Supplementary Table 1.Selected reference drugs, testing concentrations and therapeutic exposure

a Ceff, the maximal therapeutic plasma concentration reported in the literature. b As aspirin was metabolized rapidly in liver with a plasma half-life of ~ 20 min, 10 µM was the median of plasma concentration over 4 h after dosing, assuming to take one 1200 mg dose every 4 h. C The clinical efficacy exposure not established, the plasmas concentration that induced TdP in the in-house telemetry monkey study was used.

Drug Class/applications Conc. tested (µM)

Ceff (nM)a Reference

Aconitine Neurotoxin, antipyretic, analgesic 0.01-10 77 Ohta, Seto, and Tsunoda (1997), Wada et al. (2005)

Alfuzosinα1-adrenergic antagonist, anti-protatic hyperplasia

0.3-10 56 Lacerda et al. (2008)

Amiodarone Class III antiarrhythmic 0.01-10 3,874 Redfern et al. (2003)

Amoxicillin Antibiotic 1-1,000 17,036 Lawrence et al. (2006)

Aspirin Anti-inflammatory 1-1,000 10,000b Seymour & Rawlins (1982); DrugBank (protein binding)

Astemizole Antihistamine 0.001-1 8.0 Redfern et al. (2003)Blebbistatin Myosin II inhibitor 0.03-30 ~ not available

Captopril ACE inhibitor, antihypertensive 1-100 2,466 Lawrence et al. (2006)

Cisapride Prokinetic 0.01-10 129 Redfern et al. (2003)

Dofetilide Class III antiarrhythmic 0.001-1 6.0 Redfern et al. (2003)E-4031 Class III antiarrhythmic 0.003-1 13 Fossa et al. (2004)Erythromycin (i.v.) Antibiotic 0.1-100 34,064 Redfern et al. (2003)

Flecainide Class Ic antiarrhythmic 1.0 - 30 1,931 Redfern et al. (2003)Fluoxetine Antidepressant 0.03-30 485 Redfern et al. (2003)

Isoproterenolβ-adrenergic agonist, bronchodilator

0.001-1 ~ not available

Moxifloxacin Antibiotic 0.1-100 10,276 FDA label (1999) for Avelox

Nifedipine L-type Ca2+ channel blocker, antihypertensive

0.003-3 194 Redfern et al. (2003)

Ouabain Na+/K+ ATPse inhibitor 0.003-3 170 Selden & Neill (1975); DrugBank (protein binding)

Quinidine Class Ia antiarrhythmic 0.1-100 3,237 Redfern et al. (2003)Ranolazine Antianginal 0.1-100 6,009 FDA label (2006) for Ranexa

RO5657 Anti-HIV 0.1-100 5,548c Roche in-house data

Rofecoxib Nonsteroidal anti-inflammatory 0.1-100 1,021 FDA label (1999) for Vioxx

Sotalol Class III antiarrhythmic 0.1-100 14,733 Redfern et al. (2003)

Terfenadine Antihistamine 0.01-10 300 Redfern et al. (2003)

Tetrodotoxin (TTX) Na+ channel blocker 0.03-30 ~ not available

Thioridazine Antipsychotic 0.01-10 1,781 Redfern et al. (2003)

Verapamil Anithypertensive, antianginal 0.3-10 815 Redfern et al. (2003)

ZD7288 If channel blocker 1-30 ~ not available

Supplementary Figure 1.

Suppl Fig. 1: Illustration of Irregular Beat Ratio (IBR) calculation. The screen-shots were taken from a 20 s-sweep window after exposure to the vehicle (0.1% DMSO, top trace) and E-4031 at 30 nM (middle trace) and 1000 nM (bottom trace), respectively. The regular (R) and irregular (I) beats were denoted by the blue-dots and red-arrowheads, respectively. The total (T) beats was a sum of R and I in each sweeps, and the IBR was calculated as the ratio of I/T.

R I T IBR

12 0 12 0

11 3 14 0.21

4 28 32 0.88

AmoxicillinSupplementary Figure 2.

Suppl Fig. 2: Representative impedance traces for amoxicillin and captopril. No obvious irregular beats nor significant alteration in beat amplitude were observed during drug exposure from 1 to 100 µM and up to 72 h. The vertical scale bar = 0.08 CI.

APre-drug Post-drug 30 min 1 h 6 h 24 h 72 h

10 s

0 µM

1

10

100

B CaptoprilPre-drug Post-drug 30 min 1 h 6 h 24 h 72 h

10 s

0 µM

1

10

100

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E-4031Supplementary Figure 3.

0 nM

1

3

10

30

100

300

1000

Pre-drug Post-drug 30 min 1 h 6 h 24 h 72 h

Conc. (nM)RTCA Cardio (n = 3)

5 s

Suppl Fig. 3: Dose- and time-dependent induction of arrhythmic beats by E-4031. A) Representative traces of RTCA Cardio recordings illustrate the development of the single, double, triple and short-train arrhythmic beats (denoted by arrowheads) that are induced dose-dependently by E-4031. Note, synchronous beats are arrested completely at 24 h and 72 h by 1000 nM, and at 72 h by 300 nM. The vertical scale bar = 0.1 CI. B) The Irregular Beat Ratio averaged from 3 e-plates (3 wells/conc.) was plotted as a function of time, demonstrating the rapid induction of arrhythmic beats that are persistent throughout the observation period at concentrations ≥ 100 nM. The standard error bars are not graphed for better clarity.

0

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0 30 100 300 1000

Concentration (nM)

Irre

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C D

MEA (n = 7)

Supplementary Figure 3.

0 nM

30

100

300

1000

E-4031

5 s1

00

µV

Suppl Fig. 3: Dose- and time-dependent induction of arrhythmic beats by E-4031. C) Representative traces of microelectrode array (MEA) recordings illustrate the development of the single, double, triple and short-train arrhythmic beats (denoted by arrowheads) induced dose-dependently by E-4031. D) The Irregular Beat Ratio averaged from 7 MEA wells was plotted as a function of concentrations to demonstrate the dose-dependent increase in arrhythmic beats induced by E-4031.

-0.2

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RTCA Cardio (n = 5)

RO5657

Conc. (µM)

Supplementary Figure 4.

Pre-drug Post-drug 30 min 1 h 6 h 24 h 72 h

5 s

Suppl Fig. 4: Dose- and time-dependent induction of arrhythmic beats by RO5657. A) Representative traces of RTCA Cardio recordings illustrate the development of typical arrhythmic beats (denoted by arrowheads), ventricular fibrillation or tachycardia-like (dark stars) arrhythmia that are induced dose-dependently by RO5657. Note, synchronous beats are arrested completely at 72 h by 100 µM. The vertical scale bar = 0.12 CI. B) The Irregular Beat Ratio averaged from 5 e-plates (5 wells /conc.) was plotted as a function of time to demonstrate the quick induction and dynamic change of arrhythmic beats throughout the observation period up to 72 h. The standard error bars are not graphed for better clarity.

Bea

t-ra

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r R

atio

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0 3 10 30 100Concentration (µM)

RO5657

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RO5657

MEA (n = 4)

0 µM

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10

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100

Supplementary Figure 4.

5 s

10

0 µ

VSuppl Fig. 4: Dose- and time-dependent induction of arrhythmic beats by RO5657. C) Representative traces of microelectrode array (MEA) recordings illustrate the development of typical arrhythmic beats (denoted by arrowheads) induced dose-dependently by RO5657. D) The plot of Irregular Beat Ratio averaged from 4 MEA wells as a function of concentrations demonstrates the dose-dependent increase in arrhythmic beats induced by RO5657.

Irre

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at

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0.03

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0.3

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TerfenadineA

0 µM

0.01

0.03

0.1

0.3

1

3

10

Pre-drug Post-drug 6 h 12 h 24 h 48 h 72 h

RTCA Cardio (n = 3)

Conc. (µM)

Supplementary Figure 5.

B

5 s

Suppl Fig. 5: Dose- and time-dependent induction of arrhythmic beats by terfenadine. A) Representative traces of RTCA Cardio recordings illustrate the quick arrest of synchronous beatings and the development of typical arrhythmic beats (denoted by arrowheads) and ventricular fibrillation-like arrhythmia (dark stars) induced dose-dependently by terfenadine. Note, the time-dependent recovery of synchronous beats at concentrations ≥ 1 µM and the delayed induction of arrhythmic beats at concentrations ≥ 0.3 µM. The vertical scale bar = 0.1 CI. B) The Irregular Beat Ratio averaged from 3 e-plates (3 wells/conc.) was plotted as a function of time to demonstrate the delayed induction of arrhythmic beats. Arrhythmic beats occur after 12 h in exposure to terfenadine. The standard error bars are not graphed for better clarity.

Irre

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ThioridazineA

0 µM

0.01

0.03

0.1

0.3

1

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10

Pre-drug Post-drug 6 h 12 h 24 h 48 h 72 h

RTCA Cardio (n = 3)

Conc. (µM)

Supplementary Figure 6.

B5 s

Suppl Fig. 6: Dose- and time-dependent induction of arrhythmic beats by thioridazine. A) Representative traces of RTCA Cardio recordings illustrate the quick arrest of synchronous beatings and the development of typical arrhythmic beats (pointed with arrow heads) dose-dependently by terfenadine. Note, the time-dependent recovery of synchronous beats at 10 µM and the delayed induction of arrhythmic beats at concentrations ≥ 3 µM. The vertical scale bar = 0.12 CI. B) The Irregular Beat Ratio averaged from 3 e-plates (3 wells/conc.) was plotted as a function of time to demonstrate the delayed induction of arrhythmic beats. Arrhythmic beats occur after 48 h in exposure to thioridazine. The standard error bars are not graphed for better clarity.

Irre

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RanolazineA

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Pre-drug Post-drug 30 min 3 h 6 h 24 h 72 h

RTCA Cardio (n = 5)

Conc. (µM)

Supplementary Figure 7.

B5 s

Suppl Fig. 7: Dose- and time-dependent induction of arrhythmic beats by ranolazine. A) Representative traces of RTCA Cardio recordings illustrate the slow development of typical arrhythmic beats (denoted by arrowheads) induced dose-dependently by ranolazine. Note, the frequent occurrence of arrhythmic beats at 3 h after exposure to the drug. The vertical scale bar = 0.08 CI. B) The plot of Irregular Beat Ratio averaged from 5 e-plates (5 wells/conc.) as a function of time demonstrates the slowly developed and transient induction of arrhythmic beats. Arrhythmic beats occur most frequently at 3 h after drug exposure.

Verapamil

Supplementary Figure 8.

Suppl Fig. 8: Dose- and time-dependent effects of verapamil. Reduction in beat amplitude and acceleration in beat rate were observed immediately after drug addition. Both changes recovered slowly and a complete recovery achieved at 48 h post-drug exposure. The vertical scale bar = 0.08 CI.

0 nM

10

30

100

300

Pre-drug Post-drug 3 h 18 h 24 h 48 h 72 h

10 s

Supplementary Reference

1. Ogata, N., & Narahashi, T. Block of sodium channels by psychotropic drugs in single guinea-pig cardiac myocytes.

British J Pharmacol, 97, 905-913. (1989).

2. Wada, K., et al. Effects of long-term administrations of aconitine on electrocardiogram and tissue concentrations of

aconitine and its metabolites in mice. Forensic Sci International, 148, 21−29. (2005).

3. Lacerda, A.E. et al. Alfuzosin delays cardiac repolarization by a novel mechanism. J Pharmacol Exp Ther. 324(2),

427-33. (2008).

4. Redfern, W.S. et al. Relationships between preclinical cardiac electrophysiology, clinical QT interval prolongation

and torsade de pointes for a broad range of drugs: evidence for a provisional safety margin in drug development.

Cardiovascular Res, 58, 32-45. (2003).

5. Lawrence, C.L., et al. A rabbit Langendorff heart proarrhythmia model: predictive value for clinical identification of

Torsades de Pointes. British J of Pharmacol, 149, 845-860. (2006).

6. Seymour, R.A., & Rawlins, M.D. Efficacy and pharmacokinetics of aspirin in post-operative dental pain. Br J Clin

Pharmacol. 13(6), 807-10. (1982).

7. Selden, R., & Neill, W. A. Myocardial uptake of ouabain in intact dog and man. J Pharmacol Exp Ther, 193,

951−962. (1975).

8. Fossa, A. A., et al. Differential effect of HERG blocking agents on cardiac electrical alternans in the guinea pig.

Eur J Pharmacol. 486(2), 209-21. (2004).

9. FDA label (2006) for Ranexa:

http://129.128.185.122/drugbank2/drugs/DB00243/fda_labels/147

10. FDA label (1999) for Avelox:

http://129.128.185.122/drugbank2/drugs/DB00218/fda_labels/389

11. FDA label (1999) for Vioxx:

http://129.128.185.122/drugbank2/drugs/DB00533/fda_labels/980

Supplementary Video 1 (legend)

Supplementary Video 1: Representative videos depicting the contractility reduction of Blebbistatin, a myosin II Inhibitor on human iPSC-CMs. The 20 s video on the left shows pre-drug, iPSC-CMs beating at their typical rate and contraction. The 20 s video on the right depicts the same field of cells as on the left, after 15 min exposure of 1 µM Blebbistatin. While robust contractions are evident on the left, the contraction is severely reduced yet still present in the video on the right, in accordance with impedance traces. Additionally, focusing on specific cells present in both the left and right videos, it is apparent that there is no change in the beat rate, in accordance with both impedance and MEAs. For example, note the large, dark round cell cluster in the lower right corner of both videos; this cluster is beating at the same rate in both videos but the contraction is significantly muted in the right post-drug video. The cells depicted are plated onto an ordinary 96-well tissue culture plate in order to not have visuals imposed upon by impedance electrodes at 10 X magnification.

Supplementary Video 2 (legend)

Supplementary Video 2: Representative video depicting the proarrhythmic effect of E-4031, a specific hERG inhibitor on iPSC-CMs. The 17 s video on the left shows pre-drug, iPSC-CMs beating with a consistent rate, size of the contraction and duration of each individual beat. The 17 s video on the right depicts the same field of cells as on the left, after 1 h exposure of 0.03 µM E-4031. Both videos begin with the onset of the first beat, with all beats numbered in yellow text. In the pre-drug movie, the entire field of cardiomyocytes beat synchronously at a regular frequency, approximately once every two seconds, for a total of 11 beats. In contrast, after E-4031 treatment, the same field of cells exhibit asynchronous beating, with a reduced frequency of large contractions and many smaller contracting, beats or twitches of single cells for at least a combined 15 regular and irregular beats/small twitches. These differences are best observed by looping the movie playback and observing each field separately. The cells depicted are plated onto glass cover slips in order to not have visuals imposed upon by impedance electrodes at 10 X magnification.

Supplementary Video 3 (legend)

Supplementary Video 3: Representative videos depicting the fibrillation-like arrhythmia induced by ouabain, a Na+/K+-ATPase inhibitor on iPSC-CMs. The 20 s video on the left shows pre-drug, iPSC-CMs beating at their typical rate and contraction. The 20 s video on the right depicts the same field of cells as on the left, after 15 min exposure of 0.3 µM ouabain. A synchronous monolayer of cells is visible on the left video under pre-drug conditions. On the right, ouabain caused very rapid and asynchronous (fibrillation-like) contractions of individual cells. The cells depicted are plated onto an ordinary 96-well tissue culture plate in order to not have visuals imposed upon by impedance electrodes at 10 X magnification.