Assessment of pro-arrhythmic effects using Pluricyte Cardiomyocytes on the ACEA xcelligence RTCA CardioECR

Transcription

1 Assessment of pro-arrhythmic effects using Pluricyte Cardiomyocytes on the ACEA xcelligence RTCA CardioECR Version 1.2

2 CONTENTS Getting started... 3 Technical support and training Introduction Work flow Important recommendations Equipment, materials and reagents Methods Coating of the 48-well E-plate Performing background measurement Thawing Pluricyte Cardiomyocytes and seeding onto the 48-well E-plate Maintenance of the Pluricyte Cardiomyocytes in the 48-well E-plate Data acquisition during maintenance Compound assay Studying acute drug effects Studying long term drug effects Data analysis Case study: Assessment of pro-arrhythmic effects using Pluricyte Cardiomyocytes on the ACEA xcelligence RTCA CardioECR Experimental design to study acute-drug effects Results References

3 GETTING STARTED Please make sure to read the entire Pluricyte Cardiomyocyte Manual (available online at /support) carefully before you start. Pluricyte Cardiomyocytes are for in vitro life science research use only. A Material Safety Data Sheet (MSDS) for Pluricyte Cardiomyocytes is available online at /safety. TECHNICAL SUPPORT AND TRAINING Our scientists are ready to help you with any questions you may have regarding this application note or our Pluricyte Cardiomyocytes. In addition, in-lab training is available upon request. For further information please visit our website, or contact us directly by (support@pluriomics.com). 3

4 1. INTRODUCTION Pluricyte Cardiomyocytes are highly suitable for ACEA xcelligence RTCA CardioECR MEA assays Pluricyte Cardiomyocytes are fully functional human induced pluripotent stem cell (hipsc) derived ventricular cardiomyocytes that are particularly suitable for electrophysiology-based multi-electrode array (MEA) assays for predictive safety pharmacology, toxicity testing and efficacy screening in early drug discovery. The combination of Pluricyte Cardiomyocytes and the xcelligence RTCA CardioECR system enables detailed electrophysiological detection of potential cardiotoxic/pro-arrhythmic effects of test compounds in a 48-well plate format. The well-pronounced depolarization and repolarization peaks of Pluricyte Cardiomyocyte monolayer field potential signals allow an easy detection of electrophysiological parameters (e.g. depolarization/repolarization peak amplitudes, beat rate, field potential duration) and facilitate efficient data analysis and interpretation of studies performed with the xcelligence RTCA CardioECR system. Pluricyte Cardiomyocytes - strengths and characteristics Pluricyte Cardiomyocytes exhibit a relatively high level of maturity, when compared to other human stem cell-derived cardiomyocytes and present the following unique characteristics: High purity of ventricular cardiomyocytes Low resting membrane potentials (~-78 mv) Fast upstroke velocities and action potential amplitudes Organized sarcomeric structures Monolayer field potential contains well-pronounced depolarization and repolarization peaks, enabling easy detection of field potential durations in MEA assays This application note describes our recommendations for the analysis of Pluricyte Cardiomyocytes electrophysiology by MEA and impedance measurements using the xcelligence RTCA CardioECR system (ACEA Biosciences). In addition, a case study describing the assessment of the effects of a set of proarrhythmic compounds in Pluricyte Cardiomyocytes, showing the expected pharmacological responses, is provided in this document. Pluricyte Cardiomyocytes, cultured in Pluricyte Cardiomyocyte Medium, in combination with the xcelligence RTCA CardioECR system provide a highly relevant in vitro assay platform to study the cardiac safety profile of compounds during drug development. 4

5 2. WORK FLOW * Optional: in order to monitor the condition of the Pluricyte Cardiomyocyte monolayer it is advised to perform daily measurements ( 1h after refreshment). 3. IMPORTANT RECOMMENDATIONS O Prior to plating the Pluricyte Cardiomyocytes, always perform a background measurement of the E- plate according to the xcelligence RTCA CardioECR Software Manual (Section 5.2). O Carefully follow the thawing and seeding instructions. This step is essential for optimal cell survival and attachment (Section 5.3). Pluricyte Cardiomyocytes are directly seeded onto the E-plate. O We strongly recommend to use fibronectin as coating substrate for the E-plates. Other types of coatings may reduce the signal and/or impact the condition of the cells. O Always refresh the Pluricyte Cardiomyocyte Medium of the cells the day after seeding the cells (Section 5.4). Subsequently, refresh the Pluricyte Cardiomyocyte Medium of the cells every 2 days, or 3 days when refreshing on Friday and Monday to prevent weekend-work. O First contractions of Pluricyte Cardiomyocytes appear between hours post-thawing. It will take 3-4 days before the cells have formed an electrically coupled monolayer. Stable beating monolayers can be observed 7-8 days post-thawing. The optimal time window to perform electrophysiology-based assays with Pluricyte Cardiomyocytes is between 8-12 days after plating the cardiomyocytes. 5

6 4. EQUIPMENT, MATERIALS AND REAGENTS Equipment, materials and reagents are described respectively in Tables 4.1, 4.2 and 4.3. Table 4.1: Equipment Equipment xcelligence RTCA CardioECR Instrument + software Flow cabinet Incubator at 37 C, with 5% CO 2 and humidified air Manufacturer ACEA Biosciences P10, P20, P200 and P1000 pipettes 8-channel multichannel pipette RTCA Cardio Temperature Tool Hemocytometer or automated cell counter ACEA Biosciences Table 4.2: Materials Materials Manufacturer Cat# E-plate CardioECR 48 ACEA Biosciences Sterile disposable 5 ml pipettes Sterile disposable 10 ml pipettes Sterile disposable 25 ml pipettes Sterile 15ml conical tubes Sterile 50ml conical tubes Sterile 20µl filter pipette tips Sterile 300µl filter pipette tips Sterile 1000µl filter pipette tips Sterile multichannel reservoirs Table 4.3: Reagents Reagents Manufacturer Cat# Fibronectin (1 mg/ml) Sigma F1141 1x DPBS +Ca 2+ +Mg 2+ e.g. Life technologies Gibco Pluricyte Cardiomyocyte Medium Pluriomics PM ml Pluricyte Cardiomyocytes Kit Pluriomics PCK-1.5 6

7 5. METHODS 5.1 Coating of the 48-well E-plate The E-Plate is coated on the day of plating the Pluricyte Cardiomyocytes ( 3h before plating of the cells). Note: The volumes used below are calculated for one 48-well E-plate. For plating more than one 48-well E-plate, multiply the volumes used by the number of E-plates needed. Per E-Plate : 1. Dilute 30µL fibronectin solution in 3ml sterile D-PBS (incl. Ca 2+ and Mg 2+ ) in a 15ml conical tube to get a 10µg/ml fibronectin coating solution. Mix the solution carefully. Note: Fibronectin is susceptible to shear stress, do not vortex or spin the solution, and avoid harsh pipetting. 2. Add 50μl/well of the fibronectin coating solution to the E-Plate to evenly coat the bottom of each well. Note: Be careful not to touch the bottom of the plate with the pipette tips. 3. Incubate the E-plate at 37 C for 3 hours. Note: Do not to let the fibronectin coating dry out. 5.2 Performing background measurement 4. Carefully aspirate the fibronectin coating solution from the wells of the E-plate and immediately add 50μl of Pluricyte Cardiomyocyte Medium to each well using a multichannel pipette and sterile multichannel reservoirs. Incubate the E-plate for 10 minutes at 37 C, 5% CO 2. Note: Prevent the fibronectin coating from drying out and avoid touching the bottom of the wells with the pipette tips. 5. Place the E-plate into the xcelligence RTCA CardioECR instrument. 6. Perform background measurement according to the xcelligence RTCA CardioECR Software Manual. 7. Remove the E-plate from the instrument and leave it in the incubator until seeding of the cells. 5.3 Thawing Pluricyte Cardiomyocytes and seeding onto the 48-well E-plate Note: The volumes used below are calculated for one 48-well E-plate. For plating more than one 48- well E-plates, multiply the number of vials used by the number of E-plates needed. Combine the contents of the vials in the 50ml conical tube (see step 11) and adjust the volumes of Pluricyte Cardiomyocyte Medium to add accordingly. We recommend to thaw a maximum of 3 vials per operator at a time. 7

8 Per E-Plate : 8. Coat the tissue culture plate(s) with fibronectin as described in Section Warm 6ml Pluricyte Cardiomyocyte Medium to room temperature (RT). Note: make sure to mix the medium by inverting before use. 10. Take 1 vial of Pluricyte Cardiomyocytes from LN2 storage (optional: transport the vial on dry ice) and place the vial in a 37 C incubator for exactly 4 minutes. 11. Gently transfer the contents of the vial to a 50ml tube using a P1000 pipette. Avoid pipetting up and down. 12. Rinse the empty vial with 1ml Pluricyte Cardiomyocyte Medium (at RT) and add the 1ml Pluricyte Cardiomyocyte Medium drop-wise to the 50ml tube containing the cells: add 1 drop every 5 seconds using a P1000 pipette while gently swirling the cells after each drop. Note: This step is crucial for the recovery of the cardiomyocytes. We recommend to use a timer. 13. Add 4.7ml of Pluricyte Cardiomyocyte Medium drop-wise to the 50ml tube, 1 drop every 2 seconds using a 5ml pipette. Note: the total volume of the cell suspension is now 6 ml. 14. Take a 20µl sample of the homogenous cell suspension and add to a micro centrifuge tube. 15. Spin down the cell suspension for 3 minutes at 250xg. 16. Aspirate the medium and gently resuspend the cells in 1 ml Pluricyte Cardiomyocyte Medium. 17. Determine the total cell number and cell viability as follows: We highly recommend to perform the cell counting manually using a hemocytometer. For instance, by using the Fuchs Rosenthal Counting Chamber (Figure 5.1): a. Add 20µl Trypan blue solution to the 20µl cell sample (collected in step 1), mix carefully. b. Add 20µl of the Trypan blue/cell suspension mix to the counting chamber. c. Calculate the total number of cells according to equation Calculate the dilution factor to reach 30,000 cells/50µl and add Pluricyte Cardiomyocyte Medium to the cell suspension accordingly. 19. Add the solution to a multichannel reservoir using a 5ml pipette. 20. Transfer the coated plate(s) to the flow cabinet, do not aspirate medium from the E-plate but add 50μl/well of the cell suspension (=30,000 cells/well) to the side of the well using a multichannel pipette. Note: Avoid air bubbles and gently resuspend cells in the multichannel reservoir in between pipetting steps to evenly distribute the cells. 21. Incubate the E-plate in the flow cabinet at room temperature for 30 minutes to allow the cells to settle and ensure an even distribution. 22. Transfer the E-plate to the incubator (37 C, 5% CO 2). 8

9 Equation 1 Cell counting Count 4 #2 squares according to Figure 5.1 Viable cells: = (#vc) Non-viable (blue) cells: = (#nvc) /4 x 2 x 5000 = cells/ml [#vc] = (= cells in total) [# of cells/ml] [total volume after step 13] Viability = : ( + ) x 100 = % [#vc] [#vc] [#nvc] Figure 5.1. Lay-out of a Fuchs Rosenthal Counting chamber. 9

10 5.4 Maintenance of the Pluricyte Cardiomyocytes in the 48-well E-plate It is crucial to always refresh the Pluricyte Cardiomyocyte Medium of the cells one day after seeding the cells (day 1), and subsequently every 2 days (see workflow in Section 2). Per E-Plate : 23. Place the RTCA Cardio Temperature Tool in the incubator and warm the Temperature Tool to 37 C. 24. Pipette 6ml Pluricyte Cardiomyocyte Medium into a sterile 15ml conical tube and warm the medium to 37 C for minutes. 25. Immediately before use, transfer the warm medium into a multichannel reservoir. Transfer the E- plate from the incubator into the pre-warmed Temperature Tool in the flow cabinet. 26. Aspirate the medium from each well using a multichannel aspirator. Note: Avoid touching the bottom of the plate with the pipette tips to not disturb the cardiomyocyte monolayer. 27. Add 100μl/well pre-warmed medium to the side of the well using a multichannel pipette. Note: Avoid touching the bottom of the plate with the pipette tips to not disturb the cardiomyocyte monolayer. 28. Transfer the Temperature Tool and the E-plate back into the incubator. 5.5 Data acquisition during maintenance In order to monitor the condition of the Pluricyte Cardiomyocyte monolayer, it is advised to perform daily measurements during the maintenance, starting at day 1. See the xcelligence RTCA CardioECR Software Manual for specific instructions on using the software for data acquisition and analysis. First contractions of Pluricyte Cardiomyocytes appear between hours post-thawing. It will take 3-4 days before the cells have formed an electrically coupled monolayer. The amplitudes of the ExtraCellular Recording (ECR) and Cell Index (CI) signals increase with prolonged culturing. Stable beating monolayers can be observed 7-8 days post-thawing. For each measurement: 29. Place the E-plate in the xcelligence RTCA CardioECR instrument and start a measurement (e.g. 2 sweeps of 60 seconds at 5 minutes intervals). Note: Wait >1 hour after medium refreshments before measurement to avoid unstable signals caused by medium change. 10

11 5.6 Compound assay The optimal time window to perform electrophysiology-based assays with Pluricyte Cardiomyocytes is between 8-12 days after plating the cardiomyocytes. Two different protocols for drug testing, for studying acute and long term drug effects, respectively, are outlined in paragraph and below Studying acute drug effects To study acute drug effects, we recommend to dilute test compounds in Pluricyte Cardiomyocyte Medium at 10x the desired final concentration and to add the compound in a volume of maximum 10% of the final volume of medium in the well (e.g. 10 µl in a final volume of 100 µl). We recommend not to use DMSO concentrations above 0.1%. 29a. Replace the Pluricyte Cardiomyocyte Medium in the E-plate 1 hour before the compound assay as described in Section 5.4, and place the plate back into the incubator. 30a. Prepare the test compounds at 10x the desired final concentration in a 96-well plate and place this compound-plate in an incubator at 37 C, 5% CO 2 for at least 10 minutes. 31a. Transfer the E-plate from the incubator into the Temperature Tool (pre-warmed at 37 C) in the flow cabinet. 32a. Remove the chosen volume (e.g. 10 µl from a final volume of 100 µl) from each well and add the same volume from the 96-well compound plate to the E-plate. Note: Mix gently by pipetting 3 times. 33a. Place the E-plate in the xcelligence RTCA CardioECR instrument immediately following compound addition and start measurements (e.g. 5 sweeps of 60 seconds at 5 minutes intervals). Figure 5.2 provides an example of data acquisition for acute studies. Figure 5.2. Example of a measurement protocol for studying acute drug effects. See the xcelligence RTCA CardioECR Instrument Operator s Guide for specific and more optional instructions. For an example with cumulative dosing of test compounds, see the case study described in Section 6 (Figure 6.2). 11

12 5.6.2 Studying long term drug effects For a long term study, we recommend to dilute the test compounds in Pluricyte Cardiomyocyte Medium to the desired final concentration and completely replace the medium in the plate. We recommend to wait at least minutes before performing the first measurement to avoid measuring the effects of medium change. 29b. Prepare the test compounds in the desired final concentration in Pluricyte Cardiomyocyte Medium in a 96-well plate and place the plate in an incubator at 37 C, 5% CO 2 for minutes. 30b. Transfer the E-plate from the incubator into the Temperature Tool (pre-warmed at 37 C) in the flow cabinet and aspirate the medium from each well using a multichannel aspirator. Note: Avoid touching the bottom of the plate with the pipette tips to not disturb the cardiomyocyte monolayer. 31b. Add 100μl/well from the 96-well compound plate to the side of the well using a multichannel pipette. Note: Avoid touching the bottom of the plate with the pipette tips to not disturb the cardiomyocyte monolayer. 32b. Incubate the E-plate at 37 C, 5% CO 2 for at least minutes to avoid measuring the effects of the medium change. 33b. Place the E-plate in the xcelligence RTCA CardioECR instrument and start measurements. Figure 5.3 provides an example of data acquisition for long term studies. Figure 5.3. Example of an experimental timeline for studying long term drug effects. See the xcelligence RTCA CardioECR Instrument Operator s Guide for specific and more optional instructions. 5.7 Data analysis 34. Analyze the acquired data using the xcelligence RTCA CardioECR analysis software. Note: See the xcelligence RTCA CardioECR Software Manual for specific instructions for data analysis. 12

13 6. CASE STUDY: ASSESSMENT OF PRO-ARRHYTHMIC EFFECTS USING PLURICYTE CARDIOMYOCYTES ON THE ACEA XCELLIGENCE RTCA CARDIOECR SYSTEM The xcelligence RTCA CardioECR instrument records cellular impedance (presented as Cell Index (CI)) and electrical field potential (presented as extracellular recording (ECR)) of cardiomyocytes in parallel, using proprietary microelectrode array (MEA) technology. Through the ECR measurement, the xcelligence RTCA CardioECR instrument captures changes in the extracellular field potential, generated by the electrophysiological processes across the cell membrane. Drugs affecting different ion channels affect different phases of the extracellular field potential which can consequently be studied using the ECR measurement. In addition, the xcelligence RTCA CardioECR instrument records the impedance of the cells by sending a small electrical current through the cardiomyocyte monolayer. The CI measurement thereby quantifies the relative contractile state of the cell monolayer. Drugs affecting the contractile machinery of the cardiomyocyte can therefore be studied using the CI measurement. Figure 6.1 depicts a single waveform of the Cell Index and the extracellular field potential signal (ECR) of a Pluricyte Cardiomyocyte monolayer obtained using the xcelligence RTCA CardioECR instrument. Indicated are the Cell Index amplitude, a measure related to the contraction; the depolarization phase, during which an influx of sodium into the cells occurs (I Na), characterized by the sodium spike; the plateau phase, with an influx of Calcium (I Ca-L); the repolarization phase, during which an efflux of potassium occurs (I Kr/I Ks), characterized by the clear repolarization peak; and the field potential duration, the time period between the depolarization and repolarization phase. Figure 6.1 A single waveform of Cell Index (top panel) and extracellular field potential (bottom panel) signals of a Pluricyte Cardiomyocyte monolayer obtained using an ACEA xcelligence RTCA CardioECR instrument. Shown are: Cell Index amplitude, sodium spike amplitude, field potential duration, and the depolarization and repolarization phases. 13

14 6.1 Experimental design to study acute-drug effects Pluricyte Cardiomyocytes were cultured on 48-well E-plates in Pluricyte Cardiomyocyte Medium for 10 days. A set of pro-arrhythmic drugs (Table 6.1) was dissolved in DMSO at a concentration of 10mM and then diluted in Pluricyte Cardiomyocyte Medium in 10-fold serial dilutions. The Pluricyte Cardiomyocytes were then treated with this set of pre-diluted pro-arrhythmic drugs in a cumulative dose response experiment (Figure 6.2). Acute-drug effects were directly measured using the xcelligence RTCA CardioECR instrument. Compound concentrations were increased by 3-fold (Figure 6.2a) for each separate recording step (Figure 6.2b). The data were analyzed using the xcelligence RTCA CardioECR Software to determine the compound effects on beat rate, field potential duration, sodium spike amplitude, and Cell Index. Drug class Drug Expected effects on hipsc-derived cardiomyocytes electrophysiology herg channel blockers (I Kr) Sodium channel blockers (I Na) Calcium channel blockers (I Ca,L) E4031 Dofetilide Flecainide Mexiletine Nifedipine Diltiazem Delay of the repolarization phase by blocking the herg channel, resulting in prolonged FPD and ultimately arrhythmias Decrease of the sodium spike amplitude by blocking sodium channels. Higher concentrations may also block potassium channels resulting in an increased FPD Decrease of the FPD and CI amplitude by blocking calcium channels β-adrenergic receptor agonist Isoproterenol Increased beat rate due to activation of β- adrenergic receptors, resulting in decreased FPD Myosin II inhibitor Blebbistatin Inhibition of cardiomyocyte contraction, resulting in diminished CI amplitude. No effects on the field potential signal (ECR) 14

15 Table 6.1. List of pro-arrhythmic drugs and their expected effects on hipsc-derived cardiomyocytes. A. Compound concentrations tested. Compounds t=0 t=30 min t=60 min t=90 min t=120 min E nm 10 nm 30 nm 100 nm -- Dofetilide 3 nm 10 nm 30 nm 100 nm -- Flecainide 300 nm 1 µm 3 µm 10 µm -- Mexiletine 300 nm 1 µm 3 µm 10 µm 30 µm Nifedipine 3 nm 10 nm 30 nm 100 nm 300 nm Diltiazem 30 nm 100 nm 300 nm 1 µm 3 µm Isoproterenol 3 nm 10 nm 30 nm 100 nm 300 nm Blebbistatin 300 nm 1 µm 3 µm 10 µm 30 µm B. Experimental timeline. Figure 6.2. Cumulative Dose-Response Experiment. A: An overview of the compounds that were tested on Pluricyte Cardiomyocytes in the given increasing concentrations. B: The experimental procedure for this study. 15

16 6.2 Results herg potassium channel blockers block the rapid component of the delayed rectifier outward potassium current (I kr), thereby delaying the repolarization phase, resulting in an increase in field potential duration and flattening of the repolarization peak. Additionally, literature shows that, at higher concentrations, blocking of the herg channel leads to Torsade de Pointes (TdP)-like arrhythmias 3,4. Similar findings are observed in Pluricyte Cardiomyocytes. Figure 6.3 shows prolongation of the field potential duration (FPD) and flattening of the repolarization peak in Pluricyte Cardiomyocytes, induced by the herg potassium channel blockers E4031 and dofetilide. Furthermore, arrhythmias were observed occasionally at 30 nm E4031 and 30 nm dofetilide, and in all wells from 100 nm E4031 and 100 nm dofetilide. Sodium channel blockers affect the depolarization phase of the field potential by blocking I Na channels, resulting in a decrease in sodium spike amplitude 3. Figure 6.4 shows that sodium channel blockers flecainide and mexiletine indeed decrease the sodium spike amplitude in Pluricyte Cardiomyocytes in a concentration-dependent manner. In addition, flecainide and mexiletine are known to block herg (I Kr) potassium channels 5,8, which is also observed in Pluricyte Cardiomyocytes by an increase in field potential duration. Calcium channel blockers affect the resting phase between the depolarization and repolarization phase, resulting in a shortening of the field potential duration 3. As shown in Figure 6.5 the L-type calcium channel blockers nifedipine and diltiazem indeed both reduce the field potential duration of Pluricyte Cardiomyocytes in a concentration-dependent manner. In addition, by blocking calcium channels, nifedipine and diltiazem also affect Pluricyte Cardiomyocyte contraction, shown by decreased Cell Index amplitudes (Figure 6.5). Drugs which do not affect ion channels directly but affect cardiomyocyte contraction through other mechanisms, such as isoproterenol (a β-adrenergic agonist) and blebbistatin (an inhibitor of myosin II), may still cause changes in the field potential signal and/or the contractility of the cells, which can be observed using the ECR or CI measurement, respectively. Isoproterenol activates β-adrenergic receptors, resulting in increased beat rate and consequently a reduction in field potential duration 6. Figure 6.6 shows that isoproterenol indeed had a concentration-dependent effect on the firing rate of Pluricyte Cardiomyocytes, as well as on the absolute field potential duration. As explained above, the impedance measurement can be used to assess the contractility of cells. Blebbistatin is an inhibitor of myosin II, resulting in impairment of contraction 7, which is expected to be identified by a decrease in CI amplitude. As the compound does not interfere with ion channel function, the field potential is not expected to be affected. In this case study, this is clearly shown by the fact that 16

17 increasing concentrations of blebbistatin lead to significant reductions of the CI amplitudes in Pluricyte Cardiomyocytes, while the electrical field potential (ECR) remains unaffected (Figure 6.7). Figure 6.8 provides an overview of the cardio-active compounds investigated in this study and their effects on beat rate, field potential duration and sodium spike amplitude (expressed in percentage of change compared to the baseline). Concluding Remarks Pluricyte Cardiomyocytes are extremely suitable for safety pharmacology screening assays due to their unique strengths and characteristics (Section 1) including field potentials containing well-pronounced depolarization and repolarization peaks. In this case study, we assessed the effects of a set of cardio-active compounds on Pluricyte Cardiomyocytes electrophysiology by ECR (electrical field potential) and CI (Cellular Index) measurements using the ACEA xcelligence RTCA CardioECR instrument. Pluricyte Cardiomyocytes, cultured in Pluricyte Cardiomyocyte Medium, showed expected pharmacological responses in a reproducible manner, which could be readily detected with the ACEA xcelligence RTCA CardioECR instrument. Therefore, we conclude that the combination of Pluricyte Cardiomyocytes with the ACEA xcelligence RTCA CardioECR platform provides a highly suitable in vitro assay to study the cardiac safety profile of compounds at an early stage of drug development. 17

18 Figure 6.3. herg channel blockers. herg potassium channel blockers E4031 (A) and dofetilide (B) increased the field potential duration and caused a reduction in the amplitude of the repolarization peak, as shown by an overlay of waveform averages (top panel A and B). Higher concentrations induced arrhythmias (lower panel A and B). 18

19 Figure 6.4. Na + channel blockers.by blocking the INa channels, mexiletine (A) and flecainide (B) reduced the amplitude of the sodium spike, as shown by an overlay of waveform averages (top panel A and B). Mexiletine and flecainide also block herg (IKr) potassium channels, resulting in an increase in field potential duration (lower panels A and B). 19

20 Figure 6.5. L-Type Ca 2+ channel blockers.calcium channel blockers nifedipine (A) and diltiazem (B) reduced the field potential duration of Pluricyte Cardiomyocytes, as shown by an overlay of waveform averages (top panels A and B). In addition, nifedipine and diltiazem reduced the Cell Index amplitude by inhibiting the influx of calcium needed for contraction (lower panel A and B). 20

21 Figure 6.6. β-adrenergic receptor agonist isoproterenol.isoproterenol activates β-adrenergic receptors, resulting in increased beat rate and subsequently reduced field potential duration, as shown by an overlay of waveform averages (top panel) and multiple waveforms of a representative well (lower panels). 21

22 Figure 6.7. Myosin II Inhibitor blebbistatin.blebbistatin inhibits myosin II, resulting in impairment of contraction. Blebbistatin reduced the Cell Index amplitude of Pluricyte Cardiomyocytes (top panel) while the electrical field potential (ECR) remained unaffected (lower panel). 22

23 Figure 6.8. Overview of effects of cardio-active compounds on Pluricyte Cardiomyocytes. Overview of different drugs and their effects on beat rate, field potential duration (time between the detected repolarization peak and the preceding sodium spike), sodium spike amplitude and Cell Index amplitude, expressed as percentage of change compared to the baseline. Mean ± SD, N= 4 wells for each condition. 23

24 7. REFERENCES 1. Ribeiro, M. C. et al. Functional maturation of human pluripotent stem cell derived cardiomyocytes in vitro Correlation between contraction force and electrophysiology. Biomaterials 51, (2015). 2. Pluriomics. Pluricyte Cardiomyocyte product sheet. 3. Ma, J. et al. High purity human-induced pluripotent stem cell-derived cardiomyocytes: electrophysiological properties of action potentials and ionic currents. Am. J. Physiol. Heart Circ. Physiol. 301, H (2011). 4. Itzhaki, I. et al. Calcium handling in human induced pluripotent stem cell derived cardiomyocytes. PLoS One 6, e18037 (2011). 5. Paul, A. A., Witchel, H. J. & Hancox, J. C. Inhibition of the current of heterologously expressed HERG potassium channels by flecainide and comparison with quinidine, propafenone and lignocaine. Br. J. Pharmacol. 136, (2002). 6. Matsa, E. et al. Drug evaluation in cardiomyocytes derived from human induced pluripotent stem cells carrying a long QT syndrome type 2 mutation. Eur. Heart J. 32, (2011). 7. Kovács, M., Tóth, J., Hetényi, C., Málnási-Csizmadia, A. & Sellers, J. R. Mechanism of blebbistatin inhibition of myosin II. J. Biol. Chem. 279, (2004). 8. Gualdani, R. et al. Inhibition of herg potassium channel by the antiarrhythmic agent mexiletine and its metabolite m-hydroxymexiletine. Pharmacology Research & Perspectives 3(5),