Abnormal Calcium Handling Properties Underlie Familial ... · PDF fileRepresentative...

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Cell Stem Cell, Volume 12

Supplemental Information

Abnormal Calcium Handling Properties Underlie

Familial Hypertrophic Cardiomyopathy Pathology

in Patient-Specific Induced Pluripotent Stem Cells

Feng Lan, Andrew S. Lee, Ping Liang, Veronica Sanchez-Freire,Patricia K. Nguyen, Li Wang, Leng Han, Michelle Yen, Yongming Wang, Ning Sun, Oscar J. Abilez, Shijun Hu, Antje D. Ebert, Enrique G. Navarrete, Chelsey S. Simmons, Matthew Wheeler, Beth Pruitt, Richard Lewis, Yoshinori Yamaguchi, Euan A. Ashley, Donald M. Bers, Robert C. Robbins, Michael T. Longaker, and Joseph C. Wu

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Ⅲ -7Ⅲ -6 Ⅲ -5Ⅲ -3

Diastole SystoleA B

OCT4

iPSC Fibroblast

SOX2

ACTIN

NANOG

Control HCM

46, XY 46, XY

C

DTUJ1 DAPI SOX17 DAPI SMA1 DAPI

OCT4

NANOG

PAX2

SOX17

BRACHYURY

GAPDH

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Endodermal glandsGlands & Cartilage

Squamous Epithelium Cartilage

40x

G AmplificationHha I digestion

150 bp

300 bp

Ⅱ -1 Ⅱ -2 Ⅲ -1 Ⅲ -2 Ⅲ -4 Ⅲ -8

Figure S1 (Referenced from Figure 1). Genomic stability and pluripotency of patient-specific iPSCs. (A) Two dimensional echocardiographic images of the left ventricle in a 2 chamber short-axis view showing asymmetric thickening of the inferior wall and hyperdynamic function in the HCM proband. (B) Western blot of protein expression for pluripotency transcription factors OCT4, SOX2, and NANOG in patient specific iPSCs and fibroblasts derived from HCM (II-1, III-1, III-2, III-3, III-8) and control (II-2, III-4, III-5, III-6, III-7) family members. β-actin was used as control. (C) Karyotyping of patient specific iPSCs demonstrated a normal karyotype of 2n=46 (XX or XY) through passage 30 for both control and HCM cells. (D) Differentiation of patient-specific iPSCs into derivatives of 3 germ layers. Representative immunofluorescence staining photographs of EBs differentiated from patient-specific iPSCs for TUJ1 (ectoderm), SOX17 (endoderm), and SMA1 (mesoderm). (E) Representative hematoxylin and eosin staining of a solid encapsulated teratoma derived from a patient-specific iPSC line implanted into the sub-renal capsule of SCID mouse mice. (F) Semi-quantitative reverse transcription (RT)-PCR of patient-specific iPSC lines and EBs differentiated from these lines. OCT4 and NANOG were used as markers of pluripotency, whereas PAX2 (ectoderm), SOX17 (endoderm), and BRACHYURY (mesoderm) were used as markers of differentiation. (G) Genotyping of patient-specific iPSC lines derived from dermal fibroblasts of HCM subjects (II-1, III-1, III-2, III-3, III-8) and control matched family members (II-2, III-4, III-5, III-6, III-7). Restriction enzyme digestion and amplification confirmed presence and absence of the Arg663His mutation in HCM and control subjects, respectively.

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Supplementary Figure 2

0

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MLC2a MLC2v DAPI MLC2a MLC2v DAPI

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Figure S2 (Referenced from Figure 2). Characterization of patient-specific iPSC-CMs. (A) Efficiency of

cardiac differentiation for each patient-specific iPSC line as measured by the percentage of beating EBs (n=50 per

line, 10 patient lines total). (B) Percentage of differentiated cells per patient-specific EB expressing cardiac troponin

T (n=100 per line, 10 patient and control subject lines). (C) Staining of iPSC-CMs for markers of cardiac

differentiation. Sub-characterization of iPSC-CMs based on immunostaining for myosin light chain 2a (MLC2a) and

myosin light chain 2v (MLC2v) derived from family members with and without HCM. (D) Quantification of iPSC-

CM into ventricular, atrial, and double positive (immature) subtypes based on immunostaining for MLC2a and

MLC2v (n=235, 10 patient and control subject lines). * denotes P<0.001 MLC2a vs MLC2v vs double positive. (E)

Representative immunofluorescence staining for nuclear translocation of NFATc4 in HCM-iPS-CMs treated with

the calcineurin inhibitors Cs-A and FK506. (F) Quantification of HCM iPSC-CMs exhibiting nuclear translocation

of NFATc4 with and without treatment of Cs-A and Fk-506 (n=83, 5 patient lines per treatment group). (G)

Representative sarcomeric stainings for F-actin and TNNT2 in control and HCM iPSC-CMs demonstrating

organized and disorganized sarcomeres. (H) Quantification of average sarcomeric content in control and HCM

iPSC-CMs as measured by average fluorescence intensity of TNNT2 (HCM n=186, 5 patient lines, control n=173, 5

control subject lines). (I) Quantification of control and HCM iPSC-CMs exhibiting disorganized sarcomeres (HCM

n=186, 5 patient lines, control n = 173, 5 control subject lines). Cells with disorganized sarcomeres were defined as

having greater than 25% cell area of punctate troponin T distribution without striations.* denotes P<0.05 HCM vs

control, ** denotes P<0.001 HCM vs control. Error bars are standard error mean.

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Day 20 Day 30 Day 400

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Figure S3 (Referenced from Figure 3). Gene expression and electrophysiological profiling of patient-specific iPSC-CMs. (A) Changes in temporal gene expression for a panel of 12 transcripts related to HCM gene expression in control and HCM iPSC-CMs following cardiac differentiation (n=32, 5 patient and control subject lines per time point). (B) Whole cell patch clamp recordings from normal and HCM human iPSC-CMs. Single spontaneous action potentials from representative control and HCM iPSC-CMs indicating presence of myocytes exhibiting nodal, atrial, and ventricular waveforms. (C) Quantification of nodal, atrial, and ventricular sub-populations in control (n=144, 5 control subject lines) and HCM iPSC-CMs (n=131, 5 patient lines). (D) Quantification of nodal, atrial, and ventricular resting membrane potentials (RMPs) in control (n=144, 5 control subject lines) and HCM iPSC-CMs (n=131, 5 patient lines). (E) Quantification of nodal, atrial, and ventricular upstroke velocities in control (n=144, 5 control subject lines) and HCM iPSC-CMs (n=131, 5 patient lines). (F) Quantification of nodal, atrial, and ventricular action potential amplitudes in control (n=144, 5 control subject lines) and HCM iPSC-CMs (n=131, 5 patient lines). (G) Quantification of nodal, atrial, and ventricular action potential durations at 90% repolarization (APD90) for control (n=144, 5 control subject lines) and HCM iPSC-CMs (n=131, 5 patient lines). (H) Capacitance measurements of cell size using patch clamp (HCM n=131, 5 patient lines; control, n=144, 5 control subject lines,). * denotes P<0.01 HCM vs control, ** denotes P<0.001 HCM vs control. Error bars represent standard error mean.

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Control HCM0

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No

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ControlHCMD E

Time (s)

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Interval of the beating (s)

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Figure S4 (Referenced from Figure 4). Contractile analysis and calcium handling properties of patient specific iPSC-CMs. (A) Analysis of time-lapse video recordings for contractile arrhythmia in control and HCM iPSC-CMs. Representative software analysis profiles of contractile timing in single control and HCM iPSC-CMs. Red arrowheads indicate representative cell contractions as determined by the video analysis. (B) The length of time between cell contractions were quantified and mapped to demonstrate the spread of beat intervals in control (n=68, 5 control subject lines) and HCM iPSC-CMs (n=64, 5 patient lines). Control iPSC-CMs exhibit a narrow distribution of beat intervals, whereas HCM iPSC-CMs exhibit a wide distribution indicating arrhythmic contraction. (C) Quantification of control (n=68, 5 control subject lines) and HCM iPSC-CMs (n=64, 5 patient lines) exhibiting contractile arrhythmia as determined by video analysis of single beating cells. (D) Representative traces of contractile motion analysis for control and HCM iPSC-CMs. (E) Quantification of normalized contractile motion through pixel displacement analysis (HCM n=663, 5 patient lines; control n=604, 5 control subject lines). * denotes P<0.01 HCM vs control. (F-K) Quantification of parameters for frequency, amplitude, and integrated Ca2+ load of Fluo-4 AM Ca2+ dye transients, as well as irregularities in the above measurements in control (n=80, 5 control subject lines) and HCM iPSC-CMs (n=100, 5 patient lines). (L) Fluo-4 AM Ca2+ imaging of patient-specific iPSC-CMs paced at 1 Hz. Representative image of region of interest (ROI) analysis used to quantify Ca2+ transients in paced (1 Hz) iPSC-CMs derived from control and HCM subjects. (M) Representative Ca2+ waveforms of paced (1 Hz) iPSC-CMs from HCM patients and control matched family members. Red arrowheads indicate irregular transients. (N) Quantification of control (n=19, 5 control subject lines) and HCM iPSC-CMs (n=20, 5 patient lines) exhibiting irregular Ca2+ transients during 1 Hz pacing. Control iPSC-CMs were not observed to exhibit abnormal Ca2+ waveforms during pacing. * denotes P<0.05 HCM vs control, ** denotes P<0.0001 HCM vs control. Error bars represent standard error mean.

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1E-3 0.01 0.1 1 10

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Verapamil Concentration (uM)

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Verapamil Concentration (uM)

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HCMControl

IC5

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Verapamil *

% Ir

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Tra

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Untreated 100 nMDiltiazem %

Cel

ls w

ith

pu

tati

ve

DA

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Untreated 100 nMDiltiazem

* *

FP

Am

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V)

Figure S5 (Referenced from Figure 6). MEA analysis and pharmacological treatment of multicellular iPSC-CM preparations with Ca2+ inhibition. (A) Representative field potentials (FPs) from control and HCM patient-derived iPSC-CM EBs. Verapamil had a concentration-dependent effect on beating frequency that is obvious at 100 nM and 200 nM in controls. Black arrowheads indicate arrhythmia triggered by higher doses of verapamil. (B) Representative FP waveforms for control and HCM iPSC-CM EBs. (C) Plots of average beat frequencies for control and HCM cardiomyocyte EBs against verapamil concentrations fitted to the Hill equation. (D) Quantification of control and HCM IC50 values for spontaneous beat frequency against verapamil (HCM n=13, 5 patient lines; control n = 11, 5 control subject lines). (E) Quantification of irregular Ca2+ transients in HCM iPSC-CMs with and without diltiazem treatment (n=40, 5 patient lines). (F) Quantification of DADs in HCM iPSC-CMs with and without diltiazem (n=25, 5 patient lines). * denotes P<0.001 HCM vs control. Error bars represent standard error mean.

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0

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##

Figure S6 (Referenced from Figure 6). Pharmacological treatment of HCM iPSC-CMs with Na+ influx inhibition. (A) Image analysis of cardiac contraction in arrhythmic HCM iPSC-CMs treated with varying concentrations of the anti-arrhythmic drug lidocaine. (B) Frequency domain representations of beat interval in single HCM iPSC-CMs treated with varying concentrations of lidocaine demonstrates normalization of arrhythmic beat frequency. (C) Representative line-scan images and spontaneous Ca2+ transients of arrhythmic HCM iPSC-CMs before and after treatment with lidocaine. Black arrowheads indicate irregular transients (control n=10, 4 control subject lines, HCM n=10, 4 patient lines). (D) Quantification of cells exhibiting irregular Ca2+ transients before and after treatment with 50 nM lidocaine. (E) Image analyses of contractile motion in time-lapse video recordings of single arrhythmic HCM iPSC-CMs treated with varying concentrations of ranolazine (n=10, 4 patient lines per treatment group). (F) Frequency domain representations of beat interval demonstrates a more narrow range of beat intervals in arrhythmic iPSC-CMs treated with ranolazine. * denotes P<0.0001 HCM vs control, # denotes P<0.01 50 nM lidocaine vs blank. Error bars represent standard error mean.

8

Table S1 (Referenced from Figure 1). Clinical features of recruited family members

Patient ID Age

MYH7 Genotype Symptoms Holter Monitor

Ejection Fraction (%)

II-1 53 Arg663His Mutation

Exertional chest pain, palpitations, dizziness

Occasional PVCs 73.7

III-1 21 Arg663His Mutation

Exertional chest pain, palpitations, dizziness


III-2 20 Arg663His Mutation Exertional chest pain


III-3 18 Arg663His Mutation Exertional chest pain


III-4 16 Wild type Normal None 61.3

III-5 14 Wild type None None 59.2



III-8 10 Arg663His Mutation None None 76.0

Clinical features of control and HCM family members recruited for this study including patient ID, age, MYH7 genotype, reported clinical symptoms, 24 hour Holter monitor for assessment of arrhythmia, and ejection fraction.

9

Table S2 (Referenced from Figure 5). Baseline electrophysiological parameters of iPSC-derived beating EBs obtained via MEA recordings

iPSC-CM Beats Per Minute

Interspike Interval (ms)

Field Potential Duration (ms)

Corrected Field Potential Duration (ms)

Control (n=25) 75.2±41.1 1592.2±2195.8 549.8±201.6 509.9±157.8

HCM (n=21) 62.9±24.0 1111.4±450.4 513.8±128.6 491.3±122.2

Electrophysiological characteristics of iPSC-derived beating EBs as assessed by MEA including beats per minute, interspike interval, and field potential duration.

10

Table S3 (Referenced from Figure 6). Drug screening of HCM iPSC-CMs for treatment of contractile arrhythmia

Drug Class Target Video

Analysis Therapeutic

Effect Concentrations

Tested

Quinidine Ia Na+ channel blocker

(intermediate association/dissociation)

× no 0.1-20 uM

Procainamide Ia Na+ channel blocker

(intermediate association/dissociation)

× no 1-200 uM

Lidocaine Ib Na+ channel blocker (fast association/ dissociation)

× yes 1-100 nM

Mexiletine Ib Na+ channel blocker (fast association/ dissociation)

× yes 1-50 uM

Ranolazine NA Late Na+ channel blocker × yes 0.1-10 uM

Flecainide Ic Na+ channel blocker (slow association/ dissociation)

× no 0.1-5 uM

Propafenone Ic Na+ channel blocker (slow association/ dissociation)

× no 1-100 uM

Propranolol II Beta-blocker × no 1-400 uM

Metoprolol II Beta-blocker × no 0.1-20 uM

Amiodarone II K+ channel blocker × no 0.1-10 uM

Sotalol III K+ channel blocker × no 1-400 uM

Dofetilide III K+ channel blocker × no 0.1-20 uM

Verapamil

IV Ca2+ channel blocker × yes* 1-100 uM

Diltiazem IV Ca2+ channel blocker

yes* 1-100 uM

Nifedipine NA Ca2+ channel blocker

yes 1-100 uM

Information of pharmacological agents used in screening assays for HCM iPSC-CMs. * Verapamil/Diltiazem were only observed to have a therapeutic effect upon HCM iPSC-CMs following continuous addition to the culture media for 5 or more days in a row. Treatment for a period of time less than 5 days was not observed to have any therapeutic effects upon Ca2+ handling or arrhythmogenicity. All other drug screening assays were conducted by incubating cells with respective pharmaceutical compounds at the listed concentrations for 10 minutes followed by washout.

11

SUPPLEMENTAL EXPERIMENTAL PROCEDURES

Patient recruitment. Clinical evaluation of the proband and family included physical

examination, cardiac echocardiography, ECG, cardiac magnetic resonance imaging (MRI), and

24-hour Holter monitoring. Results revealed hyperdynamic ventricular systolic function with

near complete obliteration of the apical walls at end systole in the proband (II-1) and the eldest

two carriers (III-1 and III-2). No delayed enhancement was found on contrast enhanced MRI in

the proband or carriers. Ambulatory monitoring revealed occasional premature ventricular

contractions. The youngest two carriers (III-3 and III-8; ages 14 and 10) exhibited hyperdynamic

cardiac function but no other clinical features of HCM most likely due to their young age.

Isolation and maintenance of fibroblast cells. Freshly isolated skin biopsies were rinsed with

PBS and transferred into a 1.5 ml tube. Tissue was minced in collagenase I (1 mg/ml in

Dulbecco’s modified Eagle medium (DMEM), Invitrogen, Carlsbad, CA) and allowed to digest

for 6 hours at 37 . Dissociated dermal fibroblasts were plated and maintained with DMEM

containing 10% FBS (Invitrogen), Glutamax (Invitrogen), 4.5 g/L glucose (Invitrogen), 110

mg/L sodium pyruvate (Invitrogen), 50 U/mL penicillin (Invitrogen), and 50 g/mL streptomycin

(Invitrogen) at 37 °C, 95% air, and 5% CO2 in a humidified incubator. All cells were used for

reprogramming within five passages.

Lentivirus production and transduction. 293FT cells (Invitrogen) were plated at 80%

confluency on 100-mm dishes and transfected with 12 μg of the lentiviral vectors (OCT4, SOX2,

KLF4, and c-MYC) plus 8 μg of packaging pPAX2 and 4 μg of VSVG plasmids using

Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. Supernatant was

collected 48 h after transfection, filtered through a 0.45-μm pore-size cellulose acetate filter

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(Millipore, Billerica, MA), and mixed with PEG-it Virus Concentration Solution (System

Biosciences, Mountain View, CA) overnight at 4°C. Viruses were precipitated at 1,500 g the

next day and resuspended with Opti-MEM medium (Invitrogen).

Derivation of patient-specific iPSCs. Generation, maintenance, and characterization of patient-

specific iPSC lines were performed as previously described using lentivirus as produced above

on Matrigel-coated tissue culture dishes (BD Biosciences, San Jose, CA) with mTESR-1 hESC

Growth Medium (StemCell Technology, Vancouver, Canada) (Sun et al., 2009).

Alkaline phosphatase staining. Alkaline phosphatase (AP) staining was conducted as in

previous studies using the Quantitative Alkaline Phosphatase ES Characterization KitS

(Millipore) using the manufacturer’s instructions (Sun et al., 2009).

Immunofluorescence staining. Immunofluorescent stains were performed using the following

primary antibodies: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, ANF, TUJ-1 (Millipore), OCT3/4,

Nanog, AFP (Santa Cruz, CA), SOX2 (Biolegend, San Diego, CA), smooth muscle actin

(Biolegend), sarcomeric α-actinin (Sigma, St. Louis, MO), (cTNT (Thermo Scientific Barrington,

IL), Alexa Fluor 488 Phalloidin (invitrogen), Myosin light chain 2a (MLC2a), Myosin light

chain 2v (MLC2v) (Synaptic Systems, Goettingen, Germnay), NFATc4 (Santa Cruz, Santa Cruz,

CA), and AlexaFluor conjugated secondary antibodies (Invitrogen) as previously described (Sun

et al., 2009).

Bisulphite pyrosequencing. The Zymo DNA Methylation Kit (Zymo Research, Irvine, CA) was

used to treat 1 μg of sample DNA with bisulfite as per the manufacturer’s instructions. Following

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PCR, cDNA was converted to single-stranded DNA templates and sequenced by a

Pyrosequencing PSQ96 HS System (Biotage, Charlotte, NC). QCpG software (Biotage) was

used to analyze each individual locus as a T/C SNP.

Microarray hybridization and data analysis. RNA was isolated from iPSCs and hybridized to

an Affymetrix GeneChip Human Gene 1.0 ST Array (Affymetrix, Santa Clara, CA). Expression

was normalized and annotated by the Affymetrix Expression Console software (Affymetrix). The

Pearson Correlation Coefficient was calculated for each pair of samples using the expression

level of transcripts which shows standard deviation greater than 0.2 among all samples.

Spontaneous in vitro differentiation. For embroid body (EB) formation, iPSC colonies were

dissociated on Matrigel coated plates with collagenase Ⅳ (Invitrogen), and seeded into low

attachment 6-well plates in Knockout DMEM (Invitrogen) containing 15% KSR (Invitrogen),

Glutamax (Invitrogen), 4.5 g/L glucose (Invitrogen), 110 mg/L sodium pyruvate (Invitrogen), 50

U/mL penicillin (Invitrogen), and 50 g/mL streptomycin (Invitrogen) to form embroid bodies

(EBs). After 5 days, EBs were transferred to adherent, gelatin-coated chamber slides and

cultured in the same medium for another 8 days.

Teratoma formation. 1x106 undifferentiated iPSCs were suspended in 10 µL Matrigel (BD

Biosciences) and delivered by a 28.5 gauge syringe to the subrenal capsule of 8 week old SCID

Beige mice. Eight weeks after cell delivery, tumors were explanted for hematoxylin and eosin

staining.

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Western blot. Whole cell extracts were isolated using RIPA buffer and 10 µg protein was

analyzed by Western blot using specific antibodies against OCT4, c-MYCc, KLF4, ACTIN

(Santa Cruz), and SOX2 (Biolegend).

Cardiac differentiation of human ESCs and iPSCs. Human H9 ESCs and iPSCs were

differentiated into cardiomyocytes as previously described (Sun et al., 2012). Briefly, pluripotent

stem cells were dissociated with accutase (Sigma) at 80% confluence into small clumps of 10-20

cells. Cells were resuspended in 2 ml basic media containing StemPro34 (Invitrogen), 2 mM

glutamine (Invitrogen), 0.4 mM monothioglycerol (Sigma), 50 μg/ml ascorbic acid (Sigma), and

0.5 ng/ml BMP4 (R&D Systems, Minneapolis, MN) to form EBs. For days 1-4 of cardiac

differentiation, cells were treated with 10 ng/ml BMP4, 5 ng/ml human bFGF (R&D Systems),

and 3 ng/ml activin A (R&D Systems) added to the basic media. From days 4-8, EBs were

refreshed with basic media containing human 50 ng/ml DKK1 (R&D Systems) and 10 ng/ml

human VEGF (R&D Systems). From day 8 onwards, cells were treated with basic media

containing 5 ng/ml human bFGF and 10 ng/ml human VEGF. Cultures were maintained in a 5%

CO2/air environment.

Measurement of cardiomyocyte size and sarcomeric content. For single cell cardiomyocyte

analysis, beating EBs were plated on gelatin-coated dishes. Three days after plating, EBs were

trypsinized, filtered through a 40-mm size pore-size filter, and single cells re-plated at low

density on gelatin-coated chamber slides (Nalgene Nunc International, Rochester, NY). Three

days after re-plating, cells were fixed with 4% paraformaldehyde (Sigma), permeabilized in 0.3%

Triton (Sigma), blocked using 5% BSA, and stained for cardiac troponin T (1:200, Thermo

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Fisher) overnight at 4 . Stained cells were washed three times with PBS, and then incubated

with the Alexa Fluor 488 phalloidin (Invitrogen), Alexa Fluor 594 donkey-anti-mouse antibody

(Invitrogen) and DAPI (Invitrogen) for 1 h. Cellular areas and sarcomeric content of normal and

HCM iPSC-CMs were quantified using the ImageJ software package (National Institutes of

Health, Bethesda, MD). Cells with disorganized sarcomeres were defined as having greater than

25% cell area of punctate troponin T distribution without striations, suggesting disorganized

myofilament structure (Sun et al., 2012).

Measurement of contractile motion. To assess contractile movement of control and HCM

iPSC-CMs, beating cell monolayers were recorded in an AVI video format under phase contrast

microscopy. Movement was quantified using a custom MATLAB script, which measured pixel

displacements of contracting cells over contraction and relaxation. For each video frame, the

mean magnitude of displacement was measured to yield an “average contractile movement”

score. “Normalized contractile motion” was calculated for each video as the mean of all peak

contraction values observed in a 5 second period.

Single cell microfluidic PCR. Single beating iPSC-CMs were picked manually under light

microscopy and placed into separate PCR tubes for reverse transcription and cDNA

amplification with specified primers (Supplemental Table 6) as previously described (Narsinh

et al., 2011). Amplified cDNA was loaded into Biomark 48.48 Dynamic Array chips (Fluidigm,

South San Francisco, CA) for analysis by the BioMark Real-Time PCR Analysis software

(Fluidigm).

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Calcium (Ca2+) imaging. iPSC-CMs were dissociated and seeded in gelatin-coated 8-well LAB-

TEK® II chambers (Nalgene Nunc International) for calcium imaging. Cells were loaded with 5

μM Fluo-4 AM (Invitrogen) and 0.02 % Pluronic F-127 (Invitrogen) in Tyrodes solution (140

mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 10 mM glucose, 1.8 mM CaCl2, and 10 mM HEPES pH

7.4 with NaOH at 25°C) for 15 min at 37°C. Following Fluo-4 loading, cells were washed three

times with Tyrodes solution. Imaging was conducted with a confocal microscope (Carl Zeiss,

LSM 510 Meta, Göttingen, Germany) with a 63x lens using Zen software (Carl Zeiss). For paced

calcium dye imaging, fluorescence was measured at 495 + 20 nm excitation and 515 ± 20 nm

emission. Videos were taken at 20 fps for 10 s recording durations. Cells were stimulated at 1

and 2 Hz. Measurements were taken on an AxioObserver Z1 (Carl Zeiss) inverted microscope

equipped with a Lambda DG-4 300 W Xenon light source (Sutter Instruments, Novato, CA), an

ORCA-ER CCD camera (Hamamatsu, Bridgewater, NJ), and AxioVison 4.7 software (Carl

Zeiss). In each video frame, regions of interest (ROIs) were analyzed for changes in dye

intensity f/f0, with the resting fluorescence value f0 determined at the first frame of each video.

Background intensity was subtracted from all values, and plots were normalized to zero.

Measurement of basal [Ca2+]i using Indo-1-AM. Cardiomyocytes were loaded in a culture

medium containing 5 µM Indo-1 AM (Invitrogen) and 0.02% Pluronic F-127 (Invitrogen) for 20

minutes at 37C. After Indo-1 loading, cells were washed three times with 2 mM Ca2+ Ringer

(155 mM NaCl, 4.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM D-glucose, and 5 mM Na-

HEPES, pH 7.4) and incubated for 20 minutes at room temperature to allow for Indo-1 de-

esterification. Cardiomyocytes were imaged in 2 Ca2+ Ringer at 32C using a Zeiss Axiovert

200M epifluorescence microscope. Indo-1 was excited at 350 ± 10 nm using a 0.6 UVND filter

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(to attenuate excitation intensity) and a 400 DCLP. The emitted light was separated using a

Cairn Optosplit II (425 dichroic, 488/22 bandpass filter, Kent, UK). Spontaneous Ca2+ transients

were collected with 4 x 4 pixel binning in stream acquisition mode using Metamorph software

(Molecular Devices, Sunnyvale, CA) at 100 ms exposures. For image analysis, short and long

wavelength emission channels were aligned using the Cairn Image Splitter ImageJ plugin.

Caffeine treatment of iPSC-CMs. Cells were perfused with PBS containing 1.8 mM Ca2+ and 1

mM Magnesium and paced at 1 Hz to view regular transients. A two second puff of 20 mM stock

caffeine solution was delivered through a perfusion apparatus. Pacing was turned off prior to

caffeine reaching the cells in order to accurately measure Ca2+ release.

Analysis of calcium imaging linescans. Average fluorescence intensity for Ca2+ linescans was

quantified using Fiji (National Institutes of Health). Timing between transients was defined as

the time between the peaks of two successive spikes. The Ca2+ baseline was defined as the

median of all minima of transients. Irregularity for spike timing was defined as the ratio of the

standard deviation (s.d.) to the mean.

Microelectrode array (MEA) recordings. Control and HCM iPSCs were differentiated into

beating EBs ranging from 60-80% purity of CMs and seeded onto multi-electrode arrays for

recording of field potential duration (FPD) and beating frequency (beats per minute, BPM) and

interspike intervals (ISI). Beating iPSC-CM EBs were plated on gelatin-coated MEA probes

(Alpha Med Scientific, Osaka, Japan) prior to experiments 20-40 days post-differentiation.

Signals were acquired at 20 kHz with a MED64 amplifier (Alpha Med Scientific) and digitized

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using a PC with PCI -6071 A/D cards (National Instruments, Austin, TX) running MED64

Mobius QT software (Witwerx, Inc., Tustin, CA). All experiments were performed at 35.8 to

37.5°C in DMEM without serum or antibiotics. Stock verapamil solutions were made in double

distilled water at a 50 mM concentration. Dose-response experiments were performed by adding

0.4 to 2 µL of 1000x verapamil concentrations in DMEM to the 1-2 ml volume in the MEA

probe for 10 minutes at each dose. Beating frequencies and field potential waveform data were

extracted offline using Mobius QT and saved as CSV files. Waveform data was imported into

IGOR Pro (Wavemetrics, Portland, OR) for FPD and Vmax measurements. Beat frequencies

were normalized to baseline for verapamil dose-response experiments and FPDs were adjusted to

the beat frequency using the Bazett correction formula: cFPD = FPD/√Interspike interval.

Patch clamping. Whole-cell patch-clamp recordings were conducted using an EPC-10 patch-

clamp amplifier (HEKA, Lambrecht, Germany). Contracting EBs were mechanically isolated,

enzymatically dispersed into single cells and attached to gelatin-coated glass coverslips (CS-

22/40, Warner, Hamden, CT). While recordings, the coverslips containing plated cardiomyocytes

or the hERG-HEK293 cells were transferred to a RC-26C recording chamber (Warner) mounted

on to the stage of an inverted microscope (Nikon, Tokyo, Japan). The glass pipettes were

prepared using thin-wall borosilicate glass (Warner) using a micropipette puller (Sutter

Instrument, Novato, CA), polished using a microforge (Narishige, Tokyo, Japan) and had

resistances between 2-4 MΩ. Extracellular solution perfusion was continuous using a rapid

solution exchanger (Bio-logic, Grenoble, France) with solution exchange requiring 1 min. Data

were acquired using PatchMaster software (HEKA, Germany) and digitized at 1.0 kHz. Data

were analyzed using PulseFit (HEKA), Igor Pro (Wavemetrics, Portland, OR), Origin 6.1

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(Microcal, Northampton, MA), and Prism (Graphpad, La Jolla, CA). For the whole-cell patch

clamp recordings of human cardiomyocytes generated from iPSCs, temperature was maintained

constant by a TC-324B heating system (Warner) at 36-37°C. Current-clamp recordings were

conducted in normal Tyrode solution containing 140 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 10

mM glucose, 1.8 mM CaCl2 and 10 mM HEPES (pH 7.4 with NaOH at 25°C). The pipette

solution contained 120 mM KCl, 1 mM MgCl2, 10 mM HEPES, 3 mM Mg-ATP, and 10 mM

EGTA (pH 7.2 with KOH at 25°C). Verapamil (Sigma) was dissolved in H2O and prepared as a

10 mM stock in a glass vial. The stock solution was mixed vigorously for 10 min at room

temperature. For testing, the compound was diluted in a glass vial using external solution; the

dilution was prepared no longer than 30 min before using. Equal amounts of DMSO (0.1%)

were present at final dilution.

Quantitative RT-PCR. Total mRNA was isolated using TRIZOL and 1 µg was used to

synthesize cDNA using the SuperscriptⅡ cDNA synthesis kit (Invitrogen). 0.25 µL of the

reaction mixture was used to quantify gene expression by qPCR using SYBR® Green Master

Mix (Invitrogen). Expression values were normalized to the average expression of GAPDH.

Drug treatment. Single contracting iPSC-CMs were treated with pharmaceutical agents for 10

minutes for immediate analysis followed by wash out. The respective concentrations of each

drug tested are listed in Supplemental Table 5. For inotropic stimulation experiments, 200 µM

isoproterenol and 400 µM propranolol were added to the cell medium for 5 continuous days.

Verapamil treatment was conducted by adding 50 µM and 100 µM to the culture medium of

iPSC-CMs for 10-20 continuous days on a daily basis.

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SUPPLEMENTAL REFERENCES Narsinh, K.H., Sun, N., Sanchez-Freire, V., Lee, A.S., Almeida, P., Hu, S., Jan, T., Wilson, K.D., Leong, D., Rosenberg, J., et al. (2011). Single cell transcriptional profiling reveals heterogeneity of human induced pluripotent stem cells. The Journal of Clinical Investigation 121, 1217-1221. Sun, N., Panetta, N.J., Gupta, D.M., Wilson, K.D., Lee, A., Jia, F., Hu, S., Cherry, A.M., Robbins, R.C., Longaker, M.T., et al. (2009). Feeder-free derivation of induced pluripotent stem cells from adult human adipose stem cells. Proceedings of the National Academy of Sciences of the United States of America 106, 15720-15725. Sun, N., Yazawa, M., Liu, J., Han, L., Sanchez-Freire, V., Abilez, O.J., Navarrete, E.G., Hu, S., Wang, L., Lee, A., et al. (2012). Patient-specific induced pluripotent stem cells as a model for familial dilated cardiomyopathy. Science Translational Medicine 4, 130ra147.

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