The Effects of Pharmacological Compounds on Beat Rate Variations in Human Long QT-Syndrome Cardiomyocytes

The E ec s o Pha macological Compounds on Bea Ra e Va ia ions in Human Long QT-Synd ome Ca diomyocy es Jukka Kuusela 1,2 &Jiyeong Kim 3 &Esa Räsänen 3 &Ka iina Aal o-Se älä 1,2,4,5 Published online: 19 Sep embe 2016 #The Au ho (s) 2016. This a icle is published wi h open access a Sp inge link.com Abs ac Heal hy human hea a e luc ua es o e ime show- ing long- ange ac al co ela ions. In con as , a ious ca diac diseases and no mal aging show he b eakdown o ac al complexi y. Recen ly, i was shown ha human induced plu- ipo en s em cell-de i ed ca diomyocy es (hiPSC-CMs) in- insically exhibi ac al beha io as in humans. He e, we in es iga ed he ac al complexi y o hiPSC-de i ed long QT-ca diomyocy es (LQT-CMs). We eco ded ex acellula ield po en ials om hiPSC-CMs a baseline and unde he e ec o a ious compounds including β-blocke bisop olol, ML277, a speci ic and po en I Ks cu en ac i a o , as well as JNJ303, a speci ic I Ks blocke . F om he peak- o-peak-in e - als, we de e mined he long- ange ac al co ela ions by using de ended luc ua ion analysis. Elec ophysiologically, he baseline co ec ed ield po en ial du a ions (cFPDs) we e mo e p olonged in LQT-CMs han in wild ype (WT)-CMs. Bisop olol did no ha e signi ican e ec s o he cFPD in any CMs. ML277 sho ened cFPD in a dose-dependen ash- ionby11%and5–11 % in WT- and LQT-CMs, espec i ely. JNJ303 p olonged cFPD in a dose-dependen ashion by 22 % and 7–13 % in WT- and LQT-CMs, espec i ely. A baseline, all CMs showed ac al co ela ions as de e mined by sho - e m scaling exponen α. Howe e , in all CMs, he αwas inc eased when pha macological compounds we e applied in- dica ing o b eakdown o ac al complexi y. These indings sugges ha he in insic mechanisms con ibu ing o he ac- al complexi y a e no al e ed in LQT-CMs. The modula ion o I Ks channel and β1-ad eno ecep o s by pha macological com- pounds may a ec he ac al complexi y o he hiPSC-CMs. Keywo ds Induced plu ipo en s em cell .Long QT synd ome .Ca diomyocy es .Mul ielec ode a ay . De ended luc ua ion analysis .F ac als .Nonlinea dynamics In oduc ion Hea a e dynamics ha e been p e iously analyzed using con en ional linea and newe nonlinea me hods in heal hy and diseased s a es ( o e iew see Pe kiömäki [1]). The non- linea indices o he RR a ia ions include ac als, which a e geome ically de ined as objec s composed o subuni s (and sub-subuni s) ha sus ain sel -simila i y on di e en measu e- men scales [2]. The cha ac e is ic ea u e o ac als is 1/ -like luc ua ions, which has been shown o be p esen in a heal hy human hea bea [3–6]. Such luc ua ions possess long- ange co ela ions indica i e o a memo y e ec , which means ha he hea a e is no only ela ed o immedia ely p eceding alue bu also o alues in he emo e pas [2]. Elec onic supplemen a y ma e ial The online e sion o his a icle (doi:10.1007/s12015-016-9686-0) con ains supplemen a y ma e ial, which is a ailable o au ho ized use s. *Ka iina Aal o-Se älä ka iina.aal o-se ala@u a. i Jukka Kuusela jukka.kuusela@u a. i Esa Räsänen esa. asanen@ u . i 1 Ins i u e o Biomedical Technology, Uni e si y o Tampe e, Tampe e, Finland 2 BioMediTech, Tampe e, Finland 3 Depa men o Physics, Tampe e Uni e si y o Technology, Tampe e, Finland 4 School o Medicine, Uni e si y o Tampe e and Tampe e Uni e si y Hospi al, Finn-Medi 5, Bioka u 12, FI-33014 Tampe e, Finland 5 Hea Hospi al, Tampe e Uni e si y Hospi al, Tampe e, Finland S em Cell Re and Rep (2016) 12:698–707 DOI 10.1007/s12015-016-9686-0 In he hea a e ime se ies, a b eakdown o 1/ -like luc- ua ions may lead in o ei he comple ely unco ela ed andom- ness (whi e noise) o in o o al co ela ion esembling andom walk (B ownian mo ion). A numbe o s udies ha e shown ha in a ious ca diac disease s a es (e.g. conges i e hea ailu e, myoca dial in a c ion) he ac al 1/ -like long- ange co ela ions o he hea bea b eakdown p oducing mo e un- co ela ed andomness [7–13]. Fu he mo e, no mal aging has been associa ed wi h he b eakdown o ac al complexi y p oducing mo e o al co ela ion in he hea bea dynamics [6,14,15]. Thus, i is hough ha he b eakdown o ac al complexi y in hea a e dynamics may cause he sys em o be less adap able and less esponsi e o unp edic able s imuli and s esses inc easing suscep ibili y o inju y and illness [6,16]. The biological o igin o he ac al-like beha io has no been ye ully es ablished and is somewha con adic o y. In he hea , he o igin o he ac al-like beha io is hough o esul om complex in e ac ion be ween agal and sympa he - ic inpu s o he au onomous ne ous sys em [17,18]. Expe imen al obse a ions in humans ha e suppo ed his no- ion [19–23], al hough opposing iews ha e been p esen ed [24]. Howe e , e idence sugges s ha monolaye cul u es o a en icula ca diomyocy es and human induced plu ipo en s em cell-de i ed ca diomyocy es (hiPSC-CMs) lacking au o- nomic con ol exhibi ac al-like complexi y, which indica es ha he au onomous ne ous sys em inpu is no necessa y o ac al dynamics [25,26]. I has been u he demons a ed ha single ca diomyocy es also exhibi ac al-like complexi y and ha in acellula Ca 2+ -cycling mechanisms con ibu e o he ac al complexi y o he single ca diomyocy es [27]. Long QT synd ome (LQTS) is a po en ially se e e li e- h ea ening a hy hmic ca diac disease cha ac e ized by p olonged QT in e al on he elec oca diog am. LQTS is asso- cia ed wi h o sades de poin es, a special ype o en icula achy- ca dia, which may degene a e in o en icula ib illa ion and cause sudden ca diac dea h [28]. Inhe i ed o ms o LQTS a e a esul o mu a ions in he ca diac ion channel coding genes. One o he mos common LQTS genes is KCNQ1, which encodes he α-subuni o he ol age-ga ed po assium channel esponsible o he slow delayed ec i ie K + cu en (I Ks )[29,30]. In Finland, he p e alence o gene mu a ions associa ed wi h LQTS is high (0.4 % o Finnish popula ion), which has been explained o be caused by ou ounde mu a ions wi h one o hem being C- e minal G589D missense mu a ion in KCNQ1 gene [31,32]. The hiPSCs ep esen excellen esea ch ool o s udy he pa ho- physiology o inhe i ed ca diac diseases [33–41]. Un il now, he ac al dynamics o hiPSC-LQT-CMs ha e no been in es iga ed. He e, we u ilize pa ien -speci ic LQTS disease model [41] o s udy he ac al dynamics in he symp oma ic and asymp- oma ic LQTS ype 1 (LQT1)-speci ic CMs ca ying Finnish ounde mu a ion G589D. The aim o his s udy was o in es- iga e he ac al dynamics o LQT-CMs in unmedica ed and medica ed condi ions as compa ed o heal hy con ol and unde he e ec o a ious compounds a ec ing ca diac ac ion po en ial. Ma e ial & Me hods ECG Reco dings and Human Induced Plu ipo en S em Cell Gene a ion The s udy was app o ed by he e hical commi ee o Pi kanmaa Hospi al Dis ic (R08070). Pa icipan s who olun ee ed o he s udy ga e hei consen . The ECGs we e eco ded using MARS-Hol e om a heal hy indi idual, asymp oma ic LQT-mu a ion ca ie and symp oma ic LQT- pa ien . The LQT-pa ien s a e on bisop olol medica ion. The heal hy indi idual has no medica ion. Human iPSCs we e gene a ed as desc ibed ea lie [42]. The LQT1-speci ic hiPSCs we e de i ed om pa ien s’skin ib oblas s ca ying G589D missense mu a ion in KCNQ1 [41,43]. Pa ien Cha ac e is ics Skin biopsies wi h LQT1 mu a ion we e ob ained om a symp oma ic 41-yea old emale pa ien (QTc in e al, 456 ms) and om an asymp oma ic 28-yea old emale mu a- ion ca ie (QTc in e al, 428 ms). Bo h ca y he KCNQ1 G589D mu a ion. The symp oma ic 41-yea old pa ien had expe ienced seizu es, episodes o unconsciousness and synco- pe be o e β-blocke (bisop olol) medica ion. The heal hy con- ol human iPS cells we e de i ed om skin ib oblas s o a heal hy 55-yea old emale (QTc in e al, 406 ms) [44]. Human Induced Plu ipo en S em Cell Cul u e, Di e en ia ion and Cha ac e iza ion Human iPS cells we e cul u ed and di e en ia ed as p e ious- ly desc ibed [43]. All he hiPSC lines (UTA.04602.WT, UTA.00208.LQT1, UTA.00211.LQT1, UTA.00303.LQT1 and UTA.00313.LQT1) and he di e en ia ed CMs om hem ha e been p e iously cha ac e ized elsewhe e [41,43,44]. Mul ielec ode A ay Reco dings and Da a Analysis In his s udy, 30–45 days old hiPSC-CMs we e used o he expe imen s. Spon aneously bea ing ca diomyocy e 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) o 30 min a oom empe a u e and hen wi h 0.1 % gela ine (Sigma Ald ich) o 1 h a oom empe - a u e. The ca diomyocy e clus e s we e cul u ed in EB-medi- um: KO-DMEM wi h 20 % FBS, NEAA, Glu amax and pen- icillin/s ep omycin. The expe imen s we e conduc ed in 5 % S em Cell Re and Rep (2016) 12:698–707 699 FBS con aining EB-medium (5 % EB-medium). Be o e d ug es s, he ield po en ials o igina ing om he spon aneously bea ing ca diomyocy es we e eco ded o 30 min (baseline) a +37 °C wi h he MEA pla o m (MEA2100-2 × 60–2, Mul ichannel Sys ems, Reu lingen, Ge many) using 10 kHz sampling equency and MC_Rack (Mul ichannel Sys ems, Reu lingen, Ge many) so wa e. A e he 30-min baseline measu emen , he MEA pla e was pu on +37 °C he mal pla e (Tokai Hi , Japan) o keeping he empe a u e s able while adding d ugs. The ollowing d ugs we e used in he s udy: Bisop olol (Sigma-Ald ich), ML277 (Toc is Bioscience) and JNJ303 (Toc is Bioscience). The d ugs we e dissol ed in di- me hyl sul oxide (DMSO, Sigma-Ald ich) acco ding o man- u ac u e ’s ins uc ions. The bisop olol concen a ions we e chosen based on i s he apeu ic blood se um concen a ion ange [45]. Fo bisop olol, 260 nM (uppe limi o he he a- peu ic se um concen a ion) and 520 nM ( wice he uppe limi o he he apeu ic concen a ion) concen a ions we e used. ML277 (I Ks channel ac i a o ) concen a ions o 1 μMand 2μM we e chosen based on p e ious epo s [46,47]. The concen a ions o I Ks blocke JNJ303 (300 nM and 1000 nM) we e chosen based on ou p e ious s udy [41]. A e d ug addi ion, he MEA pla es we e incuba ed o 5 min a +37 °C he mal pla e be o e he 30-min measu emen ( i s d ug concen a ion). A e his, we added mo e d ugs o he cells (second d ug concen a ion) and simila ly as be o e, e- co ded he ield po en ials o 30 min. We also conduc ed ehicle con ol expe imen s wi h simila p o ocol as desc ibed abo e, wi h he excep ion ha no d ugs bu only DMSO (0.1 %) was added o he cells. The eco ding ime o baseline and o each d ug concen a ion was 30 min. The da a ob ain- ed om MEA was analyzed by ou in-house de eloped Ca dioMDA so wa e, which a e ages ield po en ial signals using c oss co ela ion algo i hms [48]. F om each eco ding, he las 2 min om he 30-min eco ding we e chosen o a e aging he ield po en ial signals. Fo de e mining he ield po en ial du a ion (FPD), he onse was de e mined as he beginning o depola izing peak and he o se as T max o he epola izing wa e. The Baze ’s and F ide icia’s o mula we e used o calcula e he co ec ed ield po en ial du a ion (cFPD). De ended Fluc ua ion Analysis We applied de ended luc ua ion analysis (DFA) o he RR- in e als. DFA is one o he mos used ime-se ies analysis me hods ha gi es a eliable es ima e o he exis ence and he cha ac e is ics o long- ange co ela ions in he da a [7]. DFA has been applied in a ious ields o science ang- ing om physiological signals such as hea bea and gai [49] o, o example, musical hy hms [50,51], ain all s a is ics [52], s uc u al p ope ies o DNA [53], and elec onic quan um anspo [54]. A de ailed desc ip ion o DFA can be ound in he abo e- lis ed e e ences and he e we only summa ize he main s eps. Fi s , we ake he peak- o-peak in e als o he hiPSC-CM o ECG da a se and sub ac he mean alue, so ha we conside he luc ua ions a ound he mean. Nex , we in eg a e he se ies by aking a cumula i e sum o he luc ua ions. The ime axis is hen di ided in o non-o e lapping windows, and in each window, a leas -squa es line ( end) is i o he da a. The oo -mean-squa e de ia ions om he end ( esiduals) a e a - e aged h ough he whole da a se . This p ocedu e is epea ed o di e en window sizes. As a esul , we ge ela ionship be ween he window size and he a e age luc ua ion (wi hin ha window size). The slope in his plo in a log-log scale co esponds o he DFA exponen α. Whi e noise wi h no co ela ion be ween consecu i e alues has α=0.5,whe eas B ownian mo ion wi h s ongly co ela ed alues gene a ed by unco ela ed consecu i e inc emen s has α=1.5.Ingen- e al, in e media e p edic abili y be ween hese limi s wi h 0.5 < α≤1.5 indica es long- ange ( ac al) co ela ions. An i-co ela ions a e cha ac e ized by −0.5 < α< 0.5. The special case o pink noise α= 1 co esponds o 1/ beha io . S a is ical Analyses F om MEA da a, one-way ANOVA ollowed by Dunne ’spos hoc es was pe o med o es di e ences in baseline alues be ween con ol and LQT cell lines (IBM e sion 22.0; SPSS Inc., Chicago, USA). I da ase s did no mee ANOVA equi e- men s (no mal dis ibu ion, equal a iances), nonpa ame ic es Mann-Whi ney U ollowed by alpha co ec ion was employed o compa e con ol cell line and LQT cell lines. In d ug expe - imen s, he baseline and he e ec o d ug concen a ions we e compa ed using pai ed sample - es . Simila ly, i da ase s did no mee he equi emen s o - es , nonpa ame ic Wilcoxon es was employed. F om DFA da a, pai ed sample - es was employed o de e mine he s a is ical di e ences be ween base- line and each d ug concen a ion. The p< 0.05 was conside ed s a is ically signi ican . The le els o signi icance a e ep esen - ed as (*) p < 0.05, (**) p<0.01and(***)p< 0.001. The cFPD p olonga ions o >10 % a e conside ed physiologically signi - ican . The da a is p esen ed as mean ± s anda d de ia ion (SD). Resul s The E ec o Pha macological Compounds o he hiPSC-CM Clus e s The 208.LQT1 and 211.LQT1 we e de i ed om symp om- a ic pa ien whe eas 303.LQT1 and 313.LQT1 we e de i ed om asymp oma ic mu a ion ca ie . The baseline cFPDs (F ide icia’s co ec ion) we e signi ican ly mo e p olonged in hiPSC-LQT-CMs han in heal hy WT-CMs (Fig. 1b, d, ). 700 S em Cell Re and Rep (2016) 12:698–707 Simila esul s we e ob ained when Baze ’s FPD co ec ion was used (Supplemen al Fig. 1). In con as , we did no ind any signi ican di e ences in he bea ing a es (BRs) be ween WT- and LQT-CMs (Fig. 1a, c, e). β-Blocke Bisop olol The bisop olol did no cause any majo di e ences in he BRs o he hiPSC-CMs (Fig. 1a). We no ed a sligh ly inc easing end o BR wi h inc easing bisop olol con- cen a ion in he hiPSC-CMs om asymp oma ic mu a ion ca - ie (15 % o 303.LQT1 and 19 % o 313.LQT1). As o he cFPD, bisop olol caused only mild cFPD p olonga ion in he WT-CMs (3–7 %) a he concen a ion ange o 260–520 nM. Simila cFPD p olonga ions we e seen in LQT-CMs om symp oma ic pa ien (2–6 % o 208.LQT1 and 5–7 % o 211.LQT1) and om asymp oma ic mu a ion ca ie (5–10 % o 303.LQT1 and 5–8 % o 313.LQT1) a he concen a ion ange o 260-520 nM (Fig. 1b). The ep esen a i e bisop olol aces o WT- and LQT-CMs a e illus a ed in Fig. 2a. I Ks Ac i a o ML277 The ML277 caused signi ican inc ease in he BRs o WT and 211.LQT1, 9 % and 14 %, espec i ely (Fig. 1c). O e all, he end o inc easing BR wi h inc easing ML277 concen a ion was obse ed al hough s a is ical sig- ni icance was no ound o all he cell lines. As expec ed, he ML277 sho ened he cFPD in a dose-dependen manne , al- hough he cFPD sho ening was ela i ely mild (Fig. 1d). In he WT, he sho ening o cFPD was 11 % whe eas in he LQTs i anged om 5 o 11 % a he concen a ion ange o 1-2 μM. The ep esen a i e ML277 aces o WT- and LQT- CMs a e illus a ed in Fig. 2b. I Ks Blocke JNJ303 Blocking he I Ks channel wi h JNJ303 did no signi ican ly change he BRs o he WT- o LQT-CMs excep in 313.LQT1, in which he 23 % inc ease in he BR was obse ed (Fig. 1e). JNJ303 showed a dose-dependen cFPD p olonga ion in he WT- and LQT-CMs (Fig. 1 ). The mos inc emen in cFPD was seen in he WT (22 %) whe eas in he LQTs, he cFPD inc ease was a ound 7–13 % a he highes concen a ion (1000 nM). The ep esen a i e JNJ303 aces o WT- and LQT-CMs a e illus a ed in Fig. 2c. De ended Fluc ua ion Analysis (DFA) o Heal hy Con ol- and LQT-Speci ic Ca diomyocy es and ECG Da a We i s de e mined he ac al scaling exponen α om hu- man subjec s who pa icipa ed o he s udy (n= 3). Resul s a e shown in Table 1. Nex , we de e mined he scaling exponen α om hiPSC-CMs de i ed om he same human subjec s pa - icipa ing o he s udy. In Fig. 3we show he DFA esul s o he peak- o-peak luc ua ions o heal hy con ol- (WT) and LQT-speci ic CMs when exposed o bisop olol (Fig. 3a), Fig. 1 The e ec s o a ious compounds o he human induced plu ipo en s em cell (hiPSC)-de i ed ca diomyocy es’ ield po en ial pa ame e s. The uppe ow depic s he bea ing a e (BR) and he lowe ow F ide icia-co ec ed ield po en ial du a ion (cFPD). The baseline cFPDs o long QT-speci ic ca diomyocy es we e signi ican ly mo e p olonged han in heal hy wild ype-ca diomyocy es. The as e isks on op o he ba s depic he s a is ical signi icance o mean BR o cFPD change compa ed o baseline alues. Signi icance le els a e indica ed by (*) p< 0.05, (**) p< 0.01 and (***) p< 0.001, espec i ely S em Cell Re and Rep (2016) 12:698–707 701 ML277 (Fig. 3b), o JNJ303 (Fig. 3c). The i s ba in all he subplo s co esponds o he baseline wi h ze o d ug con- cen a ion. The LQT da a is g ouped acco ding o symp- oma ic (208.LQT1, 211.LQT1) and asymp oma ic cases (303.LQT1, 313.LQT1) shown in he middle and in he igh column, espec i ely. We ound ha all he a e age baseline alues o he DFA scaling exponen a e close o one, i.e., α~0.9–1.1. The e was Fig. 2 The ep esen a i e aces o he human induced plu ipo en s em cell (hiPSC)-de i ed wild ype- and long QT (LQT)-speci ic ca diomyocy es unde he e ec o a ious compounds. The LQTs a e g ouped acco ding o symp oma ic (208.LQT1, 211.LQT1) and asymp oma ic cases (303.LQT1, 313.LQT1) shown in he middle and in he igh column, espec i ely. a)β-blocke Bisop olol, b)I Ks ac i a o ML277 c)I Ks blocke JNJ303. No ice, how he T max o he epola iza ion wa e is ela i ely unchanged be ween baseline and di e en bisop olol concen a ions depic ing ma ginal e ec o he β- blocke o he co ec ed ield po en ial du a ion (cFPD). Howe e , I Ks ac i a o ML277 shows clea sho ening o cFPD seen by shi in he T max . Simila ly, bu con e sely o ML277, I Ks blocke JNJ303 shows clea p olonga ion o cFPD assessed by he shi in he T max Table 1 The pa ien cha ac e is ics and de ended luc ua ion analysis om ECG da a Heal hy indi idual LQT1-pa ien (symp oma ic) LQT1-pa ien (asymp oma ic) Age 55 41 28 Medica ion - Bisop olol Bisop olol QTc (ms) 406 456 428 α1.21 ± 0.03 1.08 ± 0.02 1.07 ± 0.02 702 S em Cell Re and Rep (2016) 12:698–707 no no able di e ence in he baseline α alues be ween he WT and he LQTs. This is in line wi h ou ECG da a, which shows αo 1.08, 1.07 and 1.21 o symp- oma ic and asymp oma ic LQT pa ien as well as o heal hy indi idual, espec i ely (Table 1). Howe e , all he pha macological compounds lead o an inc ease in he DFA scaling exponen α owa d B ownian mo ion (α= 1.5) when compa ed o he baseline (Fig. 3). The end is isible bo h in he WT and in he LQTs. In mos cases a u he inc ease in he concen a ion does no a ec α. When compa ing all he α alues o WT and LQTs, we did no ind any s a is ical di e ence be ween he g oups. We also conduc ed ehicle con ol expe imen s in which no pha macological compounds bu only DMSO (0.1 %) was added o he CMs. We did no obse e any signi ican changes in he αscaling ex- ponen du ing hese eco dings. The αwas 1.024 ± 0.13 a baseline, 1.00 ± 0.12 ( i s DMSO addi ion) and 1.03 ± 0.13 (second DMSO addi ion) (n= 6). Thus, he change seen in α scaling exponen wi h a ious pha macological compounds e lec he in insic p ope ies o he pha macological com- pounds hemsel es. Fig. 3 The de ended luc ua ion analysis (DFA) αscaling exponen s o he human induced plu ipo en s em cell (hiPSC)-de i ed wild ype- and long QT (LQT)-speci ic ca diomyocy es. The LQTs a e g ouped acco ding o symp oma ic (208.LQT1, 211.LQT1) and asymp oma ic cases (303.LQT1, 313.LQT1) shown in he middle and in he igh column, espec i ely. a) ( ow) β-blocke Bisop olol, b)I Ks ac i a o ML277 c)I Ks blocke JNJ303. A baseline, he αscaling exponen s a e close o 1 in all hiPSC-CMs. The addi ion o a ious compounds o he hiPSC-CMs inc eased he αscaling exponen close o B ownian mo ion (α= 1.5). The as e isks on op o he ba s depic he s a is ical signi icance o mean αscaling exponen change compa ed o baseline alues. Signi icance le els a e indica ed by (*) p < 0.05, (**) p < 0.01 and (***) p < 0.001, espec i ely S em Cell Re and Rep (2016) 12:698–707 703 Discussion In human hea s, he ac al dynamics is hough o esul om complex in e ac ion be ween agal and sympa he ic inpu s o au onomous ne ous sys em [17,18]. Howe e , e idence sug- ges ha he hiPSC-CMs also exhibi ac al-like complexi y indica ing in insic mechanisms o he CMs con ibu ing o ac al dynamics [26,27]. This s udy u he suppo s he con- cep ha heal hy hiPSC-CMs lacking au onomous ne ous sys em inpu exhibi ac al-like complexi y a baseline condi- ion. We also obse ed ha he un ea ed (wi hou any pha - macological in e en ion) hiPSC-LQT-CMs also appea o ex- hibi ac al-like complexi y a baseline condi ion. This esul is in line wi h he p e ious s udy in un ea ed pa ien s wi h congeni al LQTS [55]. Thus, ou esul s u he expand his concep sugges ing ha he in insic mechanisms con ibu ing o he ac al-like complexi y is no al e ed in hiPSC-LQT- CMs. Howe e , i is o no e ha he esul s ob ained we e om 2 LQTS pa ien s only and u he s udies on a la ge popula- ion would be needed o de ini ely conclude he ma e . The Elec ophysiological P ope ies o he hiPSC-CMs The baseline beha io o hiPSC-CMs is in line wi h ou p e- ious s udies [41,43]. Resul s ob ained om his s udy clea ly show ha he cFPDs, co ec ed wi h ei he Baze ’so F ide icia’s o mulae, a e mo e p olonged in LQT-CMs han in heal hy WT-CMs. We also in es iga ed he heal hy and LQT-speci ic hiPSC-CMs unde he e ec o β-blocke bisop olol and pha macological compounds a ec ing speci i- cally o ca diac ion channel I Ks . O e all, he d ug e ec s in LQT-CMs appea ed o be o simila magni ude o hose o WT-CMs, simila o ou p e ious indings [43]. The E ec o Bisop olol o he F ac al Complexi y o hiPSC-CMs The β-blocke s a e he s anda d and cu en ly, he only ea men o choice o LQTS pa ien s [56]. He e, in o de o alid compa isons be ween cellula and whole hea da a, we chose o s udy bisop olol in hiPSC-CMs because he LQTS pa ien s olun ee ed o his s udy we e on bisop olol medica ion. Bisop olol, a β1-ad eno ecep o selec i e β- blocke , did no ha e physiological signi icance o he ield po en ial pa ame e s (>10 % cFPD p olonga ion) in any o he hiPSC-CMs a clinically ele an concen a ion. I is impo an o no ice ha we did no ac i a e β- ecep o s wi h β-agonis s p io o β-blocke applica ion. Howe e , he acu e applica ion o bisop olol inc eased he αscaling exponen owa d B ownian mo ion (α= 1.5) in all hiPSC-CMs a he uppe limi o he apeu ic concen a ion (260 nM). A 520 nM, he e ec was a enua ed. Al hough he baseline αda a om hiPSC-LQT-CMs co ela ed wi h he ECG da a (α~ 1.0), bisop olol αda a did no co ela e wi h he ECG da a. The disc epancy be ween cellula and hea αda a emains ye unclea bu i may be a ibu ed o e.g. acu e applica ion o he bisop olol o in he absence o au onomous ne ous sys- em in hiPSC-LQT-CMs, which may a ec o ac al complex- i y upon d ug applica ion. P e ious human s udy has shown ha he ac al complexi y (α~ 1.0) was unal e ed in pa ien s wi h congeni al LQTS ea ed o un ea ed wi h β-blocke s [55]. Thus, he β-blocke s may no ha e signi ican e ec on he αo he whole LQTS hea s. On he o he hand, he e is e idence om he human s udies ha he β-blocke ea men imp o es ac al dynamics o he hea in ad anced conges i e hea ailu e pa ien s by inc easing he αscaling exponen du ing 1–3-mon h he apy pe iod [21–23]. I is comple ely unknown whe he such di e ences esul om he di e en disease s a es, he ype o β-blocke used o ea men pe iods among o he hings. Howe e , his s udy clea ly shows ha al hough he ield po en ial pa ame e s we e no signi ican ly changed, he in insic mechanisms con ibu ing o ac al-like complexi y we e al e ed du ing acu e β-blocke ea men . On he o he hand, u he s udies would be needed o answe wha he e ec would be in long- e m (ch onic) si ua ion and wha he mechanism behind o his phenomenon is. Thesigni icanceo he ac al complexi y esembling B ownian mo ion in he hea is unclea . The loss o ac ali y owa d whi e noise (α= 0.5) has been ound o p edispose o se e e li e- h ea ening a hy hmias and ca diac dea h [9,10, 13]. Howe e , e y li le is known when he ac al dynamics o he hea become mo e co ela ed esembling B ownian mo ion (α= 1.5). Such phenomenon has been obse ed in heal hy elde ly subjec s implying dec ease o ac al complex- i y wi h age [6,14,15] and, al hough no ye p o en, i has been sugges ed ha such sys em would be mo e suscep ible o inju y and illness in he elde ly [6,16]. The E ec o I Ks A ec ing Pha macological Compounds o he F ac al Complexi y o hiPSC-CMs Nex , we in es i- ga ed he e ec s o he o he wo pha macological compounds (ML277 and JNJ303) o hiPSC-CMs. The ion channel ac i a- o ML277 has been shown o augmen speci ically I Ks cu en and sho en he ac ion po en ial du a ion in bo h heal hy and LQT-CMs [46,47]. Simila ly, in his s udy, he ML277 sho - ened he cFPD al hough he e ec was ela i ely mild in hese hiPSC-CM clus e s a 1-2 μM concen a ion. Simila ly, as was seen wi h bisop olol, also ML277 esul ed in he dys unc ion o egula o y mechanisms con ibu ing o ac al complexi y as he αscaling exponen was inc eased owa d B ownian mo ion in all hiPSC-CMs a 1 μM. Howe e , a 2 μM a small di e ence was seen ha in he WT-CMs αexponen de- c eased o baseline le el whe eas he LQT-CMs αexponen s con inued o inc ease om 1 μM, which was mos p ominen - ly seen in CMs de i ed om symp oma ic pa ien (208.LQT1 and 00,211.LQT1). Fu he mo e, he e ec o JNJ303, a po- en and speci ic I Ks blocke [57] known o e oke o sades de poin es, was assessed in hiPSC-CMs. The WT-CMs appea ed o be mo e sensi i e o JNJ303 han LQT-CMs as measu ed by 704 S em Cell Re and Rep (2016) 12:698–707 cFPD p olonga ion. This esul is in line wi h ou p e ious indings om single WT- and LQT-CMs measu ed wi h pa ch clamp using he same cell lines [41]. Al hough no ye p o en in he expe imen al se ing, his indica es ha he I Ks cu en is diminished in he LQT1-CMs ha bo ing G589D missense mu a ion compa ed o heal hy WT-CMs. Con adic o y o ou expec a ions, also he JNJ303 esul ed in simila al e - a ions o ac al complexi y in hiPSC-CMs as ML277 by in- c easing he αscaling exponen . Taken oge he , hese e i- dence imply ha he modula ion o he ion channel gene a ing I Ks cu en wi h pha macological compounds may esul in he dys unc ion o he in insic mechanisms con ibu ing o ac al complexi y. Indeed, hy hmic ion channel ac i a ion and inac- i a ion in pacemake cells has been hough o con ibu e o he ul adian hy hmici y in addi ion o spon aneous Ca 2+ -cy- cling [58]. P e ious s udy has shown ha he dis up ion o in acellula Ca 2+ handling causes al e a ions in he scaling exponen α, mos ly by dec easing i [27]. He e, we ha e shown ha a baseline, he hy hmici y o hiPSC-CMs is sus ained as de e mined by long- ange ac al co ela ions. Fu he mo e, he modula ion o he ion channel gene a ing I Ks cu en wi h speci ic pha macological compounds dis up s CM hy hmici y and ac al complexi y. Po en ial Limi a ion o he S udy In his s udy, only he acu e e ec s o he a ious compounds o he ac al-like complexi y we e assessed in hiPSC-CMs. Fu he s udies would be needed o show he ch onic, long- e m e ec s o he compounds. Mo eo e , he mechanisms behind he al e a ions in ac al complexi y we e no s udied. We did no in es iga e in de ail he e ec o β-ad ene gic agonis o he long- ange ac al co ela ions o hiPSC-CMs. Also, wi h his model we could no ake in o accoun he con inuous exposu e o sympa he ic and pa asympa he ic s imuli a ec ing CMs in i o. This s udy does no answe o wha would be he implica ions o al e ed ac ali y owa d B ownian mo ion in hiPSC-CMs in e ms o long- e m heal h, adap abili y and esponsi eness o unp edic able s imuli. Conclusions In conclusion, he hiPSC-LQT-CMs appea o exhibi ac al- like complexi y a baseline condi ion sugges ing ha he in in- sic mechanisms o LQT-CMs con ibu ing o he ac al com- plexi y a e no al e ed. Al hough he e ec s o a ious com- pounds o he ield po en ial pa ame e s we e as expec ed, he ac al-like complexi y o he hiPSC-CMs was signi ican ly al- e ed in heal hy as well as LQT-speci ic CMs. No signi ican di e ences in he αscaling exponen we e ound be ween WT- and LQT-CMs. These indings may sugges ha he ca diac ion channel gene a ing I Ks cu en as well as he modula ion o β1- ad eno ecep o s by β-blocke bisop olol may con ibu e o he ac al-like complexi y o he hiPSC-CMs. Acknowledgmen s We kindly hank Ma kus Haponen and Henna Venäläinen o hei excellen echnical expe ise wi h he s em cell cul- u e and ca diac di e en ia ion. We also kindly hank Anna Ki iaho o gene a ing he iPS cell lines, The END-2 cells we e a kind gi om Ch is ine Mumme y. On he compu a ional side, we hank Mika Sa ilah i, and Pe u Luukko o assis ance wi h DFA analysis. This s udy was unded by g an s om he Finnish Funding Agency o Technology and Inno a ion (TEKES), Finnish Founda ion o Ca dio ascula Resea ch, Finnish Cul u al Founda ion and he Academy o Finland. Compliance wi h E hical S anda ds Au ho Con ibu ions JK designed and pe o med MEA expe imen s, analyzed MEA da a, pe o med s a is ical analyses and w o e he manu- sc ip , JKim pe o med de ended luc ua ion analysis, analyzed he nu- me ical esul s and w o e he manusc ip , ER designed expe imen s, pe - o med de ended luc ua ion analysis and w o e he manusc ip , KA concei ed he s udy, designed expe imen s and w o e he manusc ip . All au ho s ead and app o ed he inal manusc ip . Con lic o In e es The au ho s decla e no po en ial con lic s o in e es s. 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