Patient-Specific Induced Pluripotent Stem-Cell Models for Long-QT Syndrom

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n engl j med 363;15 nejm.org october 7, 2010 1397

Thenew englandjournalofmedicineestablished in 1812 october 7, 2010 vol. 363 no. 15

Patient-Specific Induced Pluripotent Stem-Cell Modelsfor Long-QT Syndrome

Alessandra Moretti, Ph.D., Milena Bellin, Ph.D., Andrea Welling, Ph.D., Christian Billy Jung, M.Sc.,Jason T. Lam, Ph.D., Lorenz Bott-Flgel, M.D., Tatjana Dorn, Ph.D., Alexander Goedel, M.D.,

Christian Hhnke, M.D., Franz Hofmann, M.D., Melchior Seyfarth, M.D., Daniel Sinnecker, M.D.,Albert Schmig, M.D., and Karl-Ludwig Laugwitz, M.D.

A B S T R A C T

From the Cardiology Division, First De-partment of Medicine (A.M., M.B., C.B.J.,

J.T.L., L.B.-F., T.D., A.G., M.S., D.S., A.S.,K.-L.L.), and the Plastic Surgery Depart-ment (C.H.), Klinikum rechts der Isar; theCardiology Department, German HeartCenter Munich (A.M., M.B., C.B.J., J.T.L.,L.B.-F., T.D., A.G., M.S., D.S., A.S., K .-L.L.);and the Institute of Pharmacology andToxicology (A.W., F.H.) all at the Tech-nical University of Munich, Munich, Ger-many. Address reprint requests to Dr.

Laugwitz at the Cardiology Division, FirstDepartment of Medicine and GermanHeart Center Munich, Klinikum rechtsder Isar, Technical University of Munich,Ismaninger Str., 22, D-81675 Munich,Germany, or at [email protected].

Drs. Moretti, Bellin, and Welling contrib-uted equally to this article.

This article (10.1056/NEJMoa0908679) waspublished on July 21, 2010, at NEJM.org.

N Engl J Med 2010;363:1397-1409.Copyright 2010 Massachusetts Medical Society.

BACKGROUND

Long-QT syndromes are heritable diseases associated with prolongation of the QTinterval on an electrocardiogram and a high risk of sudden cardiac death due toventricular tachyarrhythmia. In long-QT syndrome type 1, mutations occur in theKCNQ1 gene, which encodes the repolarizing potassium channel mediating the de-layed rectifier I

Ks current.

METHODS

We screened a family affected by long-QT syndrome type 1 and identified an auto-somal dominant missense mutation (R190Q) in the KCNQ1 gene. We obtaineddermal fibroblasts from two family members and two healthy controls and infected

them with retroviral vectors encoding the human transcription factors OCT3/4,SOX2, KLF4, and c-MYC to generate pluripotent stem cells. With the use of a specificprotocol, these cells were then directed to differentiate into cardiac myocytes.

RESULTS

Induced pluripotent stem cells maintained the disease genotype of long-QT syn-drome type 1 and generated functional myocytes. Individual cells showed a ven-tricular, atrial, or nodal phenotype, as evidenced by the expression of cell-typespecific markers and as seen in recordings of the action potentials in singlecells. The duration of the action potential was markedly prolonged in ventricularand atrial cells derived from patients with long-QT syndrome type 1, as comparedwith cells from control subjects. Further characterization of the role of the R190Q

KCNQ1 mutation in the pathogenesis of long-QT syndrome type 1 revealed a dom-inant negative traff icking defect associated with a 70 to 80% reduction in I

Ks cur-rent and altered channel activation and deactivation properties. Moreover, weshowed that myocytes derived from patients with long-QT syndrome type 1 had anincreased susceptibility to catecholamine-induced tachyarrhythmia and that beta-blockade attenuated this phenotype.

CONCLUSIONS

We generated patient-specific pluripotent stem cells from members of a family af-fected by long-QT syndrome type 1 and induced them to differentiate into functionalcardiac myocytes. The patient-derived cells recapitulated the electrophysiologicalfeatures of the disorder. (Funded by the European Research Council and others.)

The New England Journal of Medicine

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Copyright 2010 Massachusetts Medical Society. All rights reserved.


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T h e n e w e n g l a n d j o u r n a l o f medicine

n engl j med 363;15 nejm.org october 7, 20101398

The long-qt syndrome is a familial,

usually autosomal dominant disease char-acterized by an abnormally prolonged ven-

tricular repolarization phase and a propensitytoward polymorphic ventricular tachycardia (of-ten termed torsades de pointes) and sudden car-diac death.1-3 At least 10 different forms of the

long-QT syndrome have been described, but inapproximately 45% of genotyped patients, the un-derlying causes are mutations in the KCNQ1 (alsoknown as KVLQT1 or Kv7.1) gene, which encodesthe pore-forming alpha subunits of the channelsgenerating I

Ks, an adrenergic-sensitive, slow out-ward potassium current.2,4,5 This form of the long-QT syndrome is designated as long-QT syndrometype 1.

Although it is believed that a reduction in IKs

is the cause of the disease phenotype of long-QTsyndrome type 1, this has not been established

in the case of KCNQ1 channels in human cardio-myocytes. Heterologous expression systems andgenetic animal models have been used to deter-mine the underlying mechanisms of the long-QTsyndrome; however, cardiac myocytes have dis-tinct and complex electrophysiological properties,and these properties differ among species.6,7 Thus,a human cell-based system would be extremelyuseful for understanding the pathogenesis of thedisease and for testing patient-specific therapies.8

The generation of pluripotent stem cells fromhuman adult somatic tissues9-13 offers the oppor-tunity to produce large numbers of patient-specif-ic stem cells. In recent studies, investigators havebeen successful in deriving pluripotent stem cellsfrom individual patients among whom there is avariety of simple and complex genetic disordersand in differentiating them into the specific celllineages affected by the diseases.14-19 The capac-ity of induced human pluripotent stem cells togenerate functional cardiac myocytes has been re-ported,20-23 but to our knowledge, the use of thisapproach to generate myocytes harboring a dis-

ease phenotype has not yet been shown. In thisstudy, we generated patient-specific pluripotentstem cells from members of a family affected bylong-QT syndrome type 1 and showed the capac-ity of these cells to give rise to functional cardio-myocytes that recapitulate the electrophysiologi-cal characteristics of the disorder.

Methods

Clinical History and Genetic Phenotype

During the clinical evaluation of an 8-year-old boy

for attention deficithyperactivity disorder, an elec-trocardiogram showed a prolonged QT interval (QT

interval corrected for heart rate [QTc], 445 msec).

Sequencing of the KCNQ1 gene revealed a hetero-

zygous single base exchange (569GA), resulting

in an R190Q missense mutation previously known

to be associated with long-QT syndrome type 124-26

Figure 1 (facing page). Generation of Pluripotent StemCells from Patients with Long-QT Syndrome Type 1.

Panel A shows the pedigree of the proband with thelong-QT syndrome (Patient III-2; QT interval corrected

for heart rate [QTc], 445 msec) and his father (Patient

II-2; QTc, 462 msec), as well as his affected aunt andgrandfather (QTc, 481 msec and 453 msec, respective-

ly). Squares indicate male family members, circles fe-male family members, solid symbols family members

with long-QT syndrome type 1, and open symbols un-affected family members. Panel B shows the results of

sequence analysis of genomic KCNQ1 obtained fromfibroblasts derived from one of the two control sub-

jects and Patient II-2, revealing a heterozygous mis-sense mutation in KCNQ1 exon 2, in position 569 of

the coding sequence (569GA; NM_000218), resultingin the substitution of the positively charged arginine for

an uncharged glutamine at position 190 of the protein

(R190Q; NP_000209). The same results were obtainedwith DNA from all the induced pluripotent stem-cell

lines derived from controls and from patients with thelong-QT syndrome. Panel C is a schematic representa-

tion of the KCNQ1 and KCNE1 proteins, with the R190Qmutation located in the cytoplasmic loop between

transmembrane segments S2 and S3 of the KCNQ1protein. The six transmembrane domains (S1 to S6)

are flanked by amino (NH2)terminal and carboxyl(COOH)terminal regions; P denotes the pore region.

Functional IKs channels result from the coassembly of

four KCNQ1 alpha subunits and at least two auxiliaryKCNE1 beta subunits. Panel D shows primary skin fi-

broblasts derived from Patient II-2 and a representativeinduced pluripotent stem-cell colony from the same

patient. Panel E shows the presence of alkaline phos-phatase activity in a representative colony of pluripo-

tent stem cells derived from Patient II-2. Panel F shows

immunofluorescence analyses of pluripotency markersNANOG (red) and TRA1-81 (green) in a representativepluripotent stem-cell clone derived from Patient III-2,

with nuclear staining (DNA, blue) of all cells, includingmouse embryonic fibroblast feeders. The lower right-

hand image is a magnification of the area framed in

the adjacent image.





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induced pluripotent stem cells and long-qt syndrome


(Fig. 1A and 1B). The mutation is located in the

cytoplasmic loop between the transmembrane seg-

ments S2 and S3 of the KCNQ1 protein24 (Fig. 1C).

Subsequent screening of members of the boysfamily revealed prolonged QT intervals in the42-year-old father (QTc, 462 msec), the 39-year-

old aunt (QTc, 481 msec), and the 70-year-oldgrandfather (QTc, 453 msec), and genetic testingshowed that these family members had the sameheterozygous mutation, confirming autosomaldominant inheritance in this family (Fig. 1A and1B). The father and son have thus far been as-

D

F

E

A B C

KCNQ1

S2S1 S3

R190Q

S4 S5 S6

P

KCNE1

NH2COOH

I

II

III

3 4

1 2

21

21

Proteinsequence

Codingsequence

E2

GG GC C TGG G

G GGC A GC T G

G GG GRC C TG

Control

569GA

KCNQ1

gene

ControlR190Q

G R

G Q L

L

PatientII-2

SkinFibroblasts

400m Phase

InducedPluripotentStem-CellColony

100m Phase 100m Phase

TRA1-81

TRA1-81NANOG DNA

NANOG100m

30m

TRA1-81NANOG DNA





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ymptomatic. The grandfather and aunt have re-ported periods of dizziness and palpitations. Allthe genetically affected family members are be-ing treated with beta-blockers.

Generation of Patient-Specific Pluripotent

Stem Cells

For the generation of pluripotent stem cells, werecruited the father and son in the family affect-

ed by long-QT syndrome type 1 along with two

healthy control subjects. The protocols for re-

search involving human subjects and for stem-

cell research were approved by the institutional

review board and the committee charged with

oversight of embryonic stem-cell research at the

Technical University of Munich. All the study

participants provided written informed consent.

Dermal-biopsy specimens were minced andplaced on culture dishes. Fibroblasts migrating

out of the explants were passaged twice andthen infected with a combination of retrovirus-es encoding the human transcription factorsOCT3/4, SOX2, KLF4, and c-MYC.9 After 6 days,infected cells were seeded on murine embryonicfibroblast feeders and cultured in standard hu-man embryonic stem-cell medium until inducedpluripotent stem-cell colonies appeared. Detailsof the study methods are provided in the Supple-mentary Appendix, available with the full text ofthis article at NEJM.org.

In Vitro Cardiac Differentiation

We differentiated induced pluripotent stem cells

as embryoid bodies by detaching the stem-cell

colonies from the feeder cells and maintaining

them for 3 days in feeder-cell-conditioned human

embryonic stem-cell medium in low attachment

plates.27,28 At day 4, the medium was replaced

with differentiation medium containing 20% fe-

tal-calf serum. Embryoid bodies were plated on

gelatin-coated dishes on day 7. Between days 20

and 30, areas that exhibited spontaneous contrac-

tion (indicative of cardiac differentiation) were mi-crodissected, plated on fibronectin-coated plates,

and maintained in culture in differentiation me-

dium containing 2% fetal-calf serum. For single-

cell analysis, microdissected areas were dissoci-

ated with the use of type II collagenase. Single

cells were plated on f ibronectin-coated slides for

immunohistochemical and electrophysiological

analysis.

Immunohistochemical Assessments

Immunostaining was performed according to

standard protocols with the use of antibodies

specific for the following: Nanog (Abcam), TRA-1-

81 (BD Pharmingen), cardiac troponin T (Lab Vi-

sion), -actinin (SigmaAldrich), myosin light

chain 2a and myosin light chain 2v (Synaptic Sys-

tems), KCNQ1 (Abcam), and protein disulfideisomerase (Abcam). Staining was also performed

for F-actin with the use of fluorescence-labeled

phalloidin (Invitrogen).

Polymerase-Chain-Reaction Assays

The polymerase chain reaction (PCR) was used

to amplify the mutated region of the KCNQ1 gene

for sequencing. Quantitative real-time PCR was

used in allelic-discrimination assays and for the

assessment of expression of pluripotency genes,

retroviral transgenes, and cell-lineage markers.

Reverse-transcriptase (RT)PCR was used to assaycardiomyocyte phenotype markers in single cells.

Detailed methods are provided in the Supple-

mentary Appendix; primers are listed in Table 1

in the Supplementary Appendix.

Electrophysiological Assessments

Whole-cell recordings were obtained with the

use of standard patch-clamp techniques.7,29 Cul-

ture differentiation medium was used as the ex-

ternal bath solution. Action potentials and cur-

rents were recorded at approximately 35C. All

currents were normalized to cell capacitance.

Statistical Analysis

Data that passed tests for normality and equal

variance were analyzed with the use of one-way

analysis of variance followed by Tukeys test, when

appropriate. The Wilcoxon signed-rank test and

the KruskalWallis test followed by Dunns test

were used to analyze the remaining data. Two-

sided P values of less than 0.05 were considered

to indicate statistical significance.

Results

Generation of Pluripotent Stem Cells

We generated pluripotent stem cells from pri-

mary fibroblasts derived from two patients with

long-QT syndrome type 1 and two control sub-

jects after retroviral transduction of the repro-

gramming factors. Starting at 3 weeks after viral





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B

A

Ventricular Atrial Nodal

MLC2a MLC2vHCN4 MLC2a MLC2vHCN4 MLC2a MLC2vHCN4

APD90APD50

msec

0

600

450

300

150

750

msec

0

250

200

150

100

50

APD90APD50

msec

APD90APD500

250

200

150

100

50Control

LQT1

P


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infection, colonies with stem-cell morphologic

characteristics appeared and were clonally ex-

panded on murine embryonic fibroblasts (Fig. 1D).

Three clones from each subject were chosen for

further characterization.

The genomic KCNQ1 locus was sequenced inall the induced pluripotent stem-cell clones, con-

firming the integrity of the locus and the ab-sence of retroviral DNA. The expected 569GAmutation was detected in all stem-cell clones andskin fibroblasts derived from the patients withlong-QT syndrome type 1 but not in cells derivedfrom control subjects. The presence of alkalinephosphatase activity (Fig. 1E), immunoreactivityfor embryonic stem-cellassociated antigens, in-cluding NANOG and TRA-1-81 (Fig. 1F), reacti-vation of endogenous pluripotency genes (OCT3/4,SOX2, REX1, NANOG, and CRIPTO/TDGF1) (Fig. 1Ain the Supplementary Appendix), and silencing

of retroviral transgenes (Fig. 1B in the Supple-mentary Appendix) indicated that there had beensuccessful reprogramming of putative inducedpluripotent stem-cell clones. On spontaneousembryoid-body differentiation, all induced plu-ripotent stem-cell clones showed up-regulationof lineage markers representative of the threeembryonic germ layers, endoderm (PDX1, SOX7,and AFP), mesoderm (CD31, DESMIN, ACTA2, SCL,MYL2, and CDH5), and ectoderm (KTR14, NCAM1,TH, and GABRR2) (Fig. 2 in the SupplementaryAppendix), confirming their pluripotent nature.28

Assessment of the Long-QT Syndrome Type 1

Phenotype

Using a specific differentiation protocol, we di-

rected induced pluripotent stem cells from both

affected family members and controls into the

cardiac lineage (see Methods, Results, and Fig. 3

in the Supplementary Appendix). Spontaneously

contracting foci started to appear after approxi-

mately 12 days of differentiation (see video 1,

available at NEJM.org) and were explanted and

then dissociated into single cells that maintainedthe expression of distinct myocyte markers (Fig.

2A) and spontaneous contraction (video 2). No

major differences in the efficiency of differentia-

tion into myocyte lineages were observed among

the three clones from each of the four subjects

(Fig. 3E in the Supplementary Appendix).

To assess whether the myocytes derived frominduced pluripotent stem cells from patients with

long-QT syndrome type 1 recapitulated the dis-ease phenotype, we recorded the action poten-tials in single cells. Both spontaneously beatingcells that had been dissociated from long-QTsyndrome type 1 explants and those that had beendissociated from control explants responded topacing and generated three distinct types of ac-

tion potentials. These were designated as ven-tricular, atrial, and nodal, on the basis oftheir similarity to the action potentials of ven-tricular, atrial, and nodal cardiomyocytes fromhuman fetal hearts30 (Fig. 2B; for detailed clas-sification, see Results, Discussion, and Fig. 4 inthe Supplementary Appendix). The classificationbased on action-potential properties correlatedwith gene-expression analysis of specific myo-cyte-lineage markers, as shown with the use ofsingle-cell RT-PCR on patched cells (Fig. 2B, andFig. 4 and 5 in the Supplementary Appendix).

Whereas the characteristics of the action po-tential of nodal myocytes were similar betweencells derived from patients with long-QT syndrometype 1 and cells derived from control subjects,the action potentials of ventricular and atri-al myocytes derived from patients with long-QTsyndrome type 1 were significantly longer andhad a slower repolarization velocity than didthose derived from controls (Fig. 2B, and Tables2 and 3 in the Supplementary Appendix). Withelectrical pacing set at 1 Hz, the mean (SE)duration of the action potential measured at90% repolarization was 554.235.6 msec and190.828.1 msec in ventricular and atrialmyocytes, respectively, in cells derived from pa-tients with long-QT syndrome type 1, as com-pared with 373.222.6 msec and 119.915.5 msecin the corresponding cells from control subjects(Fig. 2B). Increasing the stimulation frequencydecreased the duration of the ventricular myo-cyte action potential in both groups, with littlechange in other features of the action potential.However, adaptation of the action-potential du-

ration to higher pacing frequencies was signifi-cantly less pronounced in the myocytes from thepatients with long-QT syndrome type 1 than inthose from the control subjects (Fig. 6 in theSupplementary Appendix). The results were sim-ilar in all the clones from the two patients andin all the clones from the two healthy controls(Fig. 7 in the Supplementary Appendix), suggest-ing that there was phenotypic homogeneity among





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MyocytesfromControl StemCells MyocytesfromLQT1StemCells

15m

15mPDI

DNA

KCNQ1

PDIDNA

KCNQ1

KCNQ1DNA

KCNQ1DNA

20m

KCNQ1DNA

PDIDNA

PDIDNA

KCNQ1

25mPDI

DNAPDI

DNA

KCNQ1

DNAPhalloidinKCNQ1

DNA

B C

A

CellswithMembranePattern(%)

Fun

ctionalChannels(%)

60

40

30

10

50

20

0

100

50

75

25

0

0 25 50 75 100

0 25 50 75 100

R190Q-KCNQ1Plasmid(%)

R190Q-KCNQ1Subunits(%)

Wild-TypeKCNQ1

R190Q-KCNQ1

PhalloidinDNA

1

2

3

4

Figure 3. Characterization of the Role of the R190Q-KCNQ1 Mutation in the Pathogenesis of Long-QT Syndrome Type 1.

Panel A shows immunofluorescence analysis of the cellular distribution of KCNQ1 in representative myocytes derived from induced plu-

ripotent stem cells from a control subject and a patient with long-QT syndrome type 1. KCNQ1 (green) can be seen in the endoplasmic

reticulum, stained by protein disulfide isomerase (PDI, red), and on the plasma membrane (inset in upper left-hand image) of controlmyocytes but remains mostly in the endoplasmic reticulum of long-QT syndrome type 1 myocytes. Phalloidin staining (cyan) was used

to identify differentiated striated myocytes. Panel B shows immunofluorescence analysis of the cellular distribution of KCNQ1 in H9c2cells transfected with wild-type or R190Q-KCNQ1 expression vector. Wild-type KCNQ1 (green, upper images) is enriched in the plasma

membrane of the cell, whereas R190Q-KCNQ1 (green, lower images) shows aberrant membrane translocation. Panel C shows the effectof cotransfecting increasing amounts of an expression vector encoding R190Q-KCNQ1, fused to a cyan fluorescent protein, together

with a constant amount of a construct expressing wild-type KCNQ1, fused to yellow fluorescent protein, on the percentage of cells dis-playing yellow fluorescence in a plasma membrane or vesicular staining pattern (experimental data shown in red, with I bars indicating

standard errors). The expected percentage of functional channels is plotted against the percentage of available R190Q-KCNQ1 subunits,assuming that the presence of 1, 2, 3, or 4 R190Q-KCNQ1 subunits (indicated by the encircled numbers) in a channel tetramer is suffi-

cient to render the channel nonfunctional.





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induced pluripotent stem-cell lines from the sameperson and no evident difference in the diseasephenotype of the cells from the two patients with long-QT syndrome type 1. Therefore, welimited our further analysis to three clones fromone patient with long-QT syndrome type 1 andthree clones from one control subject.

Role of R190Q-KCNQ1 in the Pathogenesis

of Long-QT Syndrome Type 1

The analysis of gene expression with the use of

quantitative RT-PCR and immunoblotting revealed

thatKCNQ1 messenger RNA and protein levels

were similar between myocytes derived from

subjects with long-QT syndrome type 1 and

those derived from controls. In addition, there

was similar allelic expression of wild-type and

mutated 569GA transcripts in explants derived

from two different clones from the patient with

long-QT syndrome type 1 (Fig. 8 in the Supple-mentary Appendix). To further investigate the

functional consequences of the R190Q-KCNQ1

mutation in the induced pluripotent stem-cell

model, we examined the cellular distribution of

the KCNQ1 protein. Immunocytochemical tests

for KCNQ1 in myocytes derived from patients

with long-QT syndrome type 1 revealed a reticu-

lar, intracellular expression pattern, in which

KCNQ1 partially colocalized with the endoplas-

mic-reticulum marker protein disulfide isomerase.

In contrast, in cells from control subjects, the

channel subunit was enriched in the cell surface

compartment (Fig. 3A). Arginine 190 of KCNQ1

is known to be part of a basic endoplasmic-reticu-

lum retention signal.31,32 This suggests that the

subcellular distribution of KCNQ1 observed in

myocytes derived from patients with long-QT syn-

drome type 1 may be due to a trafficking defect.

To confirm this hypothesis, we expressedR190Q-KCNQ1 subunits and wild-type KCNQ1subunits in the cardiomyoblast H9c2 cell lineand analyzed their subcellular localization. As was

seen in the immunodetection pattern of KCNQ1in induced pluripotent stem-cellderived myo-cytes, wild-type KCNQ1 protein achieved cell-membrane targeting, whereas R190Q-KCNQ1failed to do so (Fig. 3B). Expression vectors en-coding wild-type KCNQ1, fused to a yellow fluo-rescent protein, and R190Q-KCNQ1, fused to acyan fluorescent protein, were then cotransfect-ed into H9c2 cells in various ratios. The percent-age of cells presenting a yellow fluorescent pro-tein signal in a cell membrane pattern decreased

as the proportion of mutant channel subunitsincreased, suggesting a model in which a tetra-meric channel containing more than one R190Qsubunit loses the ability to translocate to the cell

Figure 4 (facing page). Electrophysiological Analysisof IK Current in Myocytes Derived from Induced

Pluripotent Stem Cells from Control Subjects andfrom Patients with Long-QT Syndrome Type 1.

Panel A shows the isolation of the slow and rapid com-

ponents of IK (IKs and IKr) in ventricular myocytesfrom induced pluripotent stem cells. The graphs on

the left show sample traces of whole-cell current fromcontrol and long-QT syndrome type 1 (LQT1) cells and

the voltage protocol used for the recordings. The hold-ing potential was at 40 mV, and test potentials were

at +10, +30, and +50 mV, lasting 5 seconds. Tail currentwas recorded after the test potential was back to 40

mV. After a recording without drugs (a and a), thecells were perfused with 10 M chromanol 293B (b) or

with 1 M E4031 (b), and IKs and IKr were defined asthe chromanol-sensitive (c) and E4031-sensitive (c)

currents, respectively. The graphs on the right show

quantification of IKs and IKr. Current amplitudes mea-sured at the end of depolarization (IKs or IKr) and at

the peak of the tail (IKs tail or IKr tail) were plottedagainst membrane voltages. The control myocytes in-

cluded 9 cells from three induced pluripotent stem-cellclones derived from one of the control subjects, and

the LQT1 myocytes included 12 cells from three clonesderived from Patient II-2. There was a significant re-

duction in IKs current density in LQT1 cells as com-pared with control cells at the specified membrane

voltages. Asterisks denote P


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surface compartment (Fig. 3C; for details on themodel, see the Methods section in the Supple-mentary Appendix). Analysis of fluorescence res-

onance energy transfer showed that the R190Qmutation did not alter the capacity of the mutantsubunit to coassemble with wild-type subunits(Fig. 9 in the Supplementary Appendix).33

Electrophysiological Analysis of K+ Currents

To provide further mechanistic insight into the

function of the mutated KCNQ1 protein, we per-

formed single-cell electrophysiological analysis

of various repolarizing K+ currents in ventricu-

lar myocytes. With the use of a distinct voltage

protocol that preferentially elicits the outward

delayed rectifier current, IK, cardiac myocytes de-

rived from induced pluripotent stem cells from

patients with long-QT syndrome type 1, as com-pared with those from control subjects, showed a

substantial reduction in K+ current (Fig. 4A).

Further characterization with the use of chan-nel blockers specific for the slow and rapid com-ponents of I

K chromanol 293B (which blocks

IKs) and E4031 (which blocks IKr)

34 showedthat I

Ks current densities in myocytes from pa-tients with long-QT syndrome type 1, as comparedwith those from control subjects, were decreased,whereas I

Kr conductance was unaffected. At +30

Control

LQT1

B C

A

IKsDeacti

vation(msec)

3000

2000

1500

500

2500

1000

040 30 20 10

Voltage(mV)

Current

(pA/pF)

3

2

1

010 30 50

3

2

1

1

0

10 30 50

Voltage(mV)

IKr IKrtail

Curre

nt(pA/pF)

2

1

0

10 30 50

4

3

2

1

0

10 30 50

Voltage(mV)

IKs IKstail

Relative

IKs

Activation

1.2

0.8

0.6

0.2

1.0

0.4

0.00 20 40 60

Voltage(mV)

Control

Control

Control

Control Control

LQT1

LQT1

LQT1

LQT1

LQT1

a'

E4031

IKr=a'b'

IKs=ab

4 sec5pA/pF

b'

c'

Chromanol

a

b

c

LQT1

40 mV+10 mV

+50 mV

Control

4 sec5

pA/pF

40 mV+10 mV

+50 mV

* * *

40 mV

+50 mV

10 mV

*





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mV, IKs and tail IKs were diminished by approxi-

mately 75%, suggesting that in heterozygous ven-tricular myocytes from patients with long-QTsyndrome type 1, the mutant form of KCNQ1interferes with the function of the wild-type sub-unit (Fig. 4A). Furthermore, both activation anddeactivation properties of the tail I

Ks current in

cells from patients with long-QT syndrome type 1,as compared with cells from controls, appearedto be altered, with activation being slightly shift-ed toward more positive voltages (Fig. 4B) anddeactivation being decelerated (Fig. 4C). We nextanalyzed the transient outward (Ito) and inwardcurrents that function in the hyperpolarizationstate. As with the results for I

Kr, Ito and diastoliccurrent densities did not differ between ventric-ular myocytes from patients with long-QT syn-drome type 1 and those from control subjects(Fig. 10 in the Supplementary Appendix), show-

ing that there was a specific genotypepheno-type correlation.

Protective Action of Beta-Blockade

Since fatal arrhythmias are precipitated by in-

creased sympathetic tone in patients with long-

QT syndrome type 1,3,35 we tested whether ad-

renergic stimulation can affect the phenotype of

long-QT syndrome type 1 cardiomyocytes de-

rived from pluripotent stem cells. First, we ana-

lyzed the effect of catecholamines on the dura-

tion of the action potential in paced ventricular

myocytes. At 2 Hz, isoproterenol induced ap-

proximately a 20% reduction in the interval be-

tween 30% and 90% repolarization of the action

potential in control myocytes, whereas in cells

from patients with long-QT syndrome type 1 this

interval was almost unaffected (Fig. 5A). Accord-

ingly, IKs was markedly enhanced by adrenergic

stimulation in ventricular myocytes from con-

trol subjects, and the increase was significantly

smaller in cells from patients with long-QT syn-

drome type 1, suggesting that defective respon-

siveness to adrenergic challenge is due to an ab-normal IKs current (Fig. 5B).

36

We further investigated the effect of isopro-terenol on spontaneously beating myocytes. Incells from control subjects, isoproterenol had apositive chronotropic effect accompanied by ashortening of the duration of the action poten-tial, resulting in a 30% reduction in the ratio ofthe action-potential duration at 90% repolariza-

tion to the action-potential interval. In contrast,in myocytes from patients with long-QT syn-drome type 1, this ratio was increased by 15%,thereby exacerbating the long-QT syndrome type1 phenotype and increasing the risk of arrhyth-mic events (Fig. 5C). In fact, under conditions ofadrenergic stress, six of nine ventricular myo-cytes from patients with long-QT syndrome type 1developed early afterdepolarizations (4.11.5 early

Figure 5 (facing page). Adrenergic Modulationand Protective Effect of Beta-Blockade in Control

and Long-QT Syndrome Type 1 Ventricular Myocytes.

Panel A shows an overlay of single action potentials

from a representative cell from a control subject and

from a patient with long-QT syndrome type 1 (LQT1)at a 2-Hz stimulation rate before (basal) and after appli-

cation of 100 nM isoproterenol. The bar graph representsthe percent change induced by isoproterenol on the in-

terval from the action-potential duration (APD) at 30%repolarization (APD30) to APD90 (APD30APD90), on

APD90, and on APD. Data are means SE for 10 cellsin each group (from three clones derived from one of

the control subjects and three clones derived from Pa-tient II-2). Panel B shows the effect of adrenaline on IKs

tail current. On the left are representative overlappedIKs tail-current traces from a control and an LQT1 myo-

cyte before and after treatment with 1 M adrenaline

recorded at +30 mV depolarization with the use of thevoltage protocol depicted at the top of the panel. Mea-

surements were performed in presence of 1 M E4031in order to block IKr. The bar graph represents the fac-

tor increase in IKs tail current induced by 1 M adrena-line at the specified depolarization voltages in control

and LQT1 myocytes. Data are means SE for 8 cells ineach group (from two clones derived from one of the

control subjects and from two clones derived from Pa-tient II-2). Panel C shows representative recordings of

single-cell spontaneous action potentials from a con-

trol and an LQT1 ventricular myocyte before (base-line) and during exposure to a combination of 100 nM

isoproterenol and 200 nM propranolol or to 100 nMisoproterenol alone. Cells were pretreated for 2 min-

utes with 200 nM propranolol before application of thecombination of 100 nM isoproterenol and 200 nM pro-

pranolol (traces not shown). The bar graph represents

the percent change in the ratio of APD90 to the action-potential interval (APD90/AP interval) induced by thecombination of 100 nM isoproterenol and 200 nM pro-

pranolol or by 100 nM isoproterenol alone. Action po-tentials affected by early afterdepolarizations (EAD)

were excluded from the analysis. Data are means SE

for 8 control cells (from three clones derived from oneof the control subjects) and 9 LQT1 cells (from three

clones derived from Patient II-2). P values in the threepanels are for the comparison between LQT1 myocytes

and control myocytes, with the use of the Wilcoxonsigned-rank test.





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afterdepolarizations per 20 sec, with 9.22.1% ofthe beats affected by early afterdepolarizations), whereas none of the eight cells from controlsubjects did. Pretreatment with propranolol, anonselective beta-blocker, substantially bluntedthe effect of isoproterenol in myocytes from bothcontrol subjects and patients with long-QT syn-drome type 1 (0.60.3 early afterdepolarizationsper 20 sec, with 1.70.8% of the beats affected),

thus protecting the diseased cells from catechola-mine-induced tachyarrhythmia due to impairedrate adaptation of the action potential (Fig. 5C).

Discussion

Since the initial reports on induced human plu-

ripotent stem-cell technology were published,

several patient-specific induced pluripotent stem-

150 msec30mV

0 mV

Basal

100 nM isoproterenol

Control LQT1

500 msec2pA/pF

1 M adrenalin

Control

500 msec1pA/pF

1 M adrenalin

LQT1

40 mV

+30 mV

5 sec

Baseline100nMIsoproterenol200nMPropranolol 100nMIsoproterenol

1 sec40mV

Control

0 mV

LQT1

0 mV

EAD

B

A

5

4

3

0

2

1

5

0

5

10

15

20

25APD30APD90 APD90 APD

10mV 10mV 30mV 50mV

C

50

50

4030

20

10

0

10

20

30

40

100nMIsoproterenol200nMPropranolol

100nMIsoproterenol

Change(%)

FactorIncreaseinIKsTail

Current

ChangeinAPD90/APInterval(%)

Control LQT1

Control LQT1

Control LQT1

P=0.002

P=0.004

P=0.002

P=0.05P=0.004

P=0.01P


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cell lines for neurodegenerative and metabolic

disorders have been developed.14,16,18,19,37 Estab-

lishing reliable models of human disease in such

cells has remained challenging, owing to diffi-

culties in directing cell differentiation and in iden-

tifying disease-related mechanisms. In this study,

we reprogrammed fibroblasts derived from mem-

bers of a family with autosomal-dominant long-QT syndrome type 1 and used these induced plu-

ripotent stem cells to generate patient-specific

cardiomyocytes. These myocytes showed expres-

sion of specific markers and electrophysiological

characteristics that suggested that the reprogram-

ming process did not affect the ability of the cells

to function normally. Furthermore, we observed

disease-specific abnormalities in the duration of

the action potential, the action-potential rate

adaptation, and IKs currents, owing to an R190Q-

KCNQ1 trafficking defect, as well as vulnerabili-

ty to catecholaminergic stress.

To date, insights into the pathogenesis of thelong-QT syndrome have come primarily fromheterologous expression systems and genetic ani-mal models. Depending on the cell type used,both haploinsufficiency and dominant negativeeffects have been postulated as the mechanismof disease associated with the R190Q muta-tion.24-26 In our induced pluripotent stem-cellmodel of long-QT syndrome type 1, a reductionin I

Ks current density by approximately 75%, com-bined with subcellular localization, showed thatthe R190Q mutant suppresses channel traffick-ing to the plasma membrane in a dominantnegative manner. Owing to differences amongspecies in the channels that generate the maincardiac repolarizing currents, none of the avail-able mouse models of the long-QT syndromefully emulate the human disease phenotype. Re-cently, transgenic rabbit models of long-QT syn-drome type 1 and long-QT syndrome type 2 havebeen engineered by means of overexpression ofdominant negative pore mutants of the human

genes KCNQ1 and KCNH2.38

In these animals,both transgenes caused a down-regulation of thecomplementary I

Kr and IKs currents. In contrast,no alterations in repolarizing currents otherthan in I

Ks were observed in patient-specific myo-cytes derived from persons with the R190Q-KCNQ1 mutation associated with long-QT syn-drome type 1. This discrepancy, which may bemutant-dependent or model-dependent, shows theimportance of alternative systems in which hu-

man genetic disorders can be studied in thephysiologic and disease-causing contexts on apatient-specific level.

Our data provide clear evidence that the patho-genesis of the R190Q mutation can be modeledin myocyte lineages generated from pluripotentstem cells derived from patients with long-QT

syndrome type 1. Moreover, our findings sug-gest that there may be alternative approaches tothe development of candidate drugs, such as com-pounds to promote the delivery of the mutant tothe plasma membrane or I

Ks activators. The ob-served protective effects of beta-blockade showthat it is possible to investigate the therapeuticaction of medications for treating human cardiacdisease in vitro with the use of patient-specificcells. This approach is particularly attractivebecause of the pluripotent nature of these cellsand the potentially unlimited number of induced

cardiomyocytes available for high-throughputdrug development.

Even though the incidence of the long-QTsyndrome is only 1 case per 2500 live births, thissyndrome provides a platform for showing thesuitability of induced pluripotent stem-cell tech-nology as a means of exploring disease mecha-nisms in human genetic cardiac disorders. Largersets of long-QT syndrome cell lines harboringdifferent channel mutations will be needed to fur-ther validate the disease phenotype and comparepathogenetic mechanisms in diverse forms ofthe disease. Clinically, the severity of manifesta-tions of the long-QT syndrome varies among fam-ily members, and incomplete penetrance exists.39However, we did not observe any phenotypic dif-ferences in the prolongation of the action poten-tial between the myocytes from our two patients,a finding that is probably due to the similarityof the clinical phenotype in these cases.

In summary, we derived pluripotent stem cellsfrom patients with long-QT syndrome type 1 anddirected them to differentiate into cardiac myo-

cytes. As compared with myocytes derived in asimilar fashion from healthy controls, cells frompatients with long-QT syndrome type 1 exhibit-ed prolongation of the action potential, alteredIKs activation and deactivation properties, and an

abnormal response to catecholamine stimulation,with a protective effect of beta-blockade, thusshowing that induced pluripotent stem-cell mod-els can recapitulate aspects of genetic cardiac dis-eases.





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Documents

Patient-Specific Induced Pluripotent Stem-Cell Models for Long-QT Syndrom