Low extracellular potassium prolongs repolarization and evokes early afterdepolarization in human induced pluripotent stem cell-derived cardiomyocytes

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 RESEARCH ARTICLE Biology Open (2017) 6, 777-784 doi:10.1242/bio.024216 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. 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