Optogenetic technologies enable high throughput ion channel
drug discovery and toxicity screening
Jean-Francois Rolland , Riccardo Rizzetto , Viviana Agus , Sara Pizzi , Lia Scarabottolo , Susanne Renhelt , Daniela Malan , Tobias Bruegmann , Philipp Sasse ,Krisztina Juhasz3, Leo Doerr3, Matthias Beckler3, Michael George3, Andrea Brüggemann3, Niels Fertig3.
1111

12222

1. Axxam SpA, Bresso, Italy, 2. Institute of Physiology I, Medical Faculty, University of Bonn, Bonn, Germany 3. Nanion Technologies GmbH, Munich, GermanyElectrophysiology validation

Cell line validation
A. HEK293 cells stably transfected with ChR2T159C andtheir respective mock control (WT) were analyzed atFLIPRTETRA with membrane potential sensitive dye. Thepanel shows a representative fluorescence trace fromthe best performing clone expressing ChR2T159C and a
WT (red and black lines, respectively) during repetitive
light stimulation.
B. Following the final clone purification, the best
performing HEK293/ChR2T159C clone was stablytransfected with hNaV1.5 and analyzed at FLIPRTETRA,
along with their mock counterparts with membranepotential sensitive dye. The panels illustrate
representative fluorescence traces obtained either bylight stimulation or exposure to veratridine (left andright panels, respectively).

AAAA

BB

B

B

A

B

B

C

D

E

Optical Automated Electrophysiology

Introduction
The recent advances in the optogenetic field
allow the possibility to use the light stimulation of
Channelrhodopsin as appealing activating tools forin-vitro drug screening and toxicity tests.
Another major advance in the first phases of drug
discovery process, especially in the cardiac field, is thegrowing availability of iPS-derived cardiomyocytes;
however, intrinsic automaticity of these cells requires
still a fast, reproducible and clean electrical pacing toperform reliable drug characterization. The generalaim of this project was to combining the existing
technologies with optogenetics and iPS derived
cardiomyocytes to develop an integrated platform for
new reliable high-throughput screening strategies,
taking advance of the optical control of membrane
fluxes and/or depolarization via channelrhodospin
activation. Four commonly used readouts were assessed:
fluorescence, electrophysiology, impedance and EFP.

A. Representative current trace of light
induced current measured at -80 mVAD
ChR2T159C
hNaV1.5(gap-free recordings). All recordings were0 mV-100 mV
50 ms
performed in physiological ionic conditionsat room temperature. The external solution
contained (in mM): 145 NaCl, 4 KCl, 1 MgCl2,
2 CaCl2, 10 HEPES, 10 glucose, pH 7.4; whereas
the pipette was filled with an Internal solutionthat contained (in mM): 120 KCl, 5.374 CaCl2,
B
1.75 MgCl2, 10 HEPES, 10 EGTA, 5 Na2ATP,pH 7.2. Before the experiments, cells werecultured overnight in the presence of 5 M
all-trans-retinal.
B. Quantitative analysis of light inducedEC% expression
currents in three HEK293/ChR2T159C clones.
Left panel shows the percentage of cells
displaying a response to light. Right panels
show the average current density (± SEM)
measured both at peak (IPEAK) and steadystate (sustained, ISUS). 10 cells were measured
for each clone.
C. Average (± confidence intervals) I/V oflight induced currents evoked by blue light during a slow voltage ramp on the final selected clone (K1). The voltage
protocol is shown in the inset. N=7.
D. Representative traces of currents evoked by a 50 ms-long voltage step from -80 mV (Holding) to 0 mV in the best
performing clone of the HEK293/ChR2T159C/NaV1.5 cell line. 30 M TTX was used to univocally isolate theNaV1.5-dependent currents. The lower panel shows the TTX-sensitive current obtained by digital subtraction of thetraces in the upper panel. Similar results were obtained in 8 cells.
E. Percentage of cells expressing ChR2T159C and NaV1.5 in the best performing clone. Presence and functionality ofChR2 T159C were confirmed using the experimental conditions described on panel A.

Optogenetically paced field potentials (FP) at various
physiological stimulation rates.
A. Representative FP traces during (from left to right)
spontaneous beating and optical pacing (blue bars) at 1 Hz,
2 Hz and 3 Hz.
B. Overlay of representative FP paced at frequencies 1–3 Hz(from black to light blue).
C. Statistical analysis of absolute values of max.dV (N=37).
D. Statistical analysis of field potential duration during
pacing with indicated frequencies (FPD, N=42).
E, F. Overlay of representative FP optical paced at 1 Hz (left)
before (black) and after drug application (red), and statisticalanalysis of the relative changes of max. DV (middle) and FPD(right) at the frequencies 1–3 Hz.
E Effects of 10 μM Sotalol (n = 13–17);
F Effects of 0.3, 1 and 3 μM Flecainide (n = 11–12). *p < 0.05,**p < 0.01, ***p <0.001.

Opto-cell lines generation
Opto-cell lines generation
HEK293 cells stably transfected with ChR2T159C
HEK293 cellsOpto-cellstably transfectedwith ChR2T159C
lines generation
Co-expression of human NaV1.5
HEK293 cellsCo-expression
stably transfectedwith NaV1.5
ChR2T159Cof human
Validation at FLIPRTETRA (MP dye)
Co-expressionof human
NaV1.5TETRA (MPValidation at FLIPRdye)
Manual patch clamp validation
TETRA (MP dye)
Validation
at FLIPR
Manualpatch clampvalidation
Whole cell configuration
Manualpatchvalidation
Wholecellclamp
configuration
Light stimulation via CoolLed350
Lightstimulation
via CoolLed350
Wholecell configurationLight stimulation via CoolLed350

Implementation of light stimulation on
Implementationof lightplatforms
stimulation on
HTS compatible
HTS compatible platforms
Implementation of light stimulation on
HTS compatible platforms«Opto»Synchropatch«Opto»Synchropatch«Opto»Synchropatch
Cardio«Opto»Excyte
Cardio«Opto»Excyte
Cardio«Opto»Excyte«Opto» iPS-cardiomyocytes generation«Opto» iPS-cardiomyocytes generation«Opto»ofiPS-cardiomyocytes
Infection
COR.4U* cells withgeneration
ChR2H134R
Infection of COR.4U* cells with ChR2H134R
Optical pacing of ChR2-expressing COR.4U*Infectionof COR.4U*cells with ChR2H134R
Opticalpacingof ChR2-expressing
COR.4U*
Pharmacological validation
Optical pacing ofvalidation
ChR2-expressing COR.4U*
Pharmacological*COR.4U cells acquired from NCardiaTM
Pharmacological validation*COR.4U cells acquired from NCardiaTM*COR.4U cells acquired from NCardiaTM

FLUORESCENCEFLUORESCENCE

E-PHYS
E-PHYSFLUORESCENCEINTEGRATED

E-PHYSINTEGRATEDHTS-COMPATIBLE
HTS-COMPATIBLEINTEGRATEDOPTICAL
PLATFORM
OPTICAL
PLATFORM
HTS-COMPATIBLE
OPTICAL PLATFORM
EFP & IMPEDANCEEFP & IMPEDANCEEFP & IMPEDANCEThese figures show some ideas how optical stimulation of blue light (470 nM) with planar patch clamp could
be combined. A panel of 96 LED is built below the measuring chamber of a SyncroPatch 384PE. This allows thesimultaneous application of light stimuli with 384 parallel electrophysiological recordings.
A. Shows a picture of the blue light used for stimulation within the SyncroPatch 384PE.
B. HEK293/ChR2T159C cells were used in this experiment. The cells were measured in Current Clamp mode using theSyncroPatch 384PE. A Resting voltage of -90 mV was achieved by LFVC (Low Frequency Voltage Clamp) to close theNaV1.5 channels. Light pulses of 1 ms at
2Hz (470 nM) were used for stimulation.
The NaV1.5 channels opened due to theAB
depolarization by ChR2 T159C.
C. Cor4U iPS derived cardiomyocytes
transfected with ChR2 H134R were usedin this experiment. The cells weremeasured in Current Clamp modeusing the SyncroPatch 384PE. A Resting
voltage of -90 mV was achieved by LFVC
CD(Low Frequency Voltage Clamp). Lightpulses of 1 ms at 0.5Hz (470 nM) wereNav1.5
used for stimulation. The depolarization
by ChR2 H134Rwas able to elicit action
potential.
ChR-2D. This panel illustrates different
examples of this experiments displaying
1 ms Light Stimulus
various shapes of cardiac action
potentials.

FConclusions
Within the consortium, we generated a pure cell line
heterologously expressing both ChR2 and NaV1.5
whose functionality was validated on the stateof-the-art manual patch-clamp; on another hand
we develop HTS-compatible platforms based on
optogenetic tools for effective drug discovery inheterologous expression systems and iPS-derived
cardiomyocytes.
Taken together, the present results illustrate ascalable approach for preclinical screening of drug
effects on cardiac electrophysiology. Importantly,
we show that the recording and analysis can be fully
automated and the technology is readily available
using commercial products.

Acknowledgements
This work was supported by the “Ministero dell’istruzione, dell’università
e della ricerca” (MIUR) and the “Bundesministerium für Bildung undForschung” (BMBF) through the EuroTransBio project “OPTEL” (ETB-2015-32).

Axxam SpA • OpenZonevia Meucci 3 • 20091 Bresso, Milan (Italy) • phone +39 02 210561 • fax +39 02 2105602 • www.axxam.com

The drug discovery process involving ion channels needs
to rely on high-throughput screening (HTS) assays as wellas fine-tuned characterization by electrophysiologicalmeasurements. Combining optogenetic tools with induced
pluripotent stem-cells (iPSC)-derived cardiomyocytes
can provide a new reliable, cost-effective and highly
time-resolved approach to induce electrogenic proteins
activation. The aim of the OPTEL project, which combines
the expertise of two leading European companies and one
outstanding academic laboratory, was to develop integrated
HTS-compatible platforms based on optogenetic tools(Channelrhodopsin, ChR2) for effective drug discovery
in heterologous expression systems and iPS-derived
cardiomyocytes. We generated HEK293 cell lines stably
expressing ChR2 alone or with the cardiac sodium channel,
NaV1.5 which proper functional expression was validated byfluorescence and manual patch-clamp.
The best clones were used to implement light stimulation
on an HTS automated patch-clamp platform (SynchroPatch384PE, Nanion Technologies). We recorded light-induced
currents and action potential-like responses in voltage andcurrent-clamp, respectively, validating a new platform forhigh-throughput automated “opto-patch-clamp” assays.
For optogenetic drug toxicity screening, commerciallyavailable iPSC-derived cardiomyocytes were infected withadeno-associated viruses to express ChR2 and analyzed byrecording extracellular field potentials from 96 wells witha modified CardioExcyte96 system (Nanion Technologies).
This was equipped with a custom designed 96 LED lid
enabling pacing of cells and frequency-dependent drugscreening over the physiological heart rate (1-3 Hz). Thereby
we characterized adverse side effects of known ion channel
blockers and pro-arrhythmogenic drugs on Na+, Ca2+ and K+
channel function. These results validate new platforms thatallow optogenetic control of electrical activity in
HTS-compatible format with four different readouts:
fluorescence, electrophysiology, impedance and EFP.
The use of these complementary readouts provides a new
cost-effective and informative strategy in early stages of ionchannel drug discovery, especially in the cardiac field.

Opto-pacing of iPS-derived
cardiomyocytes

Data modified from Rehnelt S, et al. Int. J. Mol. Sci. 2017, DOI:10.3390/ijms18122634

Abstract