Agilent Assessing the Impact of Drug Application Note

Application Note

Cell Analysis

Assessing the Impact of Drug
Treatment on Cardiomyocyte Function

Through combined analysis of contractility, metabolic

flux, and cellular oxygenation

iPS Cardiomyocyte Contractility:

Cells cultured on RTCA E-Plate Cardio 96
Measured on xCELLigence RTCA Cardio
Allows interrogation of Contractility

Authors

Ryan McGarrigle, Conn Carey,
and James Hynes
Agilent Technologies, Inc.

Cell Metabolism:

Cells cultured on E-Plate Cardio 96
Measured on TRF Fluorescence Plate Reader
Agilent assays monitor mitochondrial function
(MitoXpress Xtra), glycolytic flux (pH-Xtra) and
cellular oxygenation (MitoXpress Intra).

Workflow Integration:

Allows measurement on E-Plates such that
metabolism and contractility can be measured

sequentially on the same test plate.

Abstract

In this application note, we demonstrate the feasibility of combining
microelectrode-based iPS cardiomyocyte contractility measurements with a
microplate-based bioenergetics assessment to better characterize cellular
responses to drug treatment. Contractility was assessed on 96-well E-Plate

Cardio96 using the Agilent xCELLigence RTCA Cardio system while cell metabolism
was measured on the same E-plate using a multiplexed fluorometric measurement

of O2 consumption with Agilent MitoXpress Xtra, glycolytic flux with Agilent pH Xtra,

and cellular oxygenation using Agilent MitoXpress Intra.

Introduction

Cardiotoxicity and related cardiac impairment remain one

of the main reasons for both drug withdrawal1 and FDA

black box warning2 and are a significant cause of compound

attrition in preclinical development. In vitro assays are capable
of better characterizing cardiac response to drug treatments
and are therefore of significant importance to better predict
such adverse effects in vivo.

Cardiac tissue requires an uninterrupted supply of respiratory
substrates to meet the very high ATP demand imposed by
continuous beating. Over 95% of this ATP is generated by

oxidative phosphorylation (OXPHOS) with the necessary
mitochondrial network taking up approximately one-third

of cardiomyocyte cell volume. Energy starvation and
mitochondrial dysfunction are therefore significant factors

in the progression of cardiotoxicity and so detection of such
metabolic dysfunction is an important aspect of cardiotoxicity

screening. This detection is best achieved by monitoring the

two main ATP generating processes, OXPHOS andglycolysis.

In vivo, the most important respiratory substrates for
ATP production are pyruvate and fatty acyl CoA, however,
cardiomyocyte metabolism is particularly adaptable
and substrates such as amino acids, lactate, and ketone

bodies can also be used. Examples of this adaptability
include hypoxia inducible factor (HIF) mediated metabolic
responses to hypoxia and ischemia and the shift from fatty
acid oxidation (FAO) to glucose metabolism that occurs

in hypertrophic cardiac tissue. These adaptions highlight
the importance of information on substrate preference

and oxygenation when designing and interpreting in vitro
cardiomyocyteanalyses.

As cardiac contraction is the main ATP consumer, the

coupling of contractility to ATP production, and by extension,

mitochondrial activity, is critically important to normal
cardiomyocyte function, particularly as the mitochondrial
reticulum also regulates intracellular calcium homeostasis
and a multitude of critical signally pathways. The ability to
relate cardiomyocyte beating to alter metabolic activity would
therefore be of significant utility.

Figure 1. A simplified schematic of the inter-relationship between cardiomyocyte metabolism and beating activity. OXPHOS produces most of the ATP needed,

with pyruvate and Acyl CoA being the main respirator y substrates. By measuring beating, OXPHOS (via O2 consumption), glycolytic flux (viaextracellular
acidification), and cellular oxygenation a more complete picture of cardiomyocyte function can be established.

2

Mitochondrial dysfunction and contractility

Metabolism Testing

Contractility is measured by culturing iPS cardiomyocytes
on E-Plate Cardio 96 and measuring them on the Agilent

xCELLigence RTCA Cardio system in real time. The E-Plate
has interdigitated impedance (IMP) microelectrode arrays

on the bottom of each well. IMP electrodes measure
cellular impedance, which is affected by the number of cells
covering the electrode, the morphology of the cells, and
the degree of the cell attachment. The fast sampling rate

of IMP measurement (12.9ms/77 Hz) allows capturing

temporal rhythmic changes in cell morphology and degree
of cell attachment to the plate associated with contraction
of cardiomyocytes. Therefore, the Cardio system is used to
predict drug-induced proarrhythmia, contractile liability, and

chronic toxicity of drugs under development.

Cell metabolism is measured using the Agilent MitoXpress

Xtra oxygen consumption assay to assess mitochondrial

function and the Agilent pH-Xtra glycolysis assay, which

uses extracellular acidification (ECA) to assess glycolytic

function. Soluble metabolic sensor reagents show a change

in fluorescence signal in response to changes in oxygen or

acidification as a result of energy production. Both reagents

can be measured using dual-read TR-F (time resolved
fluorescence) detection.3 This allows measurement on

E-Plates such that, if necessary, metabolism and contractility
can be measured sequentially on the same test plate.

Furthermore, cellular oxygenation measurements with the
Agilent MitoXpress Intra intracellular oxygen assay can
be conducted between xCELLigence RTCA time points (in
parallel but on plate reader platform) ifdesired.

Results and discussion

iPS cardiomyocytes maintain beat rates in the presence of
mitochondrial inhibitors.

To assess the effects of metabolism on beat rate,
cardiomyocytes were treated with mitochondrial modulators
on an E-Plate. Beat rates were assessed 0.5 and 24 hours

post-treatment (Figure 2A). Interestingly 1 µM FCCP

influenced the beat rate at both time points suggesting that
cardiomyocytes cannot recover following mitochondrial

uncoupling (Figure 2A). Lower concentrations did not reduce

the beat rate.

AB

0.5 h post treatment

FCCPAntimycinRotenone

Figure 2. The impact of mitochondrial impairment on cardiomyocyte beating. Beating is maintained in the presence of mitochondrial inhibitors through increased

glycolytic ATP supply. 30 s xCELLigence traces at 0.5 and 24 hours post-treatment (A). O2 consumption, extracellular acidification, and ATP were measured at
fixed concentrations (B). O2 consumption, extracellular acidification dose responses for antimycin (C)and FCCP (D). Data presented relative to untreated control.

Contractility Testing

30s

24 h post-treatment

30s

Vehicle

1 µM

0.1 µM

0.01 µM

Vehicle

10 µM

1 µM

0.1 µM

Vehicle

1 µM

100 nM

10 nM

C

600

500

400

300

% Effect

200

100

0

DMSO Antimycin ARotenoneFCCP

350

O2Consumption

300

Glycolytic Flux (ECA)

Baseline

250

200

150

% Effect

100

50

0

00.5 1

Antimycin (µM)

O2Consumption

Glycolytic Flux (ECA)

AT P

(1 µM) (1 µM) (1 µM) (2.5 µM)

900

800

700

600

500

400

300

200

100

O2Consumption

Glycolytic Flux (ECA)

Baseline

0

0510

FCCP (µM)

3

Inhibitory concentrations of antimycin A and rotenone (1 µM)

Compound treatment

Antimycin (1µM)

Reduction in
intr
driven by
respiration

Fu
depletion
caused by
Isoproterenol
treatment

MitoXpress Xtra (µs)

Time (min)

A

Isoproterenol (1µM)

did not have a significant impact on beat rates at both time

points (Figure 2A). This suggests that cardiomyocytes can

still generate ATP. High concentrations of antimycin A did
reduce beat rates after 24 hours.

Measuring oxygen consumption rates using MitoXpress

Xtra confirmed that antimycin A and rotenone decrease

mitochondrial respiration as oxygen consumption decreases
acutely upon treatment (Figure 2B). FCCP was shown to
increase oxygen consumption but as mitochondria are
uncoupled, they are unable to generate ATP (Figure 2B).

Analysis of the extracellular acidification using pH-Xtra

glycolysis assay shows that when mitochondria are inhibited

or uncoupled, glycolysis is increased (Figure 2B). There is

a clear concentration-dependent increase in acidification

(Figure 2C) suggesting that ATP depletion is ameliorated

through increased glycolysis in cardiomyocytes.

Together, this suggests that increased glycolysis supplies
the cells with enough ATP to facilitate cardiomyocyte
beating despite the lack of mitochondrial ATP from

oxidative phosphorylation. This is consistent with previous

observations on specific cell lines.4

Conversely, cells treated with isoproterenol were shown

to have an increased beat rate, and therefore oxygen
consumption experience as low as 6%oxygen as a result of
the increased oxygen consumption. This causes a significant
but temporary reduction in oxygen availability with values of

~6% observed for >15 minutes despite cells being cultured
and measured at 21% O2.

Untreated

Isoproterenol (1µM)

B

34

32

30

28

26

24

22

20

30 40 50 60

70 80 90

Untreated

Antimycin A

Cell metabolism is tightly coupled to contractile activity

The β-adrenoreceptor agonist, isoproterenol is used for the
treatment of bradycardia (slow heart rate). Figure 3A shows
beat rate traces of cardiomyocytes using the xCELLigence

RTCA, treatment with isoproterenol increased the beat rate
by ~45% compared to control 30 minutes post drug addition

(Figure 3A). Isoproterenol also caused a similar increase in
oxygen consumption (Figure3B).

These data suggest that when the beat rate is elevated,
the increased ATP demand is met by increasing aerobic
ATP production through mitochondrial respiration

(Figure3B). An antimycin A control was included to measure
non-mitochondrial oxygen consumption. Acidification rates
did not increase (data not shown) suggesting that OXPHOS

rather than glycolysis is supplying the additional ATP required.

Changes in cellular oxygenation were measured using

MitoXpress Intra. Figure 4 demonstrates that untreated

cardiomyocytes under these conditions experience ~14%
oxygen, ~7% less than ambient oxygen due to respiration
and other non-mitochondrial background oxygen-consuming

processes. When cells are treated with antimycin A,

experienced oxygen increases to around ambient levels
(~21%) as aerobic ATP production has been inhibited.

Figure 3. Impact of isoproterenol on cardiomyocyte beat rate measured

on an Agilent xCELLigence RTCA Cardio system (A) and cardiomyocyte
metabolism (B) measured on an advanced TR-F detection compatible
fluorescence plate reader. Increased oxygen consumption caused more
rapid oxygendepletion.

% O

2

20

acellular O2

rther O2

Figure 4. Impact of isoproterenol on cardiomyocyte oxygenation

measured using advanced TR-F detection fluorescence plate reader with

atmosphericcontrol.

18

16

14

12

10

8

6

10

20 30 40 50 60

Time

Untreated

Isoproterenol (1µM)

4

Contractility can be perturbed using several compounds

11

AB

(ECA)

AB

such as nifedipine or E-4031. Nifedipine is used to treat
and manage angina, high blood pressure, and several other

conditions, it acts as an L-type Ca2+ channel antagonist.
Figure5A demonstrates the dose-dependent effects of

nifedipine on contractile force, while Figure 5B illustrates a
dose-dependent decrease in cardiomyocyte O2 consumption.

Extracellular acidification was also reduced (data not shown).

The hERG channel inhibitor E-4031 causes an irregular beat

rate pattern (Figure 6A), which also causes a decrease in
oxygen consumption and a minor decrease in acidification
rates (Figure 6B). Suggesting that with a decrease in

ATP demand the cell responds by decreasing both ATP

generatingpathways.

20s

Figure 5. The impact of nifedipine on the beat rate (A) and metabolism
(B). Beating was measured 30minutes post-treatment. A range of
concentrations from 10 nM to 1 µM were assayed. Metabolism data
presented as oxygen consumption rate as a percentage of untreated control.

40

s

Figure 6. The impact of E-4031 on the beat rate (A) and metabolism (B).
Beating was measured 30minutes post-treatment. A single concentration
1µM of E-4031 was used. Metabolism data presented as oxygen

consumption rate and ECA as a percentage of untreated control.

Nifedipine

Vehicle

10 nM

25 nM

50 nM

100 nM

0.25 µM

0.5 µM

1.0 µM

E-4031

Vehicle

1.0 µM

120

100

80

60

40

% Effect

20

0

0.01

100

80

60

40

% Effect

20

0

O2Consumption

00

[Nifedipine] (nM)

Glycolytic Flux

Materials and methods

Cell culture

Induced pluripotent stem cells cardiomyocytes were supplied
by NCARDIA. Cells were plated onto fibronectin-coated

E-Plate Cardio 96 and placed in culture for 2to3 days,

performing media changes as per the manufacturer’s
instructions. Cells were plated at 4 to 5 ×104 cells/well for
pH-Xtra and MitoXpress-Xtra assays.

Oxygen consumption assay

Fresh media containing the MitoXpress Xtra reagent,

150µL/well was added before measurement. Compounds

were added directly, then all wells were sealed with
prewarmed HS oil. Plates were measured kinetically for

2.5to3.0hours at 37 °C (Ex 380 nm, Em 650 nm, and
Advanced dual-read TR-F plate readerdetection)

Glycolysis assay

The sample plate is placed in CO2 free incubator 3 hours
before measurement, to remove CO2. Samples were washed

three times using respiration buffer (1 mM phosphate)
prepared using the buffer tablet provided. 150 µL of

respiration buffer containing the pH-Xtra reagent was added
to sample wells. Compounds were added directly, and the

plate was measured kinetically for 2.5hours at 37 °C (Ex
380 nm, Em 615nm, and Advanced dual-read TR-F plate
readerdetection).

Cellular oxygenation assay

Cells were loaded with MitoXpress-Intra reagent

overnight (14hours) in a E-Plate Cardio 96 the day before
measurement. Cells were washed twice and 150 µL of fresh

media was added. The plate was measured kinetically at

37°C. (Ex380nm, Em 650 nm, and Advanced dual-read TR-F
plate reader detection).

Contractile assay

iPS-cardiomyocytes were plated on 96 well E-Plates and
impedance measurements were recorded at selected time

points (60 seconds sweep at a sampling rate of 77 Hz). Drug
treatment was initiated once the culture showed 40to60
synchronic beats/min. The data were normalized to baseline.

5

Conclusion

The combination of Agilent MitoXpress Xtra, MitoXpress Intra,

and pH-Xtra metabolic assays with the xCELLigence RTCA

Cardio system and E-Plate Cardio 96 enabled the sequential
measurement of metabolism and contractility from the
same sample using the same plate. Using the dual-read TR-F
measurement approach on conventional TR-F plate readers

informs on oxygen consumption and ECA. The combined

use of microplate-based contractility and metabolism
measurements has been demonstrated to generate a
more complete picture of cardiomyocyte response to drug
treatment and allows the delineation of inter-relationships
between cardiomyocyte beating and the underlying
bioenergetic processes. This multiparametric workflow helps
to improve data density per well of sample.

Complete impairment of OXPHOS through treatment with
electron transport inhibitors did not immediately impair
cardiomyocyte beating. Increased ECA suggests that ATP

supply is maintained through increased glycolytic flux
allowing beating to continue for >24hours post-treatment.

The β-adrenoreceptor agonist isoproterenol increased beat

rate and caused a significant increase in O2 consumption
but little change in ECA. This suggests that increased ATP
demand is being met through OXPHOS rather than glycolysis.

The L-type Ca2+ channel antagonist nifedipine reduced

contractile force and caused a dose-dependent reduction

in both oxygen consumption and ECA, indicative of reduced

OXPHOS and glycolytic activity in response to treatment.
This combined analysis of critical cardiomyocyte functions
therefore delivers a more holistic and informative in vitro

cardiotoxicity screen in that it related cellular function to

the metabolic activity driving that function. In so doing,
it provides additional mechanistic information as to the
cause of observed alterations in cardiomyocyte metabolism

orcontractility.

These highly informative workflows allow users to interrogate
metabolic modulators of cardiomyocyte function. As better
in vitro cardiac models are developed, knowing the metabolic
phenotype is essential to ensure that assays appropriately
reflect mature cardiomyocyte biology. Reliance on glycolysis
or OXPHOS shapes how these cells will respond to drugs
and how they will survive in environments that they can be

exposed to such as nutrient deprivation or hypoxia. These

workflows allow for assessment of contractility followed by
metabolic interrogation with the same biomaterial without
having to re-plate or potentially differentiate additional
cardiomyocytes for parallel measurements. This saves on
cell consumption while improving data density and delivering

multiparameter outputs from single samples. The flexibility of

these workflows makes them well-positioned to characterize
both metabolism and cardiomyocyte function under a range
of conditions including drug screening, nutrient deprivation,

hypoxia, and ischemia/reperfusion. Integrating these Agilent

Cell Analysis technologies offers a complete solution for
assessing cardio-metabolism.

References

1. Lawrence, C. L. et al. In Vitro Models of Proarrhythmia. Br.
J. Pharmacol. 2008 Aug, 154(7), 1516–2

2. Dykens J. A.; Will Y. The Significance of Mitochondrial
Toxicity Testing in Drug Development. Drug Discov. Today
2007 Sep, 12(17–18), 777–85.

3. Hynes J. et al. A High-Throughput Dual Parameter Assay
for Assessing Drug-Induced Mitochondrial Dysfunction
Provides Additional Predictivity Over Two Established

Mitochondrial Toxicity Assays. Toxicol. In Vitro 2013 Mar,
27(2), 560–9.

4. Marroquin L. D. et al. Circumventing the Crabtree Effect:
Replacing Media Glucose with Galactose Increases

Susceptibility of Hepg2 Cells to Mitochondrial Toxicants.
Toxicol. Sci. 2007 Jun, 97(2), 539–47.

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