Download Assessment of pro-arrhythmic effects using Pluricyte

Assessment of pro-arrhythmic effects
using Pluricyte® Cardiomyocytes on the
ACEA xCELLigence® RTCA CardioECR
Version 1.2
CONTENTS
Getting started ................................................................................................................................ 3
Technical support and training ....................................................................................................... 3
1.
Introduction ............................................................................................................................. 4
2.
Work flow ................................................................................................................................ 5
3.
Important recommendations ................................................................................................. 5
4.
Equipment, materials and reagents ........................................................................................ 6
5.
Methods................................................................................................................................... 7
5.1
Coating of the 48-well E-plate®........................................................................................ 7
5.2
Performing background measurement ............................................................................ 7
5.3
Thawing Pluricyte® Cardiomyocytes and seeding onto the 48-well E-plate® ................. 7
5.4
Maintenance of the Pluricyte® Cardiomyocytes in the 48-well E-plate® ...................... 10
5.5
Data acquisition during maintenance ............................................................................ 10
5.6
Compound assay ............................................................................................................ 11
5.6.1
Studying acute drug effects ........................................................................................ 11
5.6.2
Studying long term drug effects ................................................................................. 12
5.7
Data analysis................................................................................................................... 12
6. Case study: Assessment of pro-arrhythmic effects using Pluricyte® Cardiomyocytes on the
ACEA xCELLigence® RTCA CardioECR ............................................................................................ 13
7.
6.1
Experimental design to study acute-drug effects .......................................................... 14
6.2
Results ............................................................................................................................ 16
References ............................................................................................................................. 24
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GETTING STARTED
Please make sure to read the entire Pluricyte® Cardiomyocyte Manual (available online at
www.pluriomics.com/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
www.pluriomics.com/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 www.pluriomics.com, or contact us directly by e-mail
([email protected]).
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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.
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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
O
O
O
O
Prior to plating the Pluricyte® Cardiomyocytes, always perform a background measurement of the Eplate® according to the xCELLigence® RTCA CardioECR Software Manual (Section 5.2).
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®.
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.
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.
First contractions of Pluricyte® Cardiomyocytes appear between 24-48 hours post-thawing. It will take 34 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.
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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% CO2 and humidified air
P10, P20, P200 and P1000 pipettes
8-channel multichannel pipette
RTCA Cardio Temperature Tool
Hemocytometer or automated cell counter
Manufacturer
ACEA Biosciences
Various
Various
Various
Various
ACEA Biosciences
Various
Table 4.2: Materials
Materials
E-plate® CardioECR 48
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
Manufacturer
ACEA Biosciences
Various
Various
Various
Various
Various
Various
Various
Various
Various
Cat#
00300600940
Table 4.3: Reagents
Reagents
Fibronectin (1 mg/ml)
1x DPBS +Ca2++Mg2+
Pluricyte® Cardiomyocyte Medium
Pluricyte® Cardiomyocytes Kit
Manufacturer
Sigma
e.g. Life technologies
Pluriomics
Pluriomics
Cat#
F1141
Gibco 14040
PM-2100-100ml
PCK-1.5
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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. Ca2+ and Mg2+) 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% CO2.
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 48well 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.
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Per E-Plate®:
8. Coat the tissue culture plate(s) with fibronectin as described in Section 5.1.
9. 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 1.
18. 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% CO2).
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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.
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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 20-30 minutes.
25. Immediately before use, transfer the warm medium into a multichannel reservoir. Transfer the Eplate® 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 24-48 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.
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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 5.6.1 and 5.6.2 below.
5.6.1
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% CO2 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).
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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 30-60 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% CO2 for 20-30 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% CO2 for at least 30-60 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.
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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 (INa), characterized by the sodium spike; the plateau phase, with an
influx of Calcium (ICa-L); the repolarization phase, during which an efflux of potassium occurs (IKr/IKs),
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.
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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
hERG channel blockers (IKr)
E4031
Dofetilide
Sodium channel blockers (INa)
Flecainide
Mexiletine
Calcium channel blockers (ICa,L)
β-adrenergic receptor agonist
Nifedipine
Diltiazem
Isoproterenol
Myosin II inhibitor
Blebbistatin
Expected
effects
on
hiPSC-derived
cardiomyocytes´ electrophysiology
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
Increased beat rate due to activation of βadrenergic receptors, resulting in decreased FPD
Inhibition of cardiomyocyte contraction, resulting
in diminished CI amplitude. No effects on the field
potential signal (ECR)
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Table 6.1. List of pro-arrhythmic drugs and their expected effects on hiPSC-derived cardiomyocytes.
A. Compound concentrations tested.
Compounds
E4031
Dofetilide
Flecainide
Mexiletine
Nifedipine
Diltiazem
Isoproterenol
Blebbistatin
t=0
3 nM
3 nM
300 nM
300 nM
3 nM
30 nM
3 nM
300 nM
t=30 min
10 nM
10 nM
1 µM
1 µM
10 nM
100 nM
10 nM
1 µM
t=60 min
30 nM
30 nM
3 µM
3 µM
30 nM
300 nM
30 nM
3 µM
t=90 min
100 nM
100 nM
10 µM
10 µM
100 nM
1 µM
100 nM
10 µM
t=120 min
---30 µM
300 nM
3 µM
300 nM
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.
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6.2
Results
hERG potassium channel blockers block the rapid component of the delayed rectifier outward potassium
current (Ikr), 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 arrhythmias3,4. 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 INa channels,
resulting in a decrease in sodium spike amplitude3. 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 (IKr)
potassium channels5,8, 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 duration3. 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 duration6. 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 contraction7, 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
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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.
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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).
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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).
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Figure 6.5. L-Type Ca2+ 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).
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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).
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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).
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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.
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7.
1.
2.
3.
4.
5.
6.
7.
8.
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