A Sensor Based Technique for Pharmacological Safety Testing of Cardiac
Transport Proteins NCX, NaKATPase and Respiratory Chain Complexes
George Okeyo, Maria Barthmes, Andre Bazzone, Krisztina Juhasz, Sonja Stoelzle-Feix, Andrea Brüggemann,Michael George, Rodolfo Haedo, Niels Fertig
Nanion Technologies GmbH, Ganghoferstr. 70A, 80339 Munich, Germany, contact: Info@nanion.de

Cardiac Transporters in Pharmacology
Ion channels have long been targets for
cardiac safety testing. However, otherproteins involved in the transport of ions
across membrane barriers are also relevantfor pharmacological safety.
To drive the progress in pharmacologicalinvestigation of cardiac transporters we haveevaluated new methodologies to study bothdesired and unwanted effects of compounds
on several cardiac transport proteins, basedon solid supported membrane (SSM) andimpedance technologies, recording fromeither 1 or 96 wells simultaneously. As protein
containing samples we have used HEK293 celllines, mitochondrial inner membrane vesicles
from pig heart as well as iPSC-derived
cardiomyocytes on automated devices.
We have focused on two transporters thatemergedastargets
forcardiac
pharmacology and safety testing. First we
investigated the Sodium-Calcium Exchanger(NCX), which plays an important role in thecellular calcium homeostasis underSSM-based electrophysiology: A method to resolve
electric events generated by transporters

physiological and pathological conditions.
Inhibition of NCX is thought to be beneficial inischemia/reperfusion injury but also might
have an anti-arrhythmic effect. As a second
target we investigated Na+/K+-ATPase, whichplays a major role in the regulation of basic
cardiac functions and is targeted by cardiac
glycosides, illustrating its critical involvement inboth beneficial and potentially harmful
pharmacological effects. Additionally, wehave investigated the complexes of therespiratory chain as interference with these
ATP-producing proteins can lead to lethal sideeffects.

B

••
•
•

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••••••

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
Stock et al., 2013, ASSAY.
Watzke et al., 2010, Biochemistry.

BCA pig heart was homogenized, followed by the lysis of the cells
and isolation of mitochondria. After disruption of themitochondria, the inner mitochondrial membrane was isolated
and frozen. At the day of the experiment an aliquot of themembrane preparation was unfrozen, diluted and adhered toan SSM sensor.

+ CyC (ox)+ suc+ NADHCDA

D+ ATP

Upscaling:

Activity measurement of complex I in mitochondrial membranes
using the high throughput platform SURFE2R 96SE. 96 individual
measurements allow for compound testing with high efficiency.
In each well the activity of complex I can be triggered
repeatedly, allowing for baseline and control measurements aswell as application of different concentrations or even different
compounds in one well. Each measurement takes 5 minutes
before the next application can be started. This way over 1kdata points are generated in one hour.

Development of a screening tool for NCX
ACCEBD

The activity of individual respiratory chain complexes
recorded with SSM based electrophysiology.

A: Oxidation of NADH leads to transport of four protons across themembrane. B: Oxidation of succinate: Electrons are transferred tocytochrome c via complex III while four protons move to theintermembrane space. C: Reduction of oxygen by consumptionof electrons from cytochrome c and transport of two protons tothe intermembrane space. D: Complex V generates ATP fromADP plus phosphate. Here it works backwards as ATPase.

NaK-ATPase in different cellsABBDBSample preparation:

Current amplitude [nA]AC

A: Structure of an SSM sensor. The 3 mm diameter thin layer gold
electrode is coated with an SSM consisting of a thiol and a lipidmonolayer. On top liposomes or membrane vesicles containing thetarget protein are adsorbed. B: Protocol of one activation cycle. Thesensor is perfused with a passive non activating solution followed byan activating solution, containing a transported substrate or ATP.
During this step a transient current occurs, since the active
transporter moves charge into the vesicles and this generates amembrane potential. Instrumentation: We used the SURFE2R N1 (C)and the SURFE2R 96SE (D) instruments in this project. The respective
SSM sensors are depicted above the instruments. The SURFE2R N1 is asingle channel measurement instrument, whereas the SURFE2R 96SE isa robotic device measuring 96 sensors in parallel, designed forexperiments that require a high throughput.

Human iPSC derived cardiomyocytes show NCX currents andinhibition of NCX increases the beat rateAA+ CyC+ asc (red)Literature•

Solid
SupportedMembrane(SSM)-based
electrophysiology is a method, which allows theresolution of low amplitude electric events inbiologicalmembranes,bringingtheadvantages 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 bacterialmembranes. Unlike in cell-based electrophysiology, a large capacitive sensor coatedwith a high amount of membrane vesicles orproteoliposomes is used. In this way any processthat generates an electrical potential can beregistered with high amplification. The method
was established in the 90’s and has recently
been scaled-up to a 96-well format.

AWe developed several new methods toinvestigate these cardiac transporters in 96
wellformats,thus
vastlyincreasingthroughput,
acriticalfactor
inpharmacological screening. We showed dose
dependent effect of known substrates andinhibitors, thus verifying the reliability of ourmethods. As first result we could show that
inhibition of NCX increases the beat rate of
iPSC derived cardiomyocytes.

Direct measurement of respiratory chain complex
activity in cardiac mitochondrial membranes

NCX1 activity in HEK293 cells measured in 96 well format.
A: Representative experiment showing the software interface during the measurementof one 96 well plate coated with HEK293-NCX1 membrane. B: Mean signal amplitudesof one 96-well plate. Different conditions are compared. An incubation time of 4 min in
between repeated measurement was introduced. This led to a stable decrease of themaximum signal amplitude to about 70%, but ensures binding of inhibitors. C: Signalstability. Comparison of two individual sensors on a 96 well plate during a fullexperiment 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.

BNaK-ATPase in HEK293
cells:
HEK293cell

membrane was purified
and adhered to an SSM
sensor. NaK-ATPase activity
was measured using theSURFE2R N1 A: Applicationof ATP induces a dose
dependent positive current
response, reflecting a net
positive inward current. B:
Different availability
ofsodium and potassium
influences
thecurrent
amplitude. The amplitudedecreases in presence ofpotassium, indicating thatthe potassium binding ortransport is the rate limiting
step during the transport
cycle.

control

Adhering cardiomyocytes to an SSM sensor.

Transfer of the standardmethod.
A:
Cor.4U(Ncardia) iPSC derived
cardiomyocytes. B: The
cardiomyocytes were detached using trypsin. C:
The cardiomyocytes adhere to a lipid coated SSM
sensor. D: The sensor isrinsed in a fluidic systemwith high speed. This leads
to tearing of the cells –only a membrane sheet
remains.

Direct measurement of NCX activity with SSM-based electrophysiology.

A: After a rinse with a sodium containing buffer cardiac NCX was activated with 300 µM calcium.
B: A transient negative current indicates a net outward current. C: Calcium affinity of NCX1
recorded from HEK cells (74 µM) was comparable with iCell Cardiomyocytes2 (125 µM). D: NCX1 inHEK cells, iCell Cardiomyocytes2 and Cor.4U cells was blocked by KB-R7943 in a concentrationdependent manner with similar IC50 values. The IC50 values agreed well with the literature (Matsudaet al, 2001). E: SEA0400 is a specific blocker of NCX (Matsuda et al, 2001). SEA0400 blocked NCX
activated by 300 µM Ca2+ with IC50 in good agreement with the literature (Matsuda et al, 2001).

SEA inhibition increases the beat rate of iPSC
derived CMs. iCell Cardiomyocytes2 were seeded on theCardioExcyte96 well plate and the beat rate wasdetermined via impedance recordings. Addition ofSEA0400 significantly increased the beat rate in a dose
dependent manner. A: Individual traces comparing thedevelopment of the beat rate in one well shortly before
and 4h after addition of SEA0400. B: Time-dependent,long-term effect of SEA0400 on the beat rate of iCell
Cardiomyocytes2 (average of all wells, SD).

Validation using known NCX inhibitors.

IC50 determination using four described NCX inhibitors. The compounds show different activity. While Bepridil inhibits NCX with an IC50 of 9.6 µM(in good agreement with Watanabe & Kimura, 2001) and Amiodarone with an IC50 of 57 µM. Azimilide has no significant effect, whereas
SEA0400 inhibits NCX with high potency (IC50 35 nM,).

NaK ATPase in cardiac membrane and upscaling to 96
well. Plasma membrane of pig heart tissue was isolated andATPase activity measured using the SURFE2R 96 SE.