PERSPECTIVESOPINION

Evolution of strategies to improvepreclinical cardiac safety testing
Gary Gintant, Philip T. Sager and Norman Stockbridge

Abstract | The early and efficient assessment of cardiac safety liabilities isessential to confidently advance novel drug candidates. This article discusses
evolving mechanistically based preclinical strategies for detecting drug-induced
electrophysiological and structural cardiotoxicity using in vitro human ion
channel assays, human-based in silico reconstructions and human stem
cell-derived cardio­­myocytes. These strategies represent a paradigm shift fromcurrent approaches, which rely on simplistic in vitro assays that measure
blockade of the Kv11.1 current (also known as the hERG current or IKr) and on theuse of non-human cells or tissues. These new strategies have the potential to
improve sensitivity and specificity in the early detection of genuine cardiotoxicity
risks, thereby reducing the likelihood of mistakenly discarding viable drugcandidates and speeding the progression of worthy drugs into clinical trials.
Ideally, preclinical cardiotoxicity screening
efforts should efficiently detect unsafe
compounds sufficiently early on in the
drug discovery process to influencecandidate drug selection, thus reducing
later-stage attrition, risks to participantsin clinical studies, development time andcosts. However, over-simplified and highly
sensitive (but not specific) approaches forthe early detection of cardiac safety liabilities
can result in unwarranted attrition of novel
drug candidates owing to false-positive
findings. The risk of such unwarranted
attrition may also be increased by a lackof concordance between the effects ofcompounds in animals (or animal-derived
tissues) and those in humans.
At present, early screening assays typicallyinvolve simple, reductionist in vitro systems
and surrogate markers for cardiac liabilities
in animals or animal-derived tissues ororgans. A strong focus remains on avoiding
a rare, drug-induced, life-threatening
ventricular arrhythmia termed torsades
de pointes (TdP, ‘twisting of the points’)1–4.
TdP is associated with delayed repolarization,a surrogate marker of proarrhythmia that isobserved as prolongation of the QT interval
on a surface electrocardiogram (ECG).

Blockade of the Kv11.1 current (also known
as the hERG current or IKr, a repolarizing
potassium current carried by thepore-forming α-subunit of the voltage-gatedpotassium channel Kv11.1 (REFS 5–8),
commonly called the hERG channel, whichis encoded by KCNH2) is recognized as apredominant mechanism responsible for the
drug-induced delayed repolarization that is
linked to TdP (FIG. 1). Consequently, emphasis
on detecting IKr blockade early in drug
discovery has, along with mandated clinicalthorough QT (TQT) studies to quantify
QTc (the QT interval corrected for changes
in heart rate) prolongation later in drug
development, contributed to the successful
removal of TdP risk for new chemical
entities. However, there is concern that thelack of specificity of such studies may lead topotential drug candidates that otherwise havedesirable properties being wrongly discarded
from drug pipelines.
Contractile and structural cardiotoxicity,such as those seen with several
kinase-targeted cancer drugs9–12, represent
another growing safety concern. Contractile
and structural cardiotoxicity in non-humanspecies may prevent the progression ofpromising compounds, despite known

NATURE REVIEWS | DRUG DISCOVERY

species-related differences in contractile
function and calcium handling. As less isknown about the mechanisms responsible
for these more varied and complex
toxicities, more-comprehensive multiparametric approaches using phenotypic
cellular assessments are more appropriate(and informative) to detect contractile andstructural cardiotoxicity. Ideally, such effects
would be detected using in vitro preclinicalassays with human-derived tissues, before
the use of more costly, longer-term animal
toxicity studies.
This article discusses evolving
human-based preclinical cardiotoxicitytesting strategies that are based onmechanistic assessments in the context
of the simplest functional unit in the heart,the ventricular myocyte. The recently
proposed Comprehensive in vitro
Proarrhythmia Assay (CiPA) schema forthe preclinical evaluation of proarrhythmic
liabilities is provided as an example13,14.
The CiPA proposes to define proarrhythmic
risk based on in silico reconstructions ofhuman ventricular electrical activity. These
reconstructions integrate drug effects on
multiple human cardiac currents and theresults are confirmed with human stem
cell-derived cardiomyocytes (hSC-CMs).
This approach represents a paradigm
shift away from approaches that are
reliant on pragmatic, reductionist (often
single parameter) preclinical non-humanassays that use surrogate markers andapproaches that use integrated responses
derived from poorly characterized,
non-human preparations that are reliant
on downstream surrogate markers ofproarrhythmia (such as QT prolongation).
hSC-CMs can also be used to assess
structural and contractile cardiotoxicity basedon multi-parametric, integrated ‘signatures’of functional, phenotypic responses derived
from high-content screening. We assert thatthese approaches using human channels andin silico reconstructions of human cellular
proarrhythmia and hSC-CMs will reduce theinappropriate premature discontinuation of
new chemical entities during drug discovery,
reduce reliance on clinical surrogate markers
during later-stage drug development (such as
QT prolongation) and enable the rapid andefficient development of safer novel drugs.
VOLUME 15 | JULY 2016 | 457

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Voltage (mV)a Ventricular action potential
0 mV

Slowedrepolarization

Current (μA/μF)

b hERG current
Normal
LQTS

c Surface ECG

Voltage (mV)R

P-waveT-waveQ
S

Normal QT interval
Prolonged QT interval

Voltage (mV)d Schematized ECG strip*

NormalrhythmTorsades de pointes
Time (ms)Figure 1 | Link between delayed repolarization and torsades
de Reviews
pointes |proarrhythmia.
NatureDrug Discovery
a | The slowing of ventricular repolarization ona cellular level results from a decrease in net
outward current. b | This change in current may
occur as a result of a reduction in hERG current(IKr) owing to IKr blockade by a drug or inheritedchannelopathies such as the long QT syndromes(LQTS). c | Although the relationship between
ventricular repolarization and torsade depointes (TdP) is complex, on a cellular level,
delayed ventricular repolarization leads to prolongation of the QT interval, which can beobserved with a surface electrocardiogram(ECG). d | Prominent QT prolongation in thepresence of an appropriate electrophysiologicalsubstrate and rhythm may further dysregulate
repolarization, giving rise to a premature
ventricular beat before the end of the T wave
(indicated with an asterisk) to initiate TdP.
Figure is reproduced with permission from
REF. 174, Frontiers (http://dx.doi.org/10.3389/
fphar.2010.00137).

Current proarrhythmia testing approaches
The evolution, limitations and learningsof the International Committee on
Harmonization S7b (preclinical) and E14(clinical) guidelines. The late 1980s andearly 1990s witnessed a growing awarenessof the potential of non-cardiovascular drugs
to cause TdP15. Arguably, these concerns
took hold between 1988 and 1991, whenthree drugs (prenylamine, lidoflazine andtorodiline) were withdrawn from markets
in the United Kingdom and elsewhere inEurope owing to their association with QT
prolongation and TdP. Further concerns
were raised by QT prolongation causedby drugs from additional therapeuticclasses (halofantrine, an antimalarial drug,
cisapride, a gastric prokinetic drug andterfenadine, an antihistamine) and by
QT prolongation and increased mortality
in patients treated with d‑sotalol after
myocardial infarction (the SWORD study).
In response to these concerns, the European
Committee for Proprietary Medicinal
Products (CPMP) proposed a preclinicalapproach to evaluate the propensity of adrug to delay ventricular repolarization.
This approach included an in vitro
assessment of action potential prolongation
along with early after-depolarizations(EADs) and triggered activity (forms ofaltered or interrupted repolarization)
in appropriate animal tissues andspecies16. Subsequent studies emphasized
the link between delayed ventricularrepolarization and blockade of IKr by various
non-cardiovascular drugs7,17,18 and therelationship between a congenital long QT
syndrome (namely LQT2) and reduced IKr19.
These findings led to the inclusion of thefunctional IKr assay in the 2005 International
Committee on Harmonization (ICH) S7B
regulatory drug development guidance
document (The Non-Clinical Evaluationof the Potential for Delayed VentricularRepolarization (QT Interval Prolongation) by
Human Pharmaceuticals)20. The evolutionof this preclinical surrogate proarrhythmia
marker was paralleled by the codificationof QT prolongation in the ICH E14 clinicalguidance (The Clinical Evaluation of QT/QTc
Interval Prolongation and Proarrhythmic
Potential for Non-Antiarrhythmic Drugs)21.
The goal of this clinical guidance was similar
to that of S7B (“to assess the potential of adrug to delay cardiac repolarisation”) but
is applied to clinical studies of the QTcinterval rather than non-clinical studies.
This guidance established the TQT study,
a carefully controlled clinical assay toassess drug-induced QTc prolongation

458 | JULY 2016 | VOLUME 15

at therapeutic and supratherapeutic
exposure levels. Small increases in the QTcinterval (that is, >10 milliseconds for theplacebo-adjusted change from baseline(upper bound of the 90% confidence
interval)) trigger regulatory concern.
However, defining proarrhythmic liability
based on an arbitrary threshold for clinical
QT prolongation leads to a dichotomousclassification of risk that is often
misunderstood and not useful in definingrisk–benefit ratios of new drugs.
Since its inclusion in the ICH S7B
guidance, the IKr assay has assumed
a prominent role as gatekeeper in afrontloading screening approach
designed to reduce later-stage compound
attrition22–24. This strategy was bolsteredby the commercial availability of celllines with stable and heterologous hERG
channel expression, the development ofhigher-throughput automated planar
patch-clamp platforms, a growing listof drugs shown to block IKr and thedesire to avoid the cost and potential
negative consequences of a positive
TQT study (including additional clinicalmonitoring during clinical development
and the labelling implications should
the study be positive). In some cases,
structure-based computer models to detect
hERG-based liabilities have also been used
to attempt to avoid any hint of IKr blockadeeven before compound synthesis.
As a consequence of positioning the
IKr assay early in drug discovery, countlesscompounds are deprioritized (or discarded)
early on. De Ponti25 estimated that “asmany as 60% of new molecular entities
developed as potential therapeutic agents,
when assayed for hERG blocking liability,
test positive and are thus abandoned earlyin development.” This would be fine if IKr
blockade reliably predicted proarrhythmia,
but this is not the case, as mechanistic andtranslational studies demonstrate that IKr
blockade alone is not highly specific forpredicting either delayed repolarization
or clinical proarrhythmia26 (see below).
Although it might be possible to ‘design
out’ this property, this strategy incurs added
costs and project delays and unnecessary
animal testing, as well as the potential lossof efficacy or other favourable compound
attributes (for example, target specificity).
False-negative determinations also need to beconsidered; Kramer et al.27 identified 6 false
negatives in a total of 55 torsadogenic drugs
when the drugs were classified based only
on their effects on IKr. Finally, drug-induced
QT prolongation is not specific forwww.nature.com/nrd

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PERSPECTIVESNATURE REVIEWS | DRUG DISCOVERYa

As repolarization on a cellular level
represents an integrated response ofmultiple cardiac currents, pumps andexchangers, so the QT interval represents
the integrated response of temporaland spatial differences in repolarization
within and across the ventricles as filteredand recorded from the body surface38.
The relationship between changes in the
QT interval (prolongation as well asmorphological changes) and electrophysio­
logical effects across the ventricular wall
are complex and poorly understood, andsimpler ECG recordings are not necessarily
informative about spatial and temporal
effects on repolarization that are linked
to proarrhythmic liabilities39. Thus, QT
prolongation is a poor representation ofchanges in repolarization at the cellular
or regional levels. This fact was recently◀ Figure 2 | Ionic basis for ventricular repolari-40

Potential (mV)

Limitations of IKr and QT assays to define
proarrhythmic risk. On a cellular level,
cardiac repolarization is a complex processrepresenting a fine balance of multiple
time- and voltage-dependent inward andoutward currents (FIG. 2a). At an organ
level, differences in current densities
within the ventricles are responsible for theheterogeneity of ventricular repolarizationthat gives rise to the QT interval. Recent
data suggest that drugs affect morecardiac currents than previously expected:
automated patch-clamp systems havedemonstrated the promiscuity of multiple
non-cardiovascular drugs, including
blockade of cardiac IKr, ICaL (the L-type Ca2+
current) and INa (a Na+ current)27,30 (FIG. 2b).
Together, these findings demonstrate thata single-current ‘IKr-centric’ approach fails
to reflect a drug’s integrated effect on the
net outward current, which ultimately
drives repolarization.
The term ‘repolarization reserve’ hasbeen used to define the ability of ventricular
cells to sustain cardiac repolarization in theface of drug effects on single or multiple
currents31,32. This concept is related to‘multi-channel block’ or ‘multi-ion channel
effects’ — terms that are used to describe thecomplex electrophysiological effects of drugs
that result from the blockade of multiple
cardiac currents33,34. For example, despite
being a potent blocker of IKr, verapamil does
not cause meaningful QT prolongation
because of its concomitant blockade of thedepolarizing inward calcium current(ICaL, through blockade of the calcium
channel subunit Cav1.2), which mitigates theeffects of reduced IKr outward current 35,36.
Similarly, ranolazine elicits QT prolongationconsistent with blocking the repolarizing
IKr but does not elicit proarrhythmia, mostprobably because of concomitant blockade ofthe depolarizing late inward sodium current(INaLate, which flows through the sodium
channel subunit Nav1.5)37. An in vitro
study demonstrated that prolongationof Purkinje fibre action potentials by the
IKr-blocking drug dofetilide was reducedby the concomitant administration of
nifedipine (owing to ICaL blockade) or

lidocaine (owing to blockade of INaLate),demonstrating the role of multi-channel
blockade34 in modulating repolarization.
An in silico reconstruction demonstrating
the role of multi-channel blockade inmodulating the effects of repolarization isshown in FIG. 3. Such findings suggest thata drug that reduces outward (repolarizing)
IKr might still be sufficiently safe withregard to proarrhythmic risk owing to
the mitigating effects of reduced inward(depolarizing) current such as ICaL and INaLate.
From a cardiac perspective it is possiblethat drugs that affect multiple cardiac
currents (including IKr) but marginallyaffect delayed repolarization (owing tomultiple channel blockade) pose minimal
proarrhythmic liabilities compared todrugs that exert selective blockade of IKr
at therapeutic exposures.

2

1
0

0

3

–40

4

100 ms

–80

INa
ICaL
ICaT
Ito1
ICl(Ca)
IKur
IKr
IKs
IK1
If
INCX
Depolarizing current
Repolarizing currentbDrugs more potent
compared to IKr (%)

arrhythmogenesis. For example, alfuzosin,
phenobarbital and ranolazine prolong the QTinterval clinically but are not typically linked
with TdP28,29. Similarly, small increases in thehuman QTc interval commonly result in thepremature termination of drug development
programmes (or in label warnings), eventhough a number of these drugs are unlikely
to be proarrhythmic.

60
40
20
0

Cav1.2

Nav1.5

KCNQ1

Kv4.3

Cardiac current
n = 55 drugs
n = 31 drugs
Nature Reviews | Drug Discovery

zation and drug-induced proarrhythmia.
a | Multiple ionic currents define the ventricularaction potential. Voltage-dependent andtime-dependent inward (depolarizing) and outward (repolarizing) currents are displayed. The
outward repolarizing current, IKr (also known as
the Kv11.1 current, conducted by hERG channels), is only one of multiple overlapping currents that define repolarization. Numbers
indicate the phases of the action potential:
rapid depolarization (phase 0), early repolarization (phase 1), plateau (phase 2), rapid repolarization (phase 3) and resting membranepotential (or phase 4 depolarization). b | Multi­
channel blockade of cardiac currents by marketed drugs. The graph compares half-maximal
inhibitory concentration (IC50) values for theblockade of IKr with values for the blockade oftwo depolarizing currents (ICaL through Cav1.2
channels and INa through Nav1.5 channels) andtwo repolarizing currents (IKs through KCNQ1channels and Ito through Kv4.3 channels). Twopublished datasets were used: a 55-drug set27(blue bars), which characterized blockade of ICaLand INa, and a 31-drug dataset30 (red bars), whichcharacterized blockade of ICaL, INa, IKs and Ito.
Eleven compounds were common between thetwo datasets. Both datasets also characterized
potency of IKr blockade, used heterologously
expressed channels and used the same automated patch platform at the respective testsites to compare blockade potency (thus minimizing variability). The bars represent the percentage of drugs that blocked each current
more potently than IKr for each drug collection.
The results show that a single-current ‘IKr-centric’approach neglects to inform on the lack of
selectivity of many drugs, which results in blockade of multiple prominent cardiac currents.
Figure part a is reproduced with permission
from REF. 101 , Frontiers (http://dx.doi.org/
10.3389/fphys.2012.00346).

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PERSPECTIVES
highlighted in a technically challenging
noninvasive ECG imaging study in subjects
with long QT, which recorded 256 bodysurface ECGs (along with a thoracic
CT scan gated to the R–R interval) toA
Membrane potential (mV)

50

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a b

d

c

–50

–100

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500

1,000

1,500

IKr (% block)

0

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85

85

ICaL (% block)

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Outward

1.0

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0.5
0

InwardCurrent μA/μF

B

–0.5
–1.0

EAD inward
current spike
0

500

1,000

1,500

Time (ms)Figure 3 | Role of delayed repolarization andmulti-channel blockade in defining pro­Nature
Reviews | Drug
Discovery
arrhythmic risk.
Representativein silicore­constructions show action potentials (part A) andcorresponding net ionic current (part B) in thepresence of different levels of blockade of hERG
current (IKr) and L‑type calcium current (ICaL).
Under drug-free conditions (0% blockade of IKrand ICaL), repolarization proceeds normally (shortest action potential (part Aa) with net outward
current throughout repolarization (part Ba).
A 50% blockade of outward repolarizing IKr prolongs the action potential duration from 306 to
477 milliseconds (part Ab, part Bb), and, fur­ther, an 85% IKr blockade elicits an early afterdepolarization (EAD) before full repolarization,
which is associated with a prolonged action
potential duration of 1,466 milliseconds (part Ac).
The inward current responsible for the EAD isevident in the lower panel (part Bc). Concomitant
40% blockade of the inward (depolarizing) ICaL in
the setting of sustained 85% IKr blockade partiallyreverses delayed repolarization (part Bc and partAd), shortening the action potential durationfrom 1,380 to 782 milliseconds, and abolishes thenet inward current spike responsible for initiating
the EAD (part Bd). The simulation is based on the
O’Hara-Rudy model44 with slow stimulation(15 beats per minute), a recognized risk factor for
torsades de pointes.

construct maps of epicardial repolarization
dispersion40. The results showed that
subjects with long QT had epicardial
regions with steep repolarization dispersion
caused by heterogeneities of action potential
duration, a proarrhythmic substrate thatwas not detected with the surface ECG.
The steep repolarization gradients did notcorrelate with QTc values from 12‑lead
ECG recordings, highlighting a limitationof resolution of standard ECG monitoring
to assess proarrhythmic liabilities.
IKr assay performance characteristics inrelation to proarrhythmia and clinical QTc
prolongation. Redfern and colleagues41
compared potency of IKr blockade (within
the context of clinical exposures) withsubjective clinical experiences related to
QT-interval prolongation and TdP. Basedon a five-category ranking of proarrhythmic
risk, the authors suggested that most drugs
with half-maximal inhibitory concentration(IC50) values for IKr blockade that arecomparable to the highest free-drug plasma
concentrations observed clinically wereassociated with TdP and that a provisionalsafety margin of 30 (defined as IC50/Cmax,
where Cmax is the maximum free plasma
concentration a drug achieves clinically)
was generally predictive of cardiac safety(although a higher margin of 100 was
suggested in the case of less serious disease
indications). Wallis42 compared the potency
of IKr blockade (based on a 10% inhibition ofcurrent) with clinical QTc prolongation(powered to detect 7–10 millisecond QTc
prolongation) using a 19‑compound dataset(of which 11 prolonged QTc clinically).
When assessed at twofold clinical exposure,the IKr assay was shown to be 82% sensitive(in simplest terms, the proportion of true
positives correctly identified by the assay)
and 75% specific (the proportion of true
negatives correctly identified by the assay).
Sensitivity and specificity both describe
the concordance between test assay
results based on a clinical ‘gold standard’,but it can be argued that the ability of apreclinical test to predict QT prolongation
is more appropriately described by reverse
conditional probabilities (known as
positive and negative predictive values) that
describe the probabilities of clinical effects
given assay results. However, positive andnegative predictive values depend on theprevalence of the disease or condition in
the study sample and typically cannot be
generalized beyond a study. Furthermore,
highly sensitive tests may actually have
low predictive values in study populations

460 | JULY 2016 | VOLUME 15

with a low prevalence of the disease orcondition. By contrast, likelihood ratios(LRs) summarize sensitivity and specificity
in a form that is useful for defining theutility of an assay for increasing certainty
about a diagnosis — thus representing
a meaningful measure of test accuracy— and they have the advantage of being
less dependent on disease prevalence43,
a particularly important benefit for low
incidence events such as TdP. In clinicaldecision making, a positive LR (+LR) valueof 1 indicates no influence on the risk of
disease, values of 2–5 indicate a small
increase in probability and values of 10 orgreater indicate a large and often conclusive
increase in the likelihood of disease. The+LR value calculated for IKr assay resultsfrom the Wallis study 42 was 3.3, consistent
with a minimal-to-moderate influence on
decision making.
A later retrospective study 26 evaluated theperformance of an IKr assay in predicting QT
prolongation by comparing potency of IKr
blockade (in accordance with S7B) to resultsfrom strictly controlled clinical TQT studies(in accordance with E14) of a 39‑drug
dataset submitted for regulatory approval.
Based on IC50 values for IKr blockade (relative
to unbound clinical exposures) and a mean
QTc prolongation time of 5 milliseconds
defining a positive TQT study, the IKr assay
provided only moderate overall performance(sensitivity and specificity values of 64% and88%, respectively, and a positive likelihood
ratio of 3.5, based on an optimal safetymargin of 45). This study demonstrates
the overall minimal performance of an
IKr assay alone to reliably predict QT
prolongation (itself a surrogate marker ofproarrhythmia) and highlights the needto consider a more comprehensive view ofdrug effects on cardiac currents to better
assess proarrhythmia.
Limitations of animal models forpredicting delayed repolarization andproarrhythmic risk in humans. The needfor proarrhythmia evaluations based onhuman models is emphasized by species
differences in ventricular repolarization andresponses to drugs. For example, the rat is an
inappropriate species for modelling human
repolarization as it possesses a minimal
IKr (instead relying on a transient outward
current (Ito) through channels composedof Kv4.3 subunits. Another example isprovided by the guinea pig, which relies on
two plateau-repolarizing currents (IKr andIKs) to modulate ventricular repolarization,
whereas humans, dogs and rabbits rely
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primarily on IKr (REFS 44,45). Differences indrug responses across species have also beenreported: manifestation of EADs in response
to dofetilide and quinidine were readily
observed in rabbit Purkinje fibres but notin those of guinea pigs, dogs, pigs, goats, or
sheep under identical conditions in vitro46.
In a recent study, prominent differences in
the ability of combined pharmacologicalblockade of the inward rectifier current(IK1) and either IKr or IKs to elicit TdP acrossboth rabbits and dogs were attributed to astronger repolarization reserve in dogs45.
Various in vitro proarrhythmia models
have been used to screen for proarrhythmic
risk, most notably the rabbit ventricularwedge47–49 and the rabbit Langendorff heart
preparations50–52. Although such empiricalin vitro models are useful in defining
mechanisms of proarrhythmia, the technicalexpertise necessary to maintain these models
and the electrophysiological differences(both between non-human species andbetween non-human species and humans)
make the assessment of proarrhythmic
risk difficult.
Differences in the ionic current densities
of canine and human ventricle tissues havebeen documented53; although IKr density
and kinetics are similar in both species,
in humans, ICaL and Ito are approximately
30% larger and 29% smaller, respectively.
In addition, the ability to resist changes in
repolarization (repolarization reserve) is
less in humans because of reduced outward
current contributions from the potassium
currents IK1 and IKs. These later results are
consistent with the increased sensitivityof humans (versus dogs) to drug-induced
QT prolongation with sotalol, cisaprideand moxifloxacin54.
Towards a mechanistic approach toassess proarrhythmic risk using humancellular proarrhythmia assays. Knowledgeof the mechanisms responsible for TdP
provides the opportunity to develop a morecomprehensive approach for assessing
proarrhythmic risk. Briefly, impaired
and delayed cellular repolarization can,through various mechanisms, promote
net inward current during the action
potential plateau that gives rise to EADs
(FIG. 3). These emergent cellular effects
are not predicted based on effects on
individual cardiac currents. When coupled
temporally and spatially with a vulnerable
electrophysiological substrate that includes
heterogeneous repolarization, EADs may
give rise to early ventricular excitation.
Dispersion of repolarization across theventricles (which is often exaggerated underpathological conditions with remodelledor hypertrophied myocardium) may also
promote EADs and is recognized as a
substrate vulnerable to unstable re-entrant
excitation supporting TdP. Multiple studies
have identified EADs as the likely triggering
event for TdP4,55–58. QT prolongation alone
does not necessarily lead to TdP in theabsence of EADs.
Determination of the exact conditions
necessary for the initiation of TdP requires
sophisticated 3D computer modelling,which is not routinely available (andis not characterized in detail here; see
REFS 4,59–61). Although a 3D heart model
may be informative about proarrhythmicmechanisms, it is difficult to define
parameters for these models from theavailable data. This is not the case withcellular membrane models, which canbe fully parameterized from readily
available data. Reconstructions of cellular
proarrhythmic activity based on integrated
effects on multiple cardiac currents (delayed
repolarization, repolarization instability andEADs) can be assessed with precision using
appropriate in silico reconstructions62–64.
The discussions above highlight key
aspects of our understanding of TdP, an
event no longer viewed as an idiosyncratic
response to a seemingly diverse arrayof drugs. The evolution of a definedmechanistic understanding of the initiationof TdP provides the basis for a morecomprehensive mechanistic preclinicalassessment of proarrhythmic risk. Suchan approach is less reliant on surrogate
markers (such as QT prolongation) andirrational disqualification of preclinicaldrug candidates based on one-dimensional
assessments of delayed repolarization.
With this as the background, a meeting
was held at the US Food and Drug
Administration (FDA) in the summer of2013 to discuss a new paradigm for cardiac
safety evaluations based on in vitro humanmodels and mechanistic understandings of
TdP proarrhythmia. The evolving paradigm— CiPA — represents a novel preclinicalapproach focused on the evaluation of
TdP risk by the pharmaceutical industry,
regulators and academic investigators65,66.
The CiPA initiative
The three main preclinical elements ofthis proposed paradigm are: first, in vitro
evaluation of drug effects on multiple
individual currents using human ion
channels in heterologous expression systems;
second, integration of this information into

NATURE REVIEWS | DRUG DISCOVERY

an in silico computational reconstructionof a human ventricular myocyte toassess proarrhythmic liability; and third,
confirmation of effects on ionic currents
using in vitro electrophysiological responses
from hSC-CMs (FIG. 4). A clinical elementthat includes the assessment of ECGs
from Phase I studies represents a fourthcomponent of the CiPA. This component
remains an essential part of a drug’s safety
evaluation and functions to confirm theabsence of unanticipated electrophysiologicaleffects (for example, from a metabolite)
as well as detect other forms of untoward
electrophysiological effects (for example,
AV‑nodal blockade). The three preclinicalcomponents of the CiPA, along with thechallenges associated with each and progressin their development, are discussed below.
Component 1: drug effects on humancardiac ion channels in vitro. Although
there are multiple ion channels, transporters
and exchangers in the human ventricular
myocyte, the Ion Channel Working Group(ICWG, under the auspices of the Safety
Pharmacology Society) of the CiPA initiative
has proposed assessing drug effects on
7 ionic currents chosen based on theirprominent role in defining ventricularrepolarization and experiences from thepharmaceutical and safety pharmacology
communities. These currents are: ICaL
(through channels formed of Cav1.2
subunits), the fast sodium current (INaFast)and INaLate (both Nav1.5) for inward
depolarizing currents; and IKr (Kv11.1),
IKs (Kv7.1), Ito (Kv4.2) and IK1 (Kir2.1, Kir2.2,
Kir2.3 and Kir2.4) for outward repolarizing
currents. The use of automated patch-clamp
platforms is proposed to characterize theeffects of drugs on multiple human currents(expressed individually in heterologous
expression systems, such as humanembryonic kidney cells or Chinese hamster
ovary cells). This strategy will allow for
earlier and more-comprehensive ion channel
screening across multiple currents, which
is not realistically possible using manual
patch-clamp techniques67.
Challenges with component 1. Recent
studies have emphasized the importance oftime- and voltage-dependent drug–channelinteractions to characterize drug blockade
of IKr beyond simple IC50 values (for an
example see REF. 68). Indeed, differencesin channel gating between normal versus
mutated hERG channels69, as well as
differences in drug-binding kinetics andmode of action (trapped or unblocked) to
VOLUME 15 | JULY 2016 | 461

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Current (pA)cellular human ventricularelectrophysiology

50 pA

Istim = CCurrent (nA)Time (ms)

1 nA
0.5 ms0123Membrane potential (mV)1 ms
0 1 2 3 4 5 6 7 8

dVm
+ Im
dt

50

0

–50

–100

0

Time (ms)

500

1,000

Time (ms)Figure 4 | Elements of the Comprehensive in vitro Proarrhythmia
Assay. a | Drug effects on multiple currents provide input for in silico reconstructions of cellular ventricular proarrhythmia, including delayed repolarization and early after-depolarization (EAD). Two representative currents— Kv11.1 current (also known as hERG current or IKr; top panel) and Cav1.2(ICaL; bottom panel) — are shown. b | In silico reconstructions of ventricular
myocytes based on the O’Hara-Rudy model44 with progressive IKr blockade
leading to an EAD. c | The effects of drugs on ionic currents are confirmed
in vitro with human stem cell-derived ventricular cardiomyocytes, using
electrophysiological recordings of field potentials from microelectrodewild-type hERG channels, have a crucialrole in affecting a drugs’ ability to delay
repolarization70. Such factors need to beconsidered when designing standardized
patch-clamp protocols that more fully
characterize drug effects on currents. Suchdetails (especially for the crucial IKr) areessential to adequately inform in silico
reconstructions of ventricular electricalactivity. Towards this goal, the ICWG
(with input from the In Silico Working
Group (ISWG)) is developing standardized
voltage-clamp protocols to characterize
the dynamics of IKr blockade, and they
are presently testing the utility of these
protocols to best parameterize the in silico
reconstruction model. These protocols will
first be tested using manual patch-clamp
techniques, with data to be subsequently
compared to results from automated patch
platforms. Efforts are also ongoing tostandardize the voltage-clamp protocols
for the remaining six ionic currents.
An additional challenge for thisworkgroup is to define standardized
experimental conditions and operating
specifications for automated planar
patch-clamp platforms71,72. Standardization
is essential for benchmarking variability
within and across laboratories, a key
challenge for implementation of the
CiPA paradigm. Best practices need to be
defined that consider the potential loss ofc In vitro eﬀects on human stem

cell-derived ventricular myocytesd Clinical evaluation ofunanticipated electrophysiology

Voltage (mV)b In silico reconstruction ofhuman cardiac currents

Voltage (mV) Fluorescence ratioa Drug eﬀects on multiple

1 mV
200 msTime (ms)Time (ms)arrays or action potential configurations from voltage-sensitive dyes. The
Nature
Reviews| Drug Discovery
graphs show delayed repolarization resulting
from
IKr blockade,which wasrecorded from human stem cell-derived cardiomyocytes using voltagesensitive dyes (upper traces) and extracellular field potentials (lower traces).
Note the presence of an EAD clearly identified using voltage-sensing optical (VSO) techniques. d | Early clinical electrocardiogram (ECG) evaluations
are used to evaluate unanticipated electrophysiological effects. Figure parta is adapted with permission from REF. 118, American Physiological Society.
The upper trace in figure part c is courtesy of Clyde Biosciences, UK.
The lower trace in figure part c is courtesy of Axion Biosystems, USA.

compounds studied owing to compound
handling and perfusion systems73. The effects
of temperature (room temperature versus
37oC)74,75, buffer systems (serum versus
serum-free solutions), and plasma protein
binding 76 on data interpretation also need tobe addressed when translating drug effects
to in silico reconstructions. Study designs are
currently being considered to address thesepractical aspects.
Component 2: in silico reconstructionof human ventricular repolarization
changes. Several factors make it difficult,by inspection, to directly translate drug
effects on individual cardiac currents totheir effect on either delayed repolarization
or a propensity for proarrhythmia.
These factors are the integrated natureof repolarization, the dynamic time- andvoltage-dependent interplay of drug-induced
current blockade, the consequent effects onionic gradients and the emergent nature of
repolarization abnormalities such as EADs.
For these reasons, in silico reconstructionsof ventricular electrical activity are essential
to understand proarrhythmic effects. Robust
cellular models such as those describedby ten Tusscher et al.77 and O’Hara et al.78
represent reasonable initial approaches,
and the latter is based on extensive data
derived from human ventricles. Althoughwhole-heart models also exist and give462 | JULY 2016 | VOLUME 15

useful insight into how arrhythmias arise79,
they are not necessary for understanding
proarrhythmia on a cellular level andthey impose technical challenges that are
more appropriate for specialized centresof modelling excellence to tackle80.
Challenges with component 2. The abilityof in silico reconstructions to predictproarrhythmia relies on the validity of thehuman myocyte model, the quality ofvoltage-clamp data and appropriate definitionof proarrhythmic end points81. Discrepancies
between experimental and simulated electrophysiological responses could be attributed
to parameter fitting that is not exclusively
based on scarce available experimental
human ventricular data. Although different
models demonstrate different strengths
and limitations (when compared tocellular and clinical data) and may prove
instructive82–84, one based closely on extensive
human data would probably best serve thepresent objectives. The best cell models
represent the best selection of ionic current
models. Thus, it was considered reasonable
to start with the collection of human currents
from the O’Hara and Rudy model78, with thepossibility of systematically incorporating
additional features within each ioniccurrent to account for cell-specific electro­
physiological dynamics85 and drug effects(including use dependence). Furthermore,
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although it is best to understand thelimitations of each model based on the mostvaluable output parameters, the complexity
introduced by considering multiple models
probably provides little further advantage
for assessing proarrhythmic risks in normal
populations. In the future, the inclusionof known proarrhythmic risk factors(for example, hypokalaemia, bradycardia
and irregular rhythms) shown to reduce
repolarization reserve may be helpful
to further refine proarrhythmic risks ofvulnerable populations.
It is also essential to determine theoptimum number of currents to consider,based on the roles of various currents inproarrhythmia and the off-target effectsof compounds; this will affect the quality ofthe predictions as well as the time and costfor compound screening. Using in silico
reconstructions of rabbit ventricular
myocytes, Beattie et al.86 demonstrated
that blockade of IKr, ICaL and INaFast provided
better predictions of results obtained with
the in vitro rabbit left ventricular wedge
proarrhythmia model than did blockade
of IKr alone. Reconstructions of humanventricular action potential prolongation
with these three currents also provides
improved prediction of the risk of TdP
compared to IKr blockade alone87, consistent
with conclusions derived for predictions of
TdP using these three currents and logistic
regression models27. A sensitivity analysis
of preclinical biomarkers of proarrhythmic
risk to changes in the conductance andkinetics of select ionic currents that affectrepolarization (ICaL, IKr, IKs and IK1, as wellas the sodium-potassium pump current(INaK) and the sodium-calcium exchange
current (INaCa)) was performed using the ten
Tusscher and Panfilov human ventricularmodel88. The findings demonstrated thatchanges in action potential duration at 90%
repolarization (APD90) were particularly
sensitive to the inactivation kinetics of IKs andICaL and the conductances of ICaL, IKs and IKr,
whereas triangulation was shown to be mostdependent on IK1, and to a lesser extent on IKr.
A less appreciated factor to consider
is the extent to which concentrationdependent kinetics of blockade may
influence proarrhythmic risk. The use ofvoltage-clamp protocols to characterizedifferences in the time-dependent blockade
of IKr and the incorporation of a Markov
model of hERG gating into a dog epicardial
action potential model provided a better
prediction of the action-potentialprolonging effects of three drugs that wereassessed for concentration-dependence

and modulation using pacing frequency 89.
A more detailed kinetic model of INaLate wasrecently shown to better define the effectsof ranolazine on repolarization90. Ongoingstudies by the ISWG under the auspices of
the FDA have demonstrated improvements
on current methods, which were provided
by incorporating more-complex models of
IKr blockade (based on Markov models) for
defining cellular proarrhythmic risk, thus
moving beyond traditional conductance
reduction models and simpler IC50 values
to characterize IKr blockade.
Reconstructions of human cellular
ventricular repolarization delays
or instability provide an integrated
proarrhythmia risk assessment. Thiseffort would include assessing theconcentration–response relationship fordelayed repolarization or other torsadogenic
indicators (including the propensity toelicit EAD-triggered activity that gives rise
to TdP91) in relation to clinical exposures.
Recent experimental and modelling studies
highlight the complex interactions that
contribute to the initiation and perpetuationof EADs, and such studies emphasize
the involvement of ICaL reactivation and the
dynamic interplay of INaLate, late potassium
currents, INaCa and intracellular calcium
cycling 92–94. Additional proarrhythmic
biomarkers under consideration include
changes in membrane resistance,
triangulation of the shape of the action
potential plateau and final repolarization
phases, changes in refractoriness (anintegrated response reflecting the timecourses of repolarization and recovery of
cardiac excitability) and the dispersionof repolarization across different cell types.

Beat‑to‑beat variability of repolarization(BVR, a recognized marker of TdP inproarrhythmia models) could also be
considered as a proarrhythmic marker whenusing stochastic models of ion channels andtransporters95 (rather than non-deterministic
models cited above), as could heterogeneityof delayed repolarization (a recognized
substrate for TdP proarrhythmia) acrossepicardial, midmyocardial and endocardial
ventricular layers that define repolarization
gradients (and the QT interval).
It will be necessary to calibrate thein silico model for sensitivity using
appropriate positive controls based onclinical experience96,87. Within the CiPA,
the Clinical Translation Working Group,
under the auspices of the Cardiac Safety
Research Consortium, has selected a setof 28 drugs ranked as high, intermediate or
low proarrhythmic risk based on clinicalexperiences (TABLE 1). As an initial testof the in silico model, the ISWG planson evaluating the effects of a subset of
12 compounds (representing high-,
intermediate- and low-risk categories)
using ionic current data generated by the
ICWG using overexpressing heterologous
expression systems.
Component 3: confirmation using hSC-CMs.
Under ideal circumstances, the in vitro
assessment of a drug’s proarrhythmic risk
would use adult human ventricular tissueor myocytes. However, such preparations
are not readily available for the routine
evaluation of even small numbers of drugcandidates. Fortunately, hSC-CMs hold great
promise in regards to preclinical cardiac
safety (as well as efficacy) testing 97,98–101.

Table 1 | Set of 28 drugs ranked according to high, intermediate and low proarrhythmic risk
CiPA high risk

Redferncategory*

CiPAintermediaterisk

Redferncategory

CiPA lowor no risk

Redferncategory

Azimilide1

Astemizole

2

Diltiazem

5

Bepridil

3

Chlorpromazine–

Loratadine

5

Dofetilide

1

Cisapride2

Metoprolol–

Ibutilide1

Clarithromycin

4

Mexiletine–

Quinidine1

Clozapine–

Nifedipine

4

Vandetanib–

Domperidone

4

Nitrendipine

5

Methadone–

Droperidol–

Ranolazine–d,l-sotalol

1

Terfenadine2

Tamoxifen

5

Pimozide3

Verapamil

5

Risperidone

5

Ondansetron–

CiPA, Comprehensive in vitro Proarrhythmia Assay. *See REF. 41 for details.

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PERSPECTIVES
The present role of hSC-CMs in the
CiPA is to confirm drug effects identified inionic current studies. Any additional, novel
electrophysiological effects not anticipatedfrom simpler isolated current measureswith heterologous expression systems canbe readily detected using hSC-CMs. Suchdrug effects could include effects mediatedby cellular metabolism, oxidative stress oraltered calcium-handling 8,102–104. Additional
examples could include modulation of IKr
through intracellular second messengers105–107
and augmentation of INaLate through thephosphoinositide 3‑kinase pathway 108,109.
Discrepancies found between ionic current
studies (or in silico reconstructions) andhSC‑CM effects would subsequently
be investigated to improve future risk
assessments. Effects of longer-term, chronic
drug exposure on hSC-CMs — related tochannel expression, metabolic modulation
or structural changes (for example, owing to
off-target effects on kinases) — can also be
detected using hSC‑CM studies.
Various approaches have been used
to evaluate electrophysiological markers
of proarrhythmia with hSC-CMs. These
approaches include transmembrane action
potentials, microelectrode array (MEA)
recordings, impedance measurements,
calcium transients and voltage-sensitivedyes110,111 (FIG. 5). Although all theseapproaches reflect aspects of integrated
cellular responses, those describing theeffects of transmembrane potentials aremost easily comparable with in silico
reconstructions of transmembraneelectrical activity and ECG effects. Typicalend points for MEA studies include fieldpotential duration and incidence of EADs
(direct measures of altered repolarization),beat frequency (known to modulate
repolarization independently of effects on
repolarizing currents), amplitude of initial
depolarizing spike (a measure of sodium
current) and conduction patterns (a generalmeasure of experimental conditions).
Analogous measures are provided byvoltage-sensing optical (VSO) approaches,
which show changes in the duration as
well as the time course of repolarization,
thus providing measures of action
potential triangulation (a linearizationof repolarization that is recognized as ahallmark of proarrhythmia). Although it ispossible to measure action potentials (andisolated currents) directly from hSC-CMs112,
such efforts are presently technicallydifficult using automated patch-clamp
platforms and are not readily available forwidespread use. Furthermore, most currentsof particular interest (such as IKr) are

Potential (mV)a Intracellular (transmembrane)c Extracellular (ﬁeld potential)
0 mV

Time (ms)Potential (μV)

10 ×

d Arrays
Arbitrary unitsb Voltage-sensing optical

Time (ms)Time (ms)Figure 5 | Electrophysiological approaches for evaluating proarrhythmia with human stem
cell-derived cardiomyocytes. a | Intracellular transmembrane potentials
that arerecordedusing
Nature Reviews| Drug
Discovery
microelectrodes provide the most detailed characterization of electrophysiological effects on cellular
repolarization. Such studies are slow, technically demanding and not easily adapted to higherthroughput techniques. b | Voltage-sensitive dyes provide optical signals of transmembrane potential
based on changes in fluorescence170,171. Although the absolute membrane potential is not directly
measured, changes in the action potential duration and the configuration of repolarization (including
triangulation of the plateau, a recognized risk factor for early after-depolarizations (EADs)) are readily
obtained from multiple myocytes with minimal cellular disruption and reasonable throughput.
c | Micro­­electrode array (MEA) recordings provide localized extracellular field potentials from electrodes located on the chamber floor. d | Changes in the field potential duration (FPD) reflecting changes
in cellular repolarization are most reliably measured from the peak of the repolarization wave (indicated by arrows, just before full repolarization)172,173 and are easily translated to clinical repolarization
effects. Repolarization instability and EAD activity can also be recorded with this technique.

464 | JULY 2016 | VOLUME 15

quite small and difficult to measure fromhSC-CMs (as is the case with human adultventricular myocytes).
Experimental work continues to define
the roles, variability of responses and utilityof hSC‑CMs as a readout of integrated
cellular electrophysiological responses.
In late 2014, the CiPA Myocyte working
group (MWG, under the auspices of the
Health and Environmental Sciences institute(HESI)) conducted a pilot study across 12
volunteer sites to evaluate and compare theelectrophysiological effects of eight blinded
compounds on repolarization. This study
tested the effects of compounds on four
different hSC-CM types using MEA andVSO approaches (voltage-sensing dyes
and integrated voltage-sensing membraneproteins). Although analysis is ongoing,
preliminary results suggest reasonableconcordance of changes in repolarization
among the hSC-CM types (attesting to
the reproducibility of MEA and VSO
assessments), with some differences
in sensitivity noted across myocyte
preparations. Any differences in myocyte
sensitivities can easily be calibrated andcontrolled by the concomitant evaluationof known standards run alongside test
compounds on each plate, taking advantageof the higher-throughput multi-wellformats of MEA or VSO approaches to test
for a consensus of positive controls witheach assay.
Parallel to the work described above, aJapanese consortium recently reported ona prospective comparison study using the
MEA approach, a hSC‑CM line and the IKr
blocking agent E-4031 across three ‘trained’
independent Japanese research laboratories(REF. 113). Their results demonstrated
minimal inter-facility differences in theability to detect delayed repolarization.
Recently expanded as the Japan iPS Cardiac
Safety Assessment (JiCSA), this consortium
is presently conducting a multi-site study
comparing the electrophysiological effectsof 60 drugs on hSC-CMs using the MEA
platform. Recognizing their common goals,
the HESI MWG and the JiCSA have recently
formed a joint group to share results andplan future complementary hSC‑CM studies
in support of the CiPA initiative.
Challenges for component 3. As with adultventricular myocytes, it is recognized that
repolarization of hSC-CMs is affectedby heart rate, and that drug effects on
spontaneous rhythm (rate or rhythmicity)
may confound the interpretation of FPDchanges. Electrical or optogenetic pacing of
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PERSPECTIVEShSC-CMs at slow rates is not feasible, withsome preparations displaying rapid intrinsic
beating, which also limits the ability tocharacterize reverse use-dependent effects
on repolarization (the greater prolongationof repolarization at slower (rather than
rapid) stimulation rates, a recognizedhallmark of proarrhythmic risk linked to
EAD generation). Further efforts to develop
platforms that include optional pacing
protocols and myocytes with minimal
intrinsic beat frequencies should alleviate
this limitation.
Commercially available hSC‑CM
preparations represent a mix of atrial,nodal and ventricular cells (the latterbeing the predominant phenotype frommost commercial cell sources). It will bechallenging to standardize protocols acrossall hSC-CMs for either present or futurepreparations given the differences in the
distribution of electrophysiologicalphenotypes in cells from different
vendors; different plating, maturationand experimental conditions suggested
for different cell sources; and the different
requirements imposed by the different
electro­physiological approaches. Thus, it ismore amenable to consider standardizing
assay calibration efforts to confirm their
sensitivity and the functional roles of key ion
channels (for example, those carrying IKr,
ICaL, IKs and INaLate). This can be accomplishedby defining concentration–response curves
for specific current blockers on eachhigh-throughput assay plate. Such data willdefine assay sensitivity (providing a basis
for comparing drug effects across multiple
cell types, laboratories and platforms) andprovide a benchmark for future myocyte
preparations (for example, 3D structures or
myocyte–fibroblast co‑cultures).
No standardized guidelines currently
exist for the evaluation of hSC‑CM
phenotypes or functionality that may affectdrug responses114. Recent papers haveproposed a multi-parametric qualityassessment rubric to assess the ventricular
myocyte phenotype115–117. Although too
extensive for routine measurements(including genetic, structural, electrophysio­
logical and contractile measurements),
this does provide a guide for generalfurther considerations.
Although most currents found in adultventricular myocytes are also present in
hSC-CMs118,119, the electrophysiologicalphenotype of hSC-CMs is generally
characterized as being immature, as reflected
in a less-negative resting membranepotential, reduced upstroke velocities andspontaneous automaticity 120,121. This electro­
physiological phenotype is due (at least inpart) to reduced IK1 density (the current
responsible for the resting membranepotential of adult myocytes) as well as greater
density of pacemaker current IF. Despite
these limitations, hSC-CMs consistently
demonstrate delayed repolarization whenchallenged with QT‑prolonging drugs122–126.
For example, Harris and colleagues125
demonstrated relevant pharmacologicalresponses and excellent correlations tofunctional cardiac electrophysiologicalstudies based on ten compounds evaluatedusing hSC-CMs and an MEA platform.
A subsequent study correctly identified morethan 20 drugs with single and multiple ionchannel pharmacology 127.
Although single MEA parameters(such as field potential duration) may be
more easily compared with more standard
preclinical models (for example, QT
prolongation), a multi-parametric approachconsidering complex patterns of responses
to drugs may prove valuable to realize thefull breadth of cardiotoxicity. Techniquessuch as hierarchical clustering and parallel
coordinate data visualization (as used in
microarray gene expression) have been
applied to inform on mechanisms and rankordering of novel compounds with known
clinical risk.
Recent ongoing efforts to direct
maturation of hSC-CMs involve electricalstimulation, co‑culture with fibroblasts,
and 3D tissue engineering 128,129. A recent
study demonstrated that prolonged periodsof culture (up to 120 days) of hSC-CMs
derived from either embryonic or induced
stem cells resulted in the maturation of
their structural and contractile properties
towards a more adult-like phenotype130,with increased cell size and anisotropy,
greater myofibril density and alignment, an
increase in multi-nucleated cardiomyocytes,
doubling in shortening magnitude,
hyperpolarized diastolic potentials, faster
upstroke velocities and reduced beat rate.
With important advances towards thefaithful recapitulation of a mature electrophysiological (and contractile) phenotype131,
we anticipate that hSC-CMs will havea more prominent role in the direct
evaluation of proarrhythmic risk, withionic current and reconstruction studies
subsequently relegated to clarifying andconfirming myocyte study results.
As stable hSC-CMs can be maintainedin culture, it is possible to evaluate thelonger-term electrophysiological effectsof compounds that require hours to days of

NATURE REVIEWS | DRUG DISCOVERY

exposure. A classic example would be theability of drugs to reduce IKr by reducing
KCNH2 transcription or translationof KCNH2 mRNA (a process that is
termed trafficking, an example of which
is provided by the antiprotozoal drug
pentamidine)132,133. Longer-term studies
also provide the opportunity for the in vitro
evaluation of chronic drug effects. This may
be particularly important with oncologicaltherapeutics, for which debilitating
cardiotoxic effects often occur later in thecourse of therapy (and may be detected only
after expensive and longer-term non-clinical
toxicology studies with non-human models).
With hSC-CMs, it is also be possible toassess safety (and efficacy) end points
specific to monogenic cardiac diseases thatare related to cardiac rhythm abnormalities(such as congenital long QT syndromes)
using patient-derived cardiomyocytes134,135.
Additional practical considerations
regarding the CiPA. The CiPA represents
an evolving proposal that will substantiallyaffect the workflow for definitive
preclinical proarrhythmia assessment.
At its simplest levels, more-comprehensive
channel-screening efforts could be
implemented early in drug discovery using
automated planar patch-clamp techniques,with the resulting data used to feed earlyin silico reconstructions (the latter possiblyat centralized facilities or cloud based,
providing standardized software andresults). It is encouraging that elements of
the CiPA are already being used in earlydrug discovery (for example, increasing thenumber of ionic currents being interrogatedand screening for integrated responses withhuman-derived cardiomyocytes) as well as
guiding early medicinal chemistry efforts
and refining early hazard identification.
At a minimum, such efforts should reduce
the automatic exclusion of IKr -blocking
drugs from early drug discovery. It is
anticipated that the commercial costs forsuch services would be substantially reduced
as these efforts are implemented.
It is essential to correctly identify
clinically positive and negative torsadogenic
compounds to include in validation andtest sets; within the CiPA, this task wasundertaken by a group of academics,
clinicians and regulators who are familiar
with clinical QT studies, mechanisms ofproarrhythmia, drug safety assessments andregulatory submissions. Under the auspicesof the Cardiac Safety Research Consortium,
the Clinical Translation Working Group
has selected 28 compounds for evaluation
VOLUME 15 | JULY 2016 | 465

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PERSPECTIVES
in the CiPA based on the absence of active
metabolites, good solubility and defined
cellular electrophysiological effects. Basedon clinically demonstrated torsadogenic risk,proarrhythmic incidence, published reports,
the FDA Adverse Event Reporting System(AERS) database and expert opinion, thesecompounds were ranked into three categories
of TdP risk: high, intermediate and low or norisk. An initial set of 12 compounds, divided
equally between the three proarrhythmia risk
categories, will be used as a test set for the
initial in silico reconstructions and hSC‑CM
calibration studies. With the expectation ofreasonable concordance established between
the preclinical and clinical rankings, theremaining drugs will be prospectively tested
as a validation exercise.
In mid‑2015, official discussionscommenced with international regulatory
authorities regarding expectations of the
CiPA initiative, its role in future regulatory
guidelines and validation efforts. Suchdialogue is essential to support the
replacement of costly TQT studies, as drugs
demonstrating low proarrhythmic risk basedon the mechanistic-based CiPA approach,despite some degree of QTc prolongation,
could proceed with uncomplicated clinicalmonitoring for QT prolongation during
development. Although timelines forregulatory implementation of the CiPA
depend on such discussions with regulators,
a review of the progress made by the various
workgroups is planned for early 2016. Key
differences between the present ICH S7B
and E14 approaches and the evolving CiPA
paradigm are depicted in TABLE 2.

Contractile and structural cardiotoxicity
Effects on cellular cardiac contractility:
potential role of hSC-CMs. Drug effects on
cardiac contractility in vivo (defined most
broadly as the ability to maintain appropriate
cardiac output given cardiovascularhaemodynamics) represent the complex
interplay of direct drug effects on the
myocyte as well as extrinsic factors (forexample, resulting from changes in heartrate, preload, afterload and autonomic tone).
However, such extrinsic effects do not negate
the importance of the ability to directly
influence sarcomere shortening or relaxationin response to varying calcium levels, as
modulated by multiple interrelated signallingcascades. The ability to detect potential
inotropic liabilities at an early stage is usefulin guiding subsequent in vivo preclinicalinvestigations, compound selection andfirst‑in‑human studies.
Differences in cardiac contractility
between species are well known andwould be expected to support the wide
range of heart rates and haemodynamic
requirements that occur across species.
For example, whereas calcium reuptake by
the sarcoplasmic reticulum calcium pump islargely responsible for relaxation in the adultmouse myocyte, this mechanism accounts
for approximately 65% of calcium reuptake
in normal human adult myocytes (with theremainder accounted for by sodium-calcium
exchange and the sarcolemmal calcium
pump136–138). Given such differences between
species, human-derived cardiomyocytes havea potential advantage over myocytes fromother species in assessing drug effects oncontractility (assessed based on the ability ofthe drug to shorten or generate tension) at thecellular level for acute or longer-term studies.
Owing to the complexity involvedin producing cardiac contractions, it isdesirable to evaluate a drug’s inotropic andlusitropic effects with phenotypic assays.
Although it may be possible to assess theeffects of drugs on some specific regulatory
mechanisms that are involved in contraction
or relaxation (for example, calcium-induced
calcium release, or calcium reuptake into
the sarcoplasmic reticulum), such studies
do not provide an integrated perspective ofa drug’s overall effect on inotropy (just as
evaluating blockade of IKr fails to provide
a comprehensive proarrhythmic risk
assessment). In contrast to the analogy with
IKr and proarrhythmia, no single predominant
cellular mechanism is recognized as affecting
cardiac contractile performance.
A detailed review is outside the scope of
this article, but numerous reports indicate therelative heterogeneity and immaturity of
the contractile machinery of some hSC-CMs
compared to adult ventricular myocytes —with myofibrillar disorganization, lack of
T tubules and heterogenous distributionof ryanodine receptors139–141, reduced twitch
amplitude compared to native tissues142,
and leaky sarcoplasmic reticulum without
a mature terminating mechanism forthe calcium transient131,143–147. Although
adult ventricular myocytes demonstrate
a positive force–frequency relationship(increasing force with faster stimulation
rates) and post-rest potentiation ofcontraction, some hSC-CMs demonstrate

Table 2 | Key differences between the ICH S7B and E14 approaches and the evolving CiPA paradigm
ICH S7B

ICH E14

CiPA

Goals

Non-clinical studies of thepotential of compounds
to delay ventricularrepolarization

Clinical studies of the potential Non-clinical mechanistic studies to determine theof compounds to delay
proarrhythmic risk of compounds, the results of whichventricular repolarization
are confirmed with clinical studies

Focus

IKr and in vivo QTc;
supplemental
studies possible

QTc prolongation and clinical
TQT studies

Multiple human ionic currents informing in silico
reconstructions of human myocyte electrophysiology todetermine TdP risk: electrophysiological confirmation fromhuman stem cell-derived cardiomyocytes and clinical ECGs

Evidence of risk

Risk assessment of delayed
repolarization or QT interval
prolongation translated frommodels to humans

QTc prolongation: studies
give a dichotomous output
based on exclusion of a
10‑millisecond clinical effect

Proarrhythmic risk: studies provide a mechanism-based
assessment calibrated against clinical comparators ranked
according to a consensus of clinical experience

Standardization

None

Moxifloxacin as a positive
control arm

Standardized ion channel protocols; a moderated, publicly
available in silico model; a standardized, calibrated core
myocyte protocol and a standardized Phase I ECG protocol

Validation

None

None

Planned, but fundamentally limited by the inability tocategorize known drugs for TdP risk independent of thecontext of their use

CiPA, Comprehensive in vitro Proarrhythmia Assay; ECG, electrocardiogram; ICH, International Conference on Harmonization; TdP, torsades de pointes;
TQT, thorough QT, QTc, QT interval corrected for changes in heart rate.

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PERSPECTIVES
a negative force–frequency relationship andabsent or weak post-rest potentiation148,149.
Encouragingly, more-mature calcium
handling could be induced in hSC-CMs byculturing them for a longer time130, using
electrical stimulation and/or 3D cultures150,
or patterning them onto microgrooved
culture substrates151, providing promise fornear-term enhancements. As discussed withrespect to electrophysiological characteristics,
it is expected that evolving engineered
hSC‑CM preparations152 will better
recapitulate mature inotropic mechanisms,
thus allowing for a more robust evaluation of
direct inotropic effects of drugs in the future.
Some studies have demonstrated theutility of hSC-CMs for detecting positive (forexample, isoprenaline) and negative inotropic
agents116, whereas others have demonstrated
the immaturity of the sarcoplasmic reticulumand β-adrenergic response153,154. Further
work is necessary to evaluate the baselinecharacteristics of the effects of drugs oncontractility, their variability across hSC‑CM
preparations and their reproducibility, inorder to define the utility of hSC-CMs for
detecting positive and negative inotropic
effects consistent (in direction andamplitude) with clinical responses.
Drug effects on structural cardiotoxicity:
potential role of hSC-CMs. The growing
list of cardiac toxicities associated withestablished anticancer drugs (for example,
anthracyclines and trastuzumab) and
more-recently introduced classes oftyrosine kinase inhibitors poses a challenge
for the development of novel anticancer
agents10,155,156, and these toxicities could also
be of concern in other therapeutic areas.
In the past, the ability to detect structural
cardiotoxicity (defined here as morphologicaldamage or loss of viability) using in vitro
approaches has been hampered by theinability of non-human cardiac models torecapitulate the mechanisms involved inhuman toxicity, the stability of in vitro models
to evaluate longer-term effects (measured
in days rather than hours) and the inability
to simultaneously assess a broad range of
toxicity end points (thus providing a morecomprehensive assessment).
The advent of high-content screening(merging automated, high-throughput
screening techniques with cellular
image-based microscopic assessments
involving spatial, temporal and spectral
resolution with multiple probes) has enabled
the multi-parametric, quantitative assessmentof cellular toxicities for cardiac as well asother tissues157–160. Studies using hSC-CMs

and high-content screening provide theability to detect cardiotoxicity early indrug discovery, before using traditional,
histologically based (and costly) assessments.
These later studies require substantially larger
compound amounts, larger animals andextended dosing periods (days to weeks),
and such studies are not readily amenable
to early compound-selection efforts.
A recent example of using high-content
screening to assess structural cardiotoxicity
in hSC‑CMs compared responses
between rat myoblastic H9c2 cells(derived from embryonic rat hearts) andembryonic hSC-CMs to clinical structural
cardiotoxicants161. Cellular response profiles
characterizing toxicity following 3‑day
exposures were evaluated using multiple end
points (including ATP depletion to assess
cell viability, live-cell fluorescent imaging
of mitochondrial membrane potential,
endoplasmic reticulum integrity, calcium
mobilization and membrane permeability).
Mitochondrial and calcium mobilization
were the predominant effects for detectionof structural cardiotoxicity. EmbryonichSC-CMs offered somewhat improved
detection of structural toxicity, detecting
25 of the 34 structural cardiotoxicants at or
below Cmax concentrations compared with18 compounds in H9c2 cells. In anotherexample, Mioulane and colleagues162
described a high-content imaging approachto detect cardiomyocyte apoptosis andnecrosis using an automated microscopy
platform based on monitoring nuclear
remodelling, mitochondrial status andmembrane potential. They noted that thesusceptibility of hSC-CMs to chelerythrinestimulated apoptosis varied with time after
differentiation, but that hSC-CMs weregenerally more resistant to apoptosis than
rat neonatal ventricular myocytes. The
authors further commented that such studies,
although not fully able to replace in vivo
studies, would allow for a more targeted
assessment of human-based cardiotoxicity(despite challenges posed by the immature
characteristics of hSC-CMs) in early drug
discovery, with confidence increasing as theirpredictive value was confirmed.
Several multi-parametric imaging studies
have investigated the effects of tyrosine kinase
inhibitors on hSC-CMs. Doherty et al.163
found that three tyrosine kinase inhibitors
linked with clinical cardiotoxicity —crizotinib, sunitinib and nilotinib — showed
unique toxicity profiles, reflecting multiple
off-target mechanisms (including increased
ROS production, caspase activation,cholesterol accumulation and decreased

NATURE REVIEWS | DRUG DISCOVERY

myocyte viability) that may contribute to
overall cardiotoxicity. By contrast, minorchanges were observed with erlotinib,
an oncology drug not associated withcardiotoxicity. Another imaging study, which
examined the relationship between hESC‑CM
bioenergetics and the toxicity of tyrosine
kinase inhibitors164, revealed sensitization
to mitochondrial toxicity in cells utilizing
oxidative phosphorylation compared to cells
relying on glycolysis for ATP production.
This sensitization was exaggerated with
the HER2 kinase inhibitor mubritinib,
which was non-toxic in glycolytic cells but
affected mitochondrial integrity and toxicity
in myocytes with altered bioenergetics.
Complementary high-content profiling andhierarchical clustering of cell-surface nutrienttransporter expression data indicated the
involvement of bioenergetic modulationof kinase inhibitor toxicity in the altered
myocytes. These results demonstrate theimportance of considering metabolic
conditions when designing and translatingin vitro studies, as well as the utility ofmulti-parametric analysis techniques.
Collectively, the studies cited above
demonstrate the utility of hSC-CMs andimage-based high-content screening todistinguish subtle mechanistic differencesamong drugs based on drug- andclass-specific patterns of responses. The wealthof data generated from such studies poseschallenges for analysis and storage. A recent
review of high-content screening studies
revealed that the majority of high-content
screens use only a few image-based features(for example, disregarding the distributionof features among each cell population). This
oversimplification of informational content
was posited to result from more-traditional
data analysis processes; problems associatedwith larger, multi-parametric data sets; anda lack of familiarity of assay statistics165.
Defining cardiotoxicity-based high-content
screening is somewhat analogous to defining
proarrhythmia based on effects on multiple
cardiac currents. In the electrophysio­
logical example, a single ionic current (IKr)
is now recognized as an oversimplified
representation of proarrhythmia, and drug
effects on multiple human channels in the
CiPA paradigm provide multi-parametricdata for analysis, which lead to integrated
electrophysiological responses and better
predictivity guided by in silico reconstructions.
In the cardiotoxicity example, the need exists
to more fully utilize the multi-parametric data
generated from high-content screens, using
more-comprehensive integrated assessments(guided, at present, by phenotypic responses)
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that will lead to integrated cellular responses
and better predictivity. Although both
examples should benefit from the use ofhuman cells and tissues, the major distinction
between these two examples is the lesser
understanding of inputs into the more
complex interrelated mechanisms responsible
for structural and contractile cardiotoxicity.
Assay validation: overall considerations
In general, when mechanistic insights
into safety liabilities are lacking, assays aredeveloped that rely on complex interactions
to probe adverse effects (for example,
heart failure). With such assays, validationconsists of testing the effects of numerous
compounds and defining the relationshipbetween the preclinical effects or biomarkers
and the clinical observation (for example,
a study ranking the proarrhythmic effectsof 55 drugs on the spontaneous beat rate ofhSC‑CMs166). Using such an approach,one might expect diminished sensitivity or
specificity with compounds different to thosewithin the validation dataset. Importantly,
with an understanding of the response
profiles of mechanisms associated withsafety liabilities, a larger validation dataset
is not needed. Instead, the larger dataset issupplanted by demonstration of the presence(and sensitivity) of the mechanism andan understanding of assay results andlimitations in the larger context of thebiological (or pathological) system.
Conclusions
We are in the midst of an evolving paradigm
shift for preclinical cardiac safety assessments.
This shift is founded on a growing knowledgeof the basic cellular mechanisms underlying
electrophysiological, contractile and structural
cardiac toxicity; the availability of humanchannels and hSC-CMs as the most relevant
in vitro test systems; the availability of
automated, high-throughput and highcontent screening platforms for evaluating
electro­physiological and multi-parametricsubcellular and cellular responses; andin silico models that adequately describe
and integrate complex cellular electrophysio­
logical responses. These mechanistic-based
approaches could replace the more traditional‘black box’ approaches that rely on characteri­zation of the effects of a larger number ofcompounds on poorly characterized animalmodels to define assay performance.
Recent opinions regarding declining
pharmaceutical productivity have highlighted
the potential contribution of excessive focus
on target-centric approaches to the observed
decline, and, conversely, the potential forphenotypic screening strategies to help
to reverse it167–169. Similarly, we argue
that focused, ‘target-based’, reductionist
approaches for early cardiac safety screening(for example, studying hERG alone for TdP
proarrhythmia) hinder drug discovery efforts
and result in the premature termination of
drug candidates, and we also assert that amechanistic-based systems approach using
human-derived preparations provides a morecomprehensive and specific evaluation ofcardiac proarrhythmia liabilities. Although
structural cardiotoxicities are less wellunderstood, multi-parametric phenotypic
patterns (or signatures) obtained withhuman-derived cardiomyocytes will provide
valuable insights into cardiotoxic liabilities,
leading to a better understanding of novel
mechanistic pathways to be avoided in earlydrug discovery.
Despite the many practical challenges
remaining for the human-based cellularassessment of cardiotoxicity, continuing torely on more-precise measures of imperfectin vitro surrogate markers and simpler (andpoorly understood) metrics of cardiotoxicity
with non-human tissues will not lead tomore efficient development of safer drugcandidates. The proposed human-based
integrated in vitro approaches discussed
herein will guide and complement preclinicalin vivo cardiac safety (including traditional
telemetry ECG evaluations) and toxicity
studies, and these approaches provide amodel for future efforts aimed at assessing
toxicities in other organs. The growing use(and refinement) of hSC-CMs will provide
a unifying bridge for the efficient evaluationof proarrhythmic, contractile and structural
cardiotoxicity. Lessons learned from yearsof efforts avoiding proarrhythmia linked
to delayed repolarization (once considered
an idiosyncratic response) at all costs haveprovided a new paradigm for rationaland more efficient in vitro preclinicalsafety assessments.
Gary Gintant is at the Department of Integrative
Pharmacology, Integrated Science and Technology,
AbbVie, Department ZR 13, Building AP9 01,
1 North Waukegan Road, North Chicago,
Illinois 60064–6118, USA.

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Philip T. Sager is at Stanford University,
719 Carolina Street, San Francisco,California 94107, USA.
Norman Stockbridge is at the Division of
Cardiovascular and Renal Products,
Center for Drug Evaluation and Research,
US Food and Drug Administration, Silver Spring,
Maryland 20993, USA.
Correspondence to G.G.
gary.gintant@abbvie.com
doi:10.1038/nrd.2015.34
Published online 19 Feb 2016468 | JULY 2016 | VOLUME 15

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Acknowledgements

The authors wish to thank N. Thomas for helpful discussions
and insights regarding high-content screening approacheswith human stem cell-derived cardiomyocytes and forreviewing the manuscript. The authors also acknowledge
multiple discussions with numerous individuals regarding new
in vitro approaches that led to the development of theComprehensive in vitro Proarrhythmia Assay (CiPA) andJ. Green and J. Sutherland for their support with in silico reconstructions. AbbVie participated in the review and approvalof this article.

Competing interests statement

The authors declare competing interests: see Web version
for details.

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