+2+
Label-free analysis of Na /Ca -exchanger (NCX) isolated fromiPSC-derived cardiomyocytes

Maria Barthmes, Andre Bazzone, Ulrich Thomas, Andrea Brüggemann, MichaelGeorge, Niels Fertig, Alison Obergrussberger
Nanion Technologies GmbH, Ganghoferstr. 70A, 80339 Munich, Germany, contact:
Info@nanion.de

Abstract
The Sodium-Calcium Exchangers (NCX) play animportant role in the cellular calcium
homeostasis
underphysiologicalandpathological conditions. NCX has been ofinterest as a pharmacological target for manyyears, in particular because clinical trials
involving inhibitors of the sodium-proton
exchanger, NHE, have delivered mixed results.
Inhibition of the reversed mode of NCX isthought to be beneficial in ischemia/reperfusion
injury by reducing cardiac, neuronal and renal
infarct areas. Moreover, inhibition of NCX has
been proposed to exhibit an anti-arrhythmic
effect and therefore, may provide a novel
target for the treatment of a variety ofarrhythmic pathologies. So far, a number ofstudies have shown promising results but
investigations are limited by the currently
available NCX inhibitors such as KB-R7943, SEA0400 and SN-6 which are only partially specific.

Measuring NCX activity in human iPSC derived
cardiomyocytes

To drive the progress in pharmacological NCX
research, new methods to measure NCX
function are needed. At the current time,functional investigation of NCX range frompatch-clamp, calcium flux assays, Langendorffperfused hearts to studies in whole animals. We
havedeveloped
anelectrophysiologicalmethod to investigate NCX function which isbased on the solid supported membrane (SSM)
technology. HEK cells overexpressing NCX orhuman induced pluripotent stem cell-derived
cardiomyocytes (hiPSC-CMs) were used on asingle-well or 96-well SSM electrophysiology
device and NCX was recorded from these cells.
NCX was activated using Ca2+ in the buffer andinhibited by Cd2+ and other compounds.

Human iPSC derived cardiomyocytes are being
investigated as a model for cardiac safetyassessment. To measure native NCX in thesecardiomyocytes a cell based assay wasdeveloped. Cardiomyocytes were detachedfrom the culture dish and added to the lipid
coated SSM sensor. Where the cell connectedwith the lipid layer. Upon rinsing of the sensor

Cor.4U (Ncardia) iPSC derived
cardiomyocytes growing adherent on a cell culture dish.

The cardiomyocytes
detached using trypsin

wereSample preparation

Resolving low amplitude ion currents

A“Solid Supported Membrane (SSM)-based
electrophysiology” is a method, which allows
the resolution of low amplitude electric events
inbiologicalmembranes,bringing
theadvantages of electrophysiology to thetransporter field. It enables real-time activity
measurement of electrogenic SLC-transporters,
MFS-transporters and ion pumps, localized in theplasma membrane, in intracellular or bacterial
membranes.
Unlike cell-based electrophysiology, a large
capacitive sensor coated with a high amount of
membrane vesicles or proteoliposomes is used.
In this way any process that generates anelectrical potential can be registered with highamplification. The method was established in
the 90’s and has only recently been scaled-up
to a 96-well format.

AB

A: Structure of a SSM sensor. The 3 mm diameter thin layer gold
electrode is coated with a SSM consisting of a thiol and a lipid
monolayer. On top liposomes or membrane vesicles containing thetarget protein are adsorbed.
B: Protocol of one activation cycle. The sensor is perfused with apassive non activating solution followed by an activating solution,
containing a transported substrate or ATP. During this step a transientcurrent occurs, since the active transporter moves charge into thevesicles and this generates a membrane potential.

AE

The cardiomyocytes were added toa lipid coated SSM sensor. The sensor
was centrifuged to form a tightconnection of the cells with the lipidlayer

The sensor was rinsed in a fluidic
system with high speed. This leads totearing of the cells – only amembrane sheet remains. This is aworkinghypothesis,
furtherinvestigation is necessary.

After a rinse with a sodium
containing buffer cardiac
NCX was activated with 30 µM
calcium (A). A transientnegative current indicates a
net outward current (B).
Calcium affinity of NCX
recorded from HEK cells(74 µM) was comparable with
iCell
Cardiomyocytes2(125 µM) (C). NCX in HEK cells,
iCell Cardiomyocytes2 andCor.4U cells was blocked byKB-R7943 in a concentrationdependent manner (D) withsimilar IC50 values. The IC50values agreed well with theliterature (Matsuda et al,2001). SEA0400 is a specificblocker of NCX (Matsuda etal, 2001). SEA0400 blocked
NCX activated by 30 µM Ca2+
with IC50 in good agreementwith the literature (Matsuda etal, 2001) (E).

B

Development of a screening tool for NCX
To screen compounds for an effect on NCX for safety purposes or during development of novel
inhibitors a robust method with a high throughput is required. We employed the 96-well based
platform SURFE2R 96SE to record NCX activity from stably transfected HEK293 cells. The cells werecultured, harvested and a membrane preparation was performed. This has the advantage that theresulting signal amplitudes are higher, no running cell culture is required and it yields a very highreproducibility. The membrane preparation is defrosted and added to a 96-well sensor where it isrecorded by fluidic activation with calcium.

CAmino acids
PEPT1 (Slc15a1)
YdgR
YhiP/DtpB
PutP
GltP
EAAC1 (Slc1a1)
PAT1 (Slc36a1)
ArcD
CAT2B (Slc7a2)GlyT1/2 (Slc6a9/a5)

CD

Instrumentation: We used the SURFE2R N1 (A) and the SURFE2R 96SE
(B) instruments in this project. The respective SSM sensors are
depicted above the instruments. The SURFE2R N1 is a flexible single
channel measurement instrument, whereas the SURFE2R 96SE is arobotic device designed for experiments that require higher
throughput. C: Membrane proteins successfully studied using SSMbased electrophysiology

Transporters
Inorganic ions
NhaA
NhaP
NhaB
NCX1 (Slc8a1)
Clc-7
EcClc
NirC
Amt1-3
AmtB
SulPNIS (Slc5a5)
NaPi2b (Slc34a2)
MntH2

B

with high fluidic speed, the cells detachedagain, but a sheet of the cell membraneremains on the sensor. NCX currents can beevoked in these sheets. For a higher NCX signal
female cardiomyocytes were used. This method
enables the efficient investigation of the isolated
cardiac NCX current in a native membrane.

ATPasesNaK-ATPase
HK-ATPase
SERCA
V-ATPase
FoF1-ATPase
Kdp-ATPase
CopA
ATP7A/B
VrPPaseSugarsOrganic ions
SGLT1/2 (Slc5a1/a2) OCT2 (Slc22a2)
MelBCNT1 (Slc28a1)
LacY
ANT (Slc25a4)
FucP
AAC
XylE
GAT1 (Slc6a1)
GlcP
BetP
CHT (Slc5a7)
NupC
NacT (Slc13a5)

Redox-driven ion pumps
Complex I
respiratory chain complex I/III
respiratory chain complex II/III
cyctochrome c-oxidase
respiratory chain complexes I/III/V
Light-driven ion pumps
Bacteriorhodopsin (BR)Oxyrrhis marina Rhodopsin
Rhodopsin-2 (KR2)Halorhodopsin (HR)
Acerhodopsin
Channelrhodopsin (ChR)

Channels and Pores
Gramicidine
P2X2
nAChR
A/M2
UCP1 (Slc25a7)
TRPC5
TRPA1
CFTR
AQP61Enlarged view on 16
selectable wells. NCX
activation on theplate can be
repeated. Previous
current responses are
depicted in blue

56910A 1 µM

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E 1 µM

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569101223344778811111212A 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM
B 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM
C 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM
D 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM
E 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM

Overview on 96 parallel recordings
with color coding of sensor quality

F 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM
G 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM
H 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM 10 µM

Summary and Future prospects
We have developed two novel methodic
approaches for the functional investigation ofNCX and have recorded NCX from HEK cells andhiPSCs. NCX was activated by Ca2+ in the buffer
and could be inhibited by a number ofpharmacological agents.

Future Experiments• Further validation and optimization of themethods is required.
• We intend to develop a third complementary
method using a MEA-like system to investigate
the influence of NCX inhibitors and crossreactions of NCX inhibitors and pro-arrhythmic
drugs on the beating behavior of iPSC
derived cardiomyocytes.

• The developed methods shall be applied in a
project to characterize drug effects on NCX in
more detail.
• The next step intended is to screen theproarrhythmic drugs of the CiPA initiative
compound list using the high throughput
method for effects on NCX.

••••

Amran et al., Cardiovascular Drug Reviews, Vol. 21, No. 4, pp. 255–276
Antoons et al., Pharmacology & Therapeutics 134 (2012) 26–42
Herrmann et al., Cardiovascular Research (2013) 99, 780–788
Huag, JPTM 87 (2017) 11–23
Lee and Hryshko, Cardiovascular Drug Reviews, Vol. 22, No. 4, pp.
334–347

AB

Contact us if you are interested incollaborating with us to:• Apply the high throughput method for thedevelopment of novel NCX inhibitors.
• Evaluate the need for NCX safety testing ofnewdrugcandidates
using
thecardiomyocyte based method.

Literature•Detailed view of one individual well. Different measurements are displayed••••

Pignataro, Translational Stroke Research, DOI 10.1007/s12975-0130308-8
Watanabe, et al., J. Pharmacol.Sci.102, 7 – 16 (2006)
Matsuda, et al., 2001. JPET. 298(1): 249-256
Watanabe & Kimura., 2001. Jpn. J. Pharmacol. 85: 370-375

C

Different compound distributions on
the 96 well plate were applied.
Either compound 1, 2 and 3 in four
different
concentrations
percompound for IC50 determination or96 individual compounds each at aconcentration of 10 µM. Row H wasalways used as DMSO control.

Experiment setup
A: Result of one experiment with a (3compound setup). The compounds show
different activity. Bepridil inhibits NCX withan IC50 of 9.6 µM (in good agreement with
Watanabe & Kimura, 2001), Amiodarone
with an IC50 of 57 µM and SEA0400 blocked
NCX with an IC50 of 35.7 nM, slightly higher
than that obtained from hiPSC-CMs on theSURFE2R N1. The reasons for this are unclear
at this stage.
B: Mean signal amplitudes of one 96-wellplate. Different conditions are compared.
An incubation time of 4 min in between
repeated measurement in each well wasintroduced. This led to a stable decrease of
the maximum signal amplitude of about
70%, but ensures binding of inhibitors. The
decrease is presumably caused by declineof the sodium gradient.
C: Signal stability. Comparison of two
individual sensors on a 96 well plate during
a full experiment cycle. After a negative
control, NCX activity is triggered repeatedly
by application of 100 µM Ca2+. At 20 min a
4 min break is introduced and at 50 min theinhibitor is added to sensor 1, while sensor 2
servers as a DMSO control. While theamplitude of sensor 2 remains stable, sensor
1 decreases to the level of the negativecontrol.