phenotypic screening using patient-derived induced

EXPERIMENTAL STUDY

Phenotypic Screening Using Patient-Derived Induced PluripotentStem Cells Identified Pyr3 as a Candidate Compound for the

Treatment of Infantile Hypertrophic CardiomyopathyTaku Sakai,1 MD, Atsuhiko T. Naito,2 MD, Yuki Kuramoto,1 MD, Masamichi Ito,3 MD,

Katsuki Okada,1 MD, Tomoaki Higo,1 MD, Akito Nakagawa,1 MD, Masato Shibamoto,1 MD,

Toshihiro Yamaguchi,3 MD, Tomokazu Sumida,3 MD, Seitaro Nomura,3 MD, Akihiro Umezawa,4 MD,

Shigeru Miyagawa,5 MD, Yoshiki Sawa,5 MD, Hiroyuki Morita,3 MD, Jong-Kook Lee,1,6 MD,

Ichiro Shiojima,7 MD, Yasushi Sakata,1 MD and Issei Komuro,3 MD

SummaryHypertrophic cardiomyopathy (HCM) is a genetic disorder that is characterized by hypertrophy of the

myocardium. Some of the patients are diagnosed for HCM during infancy, and the prognosis of infantile HCM

is worse than general HCM. Nevertheless, pathophysiology of infantile HCM is less investigated and remains

largely unknown. In the present study, we generated induced pluripotent stem cells (iPSCs) from two patients

with infantile HCM: one with Noonan syndrome and the other with idiopathic HCM. We found that iPSC-

derived cardiomyocytes (iPSC-CMs) from idiopathic HCM patient were significantly larger and showed higher

diastolic intracellular calcium concentration compared with the iPSC-CMs from healthy subject. Unlike iPSC-

CMs from the adult/adolescent HCM patient, arrhythmia was not observed as a disease-related phenotype in

iPSC-CMs from idiopathic infantile HCM patient. Phenotypic screening revealed that Pyr3, a transient receptor

potential channel 3 channel inhibitor, decreased both the cell size and diastolic intracellular calcium concentra-

tion in iPSC-CMs from both Noonan syndrome and idiopathic infantile HCM patients, suggesting that the target

of Pyr3 may play a role in the pathogenesis of infantile HCM, regardless of the etiology. Further research may

unveil the possibility of Pyr3 or its derivatives in the treatment of infantile HCM.

(Int Heart J 2018; 59: 1096-1105)

Key words: Patient-specific iPSCs, High-content imaging, Calcium imaging, Transient receptor potential chan-

nel inhibitor

Hypertrophic cardiomyopathy (HCM) is a genetic

disorder characterized by left ventricular hy-

pertrophy with histopathological findings of car-

diomyocyte enlargement, disarray, and interstitial fibrosis.1)

The prevalence of HCM has been reported as 1 in 500 in

general population.2) The prognosis of HCM is generally

good, especially for those who are diagnosed later in life.

Mutations in genes encoding contractile myofilaments of

sarcomere or adjacent Z-disc have been identified in more

than half of the patients with HCM,3) and hypersensitivity

of the myofilament contraction against calcium that is in-

duced by gene mutation has been shown as the fundamen-

tal mechanism underlying the pathophysiology of HCM.4,5)

Most of the patients with HCM develop the symptoms

and are diagnosed at adolescents or adults, but some of

the patients develop HCM and are diagnosed at infancies.

The etiologies of these “infantile HCM” include syndro-

mic HCM caused by inborn errors of metabolism, muscu-

lar dystrophy, and malformation syndromes in addition to

idiopathic HCM. Although the gene mutation that causes

idiopathic HCM in infants is shared with adults/adoles-

cents,6) the prognosis of these infantile HCM, especially

those diagnosed before 1-year-old, is worse compared

with general HCM.7) Nevertheless, pathophysiology of the

infantile HCM, including the similarity and difference be-

tween general HCM, still remains largely unknown, and

investigation of the mechanism and therapeutic target of

infantile HCM is important for improving the prognosis of

From the 1Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Osaka, Japan, 2Department of Pharmacology, Faculty of

Medicine, Toho University, Tokyo, Japan, 3Department of Cardiovascular Medicine, The University of Tokyo Graduate School of Medicine, Tokyo, Japan,4Department of Reproductive Biology, Center for Regenerative Medicine, National Research Institute for Child Health and Development, Tokyo, Japan,5Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, Japan, 6Department of Advanced Cardiovascular Regenerative

Medicine, Osaka University Graduate School of Medicine, Osaka, Japan and 7Department of Medicine II, Kansai Medical University, Osaka, Japan.

This study is supported in part by the Program for Intractable Diseases Research utilizing Disease-specific iPS cells from Japan Agency for Medical Re-

search and Development, AMED (to I.K.), JSPS KAKENHI Grant Number JP26713028, Research Grant from Japan Foundation for Applied Enzymology and

Takeda Science Foundation (to A.T.N.).

Address for correspondence: Atsuhiko T Naito, MD, Department of Pharmacology, Faculty of Medicine, Toho University, 5-21-16 Omori-nishi, Ohta-ku,

Tokyo 143-8540, Japan. E-mail: [email protected]

Received for publication December 26, 2017. Revised and accepted January 19, 2018.

Released in advance online on J-STAGE August 11, 2018.

doi: 10.1536/ihj.17-730

All rights reserved by the International Heart Journal Association.

1096

Int Heart J

September 2018 1097TREATMENT OF HCM iPSC-CMs BY Pyr3

Table　I.　Patient Characteristics

Patient iHCM HCMRAF1

Age (years) 27 2

Sex Male Male

Cardiac phenotypes HCM at birth HCM at birth

Family history None None

Other cardiac complications Atrial fibrillation, Congestive heart failure Nonsustained ventricular tachycardia

Gene mutation Not detected 770C > T in exon 7 of RAF1 gene

the patients with infantile HCM.

Editorial p.914

Human induced pluripotent stem cells (iPSCs) are the

pluripotent stem cells that could be generated from any

individuals and are expected to be useful for many pur-

poses including drug discovery for genetic diseases. Es-

tablishments of iPSCs from HCM patients and characteri-

zation of iPSC-derived cardiomyocytes (iPSC-CMs) from

HCM patients (HCM iPSC-CMs), including those with

malformation syndrome,8-11) have already been reported

from several investigators. For example, HCM iPSC-CMs

carrying mutation in sarcomere gene, MYH7, are reported

to recapitulate the expected disease-specific phenotypes,

namely cellular hypertrophy, abnormal Ca2+ transient, and

liability to arrhythmia.9,10) Importantly, at least some of the

disease-specific phenotypes could be normalized by cur-

rent therapeutics against HCM, such as β-blocker and cal-

cium channel antagonists,9) suggesting that HCM iPSC-

CMs would be useful for drug discovery.

In the present study, we established iPSCs from two

patients with HCM who were diagnosed before birth: one

with Noonan syndrome and the other with idiopathic

HCM. We found that iPSC-CMs of idiopathic HCM pa-

tient exhibit disease-specific phenotypes, namely cellular

hypertrophy, impaired calcium homeostasis, and hypersen-

sitivity against β-adrenergic stimulation. We then per-

formed a phenotypic drug screening that could normalize

these disease-specific phenotypes and identified Pyr3, an

inhibitor of transient receptor potential channel 3 (TRPC

3).12)

Methods

Establishment of iPSCs: We established iPS cells from T

cells essentially as described previously.13) We used mito-

mycin C (Kyowa Hakko Kirin, Tokyo, Japan)-inactivated

mouse embryonic fibroblasts as a feeder layer and me-

dium consisting of Knockout DMEM/F12 supplemented

with 20% Knockout serum replacement (KSR), 100 μM

nonessential amino acids, 1 × GlutaMAX, 50 U/mL peni-

cillin, 50 μg/mL streptomycin, 0.1 mM β-mercaptoethanol

(Thermo Fisher Scientific, Tokyo, Japan), and 10 ng/mL

bFGF (WAKO, Osaka, Japan). The medium was changed

every other day. Quality of the iPSCs was verified by the

expression of pluripotency factors and teratoma formation

in nude mice. We also used 253G1 iPSCs (RIKEN BioRe-

source Center)16) and iPSCs established from healthy vol-

unteers (HC01-8) as controls. Characterization of the es-

tablished iPSCs was performed as described previously.17)

Written informed consent was obtained from the do-

nor or their guardians under the protocol approved by the

Institutional Review Board of Osaka University (Approval

number 13254(829-1)-3) and The University of Tokyo (G

10019).

Cardiac differentiation, purification, and quality analy-sis of iPSC-CMs: Differentiation of iPSCs into cardio-

myocytes was performed by stage-specific activation and

inhibition of Wnt signaling.18) Sub-confluent iPSCs colo-

nies were dissociated into small clumps to generate em-

bryoid bodies (EBs) in modified StemPro-34 (mST34)

medium (Invitrogen, Tokyo, Japan), including 1 × Glu-

taMAX, 1 mM ascorbic acid, and 0.4 mM monothioglyc-

erol (MTG) (Invitrogen, Tokyo, Japan), 2.5% KSR, and

10 μM Y-27632. Thereafter, EBs were serially treated with

CHIR99021 (4.5 μM), a GSK inhibitor that activates Wnt

signaling, and IWP2 (5 μM), a porcupine inhibitor that in-

hibits Wnt signaling, in mST34 without Y-27632 on day 1

and day 5, respectively. We also used TGF-β inhibitors

SB431542 (5 μM) and Dorsomorphin (0.5 μM) from day

3 to day 5 to enhance cardiomyocyte production. Purifica-

tion of cardiomyocytes was performed by culturing the

dissociated EBs in a glucose-free medium supplemented

with 4 mM lactic acid19) for 3-5 days, and the culture was

continued in DMEM containing 2% FBS until phenotypic

analysis. All cultures were maintained in a 37℃, 5% CO2

condition.

Immunofluorescent staining: iPSC-CMs were plated

onto GeltrexⓇ (Thermo Fisher Scientific, Tokyo, Japan)-

coated Lab-TekⓇ chamber slides (Nunc, Tokyo, Japan) or

96-well black well clear bottom plate (Cell carrier-96,

PerkinElmer, Yokohama, Japan). After the culture and

drug treatment, iPSC-CMs were fixed with 4% PFA for

15 minutes, permeabilized with 0.1% Triton X-100 for 10

minutes, blocked with 5% goat serum, 2% bovine serum

albumin (BSA) for 1 hour at room temperature, and incu-

bated with primary antibody at 4℃ overnight. Primary an-

tibodies were anti-Troponin T (clone 13-11) (1:400,

Thermo Scientific, Tokyo, Japan, Cat. # MA5-12960),

anti-MLC2a (1:400, Synaptic Systems, Goettingen, Ger-

many, Cat. # 311 011), and anti-MLC2v (1:400, Protein-

Tech, Tokyo, Japan, Cat. # 10906-1-AP). After washing

thrice in PBS, iPSC-CMs were incubated with secondary

antibodies (donkey anti-mouse IgG Alexa FluorⓇ 488,

Cat. # R37114, donkey anti-rabbit IgG Alexa FluorⓇ 594,

Cat. # R37119, 1:500, Thermo Fisher Scientific, Tokyo,

Japan) for 1 hour at room temperature. Cells on Lab-TekⓇ

chamber slides were counterstained and mounted with

ProLongⓇ Gold Antifade Reagent with DAPI (Thermo

Fisher Scientific, Tokyo, Japan). Cells on 96-well plates

were counterstained with NucBlueⓇ (Thermo Fisher Sci-

Int Heart J

September 20181098 Sakai, ET AL

Figure　1.　Hypertrophy in patient-derived iPSC-CMs. A: Representative images of the iPSC-CMs from control (Ctrl) and iHCM at days 30, 45,

and 60 of differentiation. iPSC-CMs were stained with cardiac Troponin T (cTnT) and DAPI. Scale bar, 100 μm. B: Quantification of the cell

area. The cell area of iPSC-CMs from control (Ctrl), HCMRAF1, and iHCM at days 30, 45, and 60 of differentiation was analyzed by high-content

imaging system. Each blue circle (control (Ctrl), n = 20 per each time point), green reverse triangle (HCMRAF1, n = 15 per each time point), and

red square (iHCM, n = 20 per each time point) represents the mean cell area of iPSC-CMs in 1 well of 96-well plate. C: Representative images of

the iPSC-CMs from control (Ctrl) and iHCM at day 60 of differentiation. iPSC-CMs were stained with MLC2v, MLC2a, and DAPI. Scale bar,

100 μm. D: The percentage of MLC2a+/MLC2v-, MLC2a+/MLC2v+, and MLC2a-/MLC2v+ cells in control and iHCM hiPSC-CMs was ana-

lyzed by high-content imaging system (n = 3 each). Ctrl, control. *P < 0.05, **P < 0.01, ****P < 0.0001; N.S, not significant in two-way ANO-

VA followed by Tukey’s post hoc test. The bar shows the mean, and the error bar represents SD.

entific, Tokyo, Japan) and CellMask™ Deep Red stain

(Thermo Fisher Scientific, Tokyo, Japan). Fluorescent im-

ages were acquired by LSM700 confocal microscope

(Zeiss, Tokyo, Japan) or OperettaⓇ high content imaging

system (PerkinElmer, Yokohama, Japan) and analyzed us-

ing Columbus™ image data storage and analysis system

(PerkinElmer, Yokohama, Japan) and HarmonyⓇ analysis

software (PerkinElmer, Yokohama, Japan).

Calcium imaging and absolute quantification of intra-

cellular calcium concentration: iPSC-CMs were plated

onto GeltrexⓇ (Thermo Fisher Scientific, Tokyo, Japan)-

coated 96-well black well clear bottom plate (Corning,

Tokyo, Japan) at 25,000 cells per well. On day 7, iPSC-

CMs were washed with FluoroBrite DMEM (Thermo

Fisher Scientific, Tokyo, Japan) supplemented with 1 ×

GlutaMAX™ and 10 mM HEPES, and then stained with

5 μM Indo-1 AM (Thermo Fisher Scientific, Tokyo, Ja-

pan) with 0.1% Pluronic F-127 (Thermo Fisher Scientific,

Int Heart J


Figure　2.　Impaired calcium homeostasis in iHCM iPSC-CMs. A: Representative image of the Ca2+ transient in spontaneously beating iPSC-CMs

from control (Ctrl) and iHCM. Blue line, Ctrl iPSC-CMs; Red line, iHCM iPSC-CMs. B, C: Mean (B) and coefficient of variation (CV, (C) ) of the

frequency of spontaneous contraction in iPSC-CMs, analyzed by Ca2+ transient (n = 6 each). Ctrl, control. D: Representative image of the Ca2+

transient in iPSC-CMs from control (Ctrl) and iHCM, electrically stimulated at 1 Hz. Blue, Ctrl iPSC-CMs; Red, iHCM iPSC-CMs. E, F: Analysis

of the Ca2+ transient in electrically stimulated iPSC-CMs at 1 Hz. Time to peak (E) and transient amplitude (F) were analyzed from the Ca2+ tran-

sient of iPSC-CMs (n = 6 each). Ctrl, control. G: Quantification of absolute diastolic [Ca2+]i in iPSC-CMs from control (Ctrl), HCMRAF1, and iHCM.

*P < 0.05, **P < 0.01; N.S. indicates not significant in two-tailed t-test or one-way ANOVA followed by Tukey’s post hoc test. The bar shows the

mean and the error bar represents SD.

Tokyo, Japan) for 60 minutes at 37℃. Calcium imaging

was performed by FDSS/μCELL equipped with 96-

channel multi-electrode array (Hamamatsu Photonics, Ha-

mamatsu, Japan). Fluorescent signals of the free calcium

were calculated by the ratio of fluorescent intensity at 409

nm and 495 nm.20) All experiments were performed at

37℃ under atmospheric condition. Calcium transients

from iPSC-CMs were averaged for 30 secs and analyzed

using MATLAB (Mathworks, Natick, MA) and OriginPro

8.6 (LightStone Mathworks). Quantification of absolute

intracellular calcium concentration was performed using

Calcium Calibration Buffer Kits (Thermo Fisher Scien-

tific, Tokyo, Japan). Briefly, using the fluorescent signals

of Ca2+-free and Ca2+-containing solutions, calcium con-

centration was determined according to the following for-

mula:

[Ca2+]free = KdEGTA ×

CaEGTA

K2EGTA

Calcium concentration of the iPSC-CMs was deter-

mined according to the double log plot.

Phenotypic screening and hit identification: For pheno-

typic screening, iPSC-CMs were plated on GeltrexⓇ-

coated 96-well black well clear bottom plate at 3,000 and

25,000 cells per well for high-content imaging and cal-

cium imaging, respectively. Two days after the plating,

cells were treated with various compounds at the indicated

concentrations. DMSO was used as a solvent control. Me-

dium and the compounds were refreshed every other day.

After 5 days, the cells were fixed and stained with anti-

Troponin T, Hoechst, and HCS CellMask™ Deep Red

Stain for high-content screening, or with Indo-1 for cal-

cium imaging. Experiments were repeated in triplicate us-

ing three different batches of iPSC-CMs from three inde-

pendent series of cardiac differentiations.

Statistical analysis: All data were presented as mean ±standard deviation (SD). Indicated sample sizes (n) repre-

sent biological replicates from independent series of car-

diac differentiation. Statistical significance was determined

by using unpaired two-tailed Student’s t-test for two-group

comparison or one-way or two-way ANOVA followed by

Turkey’s or Dunnett’s post hoc test for analyzing more

than two groups. Statistical analyses were done by using

GraphPad Prism 7 software. N.S indicates a nonsignificant

difference. P values of < 0.05 were considered statistically

significant. * or #, ** or ##, *** and **** or #### indicate P <

0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively.

Results

Generation of iPSCs from patients with infantileHCM: Patient-specific iPSCs were established from two

patients (Table I) who were diagnosed as HCM before

birth. One of the patient (HCMRAF1) possessed 770C > T

mutation in exon 7 of RAF1 gene21) and was diagnosed as

Noonan syndrome. The other patient (iHCM for idiopathic

HCM) showed no signs for inborn errors of metabolism,

neuromuscular disease, and malformation syndrome. The

pluripotency and the karyotype of iPSCs from both pa-

tients was confirmed as previously described (Supplemen-

Int Heart J


Figure　3.　Isoproterenol exacerbates disease-specific phenotype of iHCM iPSC-CMs. A: Representative images used for high-content analysis.

iPSC-CMs from control (Ctrl) and iHCM were treated with various concentrations of isoproterenol (ISO) and stained with cTnT and Hoechst.

Scale bar, 1 mm. B: Representative images of the Ca2+ transient in iPSC-CMs from control (Ctrl) and iHCM, electrically stimulated at 1 Hz. Left

panel, Ctrl iPSC-CMs treated with vehicle control (blue) and ISO (orange). Right panel, iHCM iPSC-CMs treated with vehicle control (black) and

ISO (red). C: Dose-response curve of ISO-induced hypertrophy. iPSC-CMs from control (Ctrl) (n = 4 for each concentration) and iHCM (n = 4 for

each concentration) were treated with various concentrations of ISO, and the cell area was analyzed by high-content imaging system. Y-axis repre-

sents the fold-change from the iPSC-CMs treated with vehicle control. The error bar represents SD. D: EC50 was calculated from the dose-response

curve in B. The bar shows the mean and the error bar represents 95% confidence interval. E: Dose-response curve of ISO-induced elevation of dia-

stolic [Ca2+]i. iPSC-CMs from control (Ctrl) (n = 4 for each concentration) and iHCM (n = 4 for each concentration) were treated with various con-

centrations of ISO and the levels of diastolic [Ca2+]i were quantified. Y-axis represents the percentage of change of diastolic [Ca2+]i from the iPSC-

CMs treated with vehicle control. The error bar represents SD. F: EC50 was calculated from the dose-response curve in E. The bar shows the mean

and the error bar represents 95% confidence interval. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 in one-way ANOVA followed by

Tukey’s post hoc test. The bar shows the mean and the error bar represents SD.

tary Figure 1A-D).17)

Hypertrophy in patient-derived iPSC-CMs: iPSCs were

differentiated into cardiomyocytes by serial activation and

inhibition of Wnt signaling18) in 3D culture, and iPSC-

CMs were purified by metabolic selection.19) Purity of the

iPSC-CMs with positive cardiac troponin T (cTnT) stain-

ing was approximately 85% in iPSC-CMs from control,

HCMRAF1, and iHCM (Supplementary Figure 2A, B). We

first investigated the hypertrophic phenotypes of iHCM

iPSC-CMs at day 30 of differentiation and followed its

temporal changes at day 45 and 60 (Figure 1A). Cell area

of iHCM iPSC-CMs was significantly larger than control

iPSC-CMs even at the early phase of differentiation (Fig-

ure 1A), and the hypertrophic phenotype of iHCM iPSC-

CMs progressively exacerbated upon time (Figure 1B, red

square). The cell area of HCMRAF1 iPSC-CMs was also

larger compared with control iPSC-CMs (Figure 1B,

green reverse triangle).

Previous studies reported that iPSC-CMs contain sev-

eral subtypes, namely, atrial, ventricular, and nodal-type

iPSC-CMs.22) To test the possibility that composition of

the subtypes affected the phenotype of iHCM iPSC-CMs,

we evaluated the subtype of iPSC-CMs by immunostain-

ing with myosin light chain (MLC) 2a and MLC2v, which

are preferentially expressed in atrial and ventricular cardi-

omyocytes, respectively. The cell area of MLC2a-/

MLC2v+ ventricular cardiomyocytes was larger than those

of MLC2a+/MLC2v- atrial cardiomyocytes both in control

and iHCM iPSC-CMs (Figure 1C). Although the fraction

of atrial, relatively small cardiomyocytes tends to be

higher in iHCM iPSC-CMs, there were no statistical sig-

nificance in the composition of cardiomyocyte-subtype be-

Int Heart J


Table　II.　List of the Compounds Used for the Phenotypic Screening

Drugs Targets Categories IC501st conc.

(μM)

2nd conc.

(μM)

3rd conc.

(μM)

4th conc.

(μM)

5th conc.

(μM)

Irbesartan GPCR antagonist Angiotensin II type

1 receptor

1.3 nM 0.03 0.1 0.3 1 3

Bisoprolol Adrenergic receptor 3.16 μM 0.1 0.3 1 3 10

BQ-123 Endothelin receptor

type A

7.3 nM 0.03 0.1 0.3 1 3

Verapamil Ion channel blocker Ca2+ channel 0.2 μM 0.01 0.03 0.1 0.3 1

Cibenzoline Na+ channel 7.8 μM 0.1 0.3 1 3 10

Ranolazine Late Na+ channel 17 μM 0.1 0.3 1 3 10

Pyr3 TRPC3 0.7 μM 0.03 0.1 0.3 1 3

Btp2 TRPC3/5/6/7 0.3 μM 0.03 0.1 0.3 1 3

PD0325901 Kinase inhibitor MEK 0.33 nM 3 × 10-5 1 × 10-4 3 × 10-4 1 × 10-3 3 × 10-3

Rapamycin mTOR 0.1 nM 3 × 10-4 1 × 10-3 3 × 10-3 0.01 0.03

ML-7 MLCK 0.3 μM 0.1 0.3 1 3 10

ETP45835 MAPK-interacting

kinase

0.575/0.646 μM

(MnK1/2)

0.1 0.3 1 3 10

LY333531 PKC 4.7/5.9 nM

(PKCβ1/2)

0.03 0.1 0.3 1 3

KN-93 CaMK2 0.37 μM 0.03 0.1 0.3 1 3

Y-27632 Rho kinase 140/220 nM

(ROCK1/2)

0.1 0.3 1 3 10

Sildenafil Phosphodiesterase inhibitor PDE5 3.5 nM 0.1 0.3 1 3 10

PF04447943 PDE9a 12 nM 0.1 0.3 1 3 10

NS-398 Arachidonic acid COX2 inhibitor 1.77 μM 0.1 0.3 1 3 10

BW B70C metabolism inhibitor 5-Lipoxygenase

inhibitor

0.2 μM 0.1 0.3 1 3 10

Lovastatin Cholesterol synthesis inhibitor HMG-CoA

reductase

3.4 nM 0.03 0.1 0.3 1 3

Cyclosporin A Phosphatase inhibitor Calcineurin 7 nM 1 × 10-3 3 × 10-3 0.01 0.03 0.1

TSA Chromatin modifier HDAC 1.8 nM 3 × 10-4 1 × 10-3 3 × 10-3 0.01 0.03

PJ34 DNA damage response modifier PARP 20 nM 0.1 0.3 1 3 10

Calpain inhibitor I Protease inhibitor Calpain 0.09 μM 0.1 0.3 1 3 10

tween iHCM iPSC-CMs and control iPSC-CMs (Figure 1

D). These results collectively suggest that iPSC-CMs from

both idiopathic HCM and Noonan syndrome exhibit hy-

pertrophic phenotype, which is exacerbated over time and

is independent of cardiomyocyte-subtype composition. As

the disease-specific phenotype became clearer over time,

we investigated other disease-specific phenotypes using

iHCM iPSC-CMs at day 50-60 of differentiation.

Impaired calcium homeostasis in patient-derived iPSC-CMs: Elevation of intracellular calcium concentration

([Ca2+]i) activates calcium-dependent signaling pathway

and plays essential role in cardiac hypertrophy.23) Altera-

tions in calcium handling are also described in mouse

models of cardiomyopathy and in human HCM.24,25) To in-

vestigate whether calcium handling is also impaired in

iHCM iPSC-CMs, we analyzed the calcium transient and

quantified the [Ca2+]i in iHCM iPSC-CMs (Figure 2A).20)

The frequency of spontaneous contraction (Figure 2B) and

its coefficient of variation (Figure 2C) were comparable

between control and iHCM iPSC-CMs. Arrhythmia in-

cluding early or delayed after depolarizations (EADs or

DADs, respectively) have been reported as one of the

disease-specific phenotypes of HCM iPSC-CMs.9,10) How-

ever, we could not observe any arrhythmia-like irregular

waveforms in either control, iHCM, or HCMRAF1 iPSC-

CMs. We next analyzed the calcium transient of iPSC-

CMs electrically stimulated at 1 Hz (Figure 2D).

Morphometric analysis revealed that time to peak and the

transient amplitude were comparable between control and

iHCM iPSC-CMs (Figure 2E, F). Quantification of dia-

stolic [Ca2+]i revealed that diastolic [Ca2+]i was signifi-

cantly higher in iHCM iPSC-CMs (Figure 2G). The dia-

stolic [Ca2+]i in HCMRAF1 iPSC-CMs tend to be higher

compared with control iPSC-CMs but did not reach statis-

tical significance (Figure 2G). These results collectively

suggest that iHCM iPSC-CMs exhibit abnormal calcium

handling as a disease-specific phenotype that recapitulates

the observations in HCM patients.23)

Isoproterenol exacerbates disease-specific phenotype ofiHCM iPSC-CMs: Patients with HCM have been re-

ported to show abnormal response against catechola-

mines.26,27) We, therefore, tested the effect of isoproterenol,

a β-adrenergic agonist, on the disease-specific phenotype

of iHCM iPSC-CMs. Administration of isoproterenol at

various concentrations for 5 consecutive days increased

the cell area and diastolic [Ca2+]i of both control and

iHCM iPSC-CMs (Figure 3A, B). Notably, the effects on

iHCM iPSC-CMs were more pronounced and observed

from the lower concentration (Figure 3C-F). These results

collectively suggest that iHCM iPSC-CMs exhibit abnor-

mal response (hypersensitivity) against catecholamine,

which resembles to the phenotype observed in the patients

with HCM.26)

Phenotypic screening using iHCM iPSC-CMs: We next

Int Heart J


Figure　4.　Phenotypic screening using iHCM iPSC-CMs. A: iHCM iPSC-CMs were treated with various com-

pounds at 5 concentrations (1st concentration (1st conc.) to 5th concentration (5th conc.), lower to higher) for 5 days.

iPSC-CMs were then stained with cTnT, Hoechst, and HCS CellMaskTM Deep Red Stain, and the images were taken,

and the cell area was analyzed by high-content imaging system. Percentage change from the iPSC-CMs treated with

vehicle control was calculated and presented by heat map. B: iHCM iPSC-CMs were treated with various compounds

at 3 concentrations (3rd concentration (3rd conc.) to 5th concentration (5th conc.), lower to higher) for 5 days, and the

levels of diastolic [Ca2+]i were quantified. Percentage change from the iPSC-CMs treated with vehicle control was

calculated and presented by heat map.

performed phenotypic screening to investigate the com-

pounds that could normalize the disease-specific pheno-

types in iHCM iPSC-CMs. We selected 24 compounds

(Table II) that could affect cardiomyocyte hypertrophy, in-

cluding calcium channel blocker (verapamil) and β-

blocker (bisoprolol), which were shown to normalize the

disease-specific phenotypes of HCM iPSC-CMs in previ-

ous studies.9,10) As cytotoxicity of the compounds may af-

fect the cell size, we first treated iHCM iPSC-CMs with

threefold serial dilutions of each compound, starting from

10 μM. Cytotoxicity was assessed by the viability of

iHCM iPSC-CMs after the treatment for 5 days. The con-

centration of each compound with cell viability less than

80% of DMSO (vehicle control)-treated, control iHCM

iPSC-CMs were judged as cytotoxic and three- or fivefold

serial dilutions at the sub-cytotoxic concentration were

used for phenotypic screening.

Cell hypertrophy and elevation of diastolic [Ca2+]i in

iHCM iPSC-CMs are presumed to represent distinct as-

pect of the disease. We, therefore, tried to search for the

compound that would normalize both morphological and

physiological disease-specific phenotypes. We used high-

content analysis system to evaluate and quantitate the

morphological phenotype in a non-biased manner and in-

cluded isoproterenol and insulin-like growth factor-1

(IGF-1) as a positive control to normalize the results be-

tween the assay plate and series of differentiation. After

the treatment with compounds for 5 days, we found that 6

compounds, namely Pyr3, rapamycin, bisoprolol, KN-93,

cyclosporin A, and PD0325901, decreased the cell area

of iHCM iPSC-CMs more than 5% (Figure 4A). On the

other hand, only 3 compounds, namely Pyr3, irbesartan,

and PD0325901 decreased the diastolic [Ca2+]i of iHCM

iPSC-CMs more than 5% (Figure 4B). Pyr3, a TRPC3

channel inhibitor,12) was the most potent compound that

was effective to both morphological and physiological

disease-specific phenotypes of iHCM iPSC-CMs. Notably,

Btp2, another TRPC inhibitor was not at all effective to

both morphological and physiological disease-specific

phenotypes of iHCM iPSC-CMs, suggesting that the tar-

Int Heart J


Figure　5.　Pyr3 normalizes disease-specific phenotypes of infantile HCM iPSC-CMs

with distinct etiology. A: Representative images of iPSC-CMs from iHCM and HC-

MRAF1 treated with Pyr3 (3 μM) for 5 days and stained with cTnT and Hoechst. Scale

bar, 1mm. B: Quantification of the cell area. iPSC-CMs from control (Ctrl), iHCM and

HCMRAF1 were treated with Pyr3 for 5 days and the cell area was analyzed by high-

content imaging system (n = 5 each). C: iPSC-CMs from control (Ctrl), iHCM and

HCMRAF1 were treated with Pyr3 for 5 days and the levels of diastolic [Ca2+]i were

quantified (n = 4 each). D: Same number of iPSC-CMs from control (Ctrl), iHCM and

HCMRAF1 were treated with Pyr3 for 5 days and the number of remaining iPSC-CMs

were counted by high-content imaging system. The cell number relative to vehicle

(DMSO)-treated Ctrl iPSC-CMs were presented by bar chart. E: Dose-response curve

of isoproterenol (ISO)-induced hypertrophy. iPSC-CMs from iHCM (n = 4 for each

concentration) were treated with various concentration of ISO together with Pyr3, and

the cell area was analyzed by high-content imaging system. Y-axis represents the fold-

change from the iPSC-CMs treated with vehicle instead of ISO and Pyr3. The error bar

represents SD. F: EC50 was calculated from the dose-response curve in E. The bar

shows the mean and the error bar represents 95% confidence interval. G: Dose-re-

sponse curve of ISO-induced elevation of diastolic [Ca2+]i. iPSC-CMs from iHCM (n =

3 for each concentration) were treated with various concentration of ISO together with

Pyr3 and the levels of diastolic [Ca2+]i were quantified. Y-axis represents the percentage

change of diastolic [Ca2+]i from the iPSC-CMs treated with vehicle instead of ISO and

Pyr3. H: EC50 was calculated from the dose-response curve in G. The bar shows the

mean and the error bar represents 95% confidence interval. *P < 0.05, **P < 0.01,

***P < 0.001, ****P < 0.0001 for comparisons within the iPSC-CMs from the same

iPSCs (versus DMSO-treated sample of indicated cell lines), #P < 0.05, ##P < 0.01, ####P < 0.0001 for comparisons between the iPSC-CMs from different iPSCs (versus

DMSO-treated control (Ctrl) iPSC-CMs); N.S. indicates not significant in one-way

ANOVA followed by Dunnett’s post hoc test. The bar shows the mean, and the error

bar represents SD.

Int Heart J


get of Pyr3 in normalizing the disease-specific phenotype

of iHCM iPSC-CMs is distinct from TRPC.

Pyr3 normalizes disease-specific phenotypes of infantileHCM iPSC-CMs with distinct etiology: We finally in-

vestigated the reproducibility of the effects of Pyr3 on

iHCM and HCMRAF1 iPSC-CMs. Pyr3 dose-dependently

decreased the cell area (Figure 5A, B) in both iHCM and

HCMRAF1 iPSC-CMs. Pyr3 also dose-dependently de-

creased the diastolic [Ca2+]i in both iHCM and HCMRAF1

iPSC-CMs (Figure 5C), although the effect on HCMRAF1

iPSC-CMs did not reach statistical significance due to

relatively poor disease-related phenotype in HCMRAF1

iPSC-CMs. Pyr3 did not affect the cell viability in both

iHCM and HCMRAF1 iPSC-CMs (Figure 5D), suggesting

that the effects on cell area and diastolic [Ca2+]i are inde-

pendent from the toxicity of Pyr3. Pyr3 also decreased the

sensitivity of iHCM iPSC-CMs against isoproterenol (Fig-

ure 5E-H). Pyr3 also did not affect the beating rate of

both iHCM and HCMRAF1 iPSC-CMs (data not shown).

These results collectively suggest that the Pyr3 or its de-

rivative may exhibit therapeutic potential against infantile

HCM, irrespective of the etiology, and also suggest that

the target molecule of Pyr3 compound may play crucial

roles in regulation of both cellular size and calcium ho-

meostasis in cardiomyocytes.

Discussion

In the present study, we established the iPSCs from

two patients with infantile HCM, one with totally un-

known etiology and the other with Noonan syndrome, and

characterized the shared disease-specific phenotypes in the

patients’ iPSC-CMs. We also performed phenotypic

screening using the iPSC-CMs and identified a compound

that can normalize both the morphological and physiologi-

cal disease-specific phenotypes in the iPSC-CMs from

both patients.

Establishment of iPSCs from the patients with HCM

is already reported from several groups.8-11) Disease-

specific phenotypes suggesting hypertrophic change of the

cardiomyocytes; i.e., enlargement of the cell size and acti-

vation of nuclear factor of activated T cells (NFAT) sig-

naling, is observed in the iPSC-CMs from patients with

LEOPARD syndrome, one of the variant of Noonan syn-

drome that exhibits infantile HCM.8) In addition to those

hypertrophic changes, arrhythmia and increased diastolic

[Ca2+]i were observed in the iPSC-CMs of HCM patients

with MYH7 mutation (Arg663His).9) Another study also

reported that hypertrophic changes, arrhythmia, and in-

creased diastolic [Ca2+]i were observed in the iPSC-CMs

of HCM patients with MYH7 mutation (Arg442Gly).10)

Importantly, these disease-specific changes were almost

completely normalized by current therapeutics against

HCM, i.e., β-blocker and calcium channel inhibitors. In

addition, trichostatin A (TSA), an inhibitor of histone

deacetylase, also normalized the disease-specific pheno-

type of iPSC-CMs with MYH7 mutation (Arg442Gly). In

the iPSC-CMs of the HCM patients with MYBPC3 muta-

tion, disease-specific phenotype was not obvious at the

baseline condition; however, stimulation with endothelin-1

enlarged the cell size and induced abnormal contraction

more prominently in iPSC-CMs of HCM patients11). An-

tagonist of endothelin receptor ETA normalized the

endothelin-1-induced disease-specific phenotypes in those

iPSC-CMs.

In the present study, we observed hypertrophic

change and increased diastolic [Ca2+]i. However, we never

observed spontaneous arrhythmia in both iHCM and

HCMRAF1 iPSC-CMs. We also observed some therapeutic

effects in β-blocker, calcium channel inhibitor, ETA an-

tagonist, and TSA on the hypertrophic change of hiPSC-

CMs, but these compounds did not show any effect on

normalizing the diastolic [Ca2+]i. As the disease-specific

phenotypes were similar in HCM iPSC-CMs with same

gene mutation (MYH7) and differed from other gene mu-

tation (MYBPC3), the discrepancies in disease-specific

phenotypes among the studies, including our study, may

reflect the differences in the type of gene mutation that

iPSCs are harboring. In addition, maturity of the iPSC-

CMs may also affect the arrhythmia phenotype because

shallower resting membrane potential increases the vulner-

ability against arrhythmia. Increasing the repertory of

HCM iPSCs with distinct types of gene mutation may

clarify which phenotype is “disease-specific” and which is

“genotype-specific.” Furthermore, drug screening using

HCM iPSC-CMs that harbors different types of gene mu-

tation may enable us to identify a drug candidate that can

treat the patients with HCM with any type of (including

unknown) etiology.

In the present study, we have identified Pyr3 as a

compound that could normalize both morphological and

physiological phenotypes of iPSC-CMs from infantile

HCM patients, irrespective of the background genotype/

etiology. There is a previous report showing that Pyr3 at-

tenuates the cardiac hypertrophy in pressure-overloaded

mice model.12) We, therefore, assume that Pyr3 acts on the

general process of cardiac hypertrophy, not the specific

process in hypertrophic cardiomyopathy. Pyr3 is a pyra-

zole derivative that was originally identified as a highly

selective inhibitor of TRPC3. However, later studies iden-

tified that Pyr3 also inhibits store-operated calcium entry

(SOCE)28), and the information about the selectivity of Pyr

3 in cardiomyocytes, not gene-overexpressing cell lines, is

still lacking. We favor the hypothesis that target of Pyr3

in cardiomyocytes is distinct from TRPC or SOCE be-

cause Btp2, another inhibitor of both TRPC and SOCE,

did not show any effect (rather showed detrimental effect)

on both morphological and physiological disease-specific

phenotypes of iPSC-CMs from infantile HCM patients.

Knockdown of TRPC3 using siRNA or animal experi-

ments using TRPC3 knockout mice may clarify the role

of TRPC3 in the pathogenesis of HCM. Alternatively, in-

vestigating the novel target of Pyr3 may unveil the novel

molecule that is essential in the pathogenesis of HCM.

Moreover, testing the therapeutic effect of Pyr3 in other

HCM iPSC-CMs, especially those with MYH7 or

MYBPC3 gene mutations, and in animal model of HCM

may lead to clinical development of Pyr3 or its derivative

in the treatment of HCM.

Int Heart J


Acknowledgments

We thank Y. Fujio (Osaka University), S. Kogaki

(Osaka University) for assistance in obtaining blood sam-

ples from the patients, M. Shimizu, H. Taniwaki, A.

Matsunaga, Y. Ueda, and K. Kawaguchi for their technical

supports.

Disclosures

Conflicts of interest: The authors declare no conflict of

interests.

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Supplemental Files

Supplemental Figures 1, 2

Please see supplemental files; https://doi.org/10.1536/ihj.17-730

phenotypic screening using patient-derived induced

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