ORIGINAL RESEARCH

Comprehensive T wave Morphology Assessment in a Randomized
Clinical Study of Dofetilide, Quinidine, Ranolazine, and Verapamil
Jose Vicente, MSc; Lars Johannesen, MSc; Jay W. Mason, MD; William J. Crumb, PhD; Esther Pueyo, PhD; Norman Stockbridge, MD, PhD;David G. Strauss, MD, PhD

Background-—Congenital long QT syndrome type 2 (abnormal hERG potassium channel) patients can develop ﬂat, asymmetric, andnotched T waves. Similar observations have been made with a limited number of hERG-blocking drugs. However, it is not known
how additional calcium or late sodium block, that can decrease torsade risk, affects T wave morphology.
Methods and Results-—Twenty-two healthy subjects received a single dose of a pure hERG blocker (dofetilide) and 3 drugs that
also block calcium or sodium (quinidine, ranolazine, and verapamil) as part of a 5-period, placebo-controlled cross-over trial. At predose and 15 time-points post-dose, ECGs and plasma drug concentration were assessed. Patch clamp experiments were
performed to assess block of hERG, calcium (L-type) and late sodium currents for each drug. Pure hERG block (dofetilide) andstrong hERG block with lesser calcium and late sodium block (quinidine) caused substantial T wave morphology changes (P<0.001).
Strong late sodium current and hERG block (ranolazine) still caused T wave morphology changes (P<0.01). Strong calcium andhERG block (verapamil) did not cause T wave morphology changes. At equivalent QTc prolongation, multichannel blockers(quinidine and ranolazine) caused equal or greater T wave morphology changes compared with pure hERG block (dofetilide).
Conclusions-—T wave morphology changes are directly related to amount of hERG block; however, with quinidine and ranolazine,
multichannel block did not prevent T wave morphology changes. A combined approach of assessing multiple ion channels, along with
ECG intervals and T wave morphology may provide the greatest insight into drug-ion channel interactions and torsade de pointes risk.
Clinical Trial Registration-—URL: http://clinicaltrials.gov/ Unique identiﬁer: NCT01873950. ( J Am Heart Assoc. 2015;4:
e001615 doi: 10.1161/JAHA.114.001615)
Key Words: electrocardiography • ion channels • long-QT syndrome • pharmacology • torsade de pointes

L

ong QT syndrome can be caused by congenital or acquired(eg, drug-induced or electrolyte) abnormalities in cardiac
ion channel currents regulating ventricular repolarization.1

From the Ofﬁce of Science and Engineering Laboratories, CDRH (J.V., L.J., D.G.S,
E.P.) and Division of Cardiovascular and Renal Products, Ofﬁce of New Drugs,
CDER (J.V., N.S.), US FDA, Silver Spring, MD; BSICoS Group, Aragon Institute forEngineering Research (I3A), IIS Aragon, University of Zaragoza, Zaragoza, Spain(J.V., E.P.); Department of Clinical Physiology, Karolinska Institutet and Karolinska
University Hospital, Stockholm, Sweden (L.J., D.G.S.); Biomedical Research
Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBERBBN), Zaragoza, Spain (E.P.); Spaulding Clinical Research, West Bend, WI (J.W.M.);
Zenas Technologies, New Orleans, Louisiana (W.J.C.).
Accompanying Figures S1 through S8 and Tables S1 and S2 are available at
http://jaha.ahajournals.org/content/4/4/e001615/suppl/DC1
Corresponding to: David G. Strauss, MD, PhD, U.S. Food and Drug
Administration, 10903 New Hampshire Avenue, WO62-1126 Silver Spring,
MD 20993. E-mail: david.strauss@fda.hhs.gov
Received January 24, 2015; accepted March 6, 2015.
ª 2015 The Authors. Published on behalf of the American Heart Association,Inc., by Wiley Blackwell. This is an open access article under the terms of theCreative Commons Attribution-NonCommercial License, which permits use,
distribution and reproduction in any medium, provided the original work is
properly cited and is not used for commercial purposes.

DOI: 10.1161/JAHA.114.001615

Long QT syndrome patients are at increased risk for torsadede pointes, a potentially fatal ventricular arrhythmia.2 Conventionally, physicians and drug regulators have focusedsolely on the QT interval in assessing risk for torsade;however, more information may be present in the electrocardiogram (ECG). Moss and colleagues3 identiﬁed different T
wave patterns associated with the 3 major congenital long QT
syndrome types. LQT1 patients (decreased IKs current) haveearly onset broad-based T waves, LQT2 patients (decreasedhERG potassium current, IKr) have low amplitude, biﬁd ornotched T waves, and LQT3 patients (increased late sodium
current, INalate) have long isoelectric ST segments with lateappearing, normal morphology T waves.
In the 1990s, there was recognition of an epidemic of druginduced QT prolongation and torsade de pointes resulting inmany drugs being withdrawn from the market.4 It was also
recognized that nearly all drugs that increased torsade risk
blocked the hERG potassium channel.5 This resulted in all new
drugs being required to be screened for their ability to block
the hERG potassium channel and prolong QT in Thorough QTstudies.6,7 However, the extreme focus on hERG and QT has
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Drug-Induced Changes in T Wave Morphology

Vicente et al

subjects between 18 and 35 years old, weighing between 50
and 85 kg and without a family history of cardiovasculardisease or unexplained sudden cardiac death were eligible forparticipation in the study. In addition, the subjects had to havefewer than 12 ventricular ectopic beats during a 3-hourcontinuous recording at screening, as well as a baseline heart
rate corrected QT (QTc) of <450 ms for men (470 ms for
women), using Fridericia’s correction.23 The study was
designed as a randomized, double-blind, 5-period crossoverclinical trial at a phase 1 clinical research unit (Spaulding
Clinical, West Bend, WI). Williams Latin square design balanced
for ﬁrst-order carryover effects24 was used for randomization.
In the morning of each period, subjects received a single
dose of 500 lg dofetilide (Tikosyn, Pﬁzer, New York, NY),
400 mg quinidine sulfate (Watson Pharma, Corona, CA),
1500 mg ranolazine (Ranexa, Gilead, Foster City, CA),
120 mg verapamil hydrochloride (Heritage Pharmaceuticals,
Edison, NJ) or placebo under fasting conditions. For dofetilideand quinidine, the selected dose was the maximum single doseallowed in the label25,26 to ensure substantial ECG changes
after a single dose. The ranolazine dose was selected toachieve plasma levels close to the chronic/steady state levels,
but with a single dose. This resulted in a dose higher than theclinical dose but lower than the maximum daily dose in thelabel.27 For verapamil, the highest initial single dose in thelabel was selected, and not pushed further due to concern for
extreme PR prolongation and heart block in healthy subjects.28
There was a 7-day washout period between each 24-hourtreatment period. During each period, continuous ECGs wererecorded at 500 Hz and with an amplitude resolution of
2.5 lV. From the continuous recording, triplicate 10-second
ECGs were extracted at predose and 15 predeﬁned timepoints post-dose (0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 12,
14, and 24 h) during which the subjects were resting in asupine position for 10 minutes. A blood sample was drawn forpharmacokinetic analysis after each ECG extraction timepoint period and plasma drug concentration was measured
using a validated liquid chromatography with tandem mass
spectroscopy method by Frontage Laboratories (Exton, Philadelphia, PA). The study was approved by the U.S. Food andDrug Administration Research Involving Human Subjects
Committee and the local institutional review board. All
subjects gave written informed consent.

ECG analysis

Methods
Clinical Study Design
The design of this clinical study has been previously
described.16 The study’s inclusion and exclusion criteria weresimilar to those used in dedicated QT studies.6 Brieﬂy, healthy
DOI: 10.1161/JAHA.114.001615

At each of the 16 time-points, 3 optimal 10-second 12-lead
ECGs were extracted with stable heart rates and maximum
signal quality using Antares software (AMPS-LLC, New York
City, NY).29 The resulting 5232 ECGs were up-sampled from500 to 1000 Hz. The semi-automatic evaluation, which
included computerized interval annotations (Figure 1A) by
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resulted in drugs being dropped from development, sometimes inappropriately, as not all drugs with hERG block or QT
prolongation cause torsade.4
There are multiple examples of drugs on the market thatblock the hERG channel (an outward potassium current), buthave minimal-to-no risk for torsade due to their concurrent
block of either L-type calcium or late sodium (inward
currents).8,9 Nonclinical studies have suggested that inward
current block can lessen torsade risk from hERG block by
decreasing dispersion of repolarization (eg, shortening action
potential duration) and preventing early after depolarizations,which are the trigger for torsade.10,11 In order to understandwhether drug-induced multi-ion channel block can bedetected and differentiated on the electrocardiogram as incongenital long QT syndrome, we conducted a prospective,
placebo-controlled clinical study of dofetilide, quinidine,ranolazine, and verapamil. Dofetilide is a pure strong hERG
potassium channel blocker12 with a high risk of torsade.5
Quinidine is a strong hERG potassium channel blocker thathas additional weaker block of calcium and late sodium.12
Torsade has been observed to occur more frequently at lower
quinidine concentrations13,14 where hERG block is present,
but there is lesser calcium and sodium block. Ranolazine
blocks the late sodium current and hERG approximately
equally and has not been associated with torsade, and may be
anti-arrhythmic.15 Verapamil blocks calcium stronger thanhERG and has low torsade risk.5,12
A separate study16 analyzed the J-Tpeak and Tpeak-Tendintervals and demonstrated that pure hERG block prolonged
both J-Tpeak and Tpeak-Tend, while additional inward current
block preferentially shortened J-Tpeak. The objective of thecurrent study was to evaluate the effect of pure hERG block
versus multichannel block on multiple previously developed T
wave morphology metrics (Figure 1). This included quantitative measures of T wave ﬂatness, asymmetry, and notching,which were developed to detect the ECG signature of LQT2
(abnormal hERG current).17 In addition, we studied the 30%
early and late repolarization duration (ERD30% and LRD30%) of
the T wave loop,18,19 previously shown to detect hERG block,and other vectorcardiographic measures of the relation
between depolarization and repolarization vectors (QRS-T
angle, ventricular gradient, and total cosine R-to-T [TCRT]),
which have been shown to predict arrhythmic risk in somepatient populations.20–22

Drug-Induced Changes in T Wave Morphology

Vicente et al

C

ORIGINAL RESEARCH

A

B

D

Figure 1. Illustration showing ECG biomarkers assessed in this study: (A) QT andsubintervals (QRS, J-Tpeak and Tpeak-Tend); (B) ﬂat, asymmetric and notched T waves (solidlines) vs. normal T waves (dotted lines); (C) 30% of early (ERD30%) and late (LRD30%)
repolarization duration in the preferential plane formed by the two-ﬁrst Eigen leads fromprincipal component analysis (PCA1 and PCA2); (D) other vectorcardiographic biomarkers(QRS-T angle, ventricular gradient and maximum magnitude of the T vector). See methods
for further details.

ECG readers blinded to treatment and time, has been
described elsewhere.16 T wave ﬂatness, asymmetry, and thepresence of notch were automatically assessed with
QTGuard+ (GE Healthcare, Milwaukee, WI). All the other T
wave morphology and vectorcardiographic biomarkers
described below were automatically assessed with ECGlib.30

T wave ﬂatness, asymmetry, and notching
The computation of T wave ﬂatness, asymmetry and thepresence of notch (Figure 1B) has been described elsewhere.17 Brieﬂy, a median beat was derived from theindependent leads (I-II, V1 to V6) of the 10-second 12-lead
ECG, and morphology measures were assessed in the ﬁrst
Eigen lead (PCA1) after applying principal component
analysis (PCA). T wave ﬂatness is based on the inverse
kurtosis (1—kurtosis) of the unit area of the T wave, thusincreasing values reﬂect increasing ﬂatness of the T wave
(dimensionless units [d.u.]). Asymmetry score (d.u.) evaluates
differences in the slope proﬁle and duration of the ascending
DOI: 10.1161/JAHA.114.001615

and descending parts of the T wave, where low values
correspond with symmetric T waves and higher values withmore asymmetric T waves. The presence of notch was
obtained from the inverse, signed radius of curvature of the
T wave, where values different from 0 correspond with thepresence of notch. Notch was labeled as present if at least 1
out of the 3 ECGs of the triplicate in the corresponding time
point had a notch score >0.

Percentage of early and late repolarization duration
Vectorcardiographic measurements 30% of early (ERD30%)
and late (LRD30%) repolarization duration were based on the
PCA transform from the ST-T segment of the independent
leads (I-II, V1 to V6) of the 12-lead ECG median beat. The
maximum amplitude of the T vector in the preferential plane
formed by the two-ﬁrst Eigen leads (PCA1 and PCA2) was
selected as reference, and ERD30% and LRD30% were thenmeasured at the time at which the amplitude decreased by
30% (Figure 1C).18
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Drug-Induced Changes in T Wave Morphology

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For each 12-lead ECG median beat, we derived the Frank
vectorcardiogram using Guldenring’s transformation matrix.31
The QRS-T angle was deﬁned as the angle between the QRS
and T vectors, which were deﬁned as the summation in the X,
Y, and Z leads from the QRS onset to QRS offset and QRS
offset to T offset, respectively. The ventricular gradient wasdeﬁned as the magnitude of the sum of the QRS and T
vectors.32 The vector with the maximum magnitude between
QRS offset and T offset (maximum T vector) was also
computed (Figure 1D).
In addition to the QRS-T angle described above, we also
assessed the concordance between ventricular depolarization
and repolarization sequences computing the total cosine R-toT (TCRT).22 This descriptor is deﬁned as the average of thecosines of the angles between a subset of QRS and T vectors
in the 3 ﬁrst Eigen leads (PCA1 to PCA3) derived from the PCA
transform of the independent leads (I to II, V1 to V6) of the
12-lead ECG median beat.

Analysis and removal of heart rate dependency
We determined if the T wave morphology biomarkers weredependent on heart rate, by considering if there was astatistically signiﬁcant heart rate relationship as well as ameaningful change.33 For biomarkers with dependency onheart rate, we developed a study-speciﬁc correction factor
using an exponential model and allowed the relationship to besex dependent. The heart rate correction was performed using
PROC MIXED in SAS 9.3 (SAS Institute, Cary, NC), wherein asigniﬁcance level of 0.05 was used to determine if there was adifference by sex.

Patch clamp experiments
Patch clamp experiments were conducted to assess therelative block of hERG, late sodium (Nav1.5) and calcium(Cav1.2) currents caused by each drug. Stably transfected
hERG, Nav1.5 cells (HEK-293), or Cav1.2 cells (CHO) wereobtained from Cytocentrics Biosciences (Rostock, Germany).
Cells were maintained in minimum essential medium with
Earle’s salts supplemented with nonessential amino acids,
sodium pyruvate, penicillin, streptomycin, and fetal bovine
serum.
Drugs were dissolved in either dimethyl sulfoxide (DMSO)
or deionized H2O to make stock solutions. Dilutions of stock
solutions were made immediately before the experiment tocreate the desired concentrations. The external solution(solution bathing the cell) had an ionic composition of (inmmol/L): 137 NaCl, 4 KCl, 1.8 CaCl2, 1.2 MgCl2, 11 dextrose,
10 HEPES, adjusted to a pH of 7.4 with NaOH. The internal(pipette) solution had an ionic composition of (in mmol/L):
130 KCl; 1 MgCl2, 5 NaATP, 7 NaCl, 5 EGTA, 5 HEPES, pH=7.2
DOI: 10.1161/JAHA.114.001615

using KOH. Experiments were performed at 361°C. Drugs
were obtained from Tocris Bioscience (Minneapolis, MN).
Currents were measured using the whole-cell variant of thepatch clamp method as previously described.34 After ruptureof the cell membrane (entering whole-cell mode), current
amplitude and kinetics were allowed to stabilize (3 to
5 minutes) before experiments were begun. All 3 currents(hERG, Nav1.5, Cav1.2) were elicited using a ventricularaction potential waveform paced at 0.1 Hz.

Statistical Analysis
The placebo-corrected change from baseline was computed
using PROC MIXED in SAS 9.3 (SAS institute, Cary, NC),
where the change from baseline for each T wave morphology
biomarker by time-point was the dependent variable.
Sequence, period, time, drug, and an interaction between
treatment and time were included as ﬁxed effects, andsubject was included as a random effect. Afterwards, alinear-mixed effects model was used to evaluate therelationship between each of the T wave morphology
biomarkers (except notch) and plasma concentrations. Thiswas done using PROC MIXED in SAS 9.3 using a random
effect on both intercept and slope (ie, allowing each subject
to have his or her own drug concentration-biomarker
relationship). A logistic regression model was used toevaluate the relationship between presence of notch anddrug concentration including a random effect on intercept inSAS (PROC GLIMMIX). P values <0.05 were considered
statistically signiﬁcant.
Patch clamp results are given as percentage of reduction ofcurrent amplitude, which was measured as current reduction
after a steady-state effect had been reached in the presence ofdrug relative to current amplitude before drug was introduced(control). Each cell served as its own control. Log-linear plots
were created from the mean percentage blockSEM at theconcentrations that were tested. A nonlinear least square ﬁtting
routine was used to ﬁt a 3-parameter Hill equation to the resultsin R 3.0.2 (R Foundation for Statistical Computing, Vienna,
Austria). The equation is of the following form:
Bð%Þ ¼ 100

Dn
ICn50 þ Dn

where B(%) is the percentage of current blockage at drugconcentration D, IC50 is the concentration of drug that causes
50% block, and n is the Hill coefﬁcient.

Results
Twenty-two healthy subjects (11 females) participated inthis randomized controlled clinical trial; see Table 1 for
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ORIGINAL RESEARCH

Other vectorcardiographic biomarkers

Drug-Induced Changes in T Wave Morphology

Vicente et al

Dofetilide: Pure hERG Potassium Channel BlockAll Subjects (N=22)

Demographics
Age, y26.95.5

Female11 (50%)Body mass index, kg/m2

23.12.7

ECG
Heart rate, beats per minute56.86.4

QTc, milliseconds

395.917.1

JTpeakc, milliseconds

225.619.8

Tpeak-Tend, milliseconds

73.16.4

T wave morphology
Flatness, d.u.

0.410.04

Asymmetry, d.u.

0.160.05

Presence of notch, %0

Repolarization duration
ERD30%, milliseconds

44.55.1

LRD30%, milliseconds

27.54.1

Vectorcardiographic
QRS-T angle, °34.59.9

TCRT, radians

0.670.24

Tmagmax, lV

578.5173.0

Ventricular gradient, mVms

111.429.5

Continuous variables are represented as meanSD of each subject’s 5-day baseline
average. d.u. indicates dimensionless units; ECG, electrocardiogram; ERD30%, 30% of
early repolarization duration; LRD30%, 30% of late repolarization duration; QRS-T, angle
between the mean QRS and T vectors; QTc, Fridericia’s heart rate corrected QT; TCRT,
total cosine R-to-T; Tmagmax, maximum magnitude of the T vector.

baseline characteristics. All subjects completed the study
except for one subject who withdrew prior to the lasttreatment period (quinidine period for that subject). There
were no unexpected treatment related adverse events(Figure S1). Placebo changes from baseline are shown in
Figure S2.

Analysis and Correction of Heart Rate
Dependency
Substantial heart rate dependent change was observed for T
wave ﬂatness, maximum magnitude of the T vector andventricular gradient. No sex-speciﬁc differences in the heart
rate dependency were found. The heart rate dependent
biomarkers were corrected for heart rate in all subsequent
analysis using an exponential model (biomarkerc=biomarker/
RRa), where the values of a coefﬁcient were 0.58 for T wave
ﬂatness, 0.96 for maximum magnitude of the T vector and0.85 for ventricular gradient.
DOI: 10.1161/JAHA.114.001615

From our ion channel patch clamp experiments, dofetilide wasassociated with 55% hERG potassium channel block at the
population’s mean maximum concentration (Cmax); see
Table 2. Dofetilide did not block calcium or late sodium
currents (Figure 2).
Figure 3 shows the relationships between plasma drugconcentration and each T wave morphology biomarker, where
the plasma concentration for each drug is normalized to the
population Cmax. The actual drug concentration ranges areshown in Table 3. In addition to causing signiﬁcant concentration-dependent QTc prolongation, pure hERG potassiumchannel block (dofetilide) caused concentration-dependent T
wave ﬂatness, asymmetry, and notching (see Figure 4 for ECG
examples), consistent with ECG ﬁndings from LQT2 patients(abnormal hERG potassium channel).35 T wave ﬂatness had amore consistent response than asymmetry, illustrated by thesmaller conﬁdence intervals (Figure 3), and 14 of 22 subjects
developed notched T waves (Table 2). There was also a strong
decrease in the maximum magnitude of the T vector(Figure 3), which is consistent with the observed T wave
ﬂatness (Figure 3).
As with prior pure hERG blockers,18,19 dofetilide caused anincrease in both ERD30% and LRD30% (Figure 3). While priorliterature has suggested that a wider QRS-T angle is associatedwith increased arrhythmic risk, pure hERG block caused a small
decrease in the QRS-T angle (Figure 3). A similar ﬁnding wasobserved for TCRT, while there was no concentration-dependent change in the ventricular gradient (Figure 3).

Quinidine: Strong hERG Block With Additional
Calcium and Sodium Block
Our patch clamp experiments (Figure 2) showed that at Cmax
quinidine was associated with 71% hERG potassium channel
block, 8% calcium current block and 3% of late sodium
current block (Table 2). Thus, quinidine had stronger hERG
potassium channel block than dofetilide, however quinidine
had additional calcium and sodium channel block, but resulted
in a similar amount of concentration-dependent QTc prolongation for the 2 drugs (Figure 3). Quinidine was associatedwith similar to greater amounts of T wave ﬂatness, asymmetry, notching (see Figure 4 for ECG examples) and a decreasein the maximum magnitude of the T vector (P<0.001 for all)compared to dofetilide (Figure 3). Notched T waves developedin 18 of 21 subjects (Table 2).
Similar to dofetilide, quinidine increased ERD30% and
LRD30% (Figure 3). In contrast to the small decrease in QRST angle with dofetilide, quinidine caused a small increase in
the QRS-T angle, while it had no change in TCRT or theventricular gradient (Figure 3).
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Table 1. Baseline Characteristics

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Vicente et al
ORIGINAL RESEARCH

Table 2. Predicted Relative Channel Block and Changes in ECG Biomarkers at Cmax
Dofetilide

Quinidine

Ranolazine

Verapamil

2.70.3 ng/mL

1.80.4 lg/mL

2.31.4 lg/mL

130.475.8 ng/mL

hERG, %

 55

 71

 26

7

Calcium, %No block8

No block 17

Late sodium, %No block3

 21

No block73.6‡ (65.8 to 81.5)

78.9‡ (68.2 to 89.7)

12.0† (7.3 to 16.7)

—

—

—

Population’s Cmax
Relative block

ECG intervals
QTc, milliseconds
JTpeakc, milliseconds
Tpeak-Tend, milliseconds‡

39.1 (31.6 to 46.6)‡†

26.1 (13.5 to 38.7)‡‡

34.4 (26.9 to 42.0)

51.2 (34.6 to 67.8)

10.0 (7.3 to 12.7)

3.6* (1.9 to 5.4)

0.16‡ (0.14 to 0.18)

0.21‡ (0.19 to 0.23)

0.06‡ (0.05 to 0.08)—

T wave morphology
Flatness, d.u.
Asymmetry, d.u.
Notch, %‡0.25 (0.16 to 0.34)‡†

0.34 (0.20 to 0.48)‡†

0.10 (0.05 to 0.15)†—

55.0 (25.2 to 81.6)

69.7 (43.2 to 87.5)

1.4 (0.2 to 9.4)—

23.3‡ (16.0 to 30.6)

22.1† (13.9 to 30.3)

10.2‡ (7.3 to 13.2)—

Repolarization duration
ERD30%, milliseconds
LRD30%, milliseconds

‡

‡

†

13.0 (7.4 to 18.6)

25.8 (15.4 to 36.3)

3.5 (0.9 to 6.0)—

3.9‡ (5.4 to 2.4)

2.7* (0.3 to 5.8)—

0.4* (1.0 to 1.9)

Vectorcardiographic
QRS-T angle, °‡TCRT, radians

0.08 (0.04 to 0.11)——

0.01* (0.04 to 0.02)

Tmagmax, lV

145.9‡ (176.5 to 115.2)

169.5‡ (209.3 to 129.7)

66.6† (90.3 to 42.9)—

Ventricular gradient, mV ms

—

—

—

—

Drug concentrations mean  SD. Relative ion channel block is from the Hill equation curve shown in Figure 2 at Cmax. ECG biomarker changes reported as mean values and 95% conﬁdent
intervals. d.u. indicates dimensionless units; ECG, electrocardiogram; ERD30%, 30% of early repolarization duration; hERG, hERG potassium channel; LRD30%, 30% of late repolarization
duration; QRS-T, angle between the mean QRS and T vectors; QTc, Fridericia’s heart rate corrected QT; TCRT, total cosine R-to-T; Tmagmax, maximum magnitude of the T vector.
*P<0.05; †P<0.01; ‡P<0.001; (—) means there was no relationship between drug concentration and the ECG biomarker.

Ranolazine: Similar hERG and Late Sodium Block
From our ion channel patch clamp experiments (Figure 2), at
Cmax ranolazine was associated with 26% hERG potassiumchannel block and 21% late sodium current block (Table 2).
Ranolazine produced modest concentration-dependent QTc
prolongation of 13 ms at Cmax (Table 2); however it still
produced signiﬁcant amounts of T wave ﬂatness andasymmetry (P<0.001) (Figure 3), along with a decrease in
the maximum magnitude of the T vector (P<0.01). Furthermore, while ranolazine produced statistically signiﬁcantnotching for the population overall (Figure 3, Table S1), only
2 of 22 subjects developed notching (Table 2). Figure 4B
shows the ECG traces of one of the subjects who developed
notching with ranolazine. Of note, this woman had thesecond highest plasma concentration of ranolazine(6.14 lg/mL) of all subjects in this study, and notches in
the T wave were observed at concentrations exceeding
2.45 lg/mL. This subject had even more severe T wave
morphology changes with dofetilide and quinidine
DOI: 10.1161/JAHA.114.001615(Figure 4B). Like dofetilide and quinidine, ranolazine caused
an increase in both ERD30% and LRD30% (Figure 3). Ranolazine did not cause a change in the QRS-T angle, TCRT or
ventricular gradient (Figure 3).

Verapamil: Calcium Block Stronger Than hERG
Block
From our ion channel patch clamp experiments (Figure 2), at
Cmax verapamil was associated with 17% of calcium
current block and 7% hERG potassium channel
block (Table 2). Verapamil did not cause a meaningfulchange in QTc (Figure 3) or any of the T wave morphology
markers (Figure 3) at the plasma concentrations observedin this study. Of note, as described previously,16 verapamil
did cause a substantial 32.1 [95% conﬁdence interval: 26.7
to 37.4] ms (P<0.001) increase in the PR interval, demonstrating that the plasma concentrations in this study were
sufﬁcient to elicit an electrophysiological effect. The lack of
T wave morphology changes observed in this study suggests
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Drug-Induced Changes in T Wave Morphology

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relative block (%)

relative block (%)

100
75
50
25
0

Quinidine concentration
100
75
50
25
0

0.01

0.10

1.00

10.00

10

100

Dofetilide (nM)

1000

10000

Quinidine (nM)

Ranolazine concentration

D

100

relative block (%)

relative block (%)

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ORIGINAL RESEARCH

Dofetilide concentration

A

75
50
25
0

Verapamil concentration
100
75
50
25
0

10

100

1000

10000

1

Ranolazine (nM)
hERG

10

100

1000

Verapamil (nM)
Calcium

Late sodium

Figure 2. Patch clamp experiment results for (A) dofetilide, (B) quinidine, (C) ranolazine, and (D) verapamilfor hERG (red dots), calcium (green triangles), and late sodium (blue squares). The lines in each plotcorrespond to a ﬁt between the observed means per concentration and data (see text). The error bars
denoteSEM. The dashed lines in each panel correspond to the range of observed clinical concentrations
and the solid line is the population average maximum concentration (Cmax). hERG indicates hERG potassiumchannel; Calcium, L-type calcium current; Late sodium, late sodium current.

that competing effects of inward current block from calcium
and outward current block from hERG potassium during
ventricular repolarization are very well balanced withverapamil.

Relation Between QTc and T Wave Morphology
Metrics
We used the concentration-response models to predict
the individual drug-induced changes for QTc and each T
wave morphology biomarker at 25% increments of the
population’s Cmax of each drug. Figure 5 shows the relationship between changes in QTc and changes in T wave
morphology biomarkers for dofetilide, quinidine, and ranolazine. At equivalent drug-induced QTc prolongation, quinidine
and ranolazine (multichannel blockers) induced similar to orslightly greater T wave morphology changes than did dofetilide (pure hERG potassium channel blocker).

DOI: 10.1161/JAHA.114.001615

Discussion
This study observed that pure hERG potassium channel blockwith dofetilide causes substantial concentration-dependent T
wave ﬂatness, asymmetry, and notching, along with a decreasein the maximum magnitude of the T vector. These quantitative T
wave biomarkers were originally developed to detect the ECG
signature of congenital LQT2 (decreased hERG potassiumchannel current), and this study strongly supports that thesebiomarkers capture that ECG phenotype. This study was novel
and unique in that the same subjects received 3 additionalhERG potassium channel blockers (quinidine, ranolazine, andverapamil) that also block inward currents (calcium or latesodium) during repolarization. hERG blocking drugs that also
block inward currents have been observed to have a lower
torsade risk, either because they decrease dispersion of
repolarization (the substrate for re-entry)10 or they prevent
early afterdepolarizations (the trigger for torsade).11 We

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B

C

D

E

F

G

H

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A

L

Figure 3. Mean baseline- and placebo-corrected plasma drug concentration-dependent changes (lines) and 95% conﬁdence intervals(shaded areas) for T wave (A) ﬂatness, (B) asymmetry, (C) probability of presence of notch, (D) maximum magnitude of T vector, (E) 30% of early
repolarization duration, (F) 30% of late repolarization duration, (G) angle between the mean QRS and T vectors (QRS-T angle), (H) total cosine of
R to T, (I) spatial ventricular gradient, (J) Fridericia’s heart rate corrected QT (QTc) interval, (K) early repolarization interval (J-Tpeakc), and (L) late
repolarization interval (Tpeak-Tend). x axis shows % of population Cmax for each drug (meanSD): dofetilide (2.70.3 ng/mL, solid gray),quinidine (1.80.4 lg/mL, dotted orange line), ranolazine (2.31.4 lg/mL, blue dashed line) and verapamil (130.475.8 ng/mL, dashedgreen line). See Table 3 for detailed drug concentration ranges. Mean slopes together with 95% conﬁdence interval values are reported inTable S1. Figures S3 through S5 show similar information in a drug-by-drug basis together with relative block of hERG, calcium, and latesodium currents from the patch clamp experiments. Corresponding time proﬁle plots of drug plasma concentration and time-matched druginduced changes are shown in Figures S6 through S8.
DOI: 10.1161/JAHA.114.001615

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ORIGINAL RESEARCH

Table 3. Drug Concentration Ranges
Minimum (Individual)

Mean (Population’s Cmax)

Maximum (Individual)

Plasma, ng/mL

0.16

2.72

3.27

Estimated unbound free fraction, nmol/L

0.1

2.2

2.6

Relative to population Cmax, %

6

100

120

Plasma, lg/mL

0.09

1.79

2.55

Estimated unbound free fraction, nmol/L

40.0

829.4

1179.1

Relative to population Cmax, %

5

100

142

Plasma, lg/mL

0.01

2.30

6.49

Estimated unbound free fraction, nmol/L

11.1

1726.74867.5

Relative to population Cmax, %

1

100

282

Plasma, ng/mL

1.55

130.35368.00Estimated unbound free fraction, nmol/L

0.5

45.1

127.4

Relative to population Cmax, %

1

100

282

Dofetilide

Quinidine

Ranolazine

Verapamil

Individual minimums were observed at 24 hours postdose for all drugs. See Figures S6 through S8 for concentration time proﬁles.

observed that quinidine and ranolazine both still caused T wave
morphology changes, despite inward current block.
This raises the question of how T wave morphology
changes relate to risk for drug-induced torsade de pointes.
While quinidine, ranolazine, and verapamil are multichannel
blockers, only quinidine is associated with a signiﬁcanttorsade risk. This is likely due to quinidine’s substantiallygreater hERG block (71%) than calcium and late sodium
block (8% and 3%, respectively) at Cmax. Furthermore, atlower concentrations (see Figure 2) quinidine is a relatively
pure hERG blocker, and torsade de pointes events withquinidine have been observed to occur more frequently at
lower concentrations.13 Compared with quinidine, ranolazine
was associated with less hERG block (26%) and more latesodium current block (21%), while verapamil was associatedwith only 7% hERG block and 17% calcium block.
Moreover, there was no increase in QTc or change in T wave
morphology associated with verapamil in this study, suggesting that verapamil has almost perfectly balanced inward(calcium) and outward (hERG) current block. Thus, it is likely
not just the presence of calcium or late sodium current blockthat can prevent torsade, but rather the balance between
inward and outward current block that is most important.
Taken together with our prior observations that calcium
and late sodium block primarily affect early repolarization by
shortening the J-Tpeakc interval,16,33 results suggest that thestrongest effect of calcium and late sodium block may be toshift the T wave to the left (shorter ST segment duration), with
DOI: 10.1161/JAHA.114.001615

a lesser effect on T wave morphology. The opposite effect isseen with LQT3, where the abnormally increased late sodium
current results in a long ST segment duration, with a late
appearing T wave of unchanged morphology.3 Overall, while
ﬂat, asymmetric, and notched T waves appear to bebiomarkers of hERG block, these T wave morphology markers
likely do not always correlate with torsade de pointes risk, as
has also been observed for QTc. The relationship between
events of torsade de pointes and the relative amount of hERG
block compared to additional calcium or late sodium blockdeserves further investigation with a large number of drugs,
as has been proposed under the Comprehensive in vitro
Proarrhythmia Assay (CiPA) initiative.36

T Wave Loop Biomarkers
In addition to assessing T wave ﬂatness, asymmetry, andnotching, we assessed a series of T wave loop biomarkers.
ERD30% and LRD30% measure the time from the peak of the T
wave loop to 30% of the baseline toward the beginning of the T
wave (ERD30%) and end of the T wave (LRD30%), respectively.
These markers were developed to be sensitive detectors ofhERG potassium channel block and were previously assessedwith moxiﬂoxacin18 and d,l-sotalol.37 The results of the present
study conﬁrm that ERD30% and LRD30% are indeed sensitivemarkers of hERG block from dofetilide, quinidine, and ranolazine. When assessing the relative changes of these biomarkers compared with the amount of QTc change (Figure 5),
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ORIGINAL RESEARCH

A

PlaceboDofetilide
Quinidine
Ranolazine
Verapamil
Lead: V3 0.5 hour post-dose

2.5 hours post-dose

4 hours post-dose

12 hours post-dose

3 hours post-dose

6 hours post-dose

12 hours post-dose

B

PlaceboDofetilide
Quinidine
Ranolazine
Verapamil
Lead: V3 1 hour post-doseFigure 4. Lead V3 of median beats of time-matched placebo (gray), dofetilide (black), quinidine (orange), ranolazine (blue), and verapamil(green). Top panel (A): ECGs at 0.5, 2.5, 4, and 12 hours post-dose from a woman with notches present in dofetilide and quinidine but not in
ranolazine. Bottom panel (B): ECGs at 1, 3, 6, and 12 hours post-dose from a woman with notches induced by ranolazine. See Table S2 for heart
rate and QT/QTc measurements. ECG indicates electrocardiogram.

there was a similar amount of change in LRD30% for dofetilide,
quinidine, and ranolazine. For ERD30%, dofetilide and quinidine
were similar, while ranolazine induced a greater relative change
in ERD30%; how this relates to cardiac safety is unknown.
Prior research has suggested that patients with a wide QRS-T
angle are at increased risk for ventricular arrhythmias andsudden death.20,21 Similar observations have been reportedwith the TCRT in other patient populations (hypertrophic
cardiomyopathy22 and post-myocardial infarction38), where adecrease in TCRT is equivalent to a wider QRS-T angle. However,in this study pure hERG potassium channel block (dofetilide) did
not cause an increase in the QRS-T angle, but rather caused asmall decrease. In contrast, quinidine caused a small increase in
the QRS-T angle. The small magnitude of the changes (5° atCmax) suggests that these changes are likely not clinicallymeaningful and these indices do not appear to have value for
DOI: 10.1161/JAHA.114.001615

detecting hERG potassium channel block. There was also nochange in the ventricular gradient with any drug, which has been
proposed as a global marker of action potential morphology
heterogeneity.32
Our results show heart rate dependency for the maximum
magnitude of the T vector, which is in concordance withprevious studies.39,40 Andersen et al40 reported T wave
ﬂatness as heart rate independent, but our results show T
wave ﬂatness being heart rate dependent. The present study
had a larger range of heart rates, and thus T wave ﬂatness
likely is heart rate dependent.

Limitations
This study involved only a single dose of each drug and it ispossible that different doses or chronic dosing would have
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B

C

D

E

F

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A

Figure 5. Relationship between predicted drug-induced baseline- and placebo-corrected changes in QTc (x axis) and T wave (A) ﬂatness, (B)asymmetry, (C) probability of presence of notch, (D) maximum magnitude of T vector, (E) 30% of early repolarization duration, (F) 30% of late
repolarization duration, (G) angle between the mean QRS and T vectors (QRS-T angle), (H) total cosine of R to T, (I) spatial ventricular gradient, (J)early repolarization interval (J-Tpeakc), and (K) late repolarization interval (Tpeak-Tend) (y axis). Average predictions (dots) and 95% conﬁdence
intervals (horizontal and vertical lines) from concentration dependent models for QTc (x axis) and the different T wave morphology biomarkers (yaxis) at baseline and 25% increments of population’s Cmax for dofetilide (dashed gray line) quinidine (dotted orange line) and ranolazine (solid
blue line). QTc, Fridericia’s heart rate corrected QT.

DOI: 10.1161/JAHA.114.001615

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Acknowledgments
QTGuard+ was provided by GE Healthcare through a material transfer
agreement. The mention of commercial products, their sources, ortheir use in connection with material reported herein is not to beconstrued as either an actual or implied endorsement of suchproducts by the U.S. Department of Health and Human Services.

Sources of Funding
This study was supported by U.S. Food and Drug Administration’s Critical Path Initiative, U.S. Food and Drug Administration’s Ofﬁce of Women’s Health, and appointments to the
Research Participation Program at the Center for Devices andRadiological Health administered by the Oak Ridge Institutefor Science and Education through an interagency agreement
between the U.S. Department of Energy and the U.S. Foodand Drug Administration. Pueyo is funded by Ministerio de
Economıa y Competitividad (MINECO), Spain, under project
TIN2013-41998-R and by Grupo Consolidado BSICoS from
DGA (Aragon) and European Social Fund (EU).

Disclosures
None.

Conclusion

References

This study demonstrated that there is a strong dose-response
relationship between hERG potassium channel block and T
wave morphology changes. While nonclinical studies havesuggested that late sodium current block can reduce dispersion of repolarization from hERG block, we observed thatranolazine still had a dose dependent relationship with T wave
morphology, in some cases having greater T wave morphology
changes at equivalent amounts of QTc prolongation compared with the pure hERG blocker dofetilide. Thus, the presenceof T wave morphology changes is not a perfect predictor of
torsade de pointes risk. However, this study supports that T
wave morphology biomarkers (eg, ﬂatness, asymmetry, andnotching, along with a decrease in the maximum magnitude of
the T vector) likely have value in determining if QT prolongation is due to hERG block. Subsequently, analysis of J-Tpeak and
Tpeak-Tend intervals may be able to inform whether a hERGblocking drug has additional late sodium or calcium current
block that can mitigate torsade risk, as with ranolazine. Overall,the ECG signatures of drug-induced ion channel block closely
parallel the ECG signatures seen with congenital long QT
syndromes and a combined approach of assessing multiple ionchannels, subintervals of the QT (eg, J-Tpeak and Tpeak-Tend) and T
wave morphology can provide the greatest insight into drug-ion
channel interactions and torsade de pointes risk.

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