Application Note

Cells:

patient hiPSC-derivedcardiomyocytes
FLEXcyte 96Tools:

iPSC-derived cardiomyocytes as a model
to dissect mechanical dysfunctions ofcaveolinopathies
Nanion Technologies GmbH, Munich.
Università degli Studi di Milano, Milano.
Summary

Results

Cardiac diseases remain one of the major causes of mor-Here, the CAV3 variant T78K, associated with Rippling

tality and morbidity in our society with enormous costs

muscle disease and HyperCKemia (HCK), was investi-for the health system. Arrhythmias and cardiomyopathy

gated. Human cardiomyocytes (CM) were differentiateddiseases are difficult to prevent/cure because the mo-from induced pluripotent stem cells (iPSC) derived fromlecular mechanisms behind their onset are in most casesa patient carrying this heterozygous mutation and fromnot fully clarified. Causes and effect are often confused,a healthy control. Among others, proteomic analysis re-even when directly studying patients’ cardiomyocytes,

vealed alteration in calcium handling and sarcomericbecause of the maladaptive remodelling imposed byproteins which promted the analysis of contraction prop-electro-mechanical alterations. To overcome this limita-erties using the CardioExcyte96 and FLEXcyte96 plat-tion, studying the arrhythmogenic risk associated withforms.

genetic cardiac diseases using patient-derived iPS-CMs,

Figure 1A shows the contraction profiles of control

provides a good model.

and T78K hiPSC-CMs monolayers as recorded on rigid

Caveolinopathies are a group of muscular diseases(CardioExcyte 96) and flexible substrate (FLEXcyte 96).

that arise from mutation in the caveolin-3 gene (CAV3).

have been partly elucidated using different models2, theimpact of such mutations on cardiomyocyte contraction
and thus on the risk of developing cardiomyopathy,

4200

although quite probable, has never been studied before.

12Time (s)Caveolin-3 along with cholesterol, forms membrane

Here, CardioExcyte 96 and FLEXcyte 96 compared
relative amplitude and kinetics of contraction andrelaxation in patient/control hiPS-lines in order to shed

40
200
control T78K

Amplitude(Ohm)signaling proteins and ion channels.

CardioExcyte 96

plasma membrane integrity and interacts with several

60

31680

CardioExcyte 968050
Amplitude (Ohm)caveolae and plays a key role in the maintenance ofFLEXcyte 96

Bcontrol
T78K

6Contraction rate(BPM)electrophysiological alterations behind caveolinopathies

AFLEXcyte 96

with both skeletal and cardiac pathologies1. While

Amplitude (Ohm)Several CAV3 variants have been found in patients

FLEXcyte 96

control

T78K

CardioExcyte 96

40302010001

23control T78K

control

T78K

Time (s)light on the relations between electrical and mechanicaldysfunctions. This analysis offered various advantages,
such as the possibility of electrical stimulation, recordings
in an environment with an elastic surface area
resembling that of the native cardiac tissue, as well as
high throughput3,4.
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Figure 1: A Contraction amplitude (top) and impedance (lower)
recordings of mean beat traces generated for control and T78K hPSCCMs B Comparison of contraction rate and amplitude of respective
hiPSC-CMs as recorded on the CardioExcyte 96 and the FLEXcyte 96.

Application NoteA40Amplitude (Ohm)plitude (at 1Hz), with faster rising and falling time in T78KhiPSC-CMs compared to control, as shown in Figure 2.

0.4

20

Lastly, the cells were paced at various rates (1Hz, 1,5Hz

0.2

10and 2Hz) and the same parameters were analyzed. Thisrevealed the inability of T78K-CM to adapt contraction

0.00
spont

0.6

and T78K hiPSC-CMs revealed increased mean beat am-Falling time (s)

0.630

B

Further analysis of the contraction properties of control

C

1Hz

1.5Hz

2Hz

Pulsewidth 50% (s)

spont

D

0.2

1.5Hz

2Hz

These results highlight the importance of use of hiPSC-

0.6

CMs, together with contractile force recording systems,

0.4

for understanding patient-specific pharmacological ef-

0.2

fects, aimed at preventing the life-threatening outcomes
associated with cardiac diseases.

0.0
spont

1Hz

1.5Hz

2Hz

to increasing rates, suggesting a possible excitation-contraction coupling dysfunction.

Rising Time (s)

0.8

0.4

1Hz

spont

1Hz

control

1.5Hz

2Hz

T78K CAV3

In summary, the CardioExcyte and FLEXcyte 96 weresuccessfuly used to record contractility of patientderived human iPSC cardiomyocytes. Here, it was shown,

Figure 2: Contraction analysis under pacing conditions indicating
mechanical dysfunction in T78K hiPSC-CMs compared to control cells.
Scatter plots represent the effects of various pacing rates on A amplitude,
B pulse width at 50%, C Falling time and D rising time of mean beats asrecorded on CardioExcyte 96.

for the first time, how the CAV3 T78K mutation determines
an altered excitation-contraction coupling and apotential proarrhythmic profile. This highlights the needfor cardiac monitoring in patients with CAV3 mutations.
This analysis could allow to deepen the understanding ofInterestingly, spontaneous beating of T78K hiPSC-CMs

these pathologies unmasking the true causes and to lay

was observed when the cells were recorded on flexible

the basis for a personalized-medicine approach aimed

substrate, corresponding with increased spontaneousat preventing/curing the life-threatening outcomes ofbeatingmaladaptive remodelling.

rate

previously

obtainedthroughpatch

clamp analysis. These results were not observed when
impedance was recorded on rigid substrate.

References

Impedance and contractility measurements

1. Gazzerro, E., et al. 2010. Eur. J. Hum. Genet. 18, 137–145

conducted according to Nanion’s standard procedures

2. Campostrini, G., et al. 2017. Cardiovasc Res.

for the CardioExcyte 96 and FLEXcyte 96, respectively. All

113(10):1256-1265

measurements have been performed in the laboratory

3. Doerr, L., et al. 2015. J Lab Autom. Apr;20(2):175-88

of Prof. Andrea Barbuti, Università degli Studi di Milano,

4. Goßmann, M., et al. 2020. J Pharmacol Toxicol Meth-as part of Nanion´s FLEXcyte research grant.

ods. 105:106892

Control and patient derived T78K mutation hiPSC-CMs

Methods

were cultured on were cultured on NSP-96 stim plates

Cellsperformed. Data are presented in scatterplots withImpedance and contraction force measurements wereand on FLX-96 for at least 8 days before recordings wereControl and patient derived T78K mutation hiPSC-CM cellmean ± SD.

lines were developed and differentiated in the laboratoryof Prof. Andrea Barbuti, Università degli Studi di Milano.

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