Circulation

Transcription

Circulation

Cardiomyocytes Derived from Pluripotent Stem Cells Recapitulate Electrophysiological
Characteristics of an Overlap Syndrome of Cardiac Sodium Channel Disease
Richard P. Davis, Simona Casini, Cathelijne W. van den Berg, Maaike Hoekstra, Carol Ann Remme,
Cheryl Dambrot, Daniela Salvatori, Dorien Ward-van Oostwaard, Arthur A.M. Wilde, Connie R.
Bezzina, Arie O. Verkerk, Christian Freund and Christine L. Mummery
Circulation. published online May 30, 2012;
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DOI: 10.1161/CIRCULATIONAHA.111.066092
Cardiomyocytes Derived from Pluripotent Stem Cells Recapitulate
Electrophysiological Characteristics of an Overlap Syndrome of
Cardiac Sodium Channel Disease
Running title: Davis et al.; Disease modelling using PSC cardiomyocytes
Richard P. Davis, PhD1,6*; Simona Casini, PhD1*; Cathelijne W. van den Berg, MSc1*;
Maaike Hoekstra, MSc2; Carol Ann Remme, MD, PhD2; Cheryl Dambrot, MSc1,4;
Daniela Salvatori, PhD1,5; Dorien Ward-van Oostwaard, BSc1; Arthur A.M. Wilde, MD, PhD2;
1
Connie R. Bezzina, PhD2; Arie O. Verkerk, PhD3; Christian Freund, Ph
PhD
hD1*
;
1,6*
Christine L. Mummery, PhD
1
Dept of Anatomy & Embryology, 4Dept of Cardiology, 5Proefdiercentrum, Leiden University Medical
Center,
Leiden;
Cent
Ce
nter
nt
er,, Le
er
Leiden
en
n; 2De
Dept
D
pt of Clinical & Experimental
Experimenta
tall Ca
ta
C
Cardiology,
rdiology, 3Dept off Anatomy,
Anat
An
a omy, Embryology &
Physiology,
Heart
Academic
Center,
University
Phys
Ph
y io
olo
ogy
gy, He
Hear
art Failure Research Center, Aca
ademic Medical Ce
ent
n er,, Un
Univ
iversity
y of Amsterdam,
Amsterdam;
Amst
Am
ster
erda
er
dam
da
m; 6Ne
Netherlands
eth
ther
e la
er
land
ndss Proteomics
nd
P ot
Pr
oteo
e mi
eo
m css Institute,
Instiitu
tute
te,, Ut
te
U
Utrecht,
reecht,
cht,, T
The
he N
Nethe
Netherlands
heerl
rlan
ands
an
ds
equally
**contributed
con
ntriibuteed
ed equ
ual
ally
ly
Correspondence:
C
orrespond
nden
dence:
Richard
Ri
h d P.
P Davis,
D i PhD
PhD or Christine
Ch i tii L.
L Mummery,
M
PhD
PhD
Department of Anatomy & Embryology
Leiden University Medical Center
Einthovenweg 20
2333 ZC Leiden
The Netherlands
Tel: 31-71-5269307
Fax: 31-30-2516464
E-mail: [email protected] or [email protected]
Journal Subject Codes: [106] Electrophysiology; [130] Animal models of human disease; [137]
Cell biology/structural biology; [152] Ion channels/membrane transport
1
Abstract:
Background - Pluripotent stem cells (PSCs) offer a new paradigm for modelling genetic cardiac
diseases but it is unclear whether mouse and human PSCs can truly model both gain and loss of
function genetic disorders affecting the Na+ current (INa) due to the immaturity of the PSCderived cardiomyocytes. To address this issue, we generated multiple PSC lines containing a Na+
channel mutation causing a cardiac Na+ channel overlap syndrome.
Method and Results - Induced PSC (iPSC) lines were generated from mice carrying the
Scn5a1798insD/+ (Scn5a-het) mutation. These mouse (m)iPSCs, along with wild-type miPSCs, were
compared to the targeted mouse embryonic stem cell (mESC) line used to generate the mutant
mice and to the wild-type mESC line. Patch-clamp experiments showed that the Scn5a-het
y y had a significant
g
y and a larger
g ppersistent INa when
cardiomyocytes
decrease in INa density
compared to Scn5a-wt cardiomyocytes. Action potential (AP) measurements sho
showed
howe
wedd a re
we
redu
reduced
duce
du
c d
ce
upstroke velocity and longer AP duration in Scn5a-het myocytes. These characteristics
ecapi
p tulated findings
g from primary cardiomyocytes
cardiomyocyt
ytes isolated directly from adult Scn5a-het mice.
recapitulated
Finally,
Fina
Fi
nall
na
lly,
ll
y, iPSCs
iPS
PS
SCs were
wer
eree generated
g ne
ge
nera
ratedd from
fr m a patient
pati
pa
t ent with
with
h tthe
he equ
equivalent
quiv
i alen
nt S
SCN5A
CN5
N5A
A1795insD/+ mut
mutation.
utat
atio
on.
Patch-clamp
P
atcchh clamp measurements
meas
me
a ur
as
urem
em
ments
ents on
on the
the derivative
derivativve cardiomyocytes
der
carddioomy
yocyt
ocyttess revealed
reveale
ledd similar
siimi
mila
larr changes
la
ch
hange
ange
g s too tho
tthose
h se
ho
the mPSC-derived
mPSCC-de
Cd rived
de
ved ca
card
rdio
iomy
myoc
my
ocyt
ytes
yt
es.
inn the
cardiomyocytes.
Conclusions
Conc
nclu
lusion
ions - Here
Herre we
H
w demonstrate
dem
emon
onst
stra
rate
t thatt bo
both
th ESCESC
S - an
andd iP
iPSC
iPSC-derived
SC-d
- er
eriv
ived
ed car
cardiomyocytes
rdi
d om
omyo
yocyte
tess ca
can
an
recapitulate
ecapi
p tulatee the
the characteristics
cha
hara
ract
ra
c er
ct
e issti
tics
c of
cs
of a combined
comb
co
mbin
mb
i ed gain
in
ggai
ainn and
ai
and loss
loss of
of function
funccti
fu
tion
on Na
Na+ cchannel
hann
ha
nnnel m
mutation
utation andd
that the electrophysiological immaturity of PSC-derived cardiomyocytes does not preclude their
use as an accurate model for cardiac Na+ channel disease.
Key words: differentiation; electrophysiology; stem cells; disease modelling; sodium ion
channels
2
Multiple cardiac arrhythmia syndromes including long-QT syndrome type 3 (LQT3), Brugada
syndrome (BrS), progressive cardiac conduction disease and sinus node dysfunction have been
linked to mutations in SCN5A, the gene encoding the Į-subunit of the cardiac sodium (Na+)
channel.1,2 Most SCN5A mutations associated with LQT3 act by disrupting fast inactivation of
the Na+ channel, resulting in a persistent inward Na+ current (INa) during the action potential
(AP) plateau phase, and subsequently delaying ventricular repolarisation and prolonging the QT
interval (gain of function mutations).3 In contrast, SCN5A mutations underlying BrS and
conduction disease are loss of function mutations and are believed to reduce the total amount of
available INa due to expression of non-functional channels, impaired intracellular trafficking and
decreased membrane surface channel expression, or through altered channel gat
gating
tin
ng pr
prop
properties.
op
per
erti
ties
ti
e .1,2
es
Initially, it was believed that these arrhythmia syndromes constituted separate clinical
en
entities
ntiiti
ties
es w
wit
with
ithh iindividual
it
ndi
diivi
vidu
d al SCN5A mutations leading
ng to
to one particular
ar ele
electrophysiological
ect
ctrrophysiological
ro
disorder.
H
However,
ow
wever, gen
genotype-phenotype
not
otyp
ype--ph
yp
p en
not
otyp
ypee sstudies
yp
tud
udie
ud
i s in
ie
n lar
large
rgee pe
pedigrees
edigreees
ees ha
have
ave
ve nnow
ow
w eestablished
sttab
bliishhed
ed tthat
hatt se
ha
sev
several
veral
ver
ral si
ssingle
ngle
SCN5A
with
multiple
manifestations
consequence
SC
CN5
N5A
A mutations
mut
m
utat
ut
atio
at
ionns present
pre
rese
sennt
nt w
wit
ithh mu
it
mult
ltip
lt
iplle
le clinical
cli
linnica
caal ma
man
niffest
fest
stat
atiions
at
ns aass a co
cons
nseq
equuen
uence
nce of the
hee vvarious
ario
ar
io
ous
us
biophysical de
defects
efe
fect
ctts as
aassociated
sociat
so
ated
at
ed
dw
with
ithh th
it
tthee mu
muta
mutations
tati
ta
tion
ti
o s (t
on
(the
h sso-called
he
o ca
ocall
l ed ooverlap
ll
veerl
rlap
ap ssyndromes).
yndr
yn
drrom
omes
es).
es
).4 T
The
he first
reported was the in-frame insertion of an aspartic acid residue at position 1795 in the human
protein (designated 1795insD), identified in a large Dutch family, with ECG features of
bradycardia, ventricular and atrial conduction slowing, LQT3, and BrS.5,6 Transgenic mice
carrying the mouse equivalent (1798insD) of the human SCN5A-1795insD mutation recapitulate
the majority of the electrophysiological and ECG characteristics observed in patients, with
Scn5a1798insD/+ (Scn5a-het) mice displaying signs of sinus node dysfunction in addition to
prolonged PQ, QRS, and QTc intervals on surface ECGs.7,8 Patch-clamp analysis in isolated
Scn5a-het cardiomyocytes demonstrated a prolonged AP duration (APD) and decreased upstroke
3
velocity (Vmax) compared with wild-type (Scn5a+/+; Scn5a-wt) myocytes.7,8 Additionally, the
mutation resulted in a reduction in INa density, underlying the observed conduction slowing in
the Scn5a-het mice. Finally, a larger persistent inward INa was measured in cardiomyocytes from
Scn5a-het mice, explaining the presence of the increased AP duration and QTc prolongation.9
Critically, in contrast to previous transfection studies using HEK cells,10 the majority of kinetic
properties of the INa remained unchanged in the isolated Scn5a-het cardiomyocytes,7,8
underscoring the challenges in interpreting the electrophysiological properties of ion channel
mutations in heterologous expression systems.
The ability to reprogram somatic cells into induced pluripotent stem cells (iPSCs) and
cardiomyocyttes, of
ooffers
fers
fe
rs a nnew
ew
differentiate these cells into many cell types, including functional cardiomyocytes,
paradigm for modelling human disease.11,12 However, the electrophysiological immaturity of
pl
lurrip
ipot
oten
ot
en
nt st
stem
tem cce
el
ell-derived
cardiomyocytes (P
PSCSC-CMs) might in
nflue
ueenc
ncee whether they are able
pluripotent
cell-derived
(PSC-CMs)
influence
too recapitulate
recapitulate
ec
eN
Naa+ cchannel
h nn
ha
nel llos
loss
osss off ffunction
os
unct
un
ctiion m
mutations
utattioons th
that
hat affect
affe
af
fect
ctt the
the rrapid
apiid uupstroke
pstr
ps
trok
okke of the
he A
AP.
P.. In
huma
hu
human
mann embryonic
ma
embr
em
bryyon
br
yonic stem
stem
m cell-derived
cel
ell-de
d ri
de
rive
vedd cardiomyocytes
ve
caard
dio
iomy
myoc
my
o ytees
oc
es (hESC-CMs),
(hE
hESC
SC-C
SC
CMs),
), the
thee relative
reelat
elattiv
i e contribution
co
ont
ntri
ribbut
bution
tion off
INa to the phase
phas
asse 0 of the
the
h AP
AP is a matter
mattte
t r of debate,
ddeb
ebaate
eb
t , wi
with
t uupstroke
th
pstr
ps
trok
tr
okee ve
ok
vvelocities
loci
lo
citi
ci
tiies aaro
around
r un
ro
undd 8 V/
V
V/ss typicallyy
detected in hESC-CMs13-16 compared with ~200V/s in adult cardiomyocytes.17 In hiPSC-derived
CMs (hiPSC-CMs) similar low upstroke velocities have also been reported.18-20 While mouse
(m)PSC-CMs show an increase in INa and upstroke velocity during prolonged in vitro
differentiation,21-23 the latter is still smaller than that observed in isolated primary adult mouse
cardiomyocytes.8 Therefore it remains uncertain whether PSC-CMs can recapitulate the
electrophysiological features of Na+ channel loss of function mutations.
In this study, we generated iPSC lines from Scn5a-het mice and from wild-type
littermates, and derived functional cardiomyocytes from these. In addition, the mESC line used
4
to introduce the Scn5a-1798insD mutation into the mice, and the parental (Scn5a-wt) control
mESC line were also differentiated into cardiomyocytes. The biophysical properties of the Na+
channel and AP characteristics in cardiomyocytes derived from these two PSC sources were
examined and compared to those observed in cardiomyocytes isolated from Scn5a-wt and -het
mice that were described previously.8,9 Both in vitro Scn5a-het models recapitulated the
electrophysiological phenotype observed in the corresponding primary adult cardiomyocytes.
Finally to ascertain whether some of the electrophysiological features underlying LQT3 and BrS
could also be detected in human cardiomyocytes, we generated a hiPSC line from a patient
carrying the SCN5A-1795insD variant. Cardiomyocytes derived from these cells also displayed
he INa and AP properties of this cardiac Na+ channelopathy. Collectively, these re
rresults
esuult
ltss
the
demonstrate the utility of PSCs in modelling even complex ion channel disorders.
Me
Met
th
thods
Methods
Methods
Supplemental
Ann eexpanded
xpan
xp
ande
dedd Me
de
eth
hod
odss sect
ssection
ec io
on is ava
aavailable
vail
ilab
able
ab
le iinn th
thee Su
Supp
upp
pleeme
ment
ntal
nt
a Material.
al
Mat
a erria
al.
24-26
26
Mouse
Mous
se and
a d human
an
h ma
hu
man iPSCs
iPSC
iP
SCss were
SC
wer generated
gen
ener
erat
er
ated
at
e essentially
ed
essse
sent
ntia
iall
ia
llly ass described
ddes
e cr
crib
ibed
ib
ed previously,
ppre
r viiou
re
ousl
sly,
sl
y,24
either
by retroviral or lentiviral transduction of vectors encoding Oct4, Sox2, Klf4 and c-Myc. The
undifferentiated PSC lines were maintained using standard procedures.27,28 Teratomas were
generated by injecting undifferentiated iPSCs into NOD-SCID mice. Mouse PSCs were
differentiated in vitro as embryoid bodies (EBs) using a “hanging drop” protocol,27 while human
iPSCs were induced to differentiate to cardiomyocytes by co-culture on visceral endoderm-like
(END-2) cells as described previously.28,29 Immunofluorescence and gene expression analyses
were performed as described previously.28,30 PSC-CMs were isolated from differentiated EBs by
enzymatic dissociation and the electrophysiological recordings obtained by using either ruptured
5
or perforated patch-clamp techniques. Results are expressed as mean±SEM. Comparisons were
made using either unpaired Student’s t-test, one-way ANOVA or two-way repeated measure
ANOVA (rmANOVA), followed by a Holm-Sidak test for post-hoc analysis. P<0.05 was
considered statistically significant. Statistical analyses were performed with SigmaStat software.
Results
Generation and characterisation of Scn5a-wt and -het mPSC lines
The mESC line in which one allele of the Scn5a locus was modified to contain the 1798insD
mutation, and the corresponding mutant mouse used to generate the miPSC lines, have been
described previously.7,8 Fibroblasts cultured from Scn5a-wt and -het littermates w
were
erre
reprogrammed
eprogrammed to generate the corresponding miPSC lines. Several independent clones for each
geno
ge
genotype
noty
no
typpe
ty
pe were
wer
w
erre obtained
ob
btain
tai ed from both adult tail-tip and
and embryonic fib
fibroblasts.
ib
brobl
bllas
astts. The 1798insD
hheterozygous
hete
e ero
r zygous m
mutation
utaatio
ionn in tthe
he Sc
SScn5a
cn5
n55a lo
llocus
ocuus was
wass confirmed
con
nfirm
fi med to
to be
be present
preeseentt inn the
th
he Scn5
SScn5a-het
cn5
n5aa-h
-heet m
mESCs
ESC
ES
Cs
and
Figure
an
nd miPSCs,
miPS
mi
PSCs
Cs,, but
Cs
but not
not in
in the
the wild-type
wil
ildd-ty
dtype
ty
pe lines
lin
ines
es (Supplemental
((Su
Su
upp
pple
leme
meenttal F
Fig
igur
ig
urre 11).
).
The miPSC
miP
iPSC
SC
C lines
lin
ness resembled
res
esem
es
embl
em
bled
bl
e mESCs
ed
mES
ESCs
Cs in
in terms
t rm
te
rmss of morphology
mor
orph
phol
ph
olog
ol
o y and
og
and expression
expr
ex
p es
pr
e si
sion
on of
of ESCassociated markers (Oct4, Nanog, and SSEA-1) (Figure 1A), and had reactivated endogenous
pluripotency genes (Oct4, Nanog, Sox2, Rex1 and ECat1) (Figure 1B). Furthermore, the miPSCs
when differentiated in vitro expressed the mesoderm marker smooth muscle actin (‫ޒ‬-SMA), the
endoderm marker Sox17, and the ectoderm marker glial fibrillary acidic protein (GFAP) (Figure
1C). Additionally, undifferentiated miPSCs injected into immunocompromised NOD-SCID mice
formed teratomas containing cell derivatives of the three germ layers (Figure 1D and 1E).
Collectively these results indicate that the adult somatic cells were reprogrammed to a
pluripotent state.
6
Scn5a-wt and –het mPSCs differentiate into cardiomyocytes
Scn5a-wt and -het mESCs and miPSCs were differentiated to cardiomyocytes in EBs (Figure
2A).27,31 One day after transferring EBs to adherent culture (day 8 of differentiation),
spontaneously contracting foci were apparent (Supplemental Movie 1). By day 10 of
differentiation, 75-90% of mESC- and miPSC-derived EBs contained spontaneously contracting
regions, indicating comparable cardiomyocyte differentiation potentials (Supplemental Figure
2A). Additionally, no significant difference in the spontaneous beating rate was observed
between Scn5a-wt and –het mPSC-EBs (Supplemental Figure 2B). Gene expression profiling
of mESC- and miPSC-derived EBs revealed a similar differentiation pattern into mesodermal
and cardiac cells for all four lines, including expression of Scn5a from day 3 of ddifferentiation
iffe
if
fereent
fe
ntia
iati
ia
tion
ti
o
on
(Supplemental
Supplemental Figure 3). Cardiomyocytes isolated from Scn5aͲwt and -het mESCs and miPSCs
disp
di
displayed
spla
sp
layyedd organised
la
orggani
or
nise
sedd cross-striations resembling sar
se
sa
sarcomeres
arcomeres that we
w
were
ree ppositive
os
ositive
for ȕͲmyosin
hheavy
eav
av
vy chain (ȕ
(ȕ-MHC),
ȕ-M
MHC)
HC), Į
Į-sarcomeric
-ssarrco
ome
meri
ricc actinin,
ri
actininn, troponin
tropoonin I and
and myosin
myosiin light
myo
lig
ght cchain
haiin
ha
in 22A
A (M
(MLC
(MLC2a)
LC2a
LC
2a))
Figu
Fi
gure
gu
re 22B
B).
) Similar
Sim
S
imil
im
illar tto th
thee fi
find
ndiings
nd
ings ooff ot
the
hers
rs,,32 Į
rs
Į-actinin-positive
-aact
c in
nin
n-p
-pos
ossit
itiv
iv
ve ce
cell
cells
llss fr
ll
from
om bbot
both
othh Scn5
ot
SScn5a-wt
cn5
n5aa-w
wt an
and
nd (Figure
2B).
findings
others,
het mPSC-CMs
mPSC-C
CMs also
als
lsoo displayed
d sp
di
s laaye
yedd Na+ cchannel
haann
nnel
el sstaining
tain
ta
inin
in
ingg ((Figure
in
Fiigu
gure
re 22B).
B).
)
Scn5a-het mESC-CMs and miPSC-CMs display reduced INa
INa electrophysiological properties were studied using the patch clamp technique in
cardiomyocytes derived from mESCs and miPSCs. As reported by others,21-23,32 we also
observed an increase in INa in mPSC-CMs with prolonged culture, reaching a plateau around day
17 of differentiation (Supplemental Figure 4A and 4B). Therefore, only cardiomyocytes
following 17 or 18 days of differentiation were analysed further. The capacitance was similar for
all mPSC-CMs, and did not change over time (Table 1; Supplemental Figure 4A and 4B).
Typical examples of INa recordings obtained from Scn5a-wt and -het mPSC-CMs are shown in
7
Figure 3A and 3B. Average INa density was significantly reduced in Scn5a-het cardiomyocytes
derived from both groups compared with their respective control cells (Figure 3C and 3D). At
the membrane potential of -30 mV, INa density was reduced by 44% in Scn5a-het mESC-CMs
and by 48% in Scn5a-het miPSC-CMs (Figure 3E and 3F; Table 1), mirroring similar
reductions observed in primary cardiomyocytes isolated from 129P2 Scn5a-het mice (54%).8
Scn5a-wt and -het miPSC lines derived from MEFs also displayed similar INa density differences
in their corresponding cardiomyocytes (Supplemental Figure 4C), indicating the
electrophysiological phenotype was conserved between different miPSC clones, independent of
whether they were obtained by reprogramming tail-tip fibroblasts or embryonic fibroblasts.
As we observed previously in primary cardiomyocytes isolated from the Scn5
SScn5a-het
cn5
n a-h
-het
et m
mic
ice,,8
mice,
voltage dependence of activation and inactivation parameters (half-maximal voltage (V
V1/2) and
loppe fa
fact
ctor
ct
or,, k)
or
k) di
didd nnot
ot significantly differ betwe
een
e either Scn5a-wt
Scn5a-w
wt and
an
nd -het
-h mESC-CMs or
slope
factor,
between
Scn5a-wt
Scn5a-wt andd -het
Scn
-h
het miPSC-CMs
miP
iPSC
SC-C
-CMs
CMs
M (F
(Figure
Figure
Fig
gure 4A
4A-4D;
A-4D; Table
Ta e 11).
). IIn
n ad
addi
addition,
dittion
on,, ffast
ast
s aand
nd sslow
nd
low
lo
w ti
time
me
constants
co
ons
nsta
tant
ta
n s ((WWf aand
nt
ndd Ws) ooff rrecovery
ecov
ec
oveery fr
from
om iinactivation
naact
ctiv
ivat
iv
atio
at
i nw
io
were
erre
re ccomparable
ompa
om
para
pa
raablle fo
for
or Scn5
SScn5a-wt
cn55a-w
-wtt an
andd -het
-het
e
cardiomyoc
cardiomyocytes
cyt
ytes
es in
in both
both
t groups
ggro
ro
oup
ups (Figure
(Fi
F gu
gure
ree 44E
E aand
nd 4F
4F;; Ta
T
Table
blee 11).
bl
) T
).
The
h ffraction
he
ract
ra
ctio
ct
ionn of
io
o cchannels
hann
ha
nnel
nn
els (A
el
((A))
entering the slow inactivated state, and the rate of development of slow inactivation (W), also did
not differ between Scn5a-wt and -het cardiomyocytes derived from either PSC source (Figure
4G and 4H; Table 1).
Scn5a-het mESC-CMs and miPSC-CMs exhibit a larger persistent INa
Persistent INa was measured as a 30 μM tetrodotoxin (TTX)-sensitive current either during a
descending voltage ramp protocol, or at the end of a 200 ms test pulse to -30 mV. Representative
examples of traces obtained from Scn5a-wt and -het mPSC-CMs illustrating the difference in INa
following application of TTX are shown (Figure 5A and 5B; Supplemental Figure 5A and 5B).
8
In both mESC and miPSC Scn5a-het cardiomyocytes, the persistent INa density was significantly
increased compared to their respective controls (Figure 5C and 5D; Supplemental Figure 5C5E). This is in agreement with the increased persistent INa density we reported in adult
cardiomyocytes isolated from Scn5a-het mice.9
Scn5a-het mESC-CMs and miPSC-CMs exhibit a reduced upstroke velocity and a
prolonged action potential
In our previous studies,8 adult cardiomyocytes isolated from Scn5a1798insD/+ mice were shown to
have prolonged APD and reduced upstroke velocity compared to wild-type cardiomyocytes. To
determine whether this was captured by mESC- and miPSC-CMs, APs were measured.
Representative examples of APs and upstroke velocities from Scn5a-wt and -hett mP
mPSC
SC
C-C
CMs
Ms,,
mPSC-CMs,
measured at 6 Hz, are shown in Figures 6A and 6B. Vmax was significantly smaller and APD at
90
0% re
repo
pola
lari
la
rissatiion ((APD
APD90) significantly prolon
prolonged
nge
g d in Scn5a-hett ca
ccardiomyocytes
rd
dio
iom
myocytes derived from
90%
repolarisation
bboth
oth
hm
mESCs
ESCs and
and miPSCs,
miP
PSC
SCs,
s compared
ccom
ompa
om
pare
r d tto
re
o ccardiomyocytes
arddioomyoocytees fr
from
om
m tthe
hee ccorresponding
orre
or
resppon
ondi
ding
di
ng ccontrols
ontr
on
trools ((Figure
Figu
Fi
ure
6D;
Table
No
membrane
potential
(RMP),
APD
6C
C aand
nd 6D
D; Ta
T
ab
ble 22).
). N
o si
ssignificant
gnif
gn
iffic
ican
antt diff
an
ddifferences
ifffer
eren
ence
cess in
ce
in rresting
esstiing m
mem
embbran
em
bran
anee po
pote
tent
ntia
nt
iaal (R
(RMP
MP)), A
PD
D aatt
20% and 50%
% re
repolarisation
epo
pola
lari
la
riisaation
on ((AP
(APD
APD
AP
D20 and
and APD
APD50
were
r oobserved
bser
bs
erve
er
veed be
between
etw
twee
eenn Sc
ee
Scn5a-wt
Scn5
n55a-w
wt an
aand
d –het
5 ) were
cardiomyocytes in both groups, although Scn5a-het mESC-CMs showed a reduction in AP
amplitude (APA) compared to Scn5a-wt mESC-CMs (Figure 6C and 6D; Table 2). This
reduction is likely due to the low Vmax observed in the Scn5a-het mESC-CMs. Overall, the
miPSC-CMs had a more hyperpolarized RMP and higher Vmax than the mESC-CMs. The higher
Vmax is also in line with the observed larger INa density (Figure 3C and 3D). At slower pacing
frequencies, the differences in Vmax and APD90 remained in both mESC- and miPSC-CMs,
although the APD90 was no longer significantly prolonged in the mESC-CMs due to the
relatively large variability between individual mESC-CMs (Supplemental Figure 6).
9
Scn5a-het hiPSC-CMs have reduced INa density and prolonged action potential
To determine whether cardiomyocytes derived from a human iPSC line carrying the SCN5A1795insD mutation (SCN5A-het) also had reduced INa and a longer AP compared to SCN5A-wt
hiPSC-CMs, we reprogrammed dermal fibroblasts from a patient with the mutation and from an
unaffected individual. Sequencing of genomic DNA confirmed heterozygosity for the SCN5A
c.5387_5389insTGA mutation in the SCN5A-het hiPSCs, resulting in the insertion of aspartic
acid after tyrosine 1795 (1795insD) (Figure 7A). There was no TGA insertion at the
corresponding position in the SCN5A-wt hiPSCs (Supplemental Figure 7A). Both hiPSC lines
maintained a normal karyotype, showed characteristic hESC morphology, and expressed the
Supp
ple
leme
ment
me
ntal
nt
al
ESC markers OCT4, NANOG, TRA-1-81 and SSEA4 (Figure 7B andd 7C
7C;; Supplemental
Figure 7B and 7C). Additionally, EB differentiation of the hiPSCs demonstrated the cells could
gene
ge
nera
ne
raate dder
e iv
er
ivattiv
ivees
es of the three embryonic germ
m llayers
ayers based on
n eexp
preession
ss
of the markers Įgenerate
derivatives
expression
SMA
SM
A, SOX177 and
and GFAP
GFA
FAP
P (Figure
(Fi
Figgure
guree 77D
D and
and S
uppleemen
ntal
nta
al Figure
Fig
gur
uree 7D).
7D)
SMA,
Supplemental
To
To differentiate
difffer
di
fferen
en
nti
tiat
atte the
t e SCN5A-wt
th
SCN5
SC
N5AN5
A-wt
Awtt and
and –het
––he
hett hiPSCs
he
hiP
PSC
Cs ttoo ca
Cs
cardiomyocytes,
ard
rdio
io
omy
myoc
occyttes
es, th
thee ce
cells
ellls w
were
erre
re cco
coocultured withh END-2
ENDEN
D 2 cells
Dcell
ce
l s and
and rhythmically
rhyt
rh
y hm
yt
h ic
ical
ally
al
ly contracting
con
ontr
trac
tr
a ti
ac
t ng
g areas
aare
reas
re
a started
as
sta
tart
rted
ed to
to appear
app
ppea
eaar after
afte
af
terr 6 days. At
te
day 12 of differentiation, the spontaneous beating frequency was not significantly different
between the SCN5A-wt and -het hiPSC-CMs (Supplemental Figure 8A; Supplemental Movie
2). Immunodetection with antibodies recognising ȕ-MHC, Į-actinin, troponin I, and MLC2A
confirmed the presence of cardiomyocytes with characteristic cardiac muscle striations within
these beating areas (Figure 8A).
Representative examples of INa recorded from SCN5A-wt and -het hiPSC-CMs are shown
in Figure 8B. Similar to our findings in mPSC-CMs (Figure 3C and D), average INa density was
significantly decreased in SCN5A-het hiPSC-CMs (Figure 8C), with peak INa density at -30 mV
10
being 46% of that observed in control hiPSC-CMs (Figure 8D; Table 3). Typical examples of
APs and upstroke velocities in SCN5A-wt and -het hiPSC-CMs paced at 1 Hz are shown in
Figure 8E. Similar to Scn5a-het mPSC-CMs, SCN5A-het hiPSC-CMs had a significantly
smaller Vmax and longer APD90 compared with the control cardiomyocytes (Figure 8F; Table 3).
No differences in APD20, APD50, RMP, and APA were detected between the two groups (Figure
8F; Table 3). The differences in Vmax and APD90 also persisted at faster pacing frequencies
(Supplemental Figure 8B). Examples of persistent INa measured during a descending ramp
protocol are shown in Figure 8G. As observed in the Scn5a-het mPSC-CMs (Figure 5), the
persistent INa was also significantly larger in the Scn5a-het hiPSC-CMs compared to the SCN5Awt cardiomyocytes (Figure 8H).
Discussion
Disc
Di
scus
sc
ussi
us
sion
si
on
Recent
advances
cardiac
Reccent
ce advance
cess inn the
the
h PSC
PSC field
fie
ield
ld have
hhaave
ave created
creattedd unprecedented
un
nprreced
eden
ed
entted opportunities
oppport
rtun
unnittiees too study
stu
udy cca
ardi
diac
di
acc
disease
dise
di
seas
se
asse development
deve
de
velo
ve
lopm
pm
ment
ent in
in vitro
vit
itro
o and
and establish
eest
stab
st
ab
bli
lish
sh novel
nnovvel
e platforms
platfo
pla
form
rm
ms for
fo
or drug
drrug
g discovery,
ddis
issco
over
ery,
y, either
eitthe
her too prevent
pre
revvennt
disease progression
confirmed
ability
prog
greess
ssio
io
on orr to
to reverse
reeve
vers
rsee it.
i . Several
it
S ve
Se
v ra
rall reports
reepo
port
r s have
rt
h vee already
ha
aalr
lrea
lr
eady
ea
dy con
onfi
on
firm
fi
rmeed the
rm
th
he ab
abil
ilit
il
itty of iPSCCMs to model K+, Ca2+ and Na+ gain of function mutations.18,19,32-34 Here we demonstrate that
cardiomyocytes derived from both ESCs and iPSCs can model a combined loss and gain of
function Na+ channel mutation that is conserved between humans and mice.7
We chose to investigate this Scn5a mutation originally in mPSC-CMs for several reasons.
Firstly, it has been previously established that while mESC-CMs do not initially express any
detectable INa, this current and the related upstroke velocity values increase as the
cardiomyocytes mature in vitro with phase 0 of the AP becoming Na+-dependent.21 Secondly, the
Scn5a1798insD/+ mouse has been extensively characterised,7,8 therefore offering the opportunity to
11
compare the biophysical properties of the Na+ channel in primary cardiomyocytes freshly
isolated from the mice with cardiomyocytes from either the ESCs used to generate the mutant
mouse, or from the miPSCs derived by reprogramming the corresponding fibroblasts. This direct
comparison is not possible with hPSC cardiac disease models because sufficient numbers of
human ventricular cardiomyocytes cannot be obtained from patients. Finally, by comparing the
cardiomyocytes from Scn5a-het mESCs to those from the unmodified mESC line, we could
confirm that the observed electrophysiological differences were due to the mutation. Similar
electrophysiological changes to those present between the Scn5a-wt and -het mESC-CMs were
also detected in both mouse and human iPSCs, suggesting that the reprogramming method did
not influence the electrophysiological phenotype.
Scn5a-het
As observed in cardiomyocytes isolated from the postnatal Scn5a-het mouse,7,8 Scn5a-he
mPSC
mP
SC-C
SC
-CMs
CMs displayed
ddispl
plaayed
pl
ay a significant reduction in
n INa density and up
upstroke
pstro
oke vvelocity. These cells
mPSC-CMs
also
allsoo ppresented
d aan
n iincreased
ncr
crea
ease
sedd pe
se
pers
persistent
rsis
iste
tennt INNaa lea
te
leading
ading tto
o a lon
llonger
onge
nger A
AP
P co
compared
omp
mpaared
ared tto
o co
cont
control
ntro
nt
ol
cardiomyocytes,
ca
ard
rdio
iomy
io
myoc
my
occyt
ytes
es,, al
although
lth
thoough
ough due
ue tto
o th
thee re
reduced
eduuce
cedd APA
APA in tthe
hee Scn5a-het
Scn5
Sc
n55a-het
-het mESC-CMs,
mES
m
ESCES
C-CM
CMs,
CM
s,, ddifferences
ifferrenc
iffe
renc
n ess iin
n
K+ current activation
act
cttiv
ivat
attio
ionn might
migh
mi
ghht also
al o contribute
ccon
on
ntr
t ib
but
utee here
here to
to the
th
he pr
prol
prolonged
olon
ol
onge
on
gedd AP
ge
APD.
D IInn ad
D.
aaddition,
diti
di
tion
ti
on,, no
on
n significan
significant
n
changes were observed in the voltage dependence of (in)activation, recovery from inactivation,
nor in the development of slow inactivation. This is in contrast to previous findings on the
biophysical properties of the 1795insD mutation in HEK 293 cells where the mutation did not
affect INa density, but altered voltage-dependence of inactivation, and enhanced slow
inactivation.10 A possible reason for this discrepancy is that only the SCN5A-1795insD channel
was expressed in these cells, rather than co-expression of both the wild-type and mutant
channels, as occurs in primary and PSC-derived cardiomyocytes. The presence of wild-type
SCN5A might compensate for small changes in current kinetics caused by the 1795insD
12
mutation. Additionally, only the ȕ1 subunit was co-expressed with SCN5A. In cardiomyocytes,
four ȕ-subunits (as well as other Na+ channel interacting proteins) associate with the Į subunit,
and play critical roles in the regulation of sarcolemmal expression and gating of the Na+
channel.35 This highlights the benefit of studying the electrophysiological properties of ion
channels using in vitro models that generate bona fide cardiomyocytes, rather than using
heterologous expression systems that lack the correct cellular context of a cardiomyocyte.
Although there was no apparent difference in cardiac differentiation efficiency between
the mESC and miPSC lines, it appears that the miPSC-CMs were more electrophysiologically
mature than the corresponding mESC-CMs based on a larger INa density, more negative RMP
and higher Vmax in the miPSC-CMs. The differences in peak INa density betweenn the
hee mESCmES
ESCC- and
aannd
miPSC-CMs was also present at day 10, and maintaining the mESC-CMs for more than 17 days
inn culture
cul
ultu
ture
tu
re did
did not
ott result
rreesult in further increases in the
he INNaa density (Su
(Supplemental
upp
p leeme
men
ntal Figure 4).
A
Additionally,
ddditionally,
di
th
the
he ccapacitance
apa
paaci
cita
tanc
ta
ncee of
of th
the
he ca
cardiomyocytes
ard
diom
myocyytes der
myo
derived
eriv
er
ived
ed ffrom
rom
ro
m al
all
ll th
thee mP
mPSC
PSC
SC llines
inees
in
es w
was
as vve
very
eryy
similar,
functional
were
cell
size.
imila
mila
lar,
r, excluding
excclu
ludi
dinng the
the possibility
poossi
oss bi
bili
lity
ty that
ttha
hatt the
ha
the fu
func
n ti
nc
tion
onnall differences
dif
i fe
fere
renc
re
nces
nc
e w
es
eree rela
er
rrelated
e ate
tedd to
o cce
ell si
ell
siz
ze.
Therefore wee believe
bel
e ie
ieve
v these
ve
the
h see differences
dif
i fe
fere
renc
re
nces
nc
ess are
are most
mos
ostt likely
l ke
li
kely
l due
ly
due to
to the
the inherent
in
nhe
here
rent
re
nt variability
vvar
aria
iabi
ia
b li
bi
lity
ty among
among ESC
C
and iPSC lines to differentiate into cardiac cell types.36 Indeed, others have also observed similar
discrepancies.23
Because the electrophysiological phenotype of the Scn5a-1798insD mutation was evident
in both mESC- and miPSC-CMs, we wished to determine whether hiPSC-CMs bearing the
SCN5A-1795insD mutation could also recapitulate the mixed biophysical properties of the
disorder. We found a significant difference between control and SCN5A-het hiPSC-CMs in INa
density, persistent INa, Vmax and APD90, reflecting the loss and gain of function phenotype of this
Na+ channel mutation and mirroring the findings in the mPSC models.
13
The Vmax of our control hiPSC-CMs (115 V/s) was markedly higher than that reported by
others (~8 – 10 V s-1),18-20 partly due to our more negative RMP (-72.4 mV compared to -63.5 &
-57.1 mV) and possible higher Na+ channel expression. The RMP plays a critical role in Na+
channel availability, which in turn determines upstroke velocity.37 Any small changes in RMP
will have a large effect on Na+ channel inactivation, with the majority of channels in their
inactivated state at more positive RMP. As a consequence, modelling Na+ channel loss of
function diseases, such as BrS, will be difficult in hiPSC-CMs that lack a rapid upstroke. The
reason for the more negative RMP observed in our hiPSC-CMs is unclear, but could possibly be
due to the different differentiation method used (co-culture with END-2 cells instead of EB
raath
ther
er tha
hann
ha
differentiation), the inherent variability between PSC lines, and that quiescent rather
than
pontaneously beating cardiomyocytes were selected for AP recordings.
spontaneously
It ha
has be
een pproposed that the SCN5A-1795insD
SCN5A-179
95insD mutation ccan
a ccause
an
ausse both LQT3 and
au
It
been
38
Bruugada
ug
synd
dro
rome
me ddepending
epen
endi
ding
di
ng oonn th
thee exi
eexisting
xistinng hea
art rat
te.38
IIndeed,
nd
dee
eedd, in
n cardiomyocytes
caard
dio
iomy
myoc
my
ocyt
y es
yt
es iiso
isolated
s la
so
late
teed
Brugada
syndrome
heart
rate.
from
fr
rom Sc
SScn5a-het
n5aa-h
n5
-het
et mice,
mice,
ice,, a prolonged
pro
rolo
ong
nged
ed APD
APD
D occurs
occ
ccur
urrs predominantly
prred
dom
min
nan
antlly att slower
sslo
lowe
lo
wer pacing
we
p ci
pa
cing
ng frequencies,
ffre
reqquen
re
quen
ncies
cies,,
while a decreased
decrea
eaase
sedd up
upst
upstroke
sttro
okee vvelocity
e oc
el
ocit
i y is
it
i oobserved
bsserrve
vedd at
a hhigher
ighe
ig
heer ra
rate
rates.
t s.7 W
te
While
Whi
h le tthe
hi
he m
mix
mixed
ix
xed pphe
phenotype
heno
he
n type wass
present at all frequencies in the cardiomyocytes derived from the mPSCs, we did not observe the
above features. This is possibly due to the limited APD90-frequency relationship in mPSC
models between 1 and 6 Hz, preventing more severe prolongation of the AP occurring at lower
frequencies. Also, because the APD90 in mPSC-CMs is shorter than in primary cardiomyocytes,
the Na+ channels might have sufficient time to recover from inactivation, thereby preventing a
more pronounced reduction in Vmax occurring at high frequencies. However, in SCN5A-het
hiPSC-CMs we did observe a tendency towards a longer APD when the cells were paced at
slower rates. Similarly, at faster rates, the relative reduction in Vmax appeared larger in the
14
SCN5A-het cardiomyocytes with respect to the corresponding wild-type cells. The ability to
detect these differences in hiPSC-CMs might be due to their longer APD compared to the mouse
models. Consequently, the time available for recovery from inactivation in the SCN5A-het
hiPSC-CMs is shortened, potentially resulting in further reduction in Na+ channel availability.8
In summary, we have shown that multiple PSC models can recapitulate the diverse
features of a SCN5A mutation whose electrophysiological characteristics are conserved in mice
and humans. Our findings establish that both mouse and human PSCs can model a cardiac Na+
channel overlap syndrome that evokes multiple cardiac rhythm disturbances. We also prove that
mouse and human iPSCs can model loss-of-function Na+ channel mutations, which to the best of
our knowledge has not yet been shown. These findings extend and complement a rec
recent
e en
nt
publication demonstrating the ability of miPSCs to model a Na+ gain-of-function mutation with
32
al
lteere
redd ga
ati
ting
ng pro
ro
operties,
pe
and suggest that hiPS
hiPSCs
SCs
C might be ablee tto
o em
emulate
mulate
ul the
altered
gating
properties,
el
electrophysiological
lecctr
t ophysiolloggiccall aspects
asppec
ectts of oother
th
her
er S
SCN5A
CN5A
Am
mutations.
uttattion
ns.. S
Su
Such
uchh m
models
ode
dels
ls cco
could
ould
d pprove
rove
ro
vee bbeneficial
eneffic
en
icia
ial
a ffor
or
testing
developing
pharmacological
treatments
for
LQT3
BrS
patients.
Overall
est
stin
ingg an
in
andd de
deve
vellopping
ping nnovel
ovel
ov
ell ppha
harm
ha
rmac
rm
acol
o og
ol
ogic
icall ttre
ic
reeatments
ents ffo
or L
or
QT3 an
QT3
andd Br
B
S pa
ati
tien
entts.
en
ts Ov
Over
eraall
al
this
his study sup
supports
uppo
up
port
po
rtts th
tthee no
nnotion
tiion tthat
hatt PS
ha
PSCs
Cs ccan
an bbee us
used
ed tto
o mo
mode
model
dell in
de
inhe
inherited
h ri
rite
teed fo
form
forms
rmss of ccar
rm
cardiac
ardi
ar
d ac disease,,
di
and offers additional lines to the rapidly expanding list of cardiac disease iPSC models available.
Acknowledgements: The authors thank D. de Jong, K. Szuhai, and H. Tanke (Molecular Cell
Biology, LUMC) for karyotyping analysis, S. Maas (Anatomy and Embryology, LUMC) for
assistance with the teratoma analysis, and S. van de Pas (iPSC core facility, LUMC) for cell
culture. We also thank S. Commandeur, A. el Ghalbzouri (Dermatology, LUMC), B.A.
Schoonderwoerd and M.P. van den Berg (UMC Groningen) for control and SCN5A patient skin
samples, and D. Atsma (Cardiology, LUMC) for obtaining approval from the Medical Ethics
Committee. S. Braam and M. Bellin (Anatomy and Embryology, LUMC) are thanked for
comments on the manuscript.
Funding Sources: Netherlands Heart Foundation (CLM: 2008B106; CRB: 2005T024), ZonMW
(CLM, SC: 114000101), EU FP7 (‘Industem’, CLM: PIAP-GA-2008- 230675), The Netherlands
15
Institute of Regenerative Medicine (CLM, CWvdB), The Netherlands Proteomics Consortium
(CLM, CF, RPD: 050-040-250) and the Netherlands Heart Institute (ICIN, CAR: 061.02).
Conflict of Interest Disclosures: None.
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6.. vvan
an den B
e gM
er
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ier
e sm
ma TJ
JW, Brouwer
Bro
ouw
wer J,
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n der
der Hout
Hou
outt AH,
ou
AH StolteSttolteDijkstra
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Crijns
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Di
ijk
jkst
stra
st
r I,
ra
I, Be
Bezz
zziina TC
CR, V
an
nL
ange
an
genn IM
IM,, Beau
B
eau
aufo
fo
ortt-K
Kro
ol GC
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orn
nel
e JJH,
H,, C
r jn
ri
ns HJ
HJ. Po
osssib
iblle
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successful
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large
brad
dyc
ycarddicc mo
mode
d ooff de
ddeath
athh an
at
nd su
uccessfu
full pa
pace
cema
m kerr tr
trea
eatm
tmen
ent in
n a la
larg
r e family
f mi
fa
mily
ly with
witth features
feat
atur
ures
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:1
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1 13
12
13..
Table
Tabl
blee 1.
1. Na+ cchannel
hann
nnel
el bio
biophysical
oph
p ys
ysic
ical
al propert
properties
rtie
iess in Sc
SScn5a-wt
n5aa-w
-wtt an
andd Sc
SScn5a-het
n5aa-h
n5
-het
e mESCmES
ESCC- andd miPSC-CMs
miP
PSC
SC-C
- Ms
M
mE
m
SC-w
SC
-w
wt
mESC-wt
22.2±2.0
22 2 2 0
Cell capacitance (pF)
Current density
-168.1±24.2
INa (pA/pF)
Activation
-45.9±1.4
V1/2 (mV)
k (mv)
5.8±0.3
Inactivation
-90.8±1.7
V1/2 (mV)
k (mv)
-6.2±0.2
Recovery from inactivation
Ĳf (ms)
57.2±9.5
378.4±78.0
Ĳs (ms)
Slow inactivation
A
0.27±0.02
Ĳ (ms)
1065.8±161.3
n
mESC
mE
SC-h
SC
-h
het
e
mESC-het
n
miP
m
iPSC
iP
C-w
wt
miPSC-wt
n
miPS
mi
PSC-het
PS
miPSC-het
n
117
19.7±3.0
19
30
11
16.4±1.6
16
4 16
13
19.3±1.5
19
3 1
14
17
-94.1±21.1*
11
-299.6±54.7
13
-154.5±43.4†
14
17
-42.5±1.6
6.6±0.4
11
-43.7±1.3
5.6±0.4
13
-42.6±1.3
6.0±0.2
14
14
-90.6±2.5
-6.4±0.2
9
-91.1±0.7
-6.2±0.2
11
-91.6±0.9
-5.8±0.2
12
10
61.1±8.6
374.1±78.2
8
51.6±4.0
478.1±83.9
11
54.3±8.3
420.6±104.4
8
9
0.28±0.03
904.3±202.5
7
0.28±0.03
1158.1±136.4
9
0.29±0.04
1405.3±192.0
8
n= number of mycoytes; INa, Na+ current density measured at -30 mV; V1/2, voltage of half-maximal (in)activation; k, slope factor
of voltage-dependence of (in)activation; Ĳf and Ĳs, fast and slow time constants of recovery from inactivation; A, fraction of
channels that enter the slow inactivated state; W, time constant for development of slow inactivation.
wt, Scn5a+/+; het, Scn5a1798insD/+; Values are mean±SEM. Statistical comparisons were performed using t-test between either
mESC-wt and -het, or miPSC-wt and -het; * P=0.04 vs mESC-wt; † P=0.04 vs miPSC-wt
19
Table 2. Action potential characteristics in Scn5a-wt and Scn5a-het mESC- and miPSC-CMs
mESC-wt
mESC-het
(n=12)
RMP (mV)
P
miPSC-wt
miPSC-het
(n=8)
(n=11)
(n=10)
-71.6±1.0
-69.9±0.7
-77.4±1.4
-76.9±2.1
APA (mV)
104.1±4.7
85.8±2.8
0.006
112.2±2.6
110.4±1.6
Vmax (V/s)
119.5±14.8
57.1±12.1
0.006
272.3±23.6
133.5±16.4
APD20 (ms)
9.4±2.2
15.3±4.4
7.6±2.2
12.9±2.6
APD50 (ms)
19.2±4.2
26.6±7.2
20.4±3.8
28.9±5.1
APD90 (ms)
52.4±7.5
76.2±8.2
70.5±6.7
92.7±9.0
0.04
P
7 x 10-5
0.04
n= number of mycoytes; RMP, resting membrane potential; APA, action potential amplitude; Vmax, maximal
upstroke velocity; APD20, APD50, and APD90, action potential duration at 20, 50 and 90% repolarisation,
respectively.
Values are mean±SEM. Statistical comparisons were performed using t-test between either mESC-wt and -het, or
miPSC-wt and -het; wt, Scn5a+/+; het, Scn5a1798insD/+
Table
Tabl
le 3.. A
Action
ctio
ct
ionn potential
p te
po
t ntial characteristics in SCN
SCN5A-wt
N5A
5A-wt and SCN5A-het
SCN5A-heet hiPSC-CMs
h PSC-CMs
hi
hiPSC-wt
hiPS
hi
P CPS
C-wt
wt
(n=16)
(n=
(n
=16))
-72.4±0.9
-7
72.4±0
0.9
9
106.0±3.2
10
06.
6 0±
0±3
3.2
2
115.7
7 ±18.4
±18
±1
8.4
4
50.7±6.2
50
0.7
.7±6
±6.2
.2
89.8±7.9
89.8
89
.8±7
±7.9
.9
173.5±12.2
RMP
RM
P (mV)
APA
AP
PA (mV)
(mV)
Vmaxx (V
(V/s)
(V/
/s)
/s)
APD20 (ms)
( )
APD
AP
D50 (ms
((ms)
ms))
APD90 (ms)
hiPSC-het
hi
iPS
PSC--heet
(n=13)
(n=1
n=13
3)
3)
-71.3±1.3
-7
71.3±
±1.3
3
103.1±3.2
10
03.1±
1±3.
32
3.
57.6±14.0*
57.6±1
57
±14.0*
0**
58.7±5.9
58.7
58
7±5
±5.9
.9
109.0
10
09.
9.0
0 ±10.1
±10.
±1
0.1
1
217.2±14.9†
n= number of mycoytes; RMP, resting membrane potential; APA, action potential amplitude; Vmax, maximal
upstroke velocity; APD20, APD50, and APD90, action potential duration at 20, 50 and 90% repolarisation,
respectively.
Values are mean±SEM. Statistical comparisons were performed using t-test; wt, SCN5A+/+; het, SCN5A1795insD/+; *
P=0.02, † P=0.03 vs hiPSC-wt
Figure Legends:
Figure 1. Characterisation of control and Scn5a-het miPSC lines. A, Shown from left to right are
brightfield images (original magnification, x125), and immunofluorescent images for Oct4,
Nanog and SSEA-1 of Scn5a-wt (top) and Scn5a-het (bottom) miPSCs. Nuclei were stained with
20
DAPI (blue). B, RT-PCR analysis of endogenous Oct4, Nanog, Sox2, Rex1 and Ecat1 expression
in Scn5a-wt and -het mouse fibroblasts (mFib) and miPSCs. Scn5a-wt mESCs were used as a
positive control, and Gapdh as a loading control. C, Immunofluorescent analysis of embryoid
bodies from Scn5a-wt (top) and Scn5a-het (bottom) miPSCs for the differentiation markers Įsmooth muscle actin (Į-SMA), Sox17 and glial fibrillary acidic protein (GFAP). Nuclei were
stained with DAPI. D, Teratoma derived from Scn5a-wt miPSCs comprising of cell types from
all 3 germ layers. Arrows indicate striated muscle fibres (I), thyroid-like follicles (II), keratinised
stratified squamous epithelium (III), and neural rosettes (IV). E, Teratoma derived from Scn5ahet miPSCs comprising of cell types from all 3 germ layers. Arrows indicate striated muscle
fibres (I), pseudostratified ciliated columnar epithelium (II), keratinised stratifiedd sq
squamous
quaamo
mous
us
epithelium (III), and neurons (IV). Scalebars represent 100 ȝm. wt, Scn5a+/+; het, Scn5a1798insD/+.
F
Figure
igu
gure
u 2. Dif
Differentiation
ffe
ferrentiaati
t onn ooff mE
mESC
mESCs
SC
Cs an
and
nd miPS
miPSCs
PSCs tto
PS
o ca
cardiomyocytes.
ard
rdio
iomy
myoc
my
ocyytes
ytes. A
A,, Sc
Sche
Schematic
hem
he
matiic de
mati
det
detailing
ta lin
tail
ingg th
the
he
procedure
proc
pr
oced
oc
edur
ed
uree to ddif
differentiate
iffferrent
rentia
iatte
te m
mESCs
ESC
Cs an
andd mi
miPS
miPSCs
PSCs
PS
Css ttoo ca
cardiomyocytes.
ard
dio
iomy
myoc
my
occyt
y es. EBs,
E s, embryoid
EB
emb
bry
ryoi
oidd bodi
bbodies;
odiiess; RA,
R A,
retinoic
etinoic acid.
d. B
B,, Im
Immunofluorescent
mmu
muno
n flluo
no
uore
r sc
scen
entt im
en
images
mag
ages
es ooff th
thee ca
cardiac
ard
rdia
iacc ma
ia
mark
markers
rker
rk
e sȕm
myosin
yosi
yo
sinn he
si
heav
heavy
avyy ch
av
cchain
ain (ȕMHC), Į-actinin, troponin I, myosin light chain 2 atrial isoform (MLC2a) and Scn5a in
cardiomyocytes derived from Scn5a-wt and Scn5a-het mESCs and miPSCs. All cells were costained with the nuclear dye DAPI (blue). Scale bars represent 25 ȝm. wt, Scn5a+/+; het,
Scn5a1798insD/+.
Figure 3. Na+ current densities in Scn5a-wt and Scn5a-het mESC- and miPSC-CMs. A and B,
Examples of Na+ current (INa) traces recorded from Scn5a-wt and Scn5a-het cardiomyocytes
derived from mESCs (A) and miPSCs (B). C and D, Average current-voltage relationships for
21
Scn5a-wt and Scn5a-het cardiomyocytes derived from mESCs (C) and miPSCs (D). Voltage
protocols shown as insets. E and F, INa density, measured at -30 mV, in Scn5a-het
cardiomyocytes relative to Scn5a-wt cardiomyocytes derived from mESCs (E) and miPSCs (F),
expressed as a percentage. * indicates statistical significance (P<0.05; two-way rmANOVA (C
and D); t-test (E and F)). wt, Scn5a+/+; het, Scn5a1798insD/+.
Figure 4. Na+ current gating properties in Scn5a-wt and Scn5a-het mESC- and miPSC-CMs. A
through H, Average voltage dependence of activation (A and B), voltage dependence of
inactivation (C and D), recovery from inactivation (E and F), and development of slow
nactivation (G and H) for Scn5a-wt and Scn5a-het cardiomyocytes derived from
m mE
m
SCss an
SC
andd
inactivation
mESCs
miPSCs. Voltage protocols are shown as insets. Data are summarised in Table 1. wt, Scn5a+/+;
1798insD/+
1798in
8 sD
D/+
D/+
he
et, S
Scn
cn5a
cn
5a179
.
het,
Scn5a
Figure
Fi
Figu
gure
gu
re 55.. Persistent
Pers
Pe
rsis
isttennt
nt Na
Na+ ccur
current
urrrent
rent m
mea
measurements
easu
suureeme
mentts in
nS
Scn5a-wt
cn
n5aa-w
-wtt an
andd Sc
Scn5a-het
cn5
n a-hhet
het mE
mESC
mESCSC
C- and
and miPS
m
miPSCiPS
SC CMs. A and B,
B, Persistent
Pers
Pe
rsis
rs
i te
is
t nt
n INa tr
ttraces
aces
ac
e obtained
es
obttai
a ne
nedd by subtraction
sub
ubtr
trac
tr
acti
tion
ti
on of
of the
the
h current
cur
urre
rent
re
nt before
bbef
e or
ef
o e and
and after
a ter
af
application of 30 μM TTX in Scn5a-wt (top) and Scn5a-het (bottom) cardiomyocytes derived
from mESCs (A) and miPSCs (B). Voltage protocols are shown above the traces. C and D,
Average values for persistent INa density in mESC-CMs (C) and miPSC-CMs (D). * indicates
statistical significance (P<0.05; two-way rmANOVA). wt, Scn5a+/+; het, Scn5a1798insD/+.
Figure 6. Action potential characteristics of Scn5a-wt and Scn5a-het mESC- and miPSC-CMs.
A and B, Examples of action potential (AP) and upstroke velocity (Vmax, inset) measured in
Scn5a-wt and Scn5a-het cardiomyocytes derived from mESCs (A) and miPSCs (B). C and D,
22
Average data at 6 Hz for Vmax, AP duration at 20, 50, and 90% repolarisation (APD20, APD50,
and APD90), resting membrane potential (RMP) and AP amplitude (APA) in Scn5a-wt and
Scn5a-het cardiomyocytes derived from mESCs (C) and miPSCs (D). * indicates statistical
significance (P<0.05; t-test). Data are summarised in Table 2. wt, Scn5a+/+; het, Scn5a1798insD/+.
Figure 7. Characterisation of SCN5A-het hiPSCs. A, Sequence analysis of SCN5A in SCN5Ahet hiPSCs showing the insertion of the nucleotides “TGA” in one allele, resulting in the addition
of aspartic acid (D) at position 1795 of the SCN5A protein. B, COBRA-FISH karyogram of
SCN5A-het hiPSCs showing a normal 46XY karyotype. C, Shown from left to right are a
brightfield image (original magnification, x16) of,
f and immunofluorescent imag
ges ffor
or O
OCT
CT3/
CT
3/44,
images
OCT3/4,
NANOG, TRA-1-81 and SSEA4 in SCN5A-het hiPSCs. Nuclei were stained with DAPI (blue).
D
Imm
muno
muno
nost
sta
taini
niing of embryoid bodies from SCN
CN55A-het hiPSCss ffor tthe
CN
he differentiation markers
D,, Im
Immunostaining
SCN5A-het
Į
-sm
mooth musc
scle
le aactin
cttin ((Į-SMA),
Į-S
-SMA
SMA
MA),
), S
SOX
OX117, an
OX
nd gli
ial fib
brilla
bri
illarry
ry acidic
aciidi
dic protein
prooteinn (GFAP).
pr
(GFA
(G
FA
AP)
P).. Nu
N
clei
cl
eii w
ere
Į-smooth
muscle
SOX17,
and
glial
fibrillary
Nuclei
were
tai
aine
nedd with
ne
with DAPI.
DAPI.
I. Scalebars
Scalleba
leb rss in
in C and
and
d D represent
rrep
epre
ep
reese
s nt
nt 100
100
00 ȝm.
ȝm.
m.
stained
Figure 8. Characterisation of cardiomyocytes derived from SCN5A-wt and SCN5a-het hiPSCs.
A, Immunofluorescent images of the cardiac markers ȕ myosin heavy chain (ȕ-MHC), Į-Actinin,
Troponin I and myosin light chain 2 atrial isoform (MLC2A) in SCN5A-wt (top) and SCN5Ahet (bottom) hiPSCs-CMs. All cells were co-stained with the nuclear dye DAPI (blue). Scalebars
represent 25 ȝm. B, Examples of Na+ current (INa) traces recorded in SCN5A-wt (top) and
SCN5A-het (bottom) hiPSCs-CMs. C, Average current-voltage relationships for SCN5A-wt and
SCN5A-het hiPSC-CMs. The capacitance (Cm) was 36.0±3.3 pF in the SCN5A-wt and 31.7±3.2
pF in the SCN5A-het cardiomyocytes. D, INa density, measured at -30 mV, in SCN5A-het hiPSC-
23
CMs (-121.4±23.8 pA/pF) relative to SCN5A-wt hiPSC-CMs (-264.4±57.0 pA/pF), expressed as
a percentage. E, Examples of action potential (AP) and upstroke velocity (Vmax, inset) recorded
in SCN5A-wt and SCN5A-het hiPSC-CMs. F, Average data at 1 Hz for Vmax, AP duration at 20,
50, and 90% repolarisation (APD20, APD50, and APD90), resting membrane potential (RMP) and
AP amplitude (APA) in SCN5A-wt and SCN5A-het hiPSC-CMs. G, Persistent INa traces
obtained by subtraction of the current before and after application of 30 PM TTX in SCN5A-wt
(top) and SCN5A-het (bottom) hiPSCs-CMs. The voltage protocol is shown above the traces. H,
Average values for persistent INa density in hiPSCs-CMs. * indicates statistical significance
(P<0.05; two-way rmANOVA (C and H); t-test (D and F)). wt, SCN5A+/+; het, SCN5A1795insD/+.
24

Circulation

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