Used in combination these tools could help predictthe risk of human clinical pro-arrhythmias more accurately.
Here we present data on the optimisation of hiPSC-vCMs on the CardioExcyte96. We determined seeding parameters and identified the optimal time pointfor analysis. Excitation-contraction coupling was then assessed in responseto three standard reference compounds from the Comprehensive in vitro Pro-arrythmia Assay (CiPA) guidelines. The three compounds tested wereverapamil, a mixed ion channel blocker acting upon both L-type calciumchannels (ICaV) and potassium channels (IKr); nifedipine, a selective calciumchannel (ICaV) blocker; and dofetilide, a selective ion channel blocker for IKr.Both verapamil and nifedipine exhibit low pro-arrhythmic risk whereasdofetilide is classified as a high risk pro-arrhythmic compound by the CardiacSafety Consortium. The addition of each of these compounds alteredcontractility and electrical excitation in the hiPSC-vCMs.
Here we have demonstrated that the CardioExcyte 96, a non-invasive, label-free, high temporal resolution tool may be used in conjunction with AxolhiPSC-vCMs to predict pro-arrythmic risk in vitro.
IN VITRO ASSESSMENT OF EXCITATION-CONTRACTION COUPLING FOR PREDICTING PRO-ARRHYTHMICRISK IN IPSC-DERIVED VENTRICULAR CARDIOMYOCYTES Priyanka Dutta-Passecker1, George Gibbons1, Krisztina Juhasz2, Zoe Allen1, Ying Shao1, Sonja Stölzle-Feix2, Corina Bot4, Ulrich Thomas2 Leo Doerr2, Matthias Beckler2, Niels Fertig2, Yichen Shi11Axol Bioscience Ltd., Cambridge, UK, [email protected]; 2Nanion Technologies, Munich, Germany, 3Technical University of Munich, Munich, Germany, 4Nanion Technologies Inc., Livingston, NJ, USA
www.axolbio.comwww.nanion.de
Figure 3. The cardiotoxic effects of cardiac ion channel modulators on Axol hiPSC-vCMsAxol hiPSC-vCMs showed compound-relevant responses (light blue) to known cytotoxic compounds. A; DMSO control,showed no change in EFP but an increase in amplitude. B; verapamil and C; nifedipine, both reduced the impedanceamplitude, increased the beat rate and reduced the EFP. D; dofetilide, a selective ion channel blocker for the rapidlyactivating delayed rectifier potassium channel (IKr), resulted in arrhythmic events typical of IKr blockers.
The CardioExcyte 96 (CE 96) is a hybridsystem combining impedance and EFPrecordings from the same monolayer ofcells.• Recordings are performed at
physiological temperature in an incubator, or using the environmental chamber.
• The CardioExcyte 96 can be used in cardiac safety screening on a variety of hiPSC-CMs to assess effects of CiPA-relevant compounds.
• The CardioExcyte 96 provides complementary data to other assays such as automated patch clamping.
Features CE 96
Number of channels 96Impedance and EFP measurements
Pacing
Cell adhesion and spreading assays
Operation in standard incubator
Environmental control (for use outside incubator)
Temporal resolution (Imp/EFP) 1ms / 0.1msDedicated analysis and recording software
Integration with CYBERNANOanalysis
Figure 1: CardioExcyte 96 sensor technologyThe CardioExcyte 96 combines impedance and Extracellular Field Potential (EFP) recordings to measure contractilityand ion channel activity. A; The 96-well sensor plate contains gold electrodes embedded in each well. B; Manual overlayof impedance (upper trace) and EFP (lower trace) trace data recorded from an autonomously beating cardiacpreparation.
stimulation impedance
culture chamber
reference electrode
sensing electrode
Figure 2: Molecular and physiological characterisation of Axol hiPSC-vCMsA; Top Immunocytochemistry data showed the expression of ventricular cardiomyocyte markers (cardiac troponin-I; 93.5%expression). Data from Dr Christian Zuppinger, University of Bern and Prof Matt Daniels, University of Oxford. BottomWestern blot data confirmed that Axol hiPSC-vCMs express more cardiac troponin-T (cTnT) and α-Actinin than humanskin fibroblasts (hSFs). Data from Abigail Robertson, University of Manchester. B; 87 % of Axol hiPSC-vCMs have aventricular phenotype determined by MLC2v expression, compared to 13 % expressing atrial MLC2a (n=1). Data from DrChristian Zuppinger, University of Bern.
A. Protein marker expression B. Atrial vs. ventricular phenotype quantification
Characterisation of Axol ventricular iPSC-CMs
MLC2v (87%) vs MLC2a (13%) Nodal population not included
A. Sensor Plate
Impedance
EFP
B. Trace Data
D. Dofetilide (IKr)
A. DMSO control
C. Nifedipine (ICaV)
B. Verapamil (ICaV & IKr)
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Compound assessmentCardioExcyte 96 technology
Conclusions
Materials and methodsCardiomyocyte culture: Human iPSC-Derived Ventricular Cardiomyocytes(ax2505, Axol Bioscience) were thawed on Fibronectin (ax0049)-coated platesaccording to the manufacturer’s protocol, and cultured for the first 24 hours inCardiomyocyte Maintenance Medium (ax2530) + 10% FBS. The next day cellswere switched to serum-free Cardiomyocyte Maintenance Medium. Thereafter,the medium was changed every 2 days.Plating and recording: After 4 days, cells were dissociated with Axol Unlock(ax0044) and re-plated at 30,000 cells per well on the CardioExcyte 96 SensorPlate. After one week the following drug compounds were applied: dofetilide(10nM), nifedipine (100nM) and verapamil (200nM).Immunocytochemistry: Cells were fixed in 3 % PFA, permeabilised with 0.2% Triton X-100 and blocked with BSA. Primary antibody was incubatedovernight 4 ˚C, and secondary antibody coupled to Alexa Fluor® dyes(Invitrogen) applied for 2 hours.Western blot: 30 µg protein run on 10 % SDS-PAGE gel for 70 minutes at 130V and transferred to PVDF membrane. Membranes incubated with primaryantibody overnight at 4 ˚C, washed and incubated with secondary antibody for1 hour.
