Article reSeArcH
the ATAC-seq analysis (Extended Data Fig. 9h, i). Consistent with our
observations in iPSC-CMs, heart tissue samples from both patients
with LMNA-related DCM showed lower LMNA expression and
higher PDGFRB expression when compared to healthy control tis-
sues (Extended Data Fig. 9j, k). Taken together, these data suggest that
PDGFRB is epigenetically activated in K117fs iPSC-CMs.
Next, we tested whether the abnormal activation of PDGFRB was
directly linked to the arrhythmic phenotype that was observed in
K117fs iPSC-CMs. Knockdown of PDGFRB expression in K117fs iPSC-
CMs by small interfering (si)RNA resulted in a reduced prevalence of
abnormal Ca^2 + transients (23.28%, n = 72) compared to the treatment
with scramble siRNA control (100%, n = 75) (Fig. 5h, i and Extended
Data Fig. 10a–c). Treatment with two specific PDGFRB inhibitors,
crenolanib and sunitinib, also ameliorated the arrhythmic phenotype
of K117fs iPSC-CMs (crenolanib 27.39%, n = 73; sunitinib 27.05%,
n = 85) compared to DMSO-treated cells (72.46%, n = 69) (Fig. 5j
and Extended Data Fig. 10d–f). As expected, the phosphorylation
of both CAMK2D and RYR2 was reduced after treatment of K117fs
iPSC-CMs with crenolanib or sunitinib (III-15 and III-3) (Fig. 5k and
Extended Data Fig. 10g). We also observed that the overexpression
of PDGFRB resulted in upregulation of CAMK2D phosphorylation,
inducing an arrhythmic phenotype in control iPSC-CMs (44.44%,aPercentage of differentiallyexpressed genesDownregulated
Upregulatedα-TubulinPDGFRBWT/cor-WTWT/MUT
ins-MUT/
MUT
del-KO/
WTPlatelet-derived growth factor binding
Arylsulfatase activity
Protein binding involved in cell–matrix adhesion
Sulfuric ester hydrolase activity
Collagen binding
Sterol esterase activity
Integrin binding
Protease binding
Endopeptidase inhibitor activity
Platelet-derived growth factor receptor binding
Combined scored
GO molecular functionbec860 257 267–5 50Fold changeUpregulatedgenes in both (93%of genes)PDGFRB
PDGFRA
DDR2
MUTLK
ACVR 1
RPS6KA 2
NEK7
LATS2
PTK7
GRK5ARCHS4 KinasesCombined score
40100150200 Min.Max.(^948371)
(^169153)
0
20
40
60
80
100 III-^3 IV-1
WT/M
UT
vs
WT/cor-WT
WT/ins-MUT vs
WT/WT
WT/MUT vs
WT/cor-WT
WT/ins-MUT vs
WT/WT
III-3
IV-1
WT/MUT vs
WT/cor-WT
WT/ins-MUT vs
WT/WT
III-3 IV-1
Scramble siRNA
PGFRB siRNA
h
0.4
0.9
1.4
1.9
0.6
0.8
1.0
1.2
F^340
/F
380
ratio
F^340
/F^380
ratio
Regulation of
cardiac conduction
(GO:1903779)
P = 3.38 × 10–2
III-15,
DMSO
III-3
, DMSOIII-15, SBIII-15,CBIII-3 ,CBIII-3 ,SB
III-15,
DMSO
III-3
, DMSOIII-15, SBIII-15,CBIII-3 ,CBIII-3 ,SB
Muscle contraction
(GO:0006936)
P = 9.21 × 10–5
m n
α-Tubulin
CAMK2D
pCAMK2D
DMSOCBSB
III-15
WT/MUT
III- 3
WT/MUT
0
1.5
k
(^40100150200) Min.Max.
Relativ
e signal
intensity
WT/cor-WT
WT/MUT
ins-MUT/MUT
del-KO/WT
Relativ
e PDGFRB
expression
f g
P = 0.0023
P = 0.0387
P < 0.0001 P < 0.0001
P < 0.0001
P = 0.0404
50
40
30
20
10
0
1.0
0.8
0.6
0.4
0.2
0
CB
III-15III-3III-15III-3III-15III-3
SB DMSO
l P = 0.05
0
1
2
–1
–2
AHCYL1
TRHRCPM 4
CAATP2B1SQ1
CATNMK2DNI3
TPACM2TA 1
TNITGB5NC 1
CSRP3LMOD 2
MYOM1KCNH2
PGHRCAM 2
SLMAMYH1P 1
SGCASSPN
ACTCAPTC 1
TNNI3
Ce
ll count (%)
Proarrhythmic
i Normal
Ce
ll count (%)
Proarrhythmic
Normal
CB
56
75
17
0
10
20
30
40
50
60
70
80
90
100
Scrambl
e
siRNA
PDGFRB
siRNA
19
53 62
50
20 23
0
10
20
30
40
50
60
70
80
90
100
DMSO
SB
j
WT/cor-WT
WT/MUT
ins-MUT/MUT
del-KO/WT
DMSOCB SB
Fig. 5 | Abnormal activation of PDGFRB is required for the arrhythmic
phenotype of mutant iPSC-CMs. a, Number of differentially expressed
genes in mutant iPSC-CMs compared to control iPSC-CMs. LMNA WT/
MUT and LMNA WT/cor-WT were derived from patient III-3. LMNA
WT/WT and WT/ins-MUT were generated form health control IV-1.
b, Venn diagram of differentially expressed genes in mutant iPSC-CMs
compared to control iPSC-CMs. c, Heat maps of log 2 -transformed fold
change in expression of 257 differentially expressed genes in mutant
iPSC-CMs compared to control iPSC-CMs. d, GO and ARCHS4 kinase
coexpression analysis of differential expressed genes. Colour codes
indicate the combined FDR and Z-score. e, Immunoblot analysis of
PDGFRB in control and mutant iPSC-CMs. f, Quantification of signal
intensity of LMNA in e. n = 4. g, qPCR analysis of PDGFRB expression
levels in control and mutant iPSC-CMs. n = 8 (WT/cor-WT), n = 4 (WT/
MUT), n = 5 (ins-MUT/MUT and del-KO/MUT). h, Representative Ca^2 +
transients of mutant iPSC-CMs treated with scramble siRNA or siRNA
against PDGFRB. All traces were recorded for 20 s. i, Quantification of
cells that exhibit arrhythmic waveforms as shown in h. j, Quantification
of cells that exhibit arrhythmic waveforms of Ca^2 + transients for
mutant iPSC-CMs treated with the PDGRB inhibitors crenolanib (CB)
(100 nM) and sunitinib (SB) (500 nM) for 24 h. k, Immunoblot analysis
of pCAMK2D and CAMK2D protein levels after treatment with
dimethyl sulfoxide (DMSO), crenolanib or sunitinib. The analyses
were independently repeated twice with similar results. l, Hierarchical
clustering of amplicon-based sequencing (AmpliSeq) transcriptome data;
analysed by one-way ANOVA (P = 0.05). Two different K117fs iPSC-CMs
lines treated with crenolanib, sunitinib or DMSO were analysed by RNA-
seq. The total number of genes is 915. m, n, GO analysis identified a set of
genes that was related with muscle contraction and regulation of cardiac
conduction. f, g, Data are mean ± s.e.m.; statistical significance was
calculated using one-way ANOVA. The Ca^2 + transients shown in h were
independently repeated as described in i with similar results.
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