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Received: November 2016;
in final form April 2017.
ROMANIAN J. BIOPHYS., Vol. 27, No. 1, P. 000 –000, BUCHAREST, 2017 NOVEL APPROACHES TO PROARRHYTHMOGENIC RISK
TESTING USING AUTOMA TED PATCH -CLAMP PLAT FORMS
B. AMUZESCU *,**#, R. AIRINI *,**, LOREDANA GHICA *, F.B. EPUREANU *,**, A.F. DEFTU *,**,
DANIELA MARCELA CUCU *,**, VIOLETA PAULA RISTOIU *,**, D.F.MIHĂ ILESCU *, BEATRICE
MIHAELA RAD U*,**#
*Department of Anatomy, Animal Physiology and Biophysics, Faculty of Biology, University of
Bucharest, 91 –95 Splaiul Independenței, Bucharest 050095, Romania , e-mail:
#[anonimizat] , #[anonimizat]
**Life, Environmental and Earth Sciences Division, Research Institute of the University of Bucharest
(ICUB), 91 –95 Splaiul Independenței, Bucharest 050095, Romania
Abstract . This mini review summarizes evolution of experimental techniques in cardiomyocyte
electrophysiology over the last 70 years and progressive development of cardiomyocyte
electrophysiology computational models. We emphasize the relationship between hERG (the human
Ether-à-go-go-Related Gene) ion channels’ susceptibility to block by pharmacological compounds
and drug -induced arrhythmias, particularly Torsades -de-Pointes , and expose the already classical
clinical and non -clinical, in vitro and in vivo assays used for card iac safety testing, included in the
S7B and E14 guidelines, constituting the so -called “hERG -centric” paradigm. We further present
several in -depth studies that pointed out the limitations of this paradigm, and the requirement for a
modern in vitro mechani stic approach, combining experimental and in silico (modeling) methods,
constituting the novel Cardiac in vitro Pro-arrhythmia Assay (CiPA) paradigm. We review the most
relevant achievements during the last three years in implementing CiPA as a new guideli ne, the
various approaches tested by researchers, particularly for evidencing proarrhythmogenic events like
early or delayed afterdepolarizations in human induced pluripotent stem cell -derived cardiomyocyte
preparations. We conclude that automated patch -clamp methods, especially those using the third
generation CytoPatch™ platforms based on the patented Cytocentering® technology, allow
development of complex assays combining all three stages of the CiPA approach in a single
experiment, leading to advanced t esting methods that will transform CiPA into a robust, highly
reliable and reproducible standard for the pharmacological industry.
Key words : cardiomyocyte, human induced pluripotent stem cell -derived cardiomyocyte,
hERG, early afterdepolarization, Torsade s-de-Pointes, drug -induced arrhythmia, cardiac safety drug
testing, Comprehensive in vitro Proarrhythmia Assay (CiPA), automated patch -clamp

B. Amuzescu et al.
INTRODUCTION
The field of cardiomyocyte electrophysiology progressed steadily over the
last seven decades, due to continuous improvement in experimental techniques and
equipments, and successive waves of innovative approaches. Thus, the 1950s were
dominated by microelectrode impalement experiments in multicellular cardiac
tissue preparations such as Purkinje network f ragments (“false tendons”). A
remarkable study of that epoch is that of Weidmann [35], who demonstrated that
the action potential (AP) plateau features a higher membrane resistance compared
to the resting state of the fiber. This fact was in contradiction with the first attempts
to mathematically model the cardiomyocyte AP starting from the Hodgkin -Huxley
model, by slowing down the time constant of K+ current activation and Na+ current
inactivation [12, 19]. Soon, the experiments of Hall, Hutter and Noble p roved the
existence of two distinct K+ current components: a delayed rectifier component,
similar to that in the Hodgkin -Huxley model, and an inward rectifier current [13,
15]. These were dubbed IK and IK1, respectively, in the Purkinje fiber model
develop ed by Sir Denis Noble in 1962 [22]. With the advent of the voltage -clamp
technique in 1964, numerous other current components were identified in
cardiomyocyte preparations and were progressively incorporated in cardiomyocyte
electrophysiology mathematical models, most notably by the group of Noble. Thus,
L-type Ca2+ currents were discovered by Harald Reuter in 1967 [27], and were
incorporated in the Beeler -Reuter ventricular cardiomyocyte model [2], a
modification of the MacAllister -Noble -Tsien model [18]. This model represents a
turning point, incorporating “slow inward” L -type Ca2+ currents, two components
of delayed rectifier K+ currents identified by Noble and Roger Tsien in 1968 [23],
known today as IKr and IKs, as well as a pacemaker current accounting for
automatic rhythmic activity. An improved model of the “funny”
(hyperpolarization -activated) pacemaker current If was included in the
DiFrancesco -Noble model [9], together with a Na+/Ca2+ exchanger (NCX), initially
considered electroneutral, the exchan ge rate of which was updated from 2:1 to 3:1.
Inclusion of one exchanger required taking into account the other exchangers and
pumps, as well as calcium buffering in different cellular subcompartments,
considered in the Hilgemann -Noble atrial cardiomyocyte model [14]. The shape of
ventricular action potential and of several ion currents provided by an updated
cardiomyocyte electrophysiology model is shown in Figure 1.
IKR INHIBITION BY DRUGS AND TORSADOGENIC RI SK
An important consequence of the peculiar I -V plot of the ca rdiac inward
rectifier current IK1 is maintenance of a prolonged, several -hundreds -milliseconds

Risk testing by automated patch -clamp

Fig. 1. Waveform of paced action potential and of several cardiac ion currents yielded by a
“humanized” Faber -Rudy 2007 [10] model with curren t densities adapted for mid -myocardial
ventricular cardiomyocytes. INa – fast voltage -dependent Na+ current; ICaL – L-type Ca2+ current; INaCa
– Na+/Ca2+ exchanger current; INaK – Na+/K+ pump current; IKr and IKs – rapid and slow delayed
rectifier K+ current; IK1 – cardiac inward rectifier K+ current; Ito – transient outward K+ current.
depolarization plateau, particularly in ventricular cardiomyocytes, with small
inward and outward currents, via a delicate equilibrium, an energy -saving
mechanism, but also a “nature’s pact with the devil”, as Denis Noble named this
phenomenon [21]. Repolarizing currents in cardiomyocytes are also faint, while
inward Na+ flow during fast depolarization (phase 0) is robust, resulting in a 100
fold increase in transmembrane co nductance. The fragility of repolarization renders
it susceptible to the influence of a variety of factors. The hERG1 channel,
generating IKr, the main repolarizing component, has some special features, such as
a very flexible glycine -based residue sequenc e in the C -terminal end of
transmembrane helix S6 instead of the Pro -X-Pro sequence, forming a kink,
encountered in the majority of other channels. This results in an unusually large
inner vestibule, accessible to a variety of drug compounds, the binding o f which is
stabilized by several aromatic residues [34]. Inhibition of IKr by pharmacological
compounds leads to repolarization impairment and proarrhythmogenic effects such
as early afterdepolarizations (EADs), which can in turn trigger by synchronization

B. Amuzescu et al.
at ventricular level [29] dangerous life -threatening arrhythmias like Torsades -de-
Pointes (TdP), the most common drug -induced arrhythmia. It is estimated that an
astounding 40 –50% of new drug candidates have to be withdrawn from research
pipelines due to proarrhythmogenic liability via hERG blockade [20].
Two guidelines for proarrhythmogenic risk assessment of drug candidates are
currently in use: S7B for non -clinical tests and E14 for clinical tests. Both were
issued in 2005 by the International Conferenc e on Harmonization of Technical
Requirements for Registration of Pharmaceuticals for Human Use and are applied
by the majority of drug regulatory agencies. The E14 guideline relies on the
“thorough QT/QTc (corrected QT interval) study” applied to human pat ients,
assuming marked QT interval prolongation induced by a compound is indicative of
torsadogenic risk. The S7B guidelines include in vitro testing of hERG inhibitory
effects, as well as in vitro and in vivo cardiac electrophysiology testing. Multiple in
vitro preparations can be used, such as dissociated cardiomyocytes from different
animal species, myocardial tissue preparations including myocardial wedge,
papillary muscle, isolated Purkinje fibers or ventricular trabeculae, isolated heart
preparations, or in vivo approaches such as the “telemeterized conscious dog” with
ECG monitoring.
THE “ hERG -CENTRIC” PARADIG M
The S7B and E14 guidelines have been tremendously successful, and since
their implementing in practice no case of withdrawal from the markets of a
commercial compound due to fatal drug -induced arrhythmia has occurred [25].
However, there are concerns that the markers proposed in these guidelines are not
accurate enough in predicting arrhythmogenic risk. Lawrence et al. [17] analyzed
three in vi tro and three in vivo pro-arrhythmia risk prediction models, based on
non-standard markers (beyond hERG blockade and APD/QTc prolongation),
including early afterdepolarizations (EADs), AP triangulation, instability, reverse
use-dependence, transmural dispe rsion of repolarization, concluding that no one of
them shows a clear superiority in prediction efficiency. In a landmark study,
Redfern et al . [26] correlated for 100 drugs published data on hERG inhibition,
APD90 and QT interval prolongation in dogs with QT effects and reports of TdP in
humans, as well as with the clinical range of effective therapeutic plasma
concentrations for unbound compound (ETPC unbound ), concluding that torsadogenic
compounds in humans broadly exert hERG inhibitory effects within ET PC unbound ,
but interactions with multiple cardiac ion channels may often occur, and thus
hERG block or APD/QT prolongation per se are not necessarily torsadogenic
markers. Starting from this multiplicity of ion channel targets for a drug, Bottino et
al. [4] developed an original testing method: they adapted parameters of a canine

Risk testing by automated patch -clamp
ventricular cardiomyocyte model to experimental canine Purkinje fiber data by
rapid non -linear parameter estimation of 14 ion current conductances via training
the model to AP data , then they “reverse -engineered” the effects of two compounds
on five cardiac ion channels ( INa sustained , ICaL, IKs, Ito1, INaCa) beyond IKr from data in
transfected cell lines and effects on APs in canine Purkinje fibers paced at 0.5 and
1 Hz, and furthe r they used the resulting IC 50 values in “forward” simulations to
predict effects on isolated cardiomyocytes, individual cardiomyocytes in a
myocardial wedge, and the combined electrocardiogram of that wedge model. For
the two tested compounds, the model p redicted no QT interval prolongation or
increased dispersion of repolarization, in spite of the presence of hERG inhibitory
effects. Another important study is that of a group at ChanTest led by Arthur M.
Brown (Kramer et al ., [16]), who proved that hERG i nhibition alone is not
predictive of torsadogenic risk. Using automated patch -clamp platforms on
heterologous expression cell lines, they obtained IC 50 values for hERG, the L -type
Ca2+ channel Cav1.2, and the cardiac voltage -dependent Na+ channel Nav1.5 fo r an
impressive list of compounds. Further, they studied five multiple logistic regression
models, showing that combined models that include inhibition data for multiple
channels yield better prediction of torsadogenic risk compared to hERG inhibition
alone. Illustrative examples of lack of effectiveness of classical markers to predict
torsadogenic risk are verapamil and amiodarone. Verapamil is a potent hERG
blocker (IC 50 0.25 M at room temperature [16]), yet it does not feature
torsadogenic risks, becaus e it also blocks Cav1.2 with similar potency (IC 50 0.2
M), and the effects of blocking an outward and an inward current compensate and
cancel each other. Amiodarone is also virtually non -torsadogenic (inducing very
unfrequently TdP episodes), despite its marked effect on QTc (prolongation
beyond 550 ms) due to effects on multiple calcium and sodium cardiac currents
that, again, compensate each other [28].
THE CIPA PARADIGM
This lack of efficiency and robustness in predicting torsadogenic risk of
classical proarrhythmogenic markers, both in vitro and in vivo , related to hERG
block and QTc prolongation, which can therefore be associated with a so -called
“hERG -centric” paradigm, led to the development of a new proarrhythmogenic risk
testing paradigm named the “CiPA paradigm” (Cardiac in vitro Pro-arrhythmia
Asssay). The proposal was first discussed at a “think tank” meeting held at the
FDA headquarters on July 23, 2013, and its rationale and methods were described
in Sager et al. 2014 [28]. The “CiPA initiative ” considers an advanced mechanistic
in vitro approach as an alternative to the tests comprising the “hERG -centric”
paradigm, composed of three stages: stage 1 – assessment of inhibitory effects of a

B. Amuzescu et al.
compound on multiple human cardiac ion channels (five or seven distinct channels)
expressed in heterologous cell lines, preferably via automated patch -clamp; stage 2
– use of inhibition data derived in previous stage with an advanced humanized
ventricular cardiomyocyte model, like the O’Hara -Rudy 2011 model [24], to assess
via in silico simulations triggering of pro -arrhytmogenic events like early or
delayed afterdepolarizations (EADs or DADs) by the tested compound; stage 3 –
validation of in silico predictions by real experiments on human cardiomyocytes
derived from induced pluripotent stem cells (hiPSC -CM), in various experimental
settings. This stepwise approach may result into an in -depth characterization of
each tested compound, and may rescue some drug candidates that were previously
rejected based on “hERG -centric” criteria, but which are in fact devoid of
torsadogenic risks. This explains the large support for the CiPA initiative by a
number of drug regulatory agencies and high profile international research
institutes, like US Food and Drug Administration (FDA), European Medicines
Agency (EMA), Pharmaceuticals and Medical Devices Agency (PMDA -Japan),
Health Canada, Japan National Institute of Health Sciences (NIHS), Safety
Pharmacology Society (SPS), International Life Sciences Institute (ILSI) Health
and Environmental Sciences Institute (HESI), Cardiac Safety Research Consortium
(CSRC), etc. ( http://cipaproject.org ).
IMPLEMENTING CIPA AS SAYS
To study feasibility of approaches included in the CiPA initiative and to
develop novel CiPA guidelines for cardiac safety drug testing, SPS has settled
three working groups, including top experts from pharmaceutical companies,
contract research organizations (CROs), and academia, each of them addressing
one stage of the CiPA pro tocol [11]. Thus, the Ion Channel Working Group
(ICWG) develops standardized testing protocols for a variety of cardiac ion
channels expressed in cell lines, including IKr (hERG), ICaL (Cav1.2), INa peak and INa
late (Nav1.5 peak and late current), Ito 1 f ast (Kv4.3), IKs (KCNQ1), and IK1 (KIR2.1)
[7]. A panel of 28 test compounds was defined, including drugs with high,
intermediate, and low torsadogenic risk. High risk compounds are: azimilide,
bepridil*, dofetilide*, ibutilide, quinidine*, vandetanib, dis opyramide, (±) -sotalol*;
intermediate risk compounds: astemizole, chlorpromazine*, cisapride*,
clarithromycin, clozapine, domperidone, droperidol, terfenadine*, pimozide,
risperidone, ondansetron*; low risk compounds: diltiazem*, loratadine, metoprolol,
mexiletine*, nifedipine*, nitrendipine, ranolazine*, tamoxifen, verapamil*
(compounds marked with * represent the test set, used to develop robust voltage –
clamp protocols for the CiPA ion channel panel, while dofetilide and nifedipine are
cardiomyocyte calib ration compounds). The compounds should be ideally tested

Risk testing by automated patch -clamp
both at room temperature (RT) and physiological temperature (PT) (35 –37șC),
using different automated patch -clamp platforms, or by manual patch -clamp. In a
recent study, Crumb et al. provided inhibi tion data against the CiPA panel of ion
channels for 30 compounds, tested by manual patch -clamp [8]. In this study, INa late
was tested upon application of veratridine 50 M in the bath solution, a method
that yields large late Na+ currents, driving many Na+ channels in the sustained burst
mode due to failure to inactivate, but is prone to experimental errors because tested
drugs may interact with veratridine [6]. The In Silico Working Group (ISWG),
acting under FDA direction, seeks to apply inhibition dat a derived by ICWG for
the 12 compounds of the test set to the O’Hara -Rudy 2011 humanized ventricular
cardiomyocyte electrophysiology model. For the first attempts, the group utilized
the inhibition data obtained by Crumb et al. [8], combined with dynamic h ERG
inhibition data generated also by manual patch -clamp at RT and PT, evaluating the
usefulness of classical proarrhythmogenic markers such as APD90, triangulation,
and EAD occurrence. A part of ICWG transformed recently into a Rapid Response
Team (RRT) t o mediate interactions between the ion channel and modeling groups.
Largely, by the end of 2016, the analysis performed by these two groups for the
CiPA panel of drugs was completed. The third group, the Cardiac Stem Cell
Working Group (SCWG), sponsored by HESI, attempts to validate modeling
predictions by experiments on hiPSC -CM preparations. A large variety of methods
are employed and there is still no consensus upon which of them is the best. Thus,
multielectrode array (MEA) extracellular AP recordings c an be obtained from 2D
layers of cardiomyocytes or 3D artificial myocardium constructs, impalement
intracellular microelectrodes such as MEA nanopillars can be used [5], optically
recorded APs using voltage -sensitive dyes can be obtained from isolated
cardiomyocytes or CM tissue layers using fluorescence plate readers or
fluorescence microscopy setups. A recent study assessed 26 drugs and 3 drug
combinations on two popular commercial hiPSC -CM ventricular cardiomyocyte
preparations (Cor.4U®, Axiogenesis, Col ogne, DE, and iCell® Cardiomyocytes,
CDI, Madison, WI) via voltage -sensitive dyes optical AP recordings and electrical
MEA recordings, along with manual patch -clamp on cell lines expressing hERG,
Nav1.5, and Cav1.2, concluding that hiPSC -CM represent an ad equate in vitro test
system for pro -arrhythmia [3]. The authors demonstrated that hiPSC -CM respond
well to hERG and Cav1.2 blockers, and to a lesser extent to INa late blockers. They
also assessed by RT -qPCR the levels of expression of SCN5A (main subunit on
Nav1.5), CACNA1C (main subunit of Cav1.2), KCNH2 (main subunit of Kv11.1 –
hERG1 – IKr), and KCNQ1 (main subunit of KvLQT – IKs) gene transcripts in these
commercial cardiomyocyte preparations compared to adult ventricular
cardiomyocytes.

B. Amuzescu et al.
CONCLUSIONS AN D PERSPECTIVES
Therefore, to date there is no consensus about the optimal method to be
employed for stage 3 of the CiPA protocol, and different research groups
worldwide eagerly test a large variety of solutions [32].
However, our opinion is that automate d patch -clamp platforms, and
particularly the CytoPatch™ platforms relying of the patented Cytocentering®
technology [31, 33] , via their high quality and stability of gigaseal whole -cell
configuration, offer the best testing method, allowing accurate recor ding of resting
and action potential, as well as of several cardiac ion channel components, in the
same cardiomyocyte [1].
In preliminary studies we tested the feasibility of mixed current -clamp and
voltage -clamp recordings in commercial hiPSC -CM preparat ions on the
CytoPatch™ platform, with application of pharmacological compounds [30].
Within a EU -funded project for Regional Development (POC no. 146/2016,
acronym CiPA3), we are attempting to combine all three stages of the CiPA
paradigm in a single exper iment performed on enhanced hiPSC -derived ventricular
cardiomyocytes, in order to provide a robust and reproducible industrial standard
for cardiac safety drug testing.
Acknowledgements . The authors gratefully acknowledge Dr. Thomas Knott and Dr. William
Crumb for instructive comments. This study was funded from Competitiveness Operational
Programme 2014 –2020 project P_37_675 (contract no. 146/2016), Priority Axis 1, Action 1.1.4, co –
financed by the European Funds for Regional Development and Romanian Gover nment funds. The
contents of this publication does not necessarily reflect the official position of the European Union or
Romanian Government.
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