Application Note

Topic:
cardiotox,
cardio-oncology

Chronic cardiotoxicity study on H9C2
cells using HESI stem cell working groupreference compounds

Cells:
H9C2
Tools:AtlaZ

Nanion Technologies GmbH, Munich.
innoVitro GmbH, Jülich.

SummaryLong-term exposure to cancer-related therapeutics has beenin culture and homogeneous response to stimuli that allows

linked to alterations of cardiac function in patients. The Stemtheir use in high throughput drug screening experiments or

Cell Working Group as part of the Health and Environmental

even in modeling various cardiovascular diseases such as

Science Institute (HESI) currently endeavores to gain further

cardiomyopathies or metabolic alternations 1.

insight into chronic cardiotoxicity. The objective of the HESI
study is to optimize non-clinical safety assessment strategies

The impedance of planar gold-film electrodes that are usedof chronic cardiotoxicity by testing prolonged exposure ofhere as growth substrate for adherent cells reveals changes incompounds on different cell-based assay systems using human

electrode coverage or cell behavior (Figure 1, lower graph).

induced pluripotent stem cell-derived cardiomyocytes (hiPSC-

Real-time impedance data provide insights in various cellCMs), by investigating e.g. contractility or contractile force.

phenotypes, such as cell morphology, proliferation, lateralmigration or cytotoxicity even over prolonged periods of time.

Here, we used a subset of the HESI chronic cardiotoxicity study

A crucial advantage over standard assays, e.g. filter basedreference compounds known to act via different mechanisms

methods using labels and either an optical or radiometricof actions (MoAs) in cardiomyocytes, affecting cardiac

detection technique, is the continuity of cell monitoring.

energetics (doxorubicin, erlotinib, sunitinib), contractility

Endpoint assays using dyes or labels quantify the potency of(BMS-986094, Nilotinib), electrophysiology (pentamidine), andmyofilament organization (endothelin-1 (ET-1), vincristine)(Figure 1, Table).
CompoundSide effect on cardiac function and MOA

Tested concentration

Using the AtlaZ platform, we investigated the toxicity of those

Pentamidine

hERG trafficking blocker. Induces QT prolongation and arrhythmias.

100nM, 300nM,1µM, 3µcompounds on H9C2 cell line, which has been differentiatedVincristine

Antimitotic agent by interacting with tubulin proteins. Causes
myocardial ischemia and infarction.

300pM, 3nM, 30nM, 300nM

BMS-986094

Induces contractility impairment by a decrease in calcium transient.

100nM, 300nM, 1µM, 3µM

ET-1

Causes fibrosis of the vascular cells and stimulates production ofreactive oxygen species.

300pM, 1nM, 3nM, 100nM

rat cardiomyoblast cell line H9C2 originally described in 1976 2,

Erlotinib

No cardiotoxic effects are expected.

230nM, 770nM, 2µM, 8µM

but also the mouse atrial myocyte derived HL-1 line and the

Nilotinib

Disruption of CM. Induces QT prolongation & myocardial ischemia.

10nM, 100nM, 300nM, 1µM

Doxorubicin

Mitochondrial toxicity, oxidative stress leading to apoptosis. Induces
arrhythmias and heart failure.

100nM, 300nM,1µM, 3µ

Sunitinib

Disruption of cardiomyocyte. Disruption of energy homeostasis andmitochondrial fusion-fission system via inhibition of AMPK signaling.
Induces long QT and myocardial infarction.

10nM, 100nM, 300nM, 1µM

towards a cardiac phenotype. Immortalized cardiomyocytelines, which are generated from primary cells, are feasible
alternatives of primary cultures 1, and amongst those are the3human AC16 line . All these cell lines show quick propagation
4

Compound-induced effect:cell detachment

Cell Signal

Figure 1: Top Reference compounds, their different

cell confluency

loss of tight junctions
loss of membrane integrity

cell proliferation
cell adhesion

mechanisms of action and tested concentrations.
Bottom The Cell Signal value offers information oncell adherence, proliferation or cell death, e.g.
upon treatment with compounds.

Electrode

Electric current

Time (h)Suspended cell

Adherent cell

toxins to impact target cells by either measuring target cellor Erlotinib at concentrations ranging between 230 nM and 7.7

viabiliy (i.e. MTT or flow cytometry) or membrane integrityµM did not prevent the H9C2 cells from proliferating, as cells(i.e. LDH). Those assays are labor-intensive and lack kinetic

remained viable and adherent to the surface. As expected,

information of the recorded effects.

no toxicity was observed upon the application of the hERG
trafficking inhibitors Pentamidine and Erlotinib, which are

Unique culture plates with integrated electrodes as used inconsidered as a cardio safe compounds.

the AtlaZ system enable long-term measurements over several
weeks. Thus real-time data on cell adhesion, proliferation and

Next, we applied Vincristine, BMS-986094 and Doxorubicin.

compound effects can be acquired.

We observed a time- and ratio-dependent decrease of the
Cell Signal, representing a structural cell toxicity (Figure 3).

Results

Vincristine is an antimitotic agent and is interacting with

In our assay, we used rat ventricular H9C2 cells (rat cardio-

Here, the two highest concentrations induced a decrease

myoblast cell line) which are developed towards a cardiacof viability, reaching the maximum effect after approx. 30 h.

phenotype. This model cell type represents a homogeneous

BMS-986094 is known to have an impact on contractility of

population of cardiac cells, offering the possibility tocardiomyocytes by decreasing the calcium transient. Here,simulate cardiac pathologies. The cells lack the capability

we observed a linear decay of the Cell Signal in a dose- andto contract, furthermore they do not express cardiac iontime-dependent manner. This means BMS-986094 not only haschannels like hERG.

a functional toxicity on cardiomoycytes 6 but also induces cell

tubulin proteins, causing myocardial ischemia and infarction 5.

death. Doxorubicin induces oxidative stress via mitochondria,
Cells were exposed to compounds for three days to identify

leading to apoptosis, arrhythmias and heart failure 7. As

chronic toxicity effects. Figure 1 shows H9C2 cells in control

expected, we observed a concentration-dependent andconditions as well as in the presence of 100 nM, 300 nM,

time-dependent decay of the Cell Signal, representing

1 µM and 3 µM Pentamidine or 230 nM, 770 nM, 2.3 µM andloss of cell integrity. ET-1 is known to cause hypertrophy via

7.7 µM Erlotinib. Raw traces of Cell Signal are shown on thecertain beta adrenergic pathways. We could not observe

left, the normalized Cell Signal (normalized to the timepoint of

ET-1 effects in H9C2 cells though (data not shown). Nilotinib

compound addition) is shown on the right. After compoundand Erlotinib are functionally impacting cardiomoycytes (cells

addition, cells continue to proliferate until a 100 % confluency

stop contracting), but here in the non-contractile H9C2 cells

is reached which is represented by the plateau phase.

we could not observe any impact on cell integrity (data notshown).

The H9C2 cell seeding density was 8 k cells/well. The cells thenstarted to proliferate and at t = 100 h the compounds wereThese results demonstrate the capability of AtlaZ to analyze

added. The impedance signal of the cell monolayer was

cardiotoxic compounds when using H9C2 cardiac-like cells.

recorded every 15 minutes, and is plotted using the unitlessparameter Cell Signal (Figure 2). The data revealed that
Pentamidine at concentrations between 100 nM and 3000 nM

Cell Signal

1200
1000
800
600
400

0

50

1.5

Compound addition

Normalized cell signal

Control
Pentamidine (100 nM)
Pentamidine (300 nM)
Pentamidine (1000 nM)
Pentamidine (3000 nM)

100

Control
Pentamidine (100 nM)
Pentamidine (300 nM)
Pentamidine (1000 nM)
Pentamidine (3000 nM)

1.0

0.5

0.0

150

Time (h)Cell Signal

1200

1.5

Compound addition

Normalized cell signal

Control
Erlotinib (230 nM )Erlotinib (770 nM )Erlotinib (2.3 µM )Erlotinib (7.7 µM )

1400

1000
800
600
400

1.0

0.5

0

0

50

100

Time (h)150

Control
Erlotinib (230 nM )Erlotinib (770 nM )Erlotinib (2.3 µM )Erlotinib (7.7 µM )Figure 2: Pentamidine and Erlotinib did not show
toxic effects on H9C2 cells at concentrations up to 3
µM or 7.7 µM, respectively. Data are shown +/- SEM.

A

raw impedance and B data normalized to the1000

timepoint of compound addition.

Cell Signal

dependent manner. Data are shown +/- SEM. A

Vincristine (3 nM)
Vincristine (30 nM)

400

BMS (300 nM)

1200

BMS (1 µM)

Compound addition

800

800

400

0

150

50

100

0

150

50

100

150

Time (h)Time (h)Time (h)Compound addition

Doxorubicin (3 µM)

600

400

100

Doxorubicin (1 µM)

1000

600

50

Doxorubicin (300 nM)

1200

BMS (3 µM)

1000

800
600

Doxorubicin( 100 nM)

BMS (100 nM)
Compound addition

Vincristine (300 nM)

0Control

Control

Control
Vincristine (300 pM)
1200

Cell Signal

on H9C2 cells in a concentration and time-

Cell Signal986094 and Doxorubicin showed toxic effects

Cell Signal

Figure 3: Chronic exposure to vincristine, BMS-

B
Control
Control

1.0

Vincristine (30 nM)**

Vincristine (300 nM)

***

***

0.5

0.0

Normalized Cell Signal

Normalized Cell Signal

Vincristine (3 nM)

BMS (300 nM)
BMS (1 µM)

BMS (3 µM)
1.0

**
***

0.5

0.0

Control

1.5

BMS (100 nM)

1.5

Vincristine (300 pM)

Doxorubicin( 100 nM)
Doxorubicin (300 nM)

Normalized Cell Signal

1.5

Doxorubicin (1 µM)
1.0

Doxorubicin (3 µM)

**
***

***
0.5

0.0

In summary, AtlaZ allows for label-free and real-time cellular

membranes at higher frequencies. The signal is dominatedresearch on cell adhesion, proliferation and cytolysis.

by the paracellular cell layer resistance between approx.

Measurements can be performed in up to 6 x 96-well plates

1 – 10 kHz and is increasingly influenced by capacitive

simultaneously or independently. The AtlaZ system provides

currents across the cell membranes between 10 – 100 kHz. Thisa versatile tool for in vitro cell monitoring addressing therenders impedance readouts at different frequencies (heredemands for versatility, physiological relevance anda spectrum of 0.1 kHz – 100 kHz) capable to further dissect

throughput.

physiological responses, zooming into changes in membrane

Methods
AtlaZ platform

topography, G-protein-coupled receptor (GPCRs) signaling
and cell-cell or cell-matrix junctions.
Cell Monitoring assay

The AtlaZ platform (Nanion Technologies) provides

Here we used the H9C2 cell line which has been differentiatedquantitative live-cell analytics by measuring the impedance

towards a cardiac phenotype. The readout displayed as(Ohm, Ω), displayed as Cell Signal, of adherent cells as grown

the Cell Signal represents raw impedance values. For theseon 96-well plates with embedded planar gold-film electrodes

specific recordings we have chosen the “Cell Monitoring”(Nanion Technologies).

mode (10 kHz recording frequency), one of the 3 recording
modes available, which is best suited for these type of toxicity

Real-time impedance raw data provide insights in various cell

investigations. When certain optional corrections are needed,phenotypes, such as cell morphology, proliferation, lateral

i.e. a correction for background values such as medium

migration or cytotoxicity even over prolonged periods of time.

control normalization, Cell “indexed” value can be calculated

A crucial benefit over standard assays is the continuity of celland displayed.

monitoring. Continuous measurements reveal the kinetics ofcell behavior and allow an in-depth mechanistic
understanding without the need for time- and labor-intensive

Key findings

endpoint assays. For example, one of the advantages of data
derived from the AtlaZ platform is the possibility to analyse
dose-responses at any time during the experiment.
Advanced information content is obtained by using multifrequency impedance readouts, which is possible with
the AtlaZ Control software. The methodology, Electrical
Impedance Spectroscopy (EIS), allows to gain a uniquerichness of information from your cells: depending on the dataacquisition frequency, the system detects predominantly
the resistive part of cell-cell contacts at lower frequencies
and predominantly capacitive currents across the cell

1. Our results demonstrate the capability of AtlaZ to analyze
cardiotoxic compounds when using H9C2 cardiac-likecells.
2. AtlaZ allows for cellular research on cell adhesion,
proliferation and cytotoxicity, label-free and in real-time.
3. Recordings can be performed in up to six 96-well plates
simultaneously or independently.

Gold electrodeanalyzing toxicity of applied treatments (here:
Cell Signal

1

Figure 4: Workflow of a cell monitoring assay
Adherent cellpotential cardiotoxic compounds). Target cells
Proliferation
Time (h)Adherence & Proliferationof cells

proliferate (1), upon reaching a plateau phase (2)the treatment can be executed (3). The integrityof target cells is measured and displayed as Cell
Signal over time.

Cell Signal

2

Confluent monolayer (entering plateau phase)are seeded in AtlaZ sensor plates, adhere andTime (h)Confluent cells develop
Cell Signal

3

Confluent cells, treatment should be
performed either before or upon entering theplateau phase

Time (h)References
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