RESEARCH ARTICLE
Low ex acellula po assium p olongs epola iza ion and e okes
ea ly a e depola iza ion in human induced plu ipo en s em
cell-de i ed ca diomyocy es
Jukka Kuusela
1,2,
*, Kim La sson
1,2,
*, Dishee Shah
1,2
, Chand a P ajapa i
1,2
and Ka iina Aal o-Se a
la

1,2,3,4,‡
ABSTRACT
Long QT synd ome (LQTS) is cha ac e ized by a p olonged QT-
in e al on elec oca diog am and by inc eased isk o sudden dea h.
One o he mos common and po en ially li e- h ea ening elec oly e
dis u bances is hypokalemia, cha ac e ized by low concen a ions o
K
+
. Using a mul ielec ode a ay pla o m and cu en clamp
echnique, we in es iga ed he e ec o low ex acellula K
+
concen a ion ([K
+
]
Ex
) on he elec ophysiological p ope ies o
hiPSC-de i ed ca diomyocy es (CMs) gene a ed om a heal hy
con ol subjec (WT) and om wo symp oma ic pa ien s wi h ype 1 o
LQTS ca ying G589D (LQT1A) o IVS7-2A>G mu a ion (LQT1B) in
KCNQ1. The baseline p olonga ions o ield po en ial du a ions
(FPDs) and ac ion po en ial du a ions (APDs) we e longe in LQT1-
CMs han in WT-CMs. Exposu e o low [K
+
]
Ex
p olonged FPDs and
APDs in a concen a ion-dependen ashion. LQT1-CMs we e ound
o be mo e sensi i e o low [K
+
]
Ex
compa ed o WT-CMs. A baseline,
LQT1A-CMs had mo e p olonged APDs han LQT1B-CMs, bu low
[K
+
]
Ex
caused mo e p onounced APD p olonga ion in LQT1B-CMs.
Ea ly a e depola iza ions in he ac ion po en ials we e obse ed in a
subse o LQT1A-CMs wi h u he p olonged baseline APDs and
iangula phase 2 p o iles. This wo k demons a es ha he hiPSC-
de i ed CMs a e sensi i e o low [K
+
]
Ex
and p o ide a pla o m o s udy
acqui ed LQTS.
KEY WORDS: Induced plu ipo en s em cells, Long QT synd ome,
Hypokalemia, Pa ch clamp, Mul ielec ode a ay
INTRODUCTION
Long QT synd ome (LQTS) p esen s as an acqui ed o inhe i ed
a hy hmic disease cha ac e ized by p olonged QT in e al on
elec oca diog am and is associa ed wi h he occu ence o syncope
o ca diac a es . A special ype o en icula achyca dia known as
o sades de poin es may a ise in LQTS, which may degene a e in o
li e- h ea ening en icula ib illa ion and cause sudden ca diac
dea h (Schwa z e al., 2013).
Congeni al o ms o LQTS ypically esul om mu a ions in he
ca diac ion channel-encoding genes. LQTS can be di ided in o
di e en sub ypes wi h LQT1 being he mos common sub ype
caused by mu a ions in he KCNQ1. The KCNQ1 encodes he α-
subuni o he ol age-ga ed po assium channel and, assembled wi h
auxilia y β-subuni s encoded by KCNE1, conduc s he slow delayed
ec i ie ou wa d K
+
cu en (I
Ks
) (Ba hanin e al., 1996; Sanguine i
e al., 1996). The high p e alence (0.4%) o LQTS in Finland has
been explained by ou ounde mu a ions in he Finnish popula ion
(Ma jamaa e al., 2009). The mos p e alen LQT1 causing ounde
mu a ions is he C- e minal KCNQ1 G589D missense mu a ion
(Piippo e al., 2001). E idence sugges s ha G589D is a mode a e
dominan -nega i e a icking mu a ion wi h no mal unc ioning bu
wi h hinde ed anspo o he cell memb ane (A omola an e al.,
2014). Ano he p e alen ounde mu a ion is a s ong dominan -
nega i e splice si e mu a ion IVS7-2A>G (Fods ad e al., 2006).
Bo h, he G589D and IVS7-2A>G mu a ion ypes esul in educed
I
Ks
cu en when co-exp essed independen ly wi h wild- ype
KCNQ1 and KCNE1 (Piippo e al., 2001; Fods ad e al., 2006).
Hypokalemia is one o he mos common elec oly e dis u bances
and is cha ac e ized by low blood se um K
+
le els. In no mokalemic
condi ions, K
+
concen a ions ange om 3.5-5.3 mM (Macdonald
and S u he s, 2004). In mode a e hypokalemia, he K
+
concen a ions ange om 2.5-3.0 mM; and in se e e hypokalemia
hey a e <2.5 mM (Unwin e al., 2011). Hypokalemia is known o
delay en icula epola iza ion, slow en icula conduc ion, cause
hype pola iza ion o he es ing po en ial in en icula myocy es as
well as cause abno mal en icula au oma ici y inc easing he isk o
en icula a hy hmia and sudden ca diac dea h (Macdonald and
S u he s, 2004; Osadchii, 2010; Schulman and Na ins, 1990).
Abou 40% o he pa ien s on hiazide diu e ics ha e been
epo ed o su e om hypokalemia (Genna i, 1998) and a en old
inc ease in hospi al mo ali y was ound in hypokalemic pa ien s
(Pal iel e al., 2001). Po assium elec oly e dis u bance in se um is
ecognized as a isk ac o among LQTS pa ien s. The mo ali y a e
o hospi alized hypokalemic pa ien s was en old highe han in
gene alized hospi al popula ion illus a ing he po en ially li e-
h ea ening consequences o hypokalemia (Pal iel e al., 2001).
O he s udies on hypokalemia ha e epo ed p olonged QT-
in e als, o sades de poin es and en icula a hy hmias in
LQT1 pa ien s (Schulman and Na ins, 1990; Roden, 1997).
Unde hypokalemic condi ions, LQTS pa ien s may be mo e
suscep ible o exagge a ed QT-in e al p olonga ion han heal hy
subjec s because he epola iza ion ese e in LQTS pa ien s is
al eady comp omised due o mu a ion(s) in he ca diac ion channel
genes (Roden, 2004; Va ó and Baczkó, 2011).
I is now possible o gene a e plu ipo en s em cells (human
induced plu ipo en s em cells; hiPSCs) om any indi idual
(Takahashi e al., 2007; Yu e al., 2007). hiPSCs ha e been
shown o be a easible esea ch ool o he s udy o inhe i ed ca diac
diseases (e.g. LQTS) (Bellin e al., 2013; Mo e i e al., 2010;
I zhaki e al., 2011; Lah i e al., 2012; Egashi a e al., 2012; Ma sa
Recei ed 12 Janua y 2017; Accep ed 11 Ap il 2017
1
Ins i u e o Biomedical Technology, Uni e si y o Tampe e, Tampe e, Finland.
2
BioMediTech, Tampe e, Finland.
3
School o Medicine, Uni e si y o Tampe e,
Tampe e, Finland.
4
Hea Hospi al, Tampe e Uni e si y Hospi al, Tampe e, Finland.
*These au ho s con ibu ed equally o his wo k
‡
Au ho o co espondence ([email p o ec ed])
This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion
License (h p://c ea i ecommons.o g/licenses/by/3.0), which pe mi s un es ic ed use,
dis ibu ion and ep oduc ion in any medium p o ided ha he o iginal wo k is p ope ly a ibu ed.
777
© 2017. Published by The Company o Biologis s L d
|
Biology Open (2017) 6, 777-784 doi:10.1242/bio.024216
Biology Open
e al., 2011; Spence e al., 2014; Malan e al., 2011; Yazawa e al.,
2011; Ma e al., 2013; Ki iaho e al., 2015). The e ec s o low
ex acellula K
+
concen a ions ([K
+
]
Ex
) ha e cu en ly no been
in es iga ed in hiPSC-de i ed LQT ca diomyocy es (CMs).
In his s udy, we ha e u ilized a hiPSC-based model (Ki iaho
e al., 2015) o s udy he e ec s o low [K
+
]
Ex
in con ol CMs a well
as in LQT1-speci ic CMs ca ying Finnish ounde mu a ions
G589D o IVS7-2A>G. The elec ophysiological p ope ies o
con ol subjec (WT), and LQT1A (ca ying G589D), LQT1B
(ca ying IVS7-2A>G) lines we e assessed by de e mining he ield
po en ial du a ions (FPDs) and ac ion po en ial du a ions (APDs),
which co espond o su oga es o he QT-in e al o he hea . The
p olonga ion o APDs, FPDs and ea ly a e depola iza ions (EADs)
we e assessed wi h low [K
+
]
Ex
using en icula -like CMs om WT,
LQT1A and LQT1B lines.
RESULTS
E ec o [K
+
]
Ex
on bea ing ca diomyocy e clus e s
Baseline elec ophysiological p ope ies o heal hy con ol (WT)
and pa ien -speci ic LQT1A and LQT1B CM clus e s we e assessed
by mul ielec ode a ay (MEA) measu emen s a 5.33 mM
ex acellula K
+
([K
+
]
Ex
). Rep esen a i e ield po en ial aces
om baseline (uppe panels) o WT, LQT1A and LQT1B,
espec i ely a e depic ed in Fig. 1A-C. Table 1A-C shows he
FPD and co ec ed ield po en ial du a ions (cFPDs) using wo
di e en co ec ion o mulae; Baze and F ide icia. The
F ide icia’s co ec ion o mula was used o desc ibe he ex ac ed
MEA da a and a compa ison o baseline p ope ies e ealed ha
cFPDs o LQT1A and LQT1B we e mo e p olonged compa ed o
WT (P=1.8E-4, one-way ANOVA) and in he same es no
di e ence in cFPDs be ween LQT1A and LQT1B we e ound
(P=0.839, one-way ANOVA).
Following he in es iga ion o baseline p ope ies on he CM
clus e s, he e ec o sequen ial educ ion o 3, 2 and 1 mM [K
+
]
Ex
we e s udied. Rep esen a i e ield po en ial aces om 1 mM
[K
+
]
Ex
(lowe panels) o WT, LQT1A and LQT1B, espec i ely a e
depic ed in Fig. 1A-C. A concen a ion-dependen inc ease in cFPD
co ela ed wi h sequen ial educ ion in [K
+
]
Ex
(Table 1A-C).
Table 1A shows ha low [K
+
]
Ex
p olonged cFPD in WT wi h
signi ican di e ence (P=0.045, pai ed - es ) a 1 mM [K
+
]
Ex
.
In con as , he cFPD was signi ican ly p olonged in LQT1A
(P=0.049, pai ed - es ) and LQT1B lines (P=0.043, pai ed - es )
al eady a 3 mM [K
+
]
ex
(Table 1B,C). The absolu e cFPD
p olonga ion in esponse o lowe ed [K
+
]
Ex
was calcula ed
(Table 1D) bu no s a is ically signi ican di e ences we e ound
be ween he lines.
E ec o [K
+
]
Ex
on single bea ing ca diomyocy es
Simila o MEA, he elec ophysiological p ope ies o single CMs
we e compa ed among WT, LQT1A and LQT1B in cu en clamp.
Table 2A-C shows ex ac ed da a om ac ion po en ials (AP)
consis ing o : ac ion po en ial du a ion (APD), ac ion po en ial
ampli ude (APA) and maximum dias olic po en ial (MDP). APD
90/50
a ios we e <1.30 indica ing en icula -like CM. The baseline APDs
o LQT1A and LQT1B we e signi ican ly longe han WT (P<0.008,
one-way ANOVA) and in he same es he APDs showed ha
LQT1A was signi ican ly longe han LQT1B (P=0.007, n=22 in
each g oup, one-way ANOVA). The baseline APD
50/10
a io was
highe in LQT1A compa ed o WT and LQT1B, which indica es ha
phase 2 o he APs in LQT1A we e signi ican ly mo e iangula in
shape ( a ios: LQT1A 2.95±0.13**, P<0.002: LQT1B 2.45±0.09 and
WT 2.28±0.07, P=0.438, one-way ANOVA, n=22, Table 2A-C).
The [K
+
]
Ex
in he pe usa e was hen lowe ed om 4.8 mM a
baseline o 3, 2 o 1 mM and he e ec s we e s udied on he WT,
LQT1A and LQT1B CMs. The APDs and APAs we e inc eased and
MDPs dec eased a low [K
+
]
Ex
(Table 2A-C). Fig. 2A-C show
ep esen a i e aces o baseline (uppe ace) and 1 mM [K
+
]
Ex
(lowe ace). The ela i e p olonga ion o APD
90
wi h low [K
+
]
Ex
is
shown in Fig. 2D and he APD
90
o LQT1B CMs we e ound o be
mo e p olonged compa ed o he LQT1A and WT CMs a 1 mM
[K
+
]
Ex
(P=0.025, one-way ANOVA). The absolu e APD
90
p olonga ion in esponse o lowe ed [K
+
]
Ex
was calcula ed in he
same da a se s and is p esen ed in Table 2D. This da a e eals a mo e
Fig. 1. Rep esen a i e aces o ield po en ials a baseline and a 1 mM
[K
+
]
Ex
in ca diomyocy e clus e s and he dose-dependen p olonga ion in
ield po en ials co esponding o [K
+
]
Ex
.(A) Reco ding o a ep esen a i e
ield po en ial e en by MEA a WT baseline (uppe ace) and i s
co esponding ield po en ial e en in he p esence o 1 mM [K
+
]
Ex
(lowe
ace). (B) and (C) a e equi alen o (A), bu show LQT1A (B) and LQT1B (C),
espec i ely. Scale ba s: 40 µV (A), 10 µV (B) 10 µV in abscissa, (C) and he
o dina e is 100 ms. Dashed lines (A-C) indica e 0 µV. A ows indica e he
endpoin o FPD.
778
RESEARCH ARTICLE Biology Open (2017) 6, 777-784 doi:10.1242/bio.024216
Biology Open
p onounced absolu e APD
90
p olonga ions wi h 1 mM in LQT1A
and LQT1B lines compa ed o WT. This sugges s ha LQT1A is
mo e p olonged in absolu e e ms while LQT1B is mo e sensi i e o
low [K
+
]
Ex
in ela i e e ms.
Single LQT1A ca diomyocy es exhibi a hy hmia
Low [K
+
]
Ex
did no e oke a hy hmias in WT, LQT1A and
LQT1B CM clus e s in MEA eco dings. Simila ly, in cu en
clamp eco dings no a hy hmias we e ound in WT and
LQT1B. Howe e , LQT1A exhibi ed cha ac e is ics shown in
Fig. 3A,B. The APDs we e u he p olonged and exhibi ed ea ly
a e depola iza ions (EADs) e oked only wi h 1 mM [K
+
]
Ex
(Table 3A). In some LQT1As, EADs we e al eady p esen in
baseline condi ions (Table 3B). The o al occu ence o LQT1A
CMs exhibi ing EADs was 37.5%. In baselines wi hou EADs (bu
wi h EADs a low [K
+
]
Ex
), he APD
90/50
we e simila (<1.30) o
CMs wi hou EADs a low [K
+
]
Ex
. Howe e , in he o me (EADs a
low [K
+
]
Ex
) he baseline APD
50/10
a ios we e highe han in he
la e (CMs wi hou he EADs) (P=1.3E-6, unpai ed - es , APD
50/10
a io: 4.83±0.68 Table 3A and 2.95±0.13 Table 2B, see also Figs 2B
and 3A). No e, he CMs analyzed in Table 3A exhibi ed EADs only
wi h 1 mM [K
+
]
Ex
. CMs wi h EADs occu ing a baseline had an
inc ease in EADs and APD p olonga ion wi h lowe [K
+
]
Ex
(Table 3B, Fig. 3B, a ows). The EAD ‘ ake o ’in baselines o
wi h 1 mM [K
+
]
Ex
occu ed solely in phase 2 and we e ound o
ange om −15 mV o −27 mV. EAD ‘ ake o ’in baselines we e
−17.8±1.7 mV (n=5) and when e oked wi h 1 mM [K
+
]
Ex
−20.3±
1.3 mV (n=7), sugges ing a ole o L- ype calcium cu en due o he
ac ha he iangula phase 2 is d i en owa ds mo e nega i e
memb ane po en ial du ing u he APD p olonga ion in hese
LQT1A CMs.
DISCUSSION
This s udy demons a es he e ec o low [K
+
]
Ex
on WT, LQT1A
and LQT1B CMs using MEA and cu en clamp. The baseline
APD and FPD o LQT1A and LQT1B CMs we e longe compa ed
o WT. P olonged epola iza ion was e oked in a concen a ion-
dependen ashion by low [K
+
]
Ex
in all ypes o CMs wi h he
LQT1B CMs being mos sensi i e o low [K
+
]
Ex
. In cu en clamp
we ound ha MDP was dec eased (hype pola ized) and APA was
highe wi h low [K
+
]
Ex
. EADs we e occasionally obse ed in
LQT1A a single cell le el and hese CMs had u he p olonged
APDs al eady a baseline compa ed o he majo i y o LQT1A
CMs. An in e es ing inding was ha he phase 2 iangula i y
(APD
50/10
a io) was mo e p onounced in he LQT1A CMs a
baseline when EADs we e p esen . In suppo o ou da a, p e ious
cu en clamp cha ac e is ics eco ded a physiological po assium
condi ions a baseline a e simila o WT, LQT1A and LQT1B
CMs desc ibed in his s udy (Ki iaho e al., 2015). This is he i s
s udy whe e low po assium concen a ion induces p olonged
epola iza ion in hiPSC-de i ed WT and LQTS CMs.
Addi ionally, EADs we e obse ed only in CMs wi h G589D
Table 1. Field po en ial du a ions om ca diomyocy e clus e s a baselines and low [K
+
]
Ex
(A)
WT nFPD (ms) cFPD (Baze ) (ms) cFPD (F ide icia) (ms)
Baseline 3 334.72±19.65 377.48±11.34 362.57±14.27
3mMK
+
358.42±28.64 407.56±19.33 390.37±22.62
Baseline 3 374,20±57.50 369.02±29.53 370.18±37.76
2mMK
+
450.23±68.03* 414.81±54.13 426.13±57.95
Baseline 3 431.63±40.56 401.78±10.21 411.03±20.06
1mMK
+
551.88±49.56* 463.57±20.11 484.30±20.72*
(B)
LQT1A nFPD (ms) cFPD (Baze ) (ms) cFPD (F ide icia) (ms)
Baseline 4 537.67±36.75 604.49±19.81 580.79±24.04
3mM K
+
604.52±17.70 644.68±19.95*** 630.47±12.02*
Baseline 5 520.96±34.91 565.97±27.62 549.81±27.27
2mM K
+
642.55±45.32 651.83±21.61* 647.70±26.51*
Baseline 4 539.81±19.78 596.36±28.18 576.30±21.06
1mM K
+
694.54±27.39* 720.79±5.84* 703.50±9.28*
(C)
LQT1B nFPD (ms) cFPD (Baze ) (ms) cFPD (F ide icia) (ms)
Baseline 5 515.04±26.59 549.79±31.79 537.56±27.90
3mMK
+
596.29±23.59 664.03±21.52* 640.16±18.81*
Baseline 5 510.08±23.39 542.23±31.31 530.84±26.20
2mMK
+
631.18±26.75* 684.68±17.52* 665.51±14.07*
Baseline 4 513.71±34.28 527.17±28.84 522.58±30.38
1mMK
+
723.26±22.90* 716.31±22.11* 718.07±15.67*
(D)
K+ concen a ion nWT (ms) LQT1A (ms) LQT1B (ms)
3 mM 3/4/5 27.80±14.80 49.68±15.45 103.00±35.13
2 mM 3/5/5 55.93±21.53 97.88±28.35 134.67±33.56
1 mM 3/4/4 73.27±16.07 127.20±23.76 195.49±42.91
(A-C) FPDs and cFPDs a baseline and low [K
+
]
Ex
ob ained om ca diomyocy e clus e s on MEA o (A) WT, (B) LQT1A and (C) LQT1B, espec i ely. (D) The
absolu e Δinc ease in he FPDs in milliseconds om baseline o i s co esponding 3, 2 and 1 mM [K
+
]
Ex
. S a is ical signi icance in (A-C) was calcula ed by using
pai ed - es , and in (D) i was calcula ed using ANOVA be ween he lines a each [K
+
]
Ex
*P<0.05, **P<0.01 and ***P<0.001. n=clus e s eco ded o each da a
poin . Da a is p esen ed as mean±s.e.m.
779
RESEARCH ARTICLE Biology Open (2017) 6, 777-784 doi:10.1242/bio.024216
Biology Open
poin mu a ion (LQT1A), bu no in hose ca ying he IVS7-
2A>G mu a ions (LQT1B).
The appea ance o EADs has been linked o he hype pola iza ion
o cell memb ane in low [K
+
]
Ex
condi ions (Zaza, 2009). In he
p esen s udy, we show ha EADs simila ly eme ge om mo e
hype pola ized MDPs a low [K
+
]
Ex
. This is suppo ed by an al e ed
K
+
conduc ance and elec omo i e o ce du ing acu e and ch onic
low [K
+
]
Ex
and is u he s eng hened by simila indings wi h da a
om animal en icula ca diomyocy es (Whi e and Te a , 1991;
Ruiz Ce e i e al., 1982; Kishida e al., 1979; Zaza, 2009), in
ansgenic abbi LQT1 and LQT2 models (Liu e al., 2012) and in
ansgenic mouse LQT2 model (Teng e al., 2004). Ou da a wi h
hiPSC-de i ed CMs hus co ela es well wi h p e ious s udies e en
hough p e iously epo ed da a we e conduc ed wi h ansgenic o
non-human CMs.
In his s udy, low [K
+
]
Ex
p olonged he FPD and APD in all ou
CMs (WT, LQT1A and LQT1B). This da a is suppo ed by p e ious
indings wi h a ious animal s udies as well as in isola ed
en icula CMs (Whi e and Te a , 1991; Aki a e al., 1998; Guo
e al., 2011; Pezhouman e al., 2015; Chan e al., 2015; Osadchii,
2010). The p olonga ion o epola iza ion in hypokalemia (low
[K
+
]
Ex
) has also been documen ed in ca diac pa ien s ecei ing
diu e ic he apy (S ewa e al., 1985). I is well es ablished ha low
[K
+
]
Ex
supp esses he K
+
cu en s such as I
K
con ibu ing o
delayed epola iza ion he eby p olonging he QT-in e al (Yang
e al., 1997; Sanguine i and Ju kiewicz, 1992; Scamps and
Ca melie , 1989; Guo e al., 2011; Osadchii, 2010). Thus, low
ex acellula K
+
is ecognized as a ac o educing he epola iza ion
ese e (Va ó and Baczkó, 2011).
The epola iza ion phase in ol es p ima ily I
K
and I
Ks
in
en icle myocy es. The LQT1 lines used in his s udy had mu a ion
in he KCNQ1 dep essing he I
Ks
cu en , which is p esen ed as FPD
and APD p olonga ions a baseline. Thus, his sugges s
p olonga ion in FPD and APD o inc ease in low [K
+
]
ex
pa ly
due o he lack o I
Ks
epola iza ion ese e. The de iciency in I
Ks
cu en in ou LQT1 lines ep oduces ou p e ious indings whe e
we show no o only a ma ginal e ec on he APDs wi h a pa ial I
Ks
blockade (JNJ303) in he LQT1 lines bu obus e ec s in he WT
line (Ki iaho e al., 2015). As expec ed, a pa ial I
K
blockade
(E4031) esul ed in a p olonga ion o FPD and APD in he WT,
LQT1A and LQT1B lines (Ki iaho e al., 2015; P adhapan e al.,
2013).
In a ansgene exp ession sys em wi h he G589D poin mu a ion,
a educed I
Ks
cu en has been epo ed (Piippo e al., 2001). The
G589D mu a ion has been ound o be a a icking de ec
(A omola an e al., 2014). The IVS7-2A>G splicing mu a ion is a
‘loss-o - unc ion’de ec (Fods ad e al., 2006). Based on ea lie
exp ession s udies in co-exp ession wi h wild- ype KCNQ1 and
KCNE1, he IVS7-2A>G mu a ion dec eases I
Ks
cu en mo e han
he G589D mu a ion (Piippo e al., 2001; Fods ad e al., 2006).
Thus, hese ea lie indings suppo ou da a wi h di e ence in
epola iza ion imes, bu do no explain he p esence o EADs. This
Table 2. Ac ion po en ial du a ions (APD) om single ca diomyocy e a baselines and low [K
+
]
Ex
(A)
WT nAPD
10
(ms) APD
50
(ms) APD
90
(ms) APA (mV) MDP (mV)
Baseline 3 156.87±16.16 315.71±44.96 374.59±49.13 113.95±4.59 −73.17±4.43
3mMK
+
182.92±32.57 358.03±51.02 417.14±57.39 117.68±5.71 −75.86±5.44
Baseline 11 133.40±10.94 313.06±27.64 366.45±31.62 112.51±2.29 −72.34±2.18
2mMK
+
154.90±15.19** 394.86±39.46*** 455.36±43.58*** 118.18±2.81*** −75.70±2.60*
Baseline 17 142.54±6.36 313.44±17.67 363.8±19.08 114.22±1.35 −73.22±1.31
1mMK
+
153.66±8.80* 397.79±26.01*** 459.05±28.09*** 119.29±1.50*** −76.58±1.30**
(B)
LQT1A nAPD
10
(ms) APD
50
(ms) APD
90
(ms) APA (mV) MDP (mV)
Baseline 13 165.38±11.80 495.43±47.59 578.58±52.55 115.50±1.43 −73.65±1.23
3mMK
+
170.77±12.09* 542.88±52.58*** 639.32±61.88*** 117.72±1.26*** −75.31±1.14***
Baseline 10 166.03±10.33 465.03±38.07 543.74±44.79 115.82±1.62 −73.37±1.39
2mMK
+
183.73±13.63* 562.11±48.62*** 665.67±61.48*** 120.73±1.91** −77.23±1.52**
Baseline 15 177.96±9.43 497.06±28.84 570.09±30.14 115.63±1.25 −74.04±1.02
1mMK
+
177.90±11.05 625.26±42.56*** 734.66±45.90*** 119.08±1.40*** −76.73±1.10***
(C)
LQT1B nAPD
10
(ms) APD
50
(ms) APD
90
(ms) APA (mV) MDP (mV)
Baseline 10 169.39±14.10 420.44±31.93 484.33±35.44 112.67±2.26 −71.54±2.10
3mMK
+
190.31±18.95* 487.19±38.42** 556.36±40.44** 115.34±2.68*** −73.63±2.40**
Baseline 8 153.72±14.54 406.09±33.07 473.80±37.36 111.29±2.65 −70.05±2.43
2mMK
+
175.27±15.32*** 507.54±34.75*** 588.40±40.32*** 115.10±3.02*** −72.99±2.67*
Baseline 16 181.38±11.86 424.92±25.85 489.65±28.02 114.35±1.51 −72.75±1.38
1mMK
+
219.11±17.97** 598.86±43.19*** 682.71±45.93*** 119.23±1.70*** −75.54±1.59**
(D)
K+ concen a ion nWT LQT1A LQT1B
3 mM 3/13/10 42.55±13.17 60.74±12.91 72.03±16.45
2 mM 11/10/8 88.91±16.52 121.93±20.77 114.60±10.59
1 mM 17/15/16 95.24±13.55 164.57±22.59* 193.06±23.79**
(A-C) APDs a baseline and low [K
+
]
Ex
ob ained om ac ion po en ials eco ded om (A) WT, (B) LQT1A and (C) LQT1B, espec i ely. (D) The absolu e Δinc ease
in he APD
90
in milliseconds om baseline o i s co esponding 3, 2 and 1 mM [K
+
]
Ex
. S a is ical signi icance (A-C) was calcula ed by using pai ed - es , and in
(D) i was calcula ed using ANOVA be ween he lines a each [K
+
]
Ex
*P<0.05, **P<0.01 and ***P<0.001. n=ca diomyocy es eco ded o each da a poin . Da a is
p esen ed as mean±s.e.m.
780
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Biology Open
e e s o complexi y o K
+
homeos asis and addi ional mechanisms
in CMs.
The baseline FPD and APD in ou da a demons a e ha he
epola iza ion ime o LQT1A is longe han ha o LQT1B, bu
LQT1B is mo e sensi i e o low [K
+
]
Ex
despi e MDP and APA
being simila in LQT1A and LQT1B CMs. Since I
K
is a majo
playe in ol ed in epola iza ion, he simples explana ion would be
ha he I
K
con ibu ion o he epola iza ion ese e pool is la ge in
LQT1B han in LQT1A. The epola iza ion ese e pool o LQT1B
would hus show g ea e K
+
sensi i i y due o supp essed K
+
conduc ance, inc eased elec omo i e o ce when acu ely exposed
o low [K
+
]
Ex
(Zaza, 2009) and no I
Ks
unc ionali y. This would also
explain why LQT1B has sho e FPD and APD baseline alues
compa ed o LQT1A in his s udy. Mo eo e , his would be in line
wi h ou p e ious inding ha EADs in LQT1B a e mo e
p onounced compa ed o LQT1A du ing an I
K
blockade wi h
E4031 a no mal physiological K
+
condi ions (Ki iaho e al., 2015).
As I
Ks
is diminished in ou LQT1 lines, he I
K
unc ionali y will
become impo an o he epola iza ion ese e du ing de elopmen
o hypokalemia. The acu e and ch onic K
+
homeos asis is complex
wi h e ec s in a a ie y o pa ame e s. Based on ou s udies we can
exclude I
Ks
as a majo con ibu o in he epola iza ion ese e in
he LQT1 lines. This is suppo ed by he ac ha only LQT1A
CMs p esen a p olonged phase 2 iangula i y and EADs, bu no
LQT1B CMs which, howe e , ha e less I
Ks
cu en based on gene
ans ec ion expe imen s (Piippo e al., 2001; Fods ad e al., 2006).
Ac ion po en ial shape has been sugges ed o play a c ucial ole in
occu ence o p o-a hy hmic e en s (Hondeghem e al., 2001;
Osadchii and Olesen, 2009). Phase 3 iangula i y is shown o be a
ma ke o p o-a hy hmia in monophasic ac ion po en ials o
guinea pig and abbi hea s (Hondeghem e al., 2001; Osadchii and
Olesen, 2009). I
K
de iciency has also been associa ed in phase 3
iangula i y (Hondeghem e al., 2001); howe e , in his s udy, we
did no ind EADs e oked in phase 3 bu in phase 2 sugges ing a
eopening o L- ype calcium channels (I
Ca,L
), i.e. in he LQT1A
CMs wi h EADs, hese epea edly occu ed be ween –15 mV and
–27 mV. I has p e iously been sugges ed ha phase 2 EADs a e
ca ied by I
Ca,L
ollowing an APD p olonga ion owa ds a mo e
nega i e memb ane po en ial while we canno exclude ha he Na
+
/
Ca
2+
-exchange wo ks in conce wi h he I
Ca,L
(Guo e al., 2007;
Sipido e al., 2007; Banyasz e al., 2012). I is cu en ly unknown
wha causes he u he p olonga ion and iangula i y in phase 2
gi ing ise o EADs ound in ou LQT1A CMs. I should be no ed
ha no a hy hmia was de ec ed and no phase 2 iangula i y was
obse ed in LQT1B and WT CMs.
Po en ial limi a ions o he s udy
The e ec s o low [K
+
]
Ex
on hiPSC-de i ed CM may no
comple ely e lec he symp oms o hypokalemia in i o. hiPSC-
de i ed CMs a e s ill mo e e al-like and his has o be conside ed
when ansla ing hese esul s o he clinics. I
Ks
and I
K
ha e no been
quan i ied in ou s udy bu hei unc ionali y has been analyzed
wi h he use o speci ic ion channel blocke s in ou p e ious s udy
(Ki iaho e al., 2015). Fu he unde s anding he in e ac ion
be ween ion channels and exchange s is needed o e ealing he
unde lying mechanisms o p olonga ion and a hy hmia obse ed
in he LQT1 lines used in his s udy. He e, we only ha e an acu e
expose and long e m exposu e o low [K
+
]
Ex
on hiPSC-de i ed
CMs may e eal di e en cha ac e is ics.
Fig. 2. Rep esen a i e cu en clamp eco dings o spon aneous ac ions
po en ials ex ac ed om single en icula -like ca diomyocy e baselines
and hei co esponding esponses o 1 mM [K
+
]
Ex
and dose-dependen
p olonga ion o APD
90
induced by [K
+
]
Ex
.(A-C) Rep esen a i e baselines
(uppe aces) and he co esponding e ec o 1 mM [K
+
]
Ex
(lowe aces) o
(A) WT, (B) LQT1A and (C) LQT1B, espec i ely. Scale ba s: abscissa 40 mV
and o dina e 2 s. Dashed lines (A-C) indica e 0 mV.
Fig. 3. Rep esen a i e cu en clamp eco dings o a hy hmia in LQT1A
ca diomyocy es. (A) A ca diomyocy e wi hou ea ly a e depola iza ion (EAD)
in baseline (uppe ace) and wi h (EAD) only obse ed a 1 mM [K
+
]
Ex
(lowe
ace). (B) Depic s a ca diomyocy e wi h EADs in baseline (uppe ace) and
EADs we e p olonged a 1 mM [K
+
]
Ex
(lowe ace). A ows poin o he
beginning and end o he a hy hmic Phase 2. Scale ba s: 40 mV in abscissa
and 2 s in o dina e. Dashed lines (A and B) indica e 0 mV. No e ha EADs we e
only ound in LQT1A ca diomyocy es wi h mo e p olonged baseline APDs (see
Fig. 2B o a compa ison).
781
RESEARCH ARTICLE Biology Open (2017) 6, 777-784 doi:10.1242/bio.024216
Biology Open

In conclusion, we ha e shown ha low [K
+
]
Ex
delays he
epola iza ion in hiPSC-de i ed CMs a single cell as well as a
mul icellula le el. The LQT1 CMs a e mo e sensi i e o po assium
elec oly e dis u bances han WT CMs, hus con i ming p e ious
clinical s udies. In LQT1 CMs wi h G589D mu a ion phase 2
iangula i y in EADs we e occasionally obse ed a baseline o
could be u he e oked by low [K
+
]
Ex
. Howe e , phase 2
iangula i y o EADs could no be e oked in LQT1 CMs wi h
IVS7-2A>G mu a ion o in WT CMs. The e ec s o low [K
+
]
Ex
has
no p e iously been in es iga ed in hiPSC-de i ed CMs. Thus, his
wo k demons a es ha he hiPSC-de i ed CMs p o ide a pla o m
o s udying he e ec s o low ex acellula K
+
and also p o ide a
pla o m o s udy acqui ed LQTS.
MATERIAL AND METHODS
E hical app o al
The s udy was app o ed by he E hics Commi ee o Pi kanmaa Hospi al
Dis ic o es ablish, cul u e, and di e en ia e hiPSC lines (R08070).
Pa icipan s dona ing skin biopsies signed an in o med consen a e
ecei ing o al and w i en desc ip ions o he s udy. Skin biopsies o hiPSC
es ablishmen we e ecei ed om he Hea Hospi al, Tampe e Uni e si y
Hospi al, Tampe e, Finland.
Long QT pa ien cha ac e is ics
The LQT pa ien cha ac e is ics ha e been p e iously desc ibed elsewhe e
(Ki iaho e al., 2015). In b ie , he LQT1 pa ien ca ying G589D mu a ion
is a 46-yea -old emale wi h co ec ed QT in e al (QTc) o 464 ms. The
o he LQT1 pa ien ca ying IVS7-2A>G is a 51-yea -old emale wi h QTc
alue o 489 ms. These pa ien s a e symp oma ic wi h episodes o syncope.
Human induced plu ipo en s em cell gene a ion and cul u e
Pa ien -speci ic hiPSCs we e gene a ed as desc ibed ea lie (Takahashi
e al., 2007). The LQT1-speci ic hiPSCs we e de i ed om pa ien s ca ying
G589D o IVS7-2A>G mu a ion in he KCNQ1 as p e iously desc ibed
(Ki iaho e al., 2015). Heal hy con ol hiPSCs (WT) we e de i ed om skin
ib oblas s o a heal hy 55-yea -old emale (Ahola e al., 2014). hiPSC lines
we e cul u ed in knockou se um eplacemen medium using mouse
emb yonic ib oblas s (Millipo e, Bille ica, MA, USA) as eede s. The
ollowing componen s we e included in he knockou se um eplacemen
medium: knockou -DMEM (In i ogen, Ca lsbad, CA, USA) con aining
20% knockou -se um eplacemen (In i ogen, Ca lsbad, CA, USA),
nonessen ial amino acids, Glu aMAX, penicillin/s ep omycin, 0.1 mM 2-
me cap oe hanol and 4 ng/ml ib oblas g ow h ac o 2 (R&D Sys ems Inc.,
Minneapolis, MN, USA). The medium was e eshed h ee imes a week and
he hiPSCs we e passaged weekly using collagenase IV (In i ogen,
Ca lsbad, CA, USA).
Cell line cha ac e iza ions and ca diomyocy e di e en ia ion
The hiPSC lines and hiPSC-de i ed CMs used in his s udy ha e been
cha ac e ized elsewhe e. The con ol cell line UTA.04602.WT (WT) and WT-
de i ed CMs ha e been cha ac e ized in de ail p e iously (Ahola e al., 2014).
The hiPSC lines UTA.00208.LQT1 (LQT1A, de i ed om pa ien ca ying
G589D mu a ion) and UTA.00118.LQT1 (LQT1B, de i ed om pa ien
ca ying IVS7-2A>G mu a ion) and CMs de i ed om hem ha e been
cha ac e ized (Ki iaho e al., 2015). The hiPSCs we e di e en ia ed in o CMs
in co-cul u e wi h mouse isce al endode m-like cells. The di e en ia ion
me hod has been desc ibed elsewhe e (Mumme y e al., 2003).
Mul ielec ode a ay eco dings and da a analysis
hiPSC-de i ed CMs (30-35 days old) we e used in his s udy. Spon aneously
bea ing CM clus e s we e manually dissec ed and pla ed on 6-well MEAs
(6-well MEA 200/30iR-Ti- c , Mul ichannel Sys ems, Reu lingen, Ge many),
which we e i s coa ed wi h e al bo ine se um (FBS, In i ogen, Ca lsbad,
CA, USA) o 30 min a oom empe a u e and hen wi h 0.1% gela in (Sigma
Ald ich, S Louis, MO, USA) o 1 h a oom empe a u e. The CM clus e s
we e pla ed in EB-medium consis ing o : knockou -DMEM wi h 20% FBS,
nonessen ial amino acids, Glu aMAX and penicillin/s ep omycin. Cus om
made K
+
ee DMEM (I ine Scien i ic, CA, USA) supplemen ed wi h a ying
concen a ions o K
+
(B. B aun, Melsungen, Ge many, KCl 150 mg/ml) and
penicillin/s ep omycin we e used in his s udy. The ollowing [K
+
]
Ex
we e
used: 5.33, 4, 3, 2 and 1 mM. The EB-medium was changed o se um- ee
cul u e medium (5.33 mM K
+
) and a e he incuba ion o 1 h, he ield
po en ials o igina ing om he CMs we e eco ded (baseline). A e baseline
eco dings, he [K
+
]
Ex
in he medium was sequen ially educed om 4 mM o
1 mM by medium exchanges wi h 45 min incuba ions in be ween each
concen a ion and eco ded a 37°C using MEAs (MEA1060-In -BC,
Mul ichannel Sys ems, Reu lingen, Ge many) using 20 kHz sampling
equency and MC_Rack so wa e (Mul ichannel Sys ems, Reu lingen,
Ge many). No e, 4 mM induced no o only ma ginal e ec s on FPD (da a
no shown), hus i has no been included o he esul sec ion o cla i y o he
p esen a ion. The MEAs we e co e ed wi h gas pe meable memb anes (ALA
MEA-Shee , ALA Scien i ic, NY, USA) du ing he eco dings. The o al
eco ding ime o each concen a ion was 2 min. The MEA da a was analyzed
by in-house-de eloped Ca dioMDA so wa e (P adhapan e al., 2013).
Ca dioMDA is a ailable om BioMediTech websi e (h p://www.
biomedi ech. i/Ca dioMDA/). The Baze ’s and F ide icia’s o mulae we e
used o calcula e he bea a e co ec ed FPD. The c i e ia o da a analysis o he
MEA eco dings was ha only he agg ega es esponding o low [K
+
]
Ex
wi h
FPD p olonga ion we e included in his s udy.
Cu en clamp
Cu en clamp eco dings we e pe o med as p e iously desc ibed (Ki iaho
e al., 2015). In b ie , he pe o a ed pa ch clamp echnique was used wi h
ampho e icin-B as pe o a ion agen wi h a inal concen a ion o 240 µg/ml
Table 3. Ac ion po en ial cha ac e is ics o LQT1A ca diomyocy es exhibi ing ea ly a e de pola iza ions
(A)
LQT1A nAPD
10
(ms) APD
50
(ms) APD
90
(ms) APA (mV) MDP (mV)
Baseline 5 286.89±59.09 1364.37±274.01 1524.56±286.56 121.89±2.72 −75.36±1.59
1mMK
+
EAD 295.81±70.51 2769.31±1124.54 3685.82±1493.54 121.86±4.42 −74.32±2.80
(B)
LQT1A nAPD
10
(ms) APD
50
(ms) APD
90
(ms) APA (mV) MDP (mV)
Baseline EAD 2 179.64±2.91 1023.49±117.23 2059.02±883.91 107.99±6.92 −65.48±5.68
3mMK
+
EAD 199.24±26.70 1194.39±365.80 3193.29±1781.73 110.62±4.13 −67.69±4.78
Baseline EAD 2 194.58±27.72 894.13±110.15 1170.46±242.60 116.66±2.39 −68.79±0.66
2mMK
+
EAD 205.33±36.64 1018.42±168.47 1637.03±572.69 116.21±1.32 −68.06±2.76
Baseline EAD 4 175.03±34.07 1048.61±98.32 1674.84±426.04 115.69±3.29 −69.45±2.07
1mMK
+
EAD 219.81±54.40 1822.10±270.95* 3088.91±709.53 121.05±3.55* −74.44±2.24*
(A) LQT1A ca diomyocy es wi hou ea ly a e depola iza ion (EAD) a baseline bu obse ed a 1 mM [K
+
]
Ex
wi h hei espec i e ac ion po en ial cha ac e is ics.
(B) LQT1A ca diomyocy es wi h EAD a baseline and low [K
+
]
Ex,
wi h hei espec i e ac ion po en ial cha ac e is ics. S a is ical signi icance (A) and (B) was
calcula ed by using pai ed - es , *P<0.05. n=ca diomyocy es eco ded o each da a poin . Da a is p esen ed as mean±s.e.m.
782
RESEARCH ARTICLE Biology Open (2017) 6, 777-784 doi:10.1242/bio.024216
Biology Open
(Hamill e al., 1981; Rae e al., 1991). Ac ion po en ials we e acqui ed wi h
an Axon Se ies 200B ampli ie and a Digida a 1440 AD/DA con e e
(Molecula de ice, LTD, USA). Dissocia ed CMs we e pla ed on 5 mmØ
co e slips and eco ded on he 6 h o 7 h day. In b ie , co e slips we e
ans e ed o a RC-24N eco ding chambe (Ha a d Ins umen s UK,
Wa ne Ins umen s, USA) and placed on an in e ed Olympus IX71
mic oscope. The pe usa e was main ained a 35-36°C using an SH-27B
inline hea e (Ha a d Appa a us L d., Ken , UK). The HEPES based
ex acellula solu ion (HBS) con ained (in mM): 143 NaCl, 1.8 CaCl
2
,
1.2 MgCl
2
, 5 glucose and 10 HEPES. Fo expe imen s KCl was added o he
HBS o inal concen a ions o 1, 2, 3 and 4.8 mM K
+
(pH was se o 7.4
wi h NaOH and osmola i y se o 300-302 mOsm wi h suc ose). HBS wi h
4.8 mM K
+
was used in baseline eco dings. The pipe e solu ion con ained
(in mM): 122 KMeSO
4
, 30 KCl, 1.2 MgCl
2
, 1 CaCl
2
(pH was se o 7.2 wi h
KOH and osmola i y se o 290-292mOsm wi h suc ose). The pipe e
esis ance was ∼3MΩa e illing wi h pipe e solu ion. The ac ion
po en ials om spon aneously bea ing CMs we e eco ded in cu en clamp
mode. Cu en clamp eco dings we e digi ally sampled a 20 kHz and
il e ed a 2 kHz using low pass Bessel il e on eco ding ampli ie . AP
du a ion a 10, 50 and 90% epola iza ion (APD
10
, APD
50
and APD
90
),
APA, and MDP we e ex ac ed om eco ded ac ion po en ials using in
house de eloped analysis modules unning on he O igin 9 pla o m
(Mic ocal O igin
TM
, No hamp on, Massachuse s, USA). Baseline da a in a
CM was ex ac ed om a minimum o 15 single APs and summed o
s a is ics. When EADs we e p esen in a CM, a minimum o 5 single APs
we e ob ained in egions wi h maximal e ec o [K
+
]
Ex
and summed o
each concen a ion o s a is ics. Acu e applica ion o [K
+
]
Ex
was es ed in
sequen ial applica ion wi hou wash ou , wi h wash ou and wi h single
applica ions om baselines o ei he 3, 2 o 1 mM [K
+
]
Ex
. Maximal e ec
was obse ed in seconds and eco dings we e ully e e sible, hus da a is
pools o hese se ies. The da a p esen ed om cu en clamp was om
en icula -like CMs. These we e g ouped as en icula -like when baseline
APs had APD
90/50
<1.3, APA >100 mV and MDP <−60 mV. The Phase 2
AP iangula i y was calcula ed as APD
50/10
.
S a is ical analyses
One-way ANOVA ollowed by Tukey’s pos hoc es was used o assess
di e ences in means o g oups o h ee pai s. Pai ed - es was used o access
di e ence be ween he means in baseline and he co esponding e ec o 3,
2 o 1 mM K
+
in he same eco ding, while unpai ed - es was uses o access
di e ences in means o wo simila eco dings (Mic ocal O igin™9.1,
No hamp on, Massachuse s, USA). Signi ican di e ence in he ables is
p esen ed as *P<0.05, **P<0.01 and ***P<0.001 and da a is p esen ed as
mean±s.e.m.
Acknowledgemen s
We kindly hank Ma kus Haponen and Henna Lappi o hei echnical expe ise wi h
he s em cell cul u e and ca diac di e en ia ion. The END-2 cells we e a kind gi om
P o Ch is ine Mumme y, Uni e si y o Twen e, Ne he lands.
Compe ing in e es s
The au ho s decla e no compe ing o inancial in e es s.
Au ho con ibu ions
Concep ualiza ion: J.K., K.L., K.A.-S.; Me hodology: J.K., K.L.; Valida ion: J.K., K.L.;
Fo mal analysis: J.K., K.L., D.S., C.P., K.A-S.; In es iga ion: J.K., K.L., C.P.; W i ing -
o iginal d a : J.K., K.L.; W i ing - e iew & edi ing: J.K., K.L., D.S., C.P., K.A.-S.;
Visualiza ion: J.K., K.L.; Supe ision: K.A.-S.; P ojec adminis a ion: K.A.-S.;
Funding acquisi ion: K.A.-S.
Funding
This s udy was suppo ed by g an s ecei ed om Aa ne Koskelon Sa
a
 io
, Cen e o
In e na ional Mobili y (CIMO), Finnish Cul u al Founda ion Pi kanmaan Rahas o,
Finnish Ca dio ascula Founda ion, TEKES- he Finnish Funding Agency o
Inno a ion and he S a e Resea ch Funding o Tampe e Uni e si y Hospi al.
Re e ences
Ahola, A., Ki iaho, A. L., La sson, K., Honkanen, M., Aal o-Se a
la
, K. and
Hy inen, J. (2014). Video image-based analysis o single human induced
plu ipo en s em cell de i ed ca diomyocy e bea ing dynamics using digi al image
co ela ion. Biomed. Eng. Online 13, 39.
Aki a, M., Kuwaha a, M., Tsubone, H. and Sugano, S. (1998). ECG changes
du ing u osemide-induced hypokalemia in he a . J. Elec oca diol. 31, 45-49.
A omola an, A. S., Sub amanyam, P., Chang, D. D., Kobe z, W. R. and
Colec a , H. M. (2014). LQT1 mu a ions in KCNQ1 C- e minus assembly domain
supp ess IKs using di e en mechanisms. Ca dio asc. Res. 104, 501-511.
Banyasz, T., Ho a h, B., Jian, Z., Izu, L. T. and Chen-Izu, Y. (2012). P o ile o
L- ype Ca(2+) cu en and Na(+)/Ca(2+) exchange cu en du ing ca diac ac ion
po en ial in en icula myocy es. Hea Rhy hm. 9, 134-142.
Ba hanin, J., Lesage, F., Guillema e, E., Fink, M., Lazdunski, M. and Romey, G.
(1996). K(V)LQT1 and lsK (minK) p o eins associa e o o m he I(Ks) ca diac
po assium cu en . Na u e 384, 78-80.
Bellin, M., Casini, S., Da is, R. P., D’Aniello, C., Haas, J., Wa d- an Oos waa d,
D., Te oolen, L. G. J., Jung, C. B., Ellio , D. A., Welling, A. e al. (2013).
Isogenic human plu ipo en s em cell pai s e eal he ole o a KCNH2 mu a ion in
long-QT synd ome. EMBO J. 32, 3161-3175.
Chan, Y.-H., Tsai, W.-C., Ko, J.-S., Yin, D., Chang, P.-C., Ruba , M., Weiss, J. N.,
E e e , T. H., Lin, S.-F. and Chen, P.-S. (2015). Small-conduc ance calcium-
ac i a ed po assium cu en is ac i a ed du ing hypokalemia and masks sho -
e m ca diac memo y induced by en icula pacing. Ci cula ion 132, 1377-1386.
Egashi a, T., Yuasa, S., Suzuki, T., Aizawa, Y., Yamakawa, H., Ma suhashi, T.,
Ohno, Y., Tohyama, S., Oka a, S., Seki, T. e al. (2012). Disease cha ac e iza ion
using LQTS-speci ic induced plu ipo en s em cells. Ca dio asc. Res. 95,
419-429.
Fods ad, H., Bendahhou, S., Rougie , J. S., Lai inen-Fo sblom, P. J., Ba hanin,
J., Ab iel, H., Schild, L., Kon ula, K. and Swan, H. (2006). Molecula
cha ac e iza ion o wo ounde mu a ions causing long QT synd ome and
iden i ica ion o compound he e ozygous pa ien s. Ann. Med. 38, 294-304.
Genna i, F. J. (1998). Hypokalemia. New Engl. J. Med. 339, 451-458.
Guo, D., Zhao, X., Wu, Y., Liu, T., Kowey, P. R. and Yan, G.-X. (2007). L- ype
calcium cu en eac i a ion con ibu es o a hy hmogenesis associa ed wi h
ac ion po en ial iangula ion. J. Ca dio asc. Elec ophysiol. 18, 196-203.
Guo, J., Wang, T., Yang, T., Xu, J., Li, W., F idman, M. D., Fishe , J. T. and Zhang,
S. (2011). In e ac ion be ween he ca diac apidly (I K ) and slowly (I Ks) ac i a ing
delayed ec i ie po assium channels e ealed by low K +-induced he g endocy ic
deg ada ion. J. Biol. Chem. 286, 34664-34674.
Hamill, O. P., Ma y, A., Nehe , E., Sakmann, B. and Sigwo h, F. J. (1981).
Imp o ed pa ch-clamp echniques o high- esolu ion cu en eco ding om cells
and cell- ee memb ane pa ches. P luge s A ch. 391, 85-100.
Hondeghem, L. M., Ca lsson, L. and Duke , G. (2001). Ins abili y and iangula ion
o he ac ion po en ial p edic se ious p oa hy hmia, bu ac ion po en ial du a ion
p olonga ion is an ia hy hmic. Ci cula ion 103, 2004-2013.
I zhaki, I., Maizels, L., Hube , I., Zwi-Dan sis, L., Caspi, O., Win e s e n, A.,
Feldman, O., Geps ein, A., A bel, G., Hamme man, H. e al. (2011). Modelling
he long QT synd ome wi h induced plu ipo en s em cells. Na u e 471, 225-229.
Kishida, H., Su awicz, B. and Fu, L. T. (1979). E ec s o K+ and K+-induced
pola iza ion on (dV/d )max, h eshold po en ial, and memb ane inpu esis ance in
guinea pig and ca en icula myoca dium. Ci c. Res. 44, 800-814.
Ki iaho, A. L., Ahola, A., La sson, K., Pen inen, K., Swan, H., Pekkanen-
Ma ila, M., Vena
la
inen, H., Paa ola, K., Hy inen, J. and Aal o-Se a
la
,K.
(2015). Dis inc elec ophysiological and mechanical bea ing pheno ypes o long
QT synd ome ype 1-speci ic ca diomyocy es ca ying di e en mu a ions.
In . J. Ca diol. Hea Vasc. 8, 19-31.
Lah i, A. L., Kujala, V. J., Chapman, H., Koi is o, A.-P., Pekkanen-Ma ila, M.,
Ke kela, E., Hy inen, J., Kon ula, K., Swan, H., Conklin, B. R. e al. (2012).
Model o long QT synd ome ype 2 using human iPS cells demons a es
a hy hmogenic cha ac e is ics in cell cul u e. Dis. Model. Mech. 5, 220-230.
Liu, G.-X., Choi, B.-R., Zi , O., Li, W., de Lange, E., Qu, Z. and Ko en, G. (2012).
Di e en ial condi ions o ea ly a e -depola iza ions and igge ed ac i i y in
ca diomyocy es de i ed om ansgenic LQT1 and LQT2 abbi s. J. Physiol. 590,
1171-1180.
Ma, D., Wei, H., Zhao, Y., Lu, J., Li, G., Sahib, N. B. E., Tan, T. H., Wong, K. Y.,
Shim, W., Wong, P. e al. (2013). Modeling ype 3 long QT synd ome wi h
ca diomyocy es de i ed om pa ien -speci ic induced plu ipo en s em cells.
In . J. Ca diol. 168, 5277-5286.
Macdonald, J. E. and S u he s, A. D. (2004). Wha is he op imal se um po assium
le el in ca dio ascula pa ien s? J. Am. Coll. Ca diol. 43, 155-161.
Malan, D., F ied ichs, S., Fleischmann, B. K. and Sasse, P. (2011).
Ca diomyocy es ob ained om induced plu ipo en s em cells wi h Long-QT
synd ome 3 ecapi ula e ypical disease-speci ic ea u es in i o. Ci c. Res. 109,
841-847.
Ma jamaa, A., Salomaa, V., New on-Cheh, C., Po han, K., Reunanen, A.,
Ka anko, H., Jula, A., Lahe mo, P., Va
a
na
nen, H., Toi onen, L. e al. (2009).
High p e alence o ou long QT synd ome ounde mu a ions in he innish
popula ion. Ann. Med. 41, 234-240.
Ma sa, E., Rajamohan, D., Dick, E., Young, L., Mello , I., S ani o h, A. and
Denning, C. (2011). D ug e alua ion in ca diomyocy es de i ed om human
induced plu ipo en s em cells ca ying a long QT synd ome ype 2 mu a ion. Eu .
Hea J. 32, 952-962.
Mo e i, A., Bellin, M., Welling, A., Jung, C. B., Lam, J. T., Bo -Flu
gel, L., Do n,
T., Goedel, A., Ho
hnke, C., Ho mann, F. e al. (2010). Pa ien -speci ic induced
783
RESEARCH ARTICLE Biology Open (2017) 6, 777-784 doi:10.1242/bio.024216
Biology Open
plu ipo en s em-cell models o long-QT synd ome. New Engl. J. Med. 363,
1397-1409.
Mumme y, C., Wa d- an Oos waa d, D., Doe endans, P., Spijke , R., an den
B ink, S., Hassink, R., an de Heyden, M., Op ho , T., Pe a, M., de la Ri ie e,
A. B. e al. (2003). Di e en ia ion o human emb yonic s em cells o
ca diomyocy es: ole o cocul u e wi h isce al endode m-like cells. Ci cula ion
107, 2733-2740.
Osadchii, O. E. (2010). Mechanisms o hypokalemia-induced en icula
a hy hmogenici y. Fundam. Clin. Pha macol. 24, 547-559.
Osadchii, O. E. and Olesen, S. P. (2009). Elec ophysiological de e minan s o
hypokalaemia-induced a hy hmogenici y in he guinea-pig hea . Ac a Physiol.
197, 273-287.
Pal iel, O., Salakho , E., Ronen, I., Be g, D. and Is aeli, A. (2001). Managemen o
se e e hypokalemia in hospi alized pa ien s: a s udy o quali y o ca e based on
compu e ized da abases. A ch. In e n. Med. 161, 1089-1095.
Pezhouman, A., Singh, N., Song, Z., Ni ala, M., Eskanda i, A., Cao, H., Bapa ,
A., Ko, C. Y., Nguyen, T. P., Qu, Z. e al. (2015). Molecula basis o hypokalemia-
induced en icula ib illa ion. Ci cula ion 132, 1528-1537.
Piippo, K., Swan, H., Pas e nack, M., Chapman, H., Paa onen, K., Vii asalo, M.,
Toi onen, L. and Kon ula, K. (2001). A ounde mu a ion o he po assium
channel KCNQ1 in long QT synd ome: Implica ions o es ima ion o disease
p e alence and molecula diagnos ics. J. Am. Coll. Ca diol. 37, 562-568.
P adhapan, P., Kuusela, J., Viik, J., Aal o-Se a
la
, K. and Hy inen, J. (2013).
Ca diomyocy e MEA da a analysis (Ca dioMDA)–a no el ield po en ial da a
analysis so wa e o plu ipo en s em cell de i ed ca diomyocy es. PLoS One 8,
e73637.
Rae, J., Coope , K., Ga es, P. and Wa sky, M. (1991). Low access esis ance
pe o a ed pa ch eco dings using ampho e icin B. J. Neu osci. Me hods. 37,
15-26.
Roden, D. M. (1997). A p ac ical app oach o o sade de poin es. Clin. Ca diol. 20,
285-290.
Roden, D. M. (2004). D ug-induced p olonga ion o he QT in e al. New
Engl. J. Med. 350, 1013-1022.
Ruiz Ce e i, E., Ponce Zumino, A., Blaney, R. and Cha ie , D. (1982).
Expe imen al a hy hmia elici ed by low K pe usion. Can. J. Physiol.
Pha macol. 60, 1533-1540.
Sanguine i, M. C. and Ju kiewicz, N. K. (1992). Role o ex e nal Ca2+ and K+ in
ga ing o ca diac delayed ec i ie K+ cu en s. P lu
ge s A ch. 420, 180-186.
Sanguine i, M. C., Cu an, M. E., Zou, A., Shen, J., Spec o , P. S., A kinson,
D. L. and Kea ing, M. T. (1996). Coassembly o K(V)LQT1 and minK (IsK)
p o eins o o m ca diac I(Ks) po assium channel. Na u e 384, 80-83.
Scamps, F. and Ca melie , E. (1989). Delayed K+ cu en and ex e nal K+ in single
ca diac Pu kinje cells. Am. J. Physiol. Cell Physiol. 257, C1086-C1082.
Schulman, M. and Na ins, R. G. (1990). Hypokalemia and ca dio ascula disease.
Am. J. Ca diol. 65.
Schwa z, P. J., Acke man, M. J., Geo ge, A. L., J and Wilde, A. A. M. (2013).
Impac o gene ics on he clinical managemen o channelopa hies. J. Am. Coll.
Ca diol. 62, 169-180.
Sipido, K. R., Bi o, V., An oons, G., Volde s, P. G. and Vos, M. A. (2007). Na/Ca
exchange and ca diac en icula a hy hmias. Ann. New Yo k Acad. Sci. 1099,
339-348.
Spence , C. I., Baba, S., Nakamu a, K., Hua, E. A., Sea s, M. A. F., Fu, C.-C.,
Zhang, J., Balijepalli, S., Tomoda, K., Hayashi, Y. e al. (2014). Calcium
ansien s closely e lec p olonged ac ion po en ials in iPSC models o inhe i ed
ca diac a hy hmia. S em Cell Rep. 3, 269-281.
S ewa , D. E., Ik am, H., Espine , E. A. and Nicholls, M. G. (1985).
A hy hmogenic po en ial o diu e ic induced hypokalaemia in pa ien s wi h mild
hype ension and ischaemic hea disease. B . Hea J. 54, 290-297.
Takahashi, K., Tanabe, K., Ohnuki, M., Na i a, M., Ichisaka, T., Tomoda, K. and
Yamanaka, S. (2007). Induc ion o plu ipo en s em cells om adul human
ib oblas s by de ined ac o s. Cell 131, 861-872.
Teng, G., Zhao, X., C oss, J. C., Li, P., Lees-Mille , J. P., Guo, J., Dyck, J. R. B.
and Du , H. J. (2004). P olonged epola iza ion and igge ed ac i i y induced by
adeno i al exp ession o HERG N629D in ca diomyocy es de i ed om s em
cells. Ca dio asc. Res. 61, 268-277.
Unwin, R. J., Lu , F. C. and Shi ley, D. G. (2011). Pa hophysiology and
managemen o hypokalemia: a clinical pe spec i e. Na . Re . Neph ol. 7, 75-84.
Va o, A. and Baczko,I.(2011). Ca diac en icula epola iza ion ese e: a
p inciple o unde s anding d ug- ela ed p oa hy hmic isk. B . J. Pha macol. 164,
14-36.
Whi e, E. and Te a , D. A. (1991). Ac ion po en ial du a ion and he ino opic
esponse o educed ex acellula po assium in guinea-pig en icula myocy es.
Exp. Physiol. 76, 705-716.
Yang, T., Snyde s, D. J. and Roden, D. M. (1997). Rapid inac i a ion de e mines
he ec i ica ion and [K+](o) dependence o he apid componen o he delayed
ec i ie K+ cu en in ca diac cells. Ci c. Res. 80, 782-789.
Yazawa, M., Hsueh, B., Jia, X., Pasca, A. M., Be ns ein, J. A., Hallmaye , J. and
Dolme sch, R. E. (2011). Using induced plu ipo en s em cells o in es iga e
ca diac pheno ypes in Timo hy synd ome. Na u e 471, 230-234.
Yu, J., Vodyanik, M. A., Smuga-O o, K., An osiewicz-Bou ge , J., F ane, J. L.,
Tian, S., Nie, J., Jonsdo i , G. A., Ruo i, V., S ewa , R. e al. (2007). Induced
plu ipo en s em cell lines de i ed om human soma ic cells. Science 318,
1917-1920.
Zaza, A. (2009). Se um po assium and a hy hmias. Eu opace 11, 421-422.
784
RESEARCH ARTICLE Biology Open (2017) 6, 777-784 doi:10.1242/bio.024216
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