Application NoteCells:
Cardiosight-S ®

FLEXcyte 96 recordings in comparison
to in vivo and in vitro cardiac muscle
contraction via MUSCLEMOTION dataTools:
FLEXcyte 96

Nanion Technologies GmbH, Munich.
Human iPSC-derived cardiomyocytes kindly provided by Nexel.

SummaryIn order to reduce cardiovascular safety liabilities of

it to FLEXcyte 96 data as recorded with Cardiosight-S®

new therapeutic agents, there is an urgent need to

cardiomyocytes (Nexel).

integrate human-relevant platforms/approaches into drug
development1. Optimizing baseline function of humaninduced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs) is essential for their effective application in models of
cardiac toxicity and disease2. Here, hiPSC-CMs were cultured
on flexible substrates using the FLEXcyte 96 system. The promaturation environment enables observation of inotropic
and chronotropic compound effects, which are typicallyhard to detect with 2D monolayers on overly stiff substrates3.
For example, the beta-adrenergic agonist isoprenaline, orisoproterenol, is well known for its positive inotropic effects on
the human heart, although common hiPSC-CM in vitro assays
fail to display this physiological response by this compound.
Sala et al. (2018)4 developed a method that allows for acomparison of cardiac contraction measurements derived
from different measurement approaches. The method, called
MUSCLEMOTION, builds on previously existing algorithms, isfully automated and can be used on videos, image stacks,
or image sequences loaded in the open-source imageprocessing program ImageJ5. It is an open-source, dynamic
platform that can be expanded, improved, and integrated forcustomized applications. Dynamic changes in pixel intensity
between image frames are determined and the output isexpressed as a relative measure of movement during muscle
contraction and relaxation. Here, we compare the previously
depicted data sets from Sala et al. 20184 and compare

Figure 1: A Contraction amplitude (top) andvelocity (lower) individual beat profile generatedusing MUSCLEMOTION for hPSC-CMs, hPSC-CM
monolayers, cardiac organoids, engineered hearttissue (EHTs) and adult rabbit CMs (adapted fromRef. 4). B Contraction and velocity of mean beats ofhiPSC-CMs recorded on the FLEXcyte 96.

Results
Mean beat contraction and velocity parameters for hiPSCCMs grown on the flexible substrate of the FLEXcyte 96 werecalculated and compared with beat parameters from theMUSCLEMOTION software program as previously published bySala et al, 20184. Figure 1A shows the contraction and velocity
profiles of isolated hPSC-CMs, hPSC-CM monolayers, cardiac
organoids, engineered heart tissue (EHTs) and adultrabbit cardiomyocytes. Figure 1B shows the contraction andvelocity profiles of hiPSC-CM monolayers on the FLEXcyte 96.
Interestingly, the contraction and velocity profiles are highly
comparable to adult cardiomyocytes indicating a moremature phenotype of these cells.
Average concentration response curves for relaxation timeand contraction amplitude for isoprenaline, a well-known
positive inotropic compound, for isolated hPSC-CMs, hPSC-CM
monolayers, cardiac organoids, EHTs and adult rabbit

Figure 2: A Average concentration responsecontraction amplitude (right) generated using

120

MUSCLEMOTION for hPSC-CMs, hPSC-CM100nMµM3nM6003006030Ve
hinM

tissue (EHTs) and adult rabbit CMs (adaptednMmonolayers, cardiac organoids, engineered heart

60cle80

from Ref. 4). B Average concentration response
curves (black) and single measurements (red)[Isoprenaline]
Contraction Amplitude
Normalized
Contraction Amplitude (%)

for isoprenaline for relaxation time (top) and200

contraction amplitude (lower) recorded on the150

FLEXcyte 96.

100
50µMnM

3600nM
300nMnM
6030cle0Vehi

Ba Contraction Amplitude (%)
se1 lin en
3 M
10 nM30nM
10 nM
030 nM0nM

NormalizednM3snM
1ebkr[Isoprenaline]

Normalized
Normalized
Contraction Amplitude (%) B Contraction Amplitude (%)
aselieb
1 nens3 M
110 nMnMnM
30
310 nM
nM0nM

Normalized
Relaxation Time (%)

seli
1 nen
3 M
10 nM
n30 M
10 nM
0nM

Ba

[Isoprenaline]

Adult CMs

[Isoprenaline][Isoprenaline][Isoprenaline]

kr

Ba
se
1 lin en
3 M
10 nM30nM
10 nM
030 nM
0nM[Isoprenaline]

EHTs

for isoprenaline for relaxation time (left) and140

Normalized

Normalized
Relaxation Time (%)

Base
1 lin en
3 M
10 nM
30nM
10 nM
030 nM
0nM
Normalized
Relaxation Time (%)

Cardiac organoids[Isoprenaline]

curves (black) and single measurements (red)hiPSC-CMs on FLEXcyte 96
Relaxation Time160

hPSC-CM monolayers[Isoprenaline]

Normalized
Relaxation Time (%)[Isoprenaline]

B

Normalized
Relaxation Time (%)[Isoprenaline]

Contraction Amplitude

Normalized
Contraction Amplitude (%)
DM
S
0. O
31 nM
n3 M
10 nM
30nM
10 nM
0nM

Normalized
Relaxation Time (%)
DM
0. SO
31 nMn
3 M
10 nM30nM
10 nM
0nM

Isolated hPSC-CMs
Relaxation TimeBa Contraction Amplitude (%)
se1 lin en
3 M
10 nM
30nM
10 nM
030 nM
0nM

A[Isoprenaline]

References
1. Pang, L., et al. 2019. Circ. Res. 125: 855–867
2. Birket, M.J., et al. 2015. Cell Rep. 13(4): 733–745.
3. Stoelzle-Feix, S., et al. 2019. White Paper: https://www.nanion.
de/en/products/flexcyte-white-paper-download.html
4. Sala, L., et al. 2018. Circ. Res. 122(3): e5–e16

cardiomyocytes obtained using the MUSCLEMOTION

5. Ribeiro, A.J., et al. 2015. PNAS. 112: 12705–12710.

analysis of optical images are shown in Figure 2A. Average
concentration response curves for relaxation time andcontraction amplitude for isoprenaline obtained for hiPSCCMs recorded on the FLEXcyte 96 are shown in Figure
2B. Importantly, a positive inotropic response is shown forcontraction amplitude only in adult rabbit CMs and EHTs,
whereas this increase in contraction amplitude can also be
detected in hiPSC-CMs recorded on the FLEXcyte 96 (Figure2B). We went up to higher concentrations according to theEC50 shown in Mathur et al. (2015)6. A reduction in relaxation
time is also seen in adult CMs and on hiPSC-CMs recordedon the FLEXcyte 96 indicating a more mature or adult-likeprofile of these cells when grown on the flexible substrate.
In summary, the FLEXcyte 96 in combination with hiPSCCMs can be used to investigate the effect of positive(and negative) inotropic compounds on parameters suchas contraction amplitude, velocity and relaxation time.
Moreover, hiPSC-CMs grown on the flexible substrate ofMethodsCells
We thank NEXEL for providing the hiPSC-CMs (Cardiosight-S®).
Contractility measurements
Contraction force measurements were conducted according
to Nanion’s standard procedures for the FLEXcyte 96.
Cardiosight-S® were cultured on FLX-96 plates for at least 6days before compound addition. Compounds were applied in
the external media. About 4-6 hours before drug applicationa final media exchange was performed, leaving 200 µl
media in each well. For compound addition, 50 μl media
was removed and replaced with 50 μl media containing
the compound at 4x concentration, resulting in the desired
final concentration. All signals were normalized to a group ofcontrol measurements on the same plate.

the FLEXcyte 96 plates (FLX-96) display more adult-like
contraction and velocity profiles compared with hiPSC-CMs
grown on rigid surfaces. In this way, hiPSC-CMs can be used
for more predictive assays for pro-arrhythmic effects.

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