APPLICATION NOTE

Functional and pharmacologicaldifferences between the contractilityof axoCellsTM iPSC-derived atrial andventricular cardiomyocytes assessedon the FLEXcyte 96
Commercial human iPSC-derived cardiomyocytes(hiPSC-CMs) have been available from multiple
providers for over a decade, with extensive use inresearch, drug development and toxicology testing
due to their appropriate modelling of primary
human ventricular cardiomyocytes. Less work has
been performed on human iPSC-derived atrial
cardiomyocytes despite the clear phenotypicand pharmacological differences between atrial
and ventricular cardiomyocytes, rendering thestandard ventricular cardiomyocytes a poor model
for atrial research. This is of particular concern as
atrial fibrillation (AF) is the most common form ofarrhythmia worldwide affecting over 33M people andrising, being a co-morbidity with obesity, stroke andcongestive heart failure (Chugh et al., 2014). Despite
the clear clinical need for better AF treatments there
are limited therapeutic options with poor successrates and the 1-year mortality rate for patients with
AF remains around 25% (Lee et al., 2018). Therefore,
there is a clear need for better, more human modelsof AF.
Axol Bioscience Ltd have developed their axoCellsTM
iPSC-derived atrial cardiomyocytes (ax2518) andhave performed extensive validation studies showing
differences in key markers, such as MLC2A, ANP and
KCNA5 and electrophysiology between their isogenic
atrial and ventricular cardiomyocytes (ax2508).
There has been less investigation carried out on thecontractility differences between the two cell types
which is of particular relevance to AF. Therefore, Axol
have partnered with innoVitro GmbH to examinethe phenotypic and pharmacological differences incontractility of Axol’s two forms of cardiomyocytes on
their FLEXcyte 96 platform.

With less than 10 μm in thickness and sophisticatedsurface modification, the polydimethylsiloxane(PDMS) membranes of the FLEXcyte 96 disposable
plates offer physiological elasticity of native humanheart tissue and strong mechanical support. While
being deflected by the weight of the culture medium,rhythmic contraction of the cardiomyocytes lifts themembranes in the 96-well upwards. By measuring
the changes in deflection, mechanical stress can becalculated.
The L-type calcium channel agonist S-Bay K8644 is
known for its positive inotropic effects on the humanheart, and also for prolonging the plateau phase of
the cardiac action potential.
The atrial-specific drugs Carbachol and4-Aminopyridine (4-AP) act on atrial-specific ionchannels. Carbachol, an agonist of IKACh, is expected
to exhibit negative chronotropic effects. 4-AP, anantagonist of IKur, is expected to shorten the action
potential duration.
Here, we show that Carbachol, 4-AP and S-Bay K8644
produced distinctly different effects on axoCellsTM
ventricular [ax2508] and atrial [ax2518] hiPSC-CMs
in terms of waveform shape, beat rate and beatduration.
Therefore, axoCellsTM human iPSC-derived atrial andventricular cardiomyocytes correctly reproduced thedifferent phenotypes and pharmacology of primary
cardiomyocyte sub-types as assessed using theFLEXcyte 96 and, as such, provide the starting point todevelop more reliable, physiological-relevant models
for research on atrial cardiomyocytes and AF.

APPLICATION NOTE

Cardiac Troponin T / Cardiac alpha Actinin / DAPIMLC2V / MLC2A / DAPI

Figure 1. Immunocytochemistry characterisation of axoCellsTM human iPSC-derived ventricular cardiomyocytes [ax2508].
Cardiac troponin T and cardiac alpha actinin are pan-cardiac, sarcomeric proteins. MLC2V is the ventricular isoform of theregulatory light chain of myosin, whilst MLC2A is the atrial isoform.

Methods
Both atrial [ax2518] and ventricular [ax2508] human
iPSC-derived cardiomyocytes were provided by Axol
Bioscience Ltd (Edinburgh, UK).
The cells were cultured on fibronectin-coatedFLEXcyte 96 well plates according to manufacturers’
guidelines in 200 μL maintenance medium per well.
Cells were seeded approximately 6 days before
compound treatment at 65k per well (195,000cells/cm2) to allow proper monolayer and network
formation. A final media change was conducted 4-6hours before drug application. For the experiments,
50 μL for the cell culture medium was removed
and replaced with 50 μL medium containing 4x
concentrated compound, resulting in the desired final
compound concentration.
The FLEXcyte 96 enables the analysis of a number ofcontractility parameters including contractile force,beat rate and beat duration (Figure 2).

Figure 2. FLEXcyte 96 contractility parameters

APPLICATION NOTEResults
Atrial hiPSC-CM

Ventricular hiPSC-CM

Figure 3. Representative waveforms of axoCellsTM atrial and ventricular hiPSC-CMs on the FLEXcyte 96 platform recorded
after 6 daysSix days post-thaw, atrial and ventricular hiPSCderived cardiomyocytes exhibited distinct
contractility waveforms on the FLEXcyte 96 platform(Figure 3). Atrial hiPSC-CMs showed a more rounded
waveform, whereas ventricular hiPSC-CMs showed aAtrial hiPSC-CM

slightly more prolonged plateau phase. This is similar
to the cardiac action potential, in which ventricularcardiomyocytes have an extended plateau phasecompared to atrial cardiomyocytes due to greater
Ca2+ influx through L-type Ca2+ channels (Grunnet,2010).

Ventricular hiPSC-CMA

B

Figure 4. The effect of S-Bay K8644, an L-type Ca2+ channel agonist, on contractility of axoCellsTM atrial and ventricular
hiPSC-CMs. (A) Beat duration. (B) Beat rate.

APPLICATION NOTE

S-Bay K8644 is an L-type Ca2+ channel agonist andcan exhibit a positive inotropic effect and prolonged
plateau phase due to calcium induced calcium
release from the sarcoplasmic reticulum Ca2+ stores.
The increased beat duration caused by S-Bay K8644
tends to be transient and is seen in the first 5 minutes

Atrial hiPSC-CM

after drug addition in both atrial and ventricular
hiPSC-CMs (Figure 4A). However, beat rate transiently
decreases in atrial hiPSC-CMs, whereas a sustained
increase in beat rate is observed in ventricular hiPSCCMs (Figure 4B). These transient effects may be dueto the calcium stores in the sarcoplasmic reticulum
becoming rapidly depleted.

Ventricular hiPSC-CM

A

B

Figure 5. The effect of Carbachol, an activator of IKACh in atrial cardiomyocytes, on the contractility of axoCellsTM atrial andventricular hiPSC-CMs. (A) Mean beat. (B) Beat rate

Atrial hiPSC-CM
A

Ventricular hiPSC-CMAPPLICATION NOTE

B

Figure 6. The effect of Carbachol, an activator of IKACh in atrial cardiomyocytes, on the contractility of axoCellsTM atrial andventricular hiPSC-CMs. (A) Beat duration. (B) Downstroke duration

The acetylcholine-activated inward-rectifying
potassium current (IKACh) is primarily found on
atrial cardiomyocytes (Heijman et al., 2018), andits activation by Carbachol is observed to a greater
extent in axoCellsTM atrial hiPSC-CMs compared toaxoCellsTM ventricular hiPSC-CMs (Figure 5, 6). At all

Atrial hiPSC-CMA

B

concentrations tested, carbachol exhibited a negative
chronotropic response and prolonged beat duration
on axoCellsTM atrial hiPSC-CMs. However, theseresponses were not observed to the same extent inaxoCellsTM ventricular hiPSC-CMs, demonstrating thefunctional activity of an atrial-specific ion channel inatrial hiPSC-CMs.

Ventricular hiPSC-CMAAPPPPLLIICC AATT II O
ONN NOTE

CFigure 7. The effect of 4-AP, an inhibitor of IKur in atrial cardiomyocytes, on the contractility of axoCellsTM atrial and ventricular
hiPSC-CMs. (A) Mean beat. (B) Beat rate. (C) Beat duration.

4-Aminopyridine (4-AP) inhibits the ultra-rapid
delayed rectifier potassium channel (IKur), primarilyfound on atrial cardiomyocytes (Dobrev et al., 2001).
When applied to axoCellsTM atrial hiPSC-CMs, 4-AP
caused a reduction in beat rate and an increased beatduration, albeit at higher concentrations of 50 µM and100 µM (Figure 7). In contrast, axoCellsTM ventricularConclusion

cardiomyocytes showed no change in beat rate anda transient reduction in beat duration at 1 µM, 5 µM
and 10 µM. Again, this demonstrates the presence offunctional atrial-specific ion channels on axoCellsTM
atrial hiPSC-CMs, that are not present to the same
extent on axoCellsTM ventricular hiPSC-CMs.

References

Here we show that distinct pharmacological effects
can be observed between axoCellsTM atrial andventricular hiPSC-derived cardiomyocytes when using
the FLEXcyte 96 platform to assess contractility.
Activation of IKACh by Carbachol reduced beat rate andprolonged beat duration in atrial cardiomyocytes but
not ventricular cardiomyocytes. In addition, inhibition
of IKur by 4-AP reduced beat rate and prolonged beatduration in atrial cardiomyocytes but not ventricularcardiomyocytes, demonstrating the inhibition ofPhase I repolarisation in atrial cardiomyocytes.
Therefore, the presence of functional IKACh and IKur
currents in axoCellsTM atrial cardiomyocytes that
respond appropriately to drugs provides a readily
available commercial resource for the in vitro
modelling of atrial-specific disorders such as atrial
fibrillation. In combination with the simultaneous
multiwell measurements of a range of contractility
parameters on the FLEXcyte 96 platform, this provides
a powerful tool to perform in vitro drug screening anddisease modelling on subtype-specific hiPSC-derivedcardiomyocytes.

[1] Chugh, S.S. et al. 2014. Worldwide epidemiology
of atrial fibrillation: a Global Burden of Disease 2010
Study. Circulation 129(8):837-47[2] Lee, E. et al. 2018. Mortality and causes of deathin patients with atrial fibrillation: A nationwide
population-based study. PLoS One 13(12): e0209687[3] Grunnet, M. 2010. Repolarization of the cardiac
action potential. Does an increase in repolarization
capacity constitute a new anti-arrhythmic principle?
Acta Physiol 198 Suppl 676:1-48[4] Heijman et al. 2018. Muscarinic type-1 receptors
contribute to IK,ACh in human atrial cardiomyocytes
and are upregulated in patients with chronic atrialfibrillation. Int J Cardiol 255:61-68[5] Dobrev, D. et al. 2001. Molecular basis ofdownregulation of G-protein-coupled inward
rectifying K(+) current (I(K,ACh) in chronic humanatrial fibrillation: decrease in GIRK4 mRNA correlateswith reduced I(K,ACh) and muscarinic receptormediated shortening of action potentials. Circulation
104(21):2551-7

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