Mutation-Specific Phenotypes in hiPSC-Derived Cardiomyocytes Carrying Either Myosin-Binding Protein C Or α-Tropomyosin Mutation for Hypertrophic Cardiomyopathy

Resea ch A icle Mu a ion-Speci ic Pheno ypes in hiPSC-De i ed Ca diomyocy es Ca ying Ei he Myosin-Binding P o ein C O 𝛼-T opomyosin Mu a ion o Hype ophic Ca diomyopa hy Ma isa Ojala,1Chand a P ajapa i,1Ris o-Pekka Pölönen,1K is iina Rajala,1 Ma i Pekkanen-Ma ila,1Jy ki Rasku,2Kim La sson,1and Ka iina Aal o-Se älä1,3,4 1BioMediTech, Uni e si y o Tampe e, 33014 Tampe e, Finland 2School o In o ma ion Sciences, Uni e si y o Tampe e, 33014 Tampe e, Finland 3Medical School, Uni e si y o Tampe e, 33014 Tampe e, Finland 4Hea Hospi al, Tampe e Uni e si y Hospi al, 33521 Tampe e, Finland Co espondence should be add essed o Ka iina Aal o-Se ¨ al¨ a; ka [email p o ec ed] Recei ed 21 May 2015; Re ised 22 July 2015; Accep ed 20 Sep embe 2015 Academic Edi o : Fa ah Sheikh Copy igh © 2016 Ma isa Ojala e al. This is an open access a icle dis ibu ed unde he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. Hype ophic ca diomyopa hy (HCM) is a gene ic ca diac disease, which a ec s he s uc u e o hea muscle issue. The clinical symp oms include a hy hmias, p og essi e hea ailu e, and e en sudden ca diac dea h bu he mu a ion ca ie can also be o ally asymp oma ic. To da e, o e 1400 mu a ions ha e been linked o HCM, mos ly in genes encoding o sa come ic p o eins. Howe e , he pa hophysiological mechanisms o he disease a e s ill la gely unknown. Two ounde mu a ions o HCM in Finland a e loca ed in myosin-binding p o ein C (MYBPC3-Gln1061X)and𝛼- opomyosin (TPM1-Asp175Asn) genes. We s udied he p ope ies o HCM ca diomyocy es (CMs) de i ed om pa ien -speci ic human induced plu ipo en s em cells (hiPSCs) ca ying ei he MYBPC3-Gln1061X o TPM1-Asp175Asn mu a ion. Bo h ypes o HCM-CMs displayed pa hological pheno ype o HCM bu , mo e impo an ly, we ound di e ences be ween CMs ca ying ei he MYBPC3-Gln1061X o TPM1-Asp175Asn gene mu a ion in hei cellula size, Ca2+ handling, and elec ophysiological p ope ies, as well as hei gene exp ession p o iles. These indings sugges ha e en hough he clinical pheno ypes o he pa ien s ca ying ei he MYBPC3-Gln1061X o TPM1-Asp175Asn gene mu a ion a e simila , he gene ic backg ound as well as he unc ional p ope ies on he cellula le el migh be di e en , indica ing ha he pa hophysiological mechanisms behind he wo mu a ions would be di e gen as well. 1. In oduc ion Hype ophic ca diomyopa hy (HCM) is one o he mos common gene ic ca diac diseases wi h wo ldwide p e alence o 1 : 500, as well as he mos common cause o sudden ca diac dea h (SCD) among young compe ing a hle es. HCM isinhe i edinanau osomaldominan pa e n.Ne e heless, a la ge clinical di e si y and age- ela ed pene ance a e ypical o HCM. On he issue le el, HCM is cha ac e ized by he disa ay o ca diomyocy es (CMs) and ib osis o ca diac issue, as well as hickened in e en icula sep um o ee le en icula wall. Clinical symp oms include a hy hmias, p og essi ehea ailu e,ande enSCD,bu on heo he hand hemu a ionca ie canbecomple elyasymp oma ic. Al oge he mo e han 1400 mu a ions in 11 genes encoding o he sa come ic p o eins ha e been iden i ied and ela ed o HCM. The majo i y o he mu a ions a e ound ei he in he 𝛽-myosin hea y chain (MYH7) o in he myosin-binding p o ein C (MYBPC3) genes [1]. In Finland, wo ounde mu a ions loca ed in MYBPC3 and 𝛼- opomyosin (TPM1) genes and one common mu a ion in MYH7 gene oge he accoun a ound 24% o all Finnish HCM cases [2, 3]. Al hough he gene ic in o ma ion ela ed o HCM has been g owing in he ecen yea s due o he de elopmen o sequencing echnologies, exac in o ma ion o he disease mechanisms emains unclea . Thus, cu en medica ion o he diseaseisdi ec ed owa d hesymp om elie and he eisno speci ic he apy o p e en he onse o p og ession o he Hindawi Publishing Co po a ion S em Cells In e na ional Volume 2016, A icle ID 1684792, 16 pages h p://dx.doi.o g/10.1155/2016/1684792 2S em Cells In e na ional disease [1]. Mos o he HCM s udies ha e been conduc ed wi h model sys ems, mainly ei he wi h ansgenic mice o by s udying human issues ob ained om su gical myec omy om end-s age HCM pa ien s [4]. Howe e , animal models ca y only he mu a ed gene lacking he es o he genome and myec omy samples a e ob ained om pa ien s in he la e s age o HCM de elopmen . The e o e, he disco e y o he human induced plu ipo en s em cells (hiPSCs) has o e ed a new aluable ool o model HCM and o he ca diac diseases and os udy heunde lyingdiseasemechanisms[5].Toda e, hiPSCs ha e al eady been used o model a a ie y o ca diac diseases: elec ical de ec s, o example, long-QT synd ome [6–8] and ca echolamine gic polymo phic en icula achy- ca dia (CPVT) [9, 10] as well as ca diomyopa hies including dila ed ca diomyopa hy (DCM) [11] and HCM [12–14]. He e we ha e de i ed hiPSCs om pa ien s ca ying wo o he Finnish HCM ounde mu a ions ei he in MYBPC3 (MYBPC3-Gln1061X)o inTPM1 (TPM1-Asp175Asn)gene. We ha e di e en ia ed he pa ien -speci ic hiPSCs in o CMs and compa ed he pheno ypes o he diseased and con ol CMs. 2. Ma e ials and Me hods 2.1. E hical Issues. This s udy was conduc ed in acco dance wi h 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). Skin biopsies o hiPSC es ablishmen we e ecei ed om he Hea Hospi al, Tampe e Uni e si y Hospi al, Tam- pe e, Finland. Pa ien s dona ing skin biopsies signed an in o med consen a e ecei ing bo h o al and w i en desc ip ions o he s udy. The e a oma assay, desc ibed in Sec ion 2.3.6, was app o ed by ELLA-Animal Expe imen Boa d o Regional S a e Adminis a i e Agency o Sou he n Finland (ESAVI/6543/04.10.03/2011). 2.2. Gene a ion and Cul u e o Pa ien -Speci ic hiPSC Lines. hiPSC lines we e gene a ed om skin’s ib oblas s ei he wi h Sendai ep og amming ec o s OCT4,KLF4,c-MYC,and SOX2 using Cy oTune-iPS Rep og amming Ki (Li e Tech- nologies L d., Paisley, UK) acco ding o he manu ac u e ’s ins uc ions o by using pMX e o i al ec o s OCT4,KLF4, c-MYC,andSOX2 wi h o wi hou C e-LoxP si e as desc ibed ea lie [6, 15]. UTA.13602.HCMT, UTA.02912.HCMT, and UTA.04511.WT hiPSC lines we e gene a ed by using Sendai ec o s and UTA.07801.HCMM and UTA.06108.HCMM by using pMX e o i al ec o s wi h C e-LoxP si e and UTA.04602.WT was gene a ed by using pMX e o i al ec o s wi hou C e-LoxP si e. In he p esen s udy, one line o each pa ien was used. hiPSC lines we e de i ed and cul u ed on mouse emb yonic ib oblas (MEF) eede cell laye s (26000 cells/cm2, CellSys ems Bio echnologie Ve ieb GmbH, T oisdo , Ge many) in human plu ipo en s em cell (hPSC) cul u e medium consis ing o knockou -DMEM (ko- DMEM,Gibco,Li eTechnologiesL d.)supplemen edwi h 20% knockou se um eplacemen (ko-SR, Gibco, Li e Tech- nologies L d.), 1% nonessen ial amino acids (NEAA, Lonza G oup L d., Basel, Swi ze land), 2 mM Glu aMax (Gibco, Li e Technologies L d.), 50 U/mL penicillin/s ep omycin (Lonza G oup L d.), 0.1 mM 2-me cap oe hanol (Gibco, Li e Tech- nologies L d.), and 4 ng/mL basic ib oblas g ow h ac o (bFGF, Pep oTech, Rocky Hill, NJ, USA). 2.3. Cha ac e iza ion o hiPSC Lines 2.3.1. Mu a ion Analysis by Geno yping. DNA samples om hehiPSClineswe ep epa edwi hTaqManSample- o- SNP Ki (Applied Biosys ems, Li e Technologies L d.) and he p esence o MYBPC3-Gln1061X and TPM1-Asp175Asn mu a ion in he pa ien -speci ic hiPSC lines was con i med by cus om TaqMan SNP Geno yping Assays (Applied Biosys- ems, Li e Technologies L d.) acco ding o he manu ac u e ’s ins uc ions. In he geno yping assays, MYBPC3-gene as well as TPM1-gene was ampli ied wi h speci ic p ime s. Fu he mo e, he p esence o he mu a ions was assessed wi h mu a ion-speci ic FAM labeled p obes. VIC labeled p obes we e used o assess he p esence o he wild ype allele. Sequences o hep ime sandp obesusedin heassaya e lis ed in Supplemen a y Table 1 (see Supplemen a y Ma e ial a ailable online a h p://dx.doi.o g/10.1155/2016/1684792). 2.3.2. The Exp ession o Mu an and Wild Type Alleles in hiPSC-De i ed CMs. RNA samples we e collec ed and ex ac ed om hiPSC-de i ed CMs (UTA.04511.WT, UTA.02912.HCMT, UTA.07801.HCMM, and UTA.06108. HCMM) wi h No gen’s To al RNA Pu i ica ion Plus Ki (No gen Bio ek Co p., On a io, Canada) acco ding o manu- ac u e ’s ins uc ions. 50–100 ng o RNA was ansc ibed o cDNA by High-Capaci y cDNA Re e se T ansc ip ion Ki (Applied Biosys ems, Li e Technologies L d.). The exp ession o TPM1-Asp175Asn o MYBPC3-Gln1061X mu a ion on mRNA le el in he hiPSC-de i ed CMs was s udied by Cus om TaqMan SNP Geno yping Assays (Applied Bio- sys ems, Li e Technologies L d.) simila ly as ha o geno yping desc ibed abo e. Sequences o he p ime s and p obesusedin heassaya elis edinSupplemen a yTable1. 2.3.3. Immunocy ochemis y. Undi e en ia ed hiPSC colo- nies we e ixed wi h 4% pa a o maldehyde (PFA, Sigma- Ald ich, Sain Louis, USA), s ained wi h p ima y an ibodies o Nanog (R&D sys ems Inc., Minneapolis, MN, USA), OCT4 (R&D sys ems Inc.), SOX2 (San a C uz Bio echnol- ogy, San a C uz, CA, USA), TRA-1-60 (Millipo e, Bille ica, MA, USA), and TRA-1-81 (Millipo e), and isualized wi h seconda y an ibodies as desc ibed be o e [16]. Finally, he cells we e moun ed wi h Vec ashield (Vec o Labo a o ies Inc., Bu lingame, CA, USA) con aining 40,6-diamidino-2- phenylindole (DAPI) o he nuclei s aining and imaged wi h an Olympus IX51 phase con as mic oscope equipped wi h luo escence op ics and Olympus DP30BW came a (Olympus Co po a ion, Hambu g, Ge many). 2.3.4. RT-PCR. The RNA was ex ac ed om he hiPSC lines by NucleoSpin RNA II Ki (Mache ey-Nagel GmbH & Co., D¨ u en, Ge many) and 500–1000 ng o RNA was ansc ibed o cDNA by High-Capaci y cDNA Re e se T ansc ip ion Ki S em Cells In e na ional 3 (Applied Biosys ems, Li e Technologies L d.). The p esence o plu ipo ency genes Nanog,SOX2,REX1,OCT4,andc-MYC and heabsenceo i allyimpo edexogenes(OCT4,SOX2, c-MYC,andKLF4) we e con i med by RT-PCR. GAPDH was used as an endogenous con ol. The p ime sequences o plu ipo ency genes and i ally impo ed exogenes ha e been published ea lie [5]. The p ime sequences used o de ec ion o Sendai ansgenes a e desc ibed in Cy oTune-iPS Rep og amming Ki ’s manual (Li e Technologies L d.). 2.3.5. Ka yo ype Analysis. The ka yo ypes o hiPSC lines we e s udied by G-banding (Medix Labo a o ies, Espoo, Finland) o by Ka yoLi e assay [17] (Tu ku Cen e o Bio echnology, Uni e si y o Tu ku, Tu ku, Finland). 2.3.6. Plu ipo ency Analysis. The plu ipo ency o hiPSC lines was con i med in i o by emb yoid body (EB) o ma ion and in i o by e a oma assay. hiPSCs we e emo ed om eede cell laye and cul u ed in suspension o o m EBs. The EBs we e cul u ed in EB medium consis ing o ko-DMEM supplemen ed wi h 20% e al bo ine se um (FBS, Biose a, Boussens,F ance),1%NEAA(LonzaG oupL d.),2mM Glu aMax (In i ogen, Li e Technologies L d.), and 50 U/mL penicillin/s ep omycin (Lonza G oup L d) o 4–6 weeks be o e RNA ex ac ion. 200 ng o RNA was ansc ibed o cDNA o heRT-PCRanalysis.Thep esenceo all h eege m laye s, endode m (AFP, SOX17), ec ode m (SOX1, NESTIN, and Musashi), and mesode m (KDR, alpha ca diac ac in), was s udied wi h RT-PCR. Fo in i o plu ipo ency assay, hiPSCs we e injec ed unde he es is capsule o nude mice and he o med e a omas we e collec ed and ixed wi h 4% PFA 8 weeks a e he injec ion. Te a omas we e embedded in pa a in, cu in sec ions, and s ained wi h haema oxylin and eosin. 2.4. Di e en ia ion o Ca diomyocy es. hiPSCs we e di - e en ia ed in o CMs by cocul u ing wi h mouse isce al endode mal-like cells (END-2) (Hub ech Ins i u e, U ech , Ne he lands) as desc ibed be o e [18]. A e 15–30 days bea - ing a eas we e cu om cocul u es and dissocia ed in o single cells in EB medium by Collagenase A (Roche Diagnos ics, Mannheim, Ge many) as desc ibed ea lie [18] and pla ed o 0.1% gela in-coa ed co e slips o well pla es o u he analysis. 2.5. Cha ac e iza ion o hiPSC-De i ed Ca diomyocy es 2.5.1. Immunocy ochemis y and Image Analysis. Dissocia ed CMs we e ixed wi h 4% PFA and s ained wi h T oponin T(cTnT,1:2000,ab64623,Abcam,Camb idge,MA,USA), MYBPC (1 : 400, sc-166081, San a C uz Bio echnology), and TPM1 (1 : 200, sc-73225, San a C uz Bio echnology) p ima y an ibodies, ollowed by labeling wi h seconda y an ibodies. Images we e ob ained wi h Olympus IX51 phase con as mic oscope equipped wi h luo escence op ics and Olympus DP308W came a (Olympus Co po a ion) o wi h Zeiss Axio- Scope A1 luo escen mic oscope and Zeiss AxioCam MRc5 came a (Ca l Zeiss, Jena, Ge many). Size o he T oponin T s ainedCMswasanalyzed om46 o50CMsineachcellline by in-house made so wa e (unpublished me hod). CMs we e analyzed om pic u es ob ained wi h Olympus IX51 phase con as mic oscope. The p opo ion o mul inuclea ed CMs was de e mined om he same images (46–50 CMs/cell line). 2.5.2. Ca2+ Imaging. The clus e s o CMs we e cu , dissoci- a ed, pla ed on 0.1% gela in-coa ed co e slips, and cul u ed o 1,3,and6weeks.Tos udy heCa 2+ handling p ope ies o hiPSC-de i ed CMs, cells we e loaded wi h 4 𝜇M Fu a-2 AM (Molecula P obes, Li e Technologies L d.) o 30 minu es in pe usa e medium. The pe usa e medium consis ed o (in mM) 137 NaCl, 5 KCl, 0.44 KH2PO4, 20 HEPES, 4.2 NaHCO3, 5 D-glucose, 2 CaCl2,1.2MgCl 2,and1Na-py u a e dissol edinH 2O. pH o he pe usa e medium was adjus ed o 7.4 wi h NaOH. The co e slip, con aining he dissocia ed hiPSC-de i ed CMs, was moun ed o an RC-25 eco ding chambe and con inuously pe used wi h pe usa e medium p ehea ed o 35-36∘C by an SH-27B inline-hea e con olled byaTC-324Buni (all omWa ne Ins umen sInc., Hamden, USA). The pe usion was con olled by a g a i y d i en VC38 applica ion sys em (ALA Scien i ic Ins umen s Inc., NY, USA). Co e slip was pe used o 15 minu es o Fu a-2 AM dees e i ica ion be o e expe imen al eco dings. Ca2+ handling o spon aneously bea ing CMs was imaged wi h an in e ed IX70 mic oscope using UApo/340 x20 ai objec i e (Olympus Co po a ion) and ANDOR iXon 885 CCD came a (Ando Technology, Bel as , No he n I eland) synch onized wi h a Polych ome V ligh sou ce by a eal ime DPScon oluni .TILL isIONo Li eAcquisi ionso wa e (TILL Pho onics, Munich, Ge many) was used o eco ding. Fu a-2 AM was exci ed a 340 nm and 380 nm ligh and he emission was eco ded o 10–30 seconds a 505 nm. Fo Ca2+ imaging analysis, single bea ing CMs we e selec ed as egions o in e es s and backg ound noise, eco ded om a cell- ee a ea in he same co e slip, was sub ac ed be o e u he p ocessing. Da a is p esen ed as a ios o 340/380 nm (F340/F380). The spon aneously bea ing CMs we e di ided in o i e di e en hy hm ca ego ies based on he abno mali ies obse ed in hei Ca2+signals: no mal bea ing wi h egula peaks (no mal); mo e han h ee peaks which do no e u n o he baseline (oscilla ion); signals wi h small o middle sized ampli ude e en s in he beginning, in heend,o inbe ween woCa 2+ spikes (low/middle peaks); wo o h ee peaks which do no e u n o he baseline (double peaks); Ca2+ spikes wi h p olonged ise o decay ime (pla eau abno mali y). In he low/middle peaks ca ego y, he small ampli ude was a leas 10% om he p eceding Ca2+ spike ampli ude. Full-leng h, 10–30 seconds long eco dings we e analyzed, while mos o he analyzed eco dings we e 12 seconds long. The dis ibu ion o CMs in di e en ca ego ies is p esen ed o each cell lines sepa a ely. 2.5.3. Elec ophysiological Measu emen s: Reco ding and Anal- ysis o Ac ion Po en ials. The ac ion po en ials (APs) we e eco ded by con en ional pa ch clamp [19] in pe o a ed pa ch con igu a ion using Ampho e icin B [20] in inal concen a ion o 0.24 mg/mL [8]. Da a acquisi ion was 4S em Cells In e na ional Table1:TaqManassaysusedinqRT-PCRexpe imen s. Gene Desc ip ion/alias Func ion TaqMan assay ID EEF1A1 Euka yo ic ansla ion elonga ion ac o 1 alpha 1 Housekeeping gene Hs00265885 g1 GAPDH Glyce aldehyde-3-phospha e dehyd ogenase Housekeeping gene Hs02758991 g1 TNNT2 T oponin T Sa come ic gene Hs00165960 m1 MYH6 Myosin hea y chain 6 Sa come ic gene Hs01101425 m1 ACTN2 𝛼-ac inin 2 Sa come ic gene Hs00153809 m1 TPM1 𝛼- opomyosin Sa come ic gene Hs00165966 m1 MYBPC3 Myosin-binding p o ein C Sa come ic gene Hs00165232 m1 TTN Ti in Sa come ic gene Hs00399225 m1 TNNC1 T oponin C ype 1 Sa come ic gene Hs00896999 g1 MYL9 Myosin, ligh chain 2/MLC2 Sa come ic gene Hs00697086 m1 MYL7 Myosin, ligh chain 7 Sa come ic gene Hs01085598 g1 NPPA Na iu e ic pep ide A Hype ophy ma ke Hs01081097 m1 NPPB Na iu e ic pep ide B Hype ophy ma ke Hs01057466 g1 HCN4 Hype pola iza ion ac i a ed cyclic nucleo ide-ga ed po assium channel 4 Po assium channel Hs00975492 m1 KCNQ1 Vol age-ga ed po assium channel, KQT-like sub amily, membe 1 Po assium channel Hs00923522 m1 CACNA1C Vol age-dependen calcium channel, L ype, alpha 1C subuni /CaCNA1.2 Calcium channel Hs00167681 m1 SCN5A Vol age-ga ed sodium channel, V ype, alpha subuni Sodium channel Hs00165693 m1 SLC8A1 Solu e ca ie amily 8, membe 1/NCX1 Na+/Ca2+ exchange Hs01062258 m1 PLN Phospholamban/PLB P o ein kinase subs a e Hs01848144 s1 ATP2A2 ATPase, Ca2+ anspo ing, ca diac muscle, slow wi ch 2/SERCA2a Ca2+-ATPase Hs00544877 m1 CASQ2 Calseques in Ca2+ binding p o ein in SR Hs00154286 m1 ITPR2 Inosi ol 1,4,5- isphospha e ecep o , ype 2/IP3R2 Ca2+ ecep o Hs00181916 m1 RYR2 Ryanodine ecep o 2 (ca diac) Ryanodine ecep o Hs00892883 m1 conduc ed using Axon Se ies 200B pa ch-clamp ampli ie connec ed o Digida a 1440a AD/DA con e e d i en by pCLAMP 10.2 so wa e (all om Molecula de ices LLC). On he day o use, he co e slips con aining dissocia ed hiPSC-de i ed CMs we e ans e ed o RC-24N eco ding chambe (Wa ne Ins umen s Inc.) and moun ed on an in e ed Olympus IX71 mic oscope (Olympus Co po a ion). The pa ch elec odes had ip esis ance o 3.0–3.5 MΩand con ained he ollowing in acellula solu ion (in mM): 132 KMeSO4,20KCl,1MgCl 2, and 1 CaCl2(pH was adjus ed o 7.2 wi h KOH). The ex acellula solu ion con ained (in mM) 143 NaCl, 4.8 KCl, 1.8 CaCl2, 1.2 MgCl2,5glucose,and10 HEPES (pH was adjus ed o 7.4 wi h NaOH). The p ehea ed ex acellula solu ion was con inuously pe used wi h simila se upcompa ed owha isp esen edinSec ion2.5.2.Pa ch pipe es (Ha a d Appa a us L d., Hollis on, MA, USA) we e eshly p epa ed using PC-10 mic opipe e pulle and hen lame-polished wi h MF-830 mic o o ge (bo h om Na ishige In ., Tokyo, Japan). APs we e eco ded in he gap- ee mode in he cu en clamp om he spon aneously bea ing hiPSC-de i ed CMs. 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 . Bea s pe minu e (BPM), AP du a ion (APD50 and APD90), AP ampli ude (APA), and maximum dias olic po en ial (MDP) we e analyzed om he eco ded APs by using O igin 9.1 (O iginLab Co p., No hamp on, USA). Only en icula -like wa e o ms a e p esen ed he e o a oid any biasness among di e en hiPSC lines. The en icula - like CMs we e cha ac e ized by APD90/APD50 <1.3 and APA >90 mV. 2.5.4. Real-Time qRT-PCR Analysis. A e one week o cul- u e, dissocia ed CMs we e collec ed in o a lysis solu ion bu e o CellsDi ec One-S ep qRT-PCR Ki (Li e Technolo- gies L d.) acco ding o he manu ac u e ’s ins uc ions. Two eplica e samples we e collec ed and s o ed a −70∘Cun il he DNase I diges ion and e e se ansc ip ion-speci ic a ge ampli ica ion (RT-STA) by using CellsDi ec One-S ep qRT- PCR Ki . Real-Time qPCR was pe o med wi h Bioma k HD sys em (Fluidigm Co p., San F ancisco, USA) acco ding o he manu ac u e ’s ins uc ions. The TaqMan assays (Li e TechnologiesL d.)usedin heqRT-PCRa ecollec edin Table1.Allsampleswe eanalyzedinduplica eand he old changeswe ecalcula edby he2−ΔΔCT me hod [21]. EEF1A1 and GAPDH genes we e used as endogenous con ol genes and UTA.04511.WT cell line was used as a calib a o . 2.5.5. Wes e n Blo . hiPSC-de i ed CMs we e lysed in M- PER p o ein ex ac ion eagen (The mo Scien i ic, Li e Technologies L d.), supplemen ed wi h comple e p o ease inhibi o cock ail (Roche Diagnos ics). The p o ein concen- a ion was quan i ied wi h BCA p o ein assay ki (The mo Scien i ic, Li e Technologies L d.). 10 𝜇go p o einwas un o 4–15% mini-PROTEAN TGX p ecas polyac ylamide gel S em Cells In e na ional 5 (Bio-Rad, He cules, CA, USA) and ans e ed o PVDF memb ane (Ame sham Hybond-P, GE Heal hca e, Li le Chal on , UK). Memb anes we e blocked wi h 5% milk o 1 h a RT and p o eins we e s ained wi h MYBPC (1 : 1500, sc- 166081, San a C uz Bio echnology), cTnT (1 : 2000, ab64623, Abcam), TPM1 (1 : 200, sc-73225, San a C uz Bio echnology), o 𝛽-ac in (1 : 1000, sc-47778, San a C uz Bio echnology) p i- ma y an ibodies o e nigh a +4∘C. Ho se adish pe oxidase- (HRP-) conjuga ed polyclonal abbi an i-mouse (DAKO, P0260) and abbi an i-goa IgG (San a C uz Bio echnology, sc-2922) we e used as seconda y an ibodies. S ained p o eins we e de ec ed by using Cla i y ECL subs a e (Bio-Rad) and isualized by Molecula Image ChemiDOc XRS+ (Bio-Rad). ImageJ so wa e (Na ional Ins i u es o Heal h, USA) was used o compa e he exp ession o MYBPC, cTnT, and TPM1 wi h he 𝛽-ac in exp ession om he same cell line. 2.6. S a is ical Analysis. Fo s a is ical analysis, con ol cell lines and cell lines in each mu a ion we e combined in g oups: UTA.04602.WT and UTA.04511.WT hiPSC lines in WT-CM g oup, UTA.02912.HCMT and UTA.13602.HCMT in HCMT- CM g oup, and UTA.07801.HCMM and UTA.06108.HCMM in HCMM-CM g oup. Mann-Whi ney 𝑈 es wi h Bon e - oni’s co ec ion was used o analyze he di e ences be ween WT-, HCMT-, and HCMM-CMs in cell size analysis, p opo - ion o mul inuclea ed CMs, and Ca2+ imaging expe imen s as well as in gene exp ession analysis. Fo he s a is ical compa ison be ween he h ee g oups, one-way ANOVA ollowed by Tukey es was used o he pa ch-clamp esul analysis. 𝑝 < 0.05 was conside ed s a is ically signi ican . All e o ba s a e p esen ed as s anda d e o o he mean (SEM). 3. Resul s 3.1. hiPSCs We e De i ed om HCM Pa ien s wi h Di e en Backg ounds. We de i ed hiPSCs om ou pa ien s ca ying a HCM causing mu a ion ei he in TPM1 (TPM1-Asp175Asn) o in MYBPC3 (MYBPC3-Gln1061X). UTA.13602.HCMT and UTA.02912.HCMTs ca y TPM1-Asp175Asn and UTA.07801. HCMM and UTA.06108.HCMM MYBPC3-Gln1061X mu a- ion. The hiPSC lines and hei mu a ions and abb e ia ions, used below, a e p esen ed in Table 2. UTA.13602.HCMT (46, XX) is de i ed om a 48-yea -old emale, whose mo he died suddenly a he age o 51. Ou pa ien has had one collapse a he age o 20 wi h no mal hea s uc u e, bu la e sligh hickening o sep um (16 mm) has been obse ed. Cu en ly she is no on medica ion due o low blood p essu e. UTA.02912.HCMT (46, XY) is de i ed om a 33-yea -old male whose amily membe has died suddenly a he age o 21. The maximal sep al hickness o ou pa ien has been measu ed o be 26 mm on echoca diog am. The pa ien has been asymp oma ic bu is cu en ly on 𝛽-blocke medica ion. UTA.07801.HCMM (46, XY) is de i ed om a 61-yea -old male wi h no SCDs in he amily. On echoca diog am, his myoca dial sep um has been obse ed o be 25 mm. He has a ial ib illa ion and he is on 𝛽-blocke medica ion. Due o b adyca dia and equen nonsus ained en icula achyca - dia episodes, an implan able ca dio e e de ib illa o (ICD) Table 2: The hiPSC lines and hei mu a ions and abb e ia ions used in he s udy. G oup Cell line Mu a ion Name in Figu e 2( ) WT UTA.04602.WT — WT1 UTA.04511.WT — WT2 HCMT UTA.02912.HCMT TPM1-Asp175Asn HCMT1 UTA.13602.HCMT TPM1-Asp175Asn HCMT2 HCMM UTA.07801.HCMM MYBPC3-Gln1061X HCMM1 UTA.06108.HCMM MYBPC3-Gln1061X HCMM2 has been implan ed. UTA.06108.HCMM (46, XY) is de i ed om a 55-yea -old male whose a he died suddenly a he age o 36 and uncle a he age o 38. Ou pa ien has been asymp oma ic wi h maximal sep al hickness o 22 mm on echoca diog am. He is no on medica ion due o low blood p essu e. Con ol hiPSC lines we e de i ed om heal hy indi iduals: UTA.04602.WT (46, XX) om a 56-yea -old emale and UTA.04511.WT (46, XY) om a 34-yea -old male. The plu ipo en cha ac e is ics o he hiPSC-lines used we e assessed (Figu e 1 and Supplemen a y Figu es 1–5). UTA.04602.WT cell line has been cha ac e ized ea lie [22]. All he lines o med colonies, which exp essed p o eins and genes ypical o hPSCs. The i ally ans e ed exogenous genes we e silenced and ka yo ypes o he hiPSC lines we e no mal. The plu ipo ency o hiPSC lines was p o en in i o by EB o ma ion o in i o by e a oma o ma ion. The p esence o TPM1-Asp175Asn and MYBPC3-Gln1061X mu a ions in he pa ien -speci ic hiPSC lines was con i med by cus om TaqMan SNP Geno yping Assays (Supplemen a y Figu e 6). 3.2. Mu a ion-Speci ic HCM Pheno ypes We e Obse ed in hiPSC-De i ed Ca diomyocy es. All he cell lines used in he p esen s udy di e en ia ed in o ca diomyocy es simila ly. Bea ingagg ega eswe e o med14–20daysa e heini ia ion o cocul u ing wi h END-2 cells and he e was no di e ence be ween he lines when he bea ing a eas appea ed. A e ca diac di e en ia ion, bea ing clus e s we e dissocia ed in o single cells and cul u ed o 1, 3, and 6 weeks. The di e ences in he cell sizes and Ca2+ handling p ope ies be ween wo di e en mu a ions and con ol cells we e analyzed in each ime poin . When compa ing di e en ypes o CMs, HCMM-CMs we e signi ican ly la ge han HCMT-CMs and WT-CMs in all h ee ime poin s (Figu es 2(a) and 2(b), 𝑛=96–100, 𝑝 < 0.005 in all cases). The enla gemen o HCMT-CMs was de ec ed a e h ee weeks o cul u e when hey we e signi ican ly la ge han WT-CMs (𝑝< 0.005). Gene ally, he size o he hiPSC-de i ed CMs in all g oups inc eased when he cells cul u ed o h ee weeks. Wi hin each g oup, he e we e no di e ences in cell sizes be ween h ee and six weeks, excep wi h HCMT-CMs, which seemed o be smalle in size 6-week ime poin . Howe e , du ing 6 weeks o cul u e, cell ypes o he han CMs had he endency o o e g ow he CM cul u e, which migh ha e a ec ed he cellula enla gemen . This phenomenon was 6S em Cells In e na ional OCT4 TRA-1-60 Nanog SOX2 TRA-1-81 (a) 500 250 500 250 Exogenous Endogenous OCT4 OCT4 KLF4 SOX2 SOX2 c-MYC c-MYC ++++ GAPDH Nanog H2O H2O H2O REX1 (b) 123 45 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 XY (c) Mesode m Endode m Ec ode m (d) Figu e 1: Cha ac e iza ion o UTA.13602.HCMT cell line. (a) The hiPSCs o med colonies exp essing Nanog, OCT4, SOX2, TRA-1-60, and TRA-1-81.Scaleba s:200𝜇m. (b) The i ally ans e ed Sendai exogenes, exo-OCT4 (483 bp), exo-KLF4 (410 bp), exo-SOX2 (451 bp), and exo- c-MYC (532 bp), we e silenced in he hiPSCs. + indica es posi i e con ols, o which RNA was ex ac ed om cells 1 week a e ansduc ion. hiPSCs exp essed endogenous Nanog (287 bp), OCT4 (144 bp), REX1 (306 bp), SOX2 (151 bp), and c-MYC (328 bp). GAPDH (302 bp) was used as a housekeeping con ol. (c) The hiPSC line was ka yo ypically no mal, 46 XX. (d) The plu ipo ency o hiPSCs was con i med by in i o e a oma assay, in which hiPSCs o med all h ee ge m laye s (mesode m, endode m, and ec ode m). S em Cells In e na ional 7 WT HCMT HCMM (a) 12 10 8 6 4 2 ×104 (pixels) n = 100 n = 100 n = 100 n = 100 n = 100 n=96 n = 100 n = 100 n = 100 ∗∗ ## WT 1w WT 3w WT 6w HCMT 1w HCMT 3w HCMT 6w HCMM 1w HCMM 3w HCMM 6w $$ && (b) ∗∗ 50 40 30 20 10 (%) WT 1w WT 3w WT 6w HCMT 1w HCMT 3w HCMT 6w HCMM 1w HCMM 3w HCMM 6w (c) ∗∗ 50 40 30 20 10 (%) WT 1w WT 3w WT 6w HCMT 1w HCMT 3w HCMT 6w HCMM 1w HCMM 3w HCMM 6w (d) No mal 246810 Time (s) F340/F380 Oscilla ion 246810 Time (s) F340/F380 Low/middle peaks 246810 Time (s) F340/F380 Pla eau abno mali y 246810 Time (s) F340/F380 Double peaks 246810 Time (s) F340/F380 (e) Figu e 2: Con inued. 8S em Cells In e na ional 50 60 70 80 90 40 30 20 10 (%) n= WT11w WT13w WT16w WT21w WT23w WT26w HCMT11w HCMT13w HCMT16w HCMT21w HCMT23w HCMT26w HCMM11w HCMM13w HCMM16w HCMM21w HCMM23w HCMM26w 89 24 20 99 66 50 99 104 19 145 24 97 78 141 83 90 29 42 ( ) Figu e 2: The cell size and Ca2+ handling o hiPSC-de i ed CMs a e 1-, 3-, and 6-week cul u e as single cells. (a) Rep esen a i e images o WT-CMs (WT), HCMT-CMs (HCMT), and HCMM-CMs (HCMM) s ained wi h an ibodies o cTnT ( ed) and MYBPC (g een) p o eins. Scale ba s a e 100 𝜇m. (b) The size o he HCMM-CMs was signi ican ly la ge in all h ee ime poin s when compa ed o WT- and HCMT- CMs ($$𝑝< 0.005 when compa ed o WT-CMs o HCMT-CMs in he 1-week ime poin , ∗∗𝑝< 0.005 when compa ed o WT-CMs o HCMT-CMs in 3-week ime poin , and ##𝑝< 0.005 when compa ed o WT-CMs o HCMT-CMs in 6-week ime poin ). HCMT-CMs we e signi ican ly la ge han WT-CMs in 3-week ime poin (&&𝑝< 0.005 when compa ed o WT-CMs. 𝑛 = 100, excep in HCMT 6 w 𝑛=96.) (c) The p opo ion o he mul inuclea ed CMs was signi ican ly highe in HCMT-CMs han in WT-CMs and HCMM-CMs when bo h cell lines and all ime poin s we e combined o each g oup (in s a is ical analysis 𝑛=6,∗𝑝< 0.05). The a e ages o mul inuclea ed CMs we e de e mined om he same cells, whose sizes and 𝑛numbe s a e p esen ed in (b). (d) Signi ican ly mo e CMs wi h Ca2+ handling abno mali ies we e obse ed in HCMT-CMs han in WT-CMs and HCMM-CMs when bo h cell lines and all ime poin s we e combined o each g oup (in s a is ical analysis 𝑛=6,∗𝑝< 0.05). The p opo ions o CMs wi h abno mali ies in hei Ca2+ handling we e de e mined om he same Ca2+ imaging esul s p esen ed in ( ). The o al 𝑛numbe s o he analyzed CMs a e p esen ed in ( ). (e) Rep esen a i e images o Ca2+ hy hm ca ego ies. ( ) Dis ibu ions o hiPSC-de i ed CMs in di e en Ca2+ hy hm ca ego ies (e) in each ime poin . WT1 = UTA.04602.WT, WT2 = UTA.04511.WT, HCMT1 = UTA.02912.HCMT, HCMT2 = UTA.13602.HCMT, HCMM1 = UTA.07801.HCMM, and HCMM2 = UTA.06108.HCMM. obse ed especially wi h UTA.13602.HCMT cell line (da a no shown). When all ime poin s in each g oup (WT-CM, HCMT-CM, and HCMM-CM) we e combined, he numbe o mul inuclea ed CMs was signi ican ly highe in HCMT- CMs han in WT-CMs and HCMM-CMs (𝑛=6in s a is ical analysis, 𝑝 < 0.05, Figu e 2(c)). The Ca2+handling p ope ies o hiPSC-de i ed CMs we e s udied by Ca2+ imaging. HCMT-CMs had signi ican ly mo e abno mali ies han WT-CMs and HCMM-CMs, when all ime poin s in each g oup we e combined (Figu e 2(d), 𝑛= 6in s a is ical analysis, 𝑝 < 0.05). Spon aneously bea ing CMs we e di ided in o i e di e en hy hm ca ego ies (no mal, oscilla ion, low/middle peaks, double peaks, and pla eau abno mali y) based on he abno mali ies obse ed in hei Ca2+ signals (Figu es 2(e) and 2( )). HCMT-CMs had signi ican ly highe numbe o double peaks han HCMM- CMs (𝑝 < 0.05,𝑛=6in s a is ical analysis) when bo h cell lines and all ime poin s we e combined o each g oup. 3.3. Ac ion Po en ial Cha ac e is ics o WT and HCM hiPSC- De i ed Ca diomyocy es. The spon aneous ac ion po en ials we e eco ded om he bea ing hiPSC-de i ed CMs o es ablish he elec ophysiological baselines. Mos o he cells (>80%) we e en icula -like CMs in all he hiPSC lines s udied. Fo his eason, only en icula -like wa e o ms a e p esen ed he e. We i s analyzed he pe cen age o he a hy hmias in each cell line (Figu es 3(a)–3( )) and ound simila pe cen age in bo h cell lines wi hin he g oups (UTA.04602.WT (13%) e sus UTA.04511.WT (15%), UTA.02912.HCMT (42%) e sus UTA.13602.HCMT (47%), and UTA.07801.HCMM (50%) e sus UTA.06108.HCMM (50%)). Based on he pe cen age o he a hy hmias, we com- bined hiPSC-de i ed CMs in o g oups (WT-CM, HCMT- CM, and HCMM-CM) o u he analysis. Bo h HCMT-CMs and HCMM-CMs had mo e a hy hmic e en s including delayed a e depola iza ions (DADs) and ea ly a e depola iza ions (EADs) when compa ed o he WT-CMs (WT-CM (14%), HCMT- CM (45%), and HCMM-CM (50%)). We quan i ied he occu ence o DADs in hiPSC-de i ed CMs as a a e (DADs/min) calcula ed as o al numbe o DADs/ o al numbe o APs.We ound ha heDAD a einHCMM- CMs was signi ican ly highe han in WT-CMs (Figu e 3(g), 𝑝 < 0.005). S em Cells In e na ional 9 WT (a) HCMT (b) HCMM (c) HCMT (d) HCMM (e) n = 23 n = 20 n = 24 n = 47 n = 32 n = 22 80 60 40 20 (%) DADs and EADs EADs DADs No mal UTA.04602.WT UTA.04511.WT UTA.02912.HCMT UTA.13602.HCMT UTA.07801.HCMM UTA.06108.HCMM ( ) 2 4 6 8 DADs/min n=43 WT n=71 HCMT n=54 HCMM ∗∗ (g) Figu e 3: A hy hmogenic e en s (DADs and EADs) we e obse ed in HCM-CMs. (a)–(e) Rep esen a i e eco dings o con ol hiPSC- de i ed CMs (WT) and hiPSC-de i ed CMs ca ying TPM1-Asp175Asn (HCMT) o MYBPC3-Gln1061X (HCMM) mu a ions. Typical DADs (a ows) a e p esen ed in (b) and (c) and EADs (a ows) in (d) and (e) o HCMT-CMs and HCMM-CMs, espec i ely. Scale ba s ep esen 40 m and 5 seconds, espec i ely. Scale ba s in (a) a e ep esen a i e o (b) and (c), and scale ba s in (d) a e ep esen a i e o (e). ( ) Dis ibu ion o CMs exhibi ing a hy hmogenic e en s in each cell line. (g) DAD a e was signi ican ly highe in HCMM-CMs han in WT- CMs (∗∗𝑝< 0.005). 16 S em Cells In e na ional [28] B. J. Ma on, “Hype ophic ca diomyopa hy: a sys ema ic e iew,” The Jou nal o he Ame ican Medical Associa ion, ol. 287, no. 10, pp. 1308–1320, 2002. [29] P. Liang, F. Lan, A. S. Lee e al., “D ug sc eening using a lib a y o human induced plu ipo en s em cell-de i ed ca diomyocy es e eals disease-speci ic pa e ns o ca dio oxici y,” Ci cula ion, ol.127,no.16,pp.1677–1691,2013. [30] P. J¨ a¨ askel¨ ainen, R. Mie inen, P. K¨ a kk¨ ainen, L. Toi onen, M. Laakso, and J. Kuusis o, “Gene ics o hype ophic ca diomy- opa hy in eas e n Finland: ew ounde mu a ions wi h benign o in e media y pheno ypes,” Annals o Medicine, ol.36,no.1, pp.23–32,2004. [31] P. J¨ a¨ askel¨ ainen, J. Kuusis o, R. Mie inen e al., “Mu a ions in he ca diac myosin-binding p o ein C gene a e he p edominan cause o amilial hype ophic ca diomyopa hy in eas e n Fin- land,” Jou nal o Molecula Medicine, ol.80,no.7,pp.412–422, 2002. [32] S. Ma s on, O. Copeland, A. Jacques e al., “E idence om human myec omy samples ha MYBPC3 mu a ions cause hype ophic ca diomyopa hy h ough haploinsu iciency,” Ci - cula ion Resea ch, ol.105,no.3,pp.219–222,2009. [33] W. Ro baue , M. Gau el, J. Zehelein e al., “No el splice dono si e mu a ion in he ca diac myosin-binding p o ein-C gene in amilial hype ophic ca diomyopa hy. Cha ac e iza ion o ca diac ansc ip and p o ein,” Jou nal o Clinical In es iga ion, ol. 100, no. 2, pp. 475–482, 1997. [34] S. J. Van Dijk, D. Dooijes, C. Dos Remedios e al., “Ca - diac myosin-binding p o ein C mu a ions and hype ophic a diomyopa hy haploinsu iciency, de anged phospho yla ion, and ca diomyocy e dys unc ion,” Ci cula ion, ol. 119, no. 11, pp. 1473–1483, 2009. [35] A. S. Helms, F. M. Da is, D. Coleman e al., “Sa come e mu a ion-speci ic exp ession pa e ns in human hype ophic ca diomyopa hy,” Ci cula ion: Ca dio ascula Gene ics, ol.7, no. 4, pp. 434–443, 2014. [36]J.L.Theis,J.M.Bos,J.D.Theise al.,“Exp essionpa e ns o ca diac myo ilamen p o eins: genomic and p o ein analysis o su gical myec omy issue om pa ien s wi h obs uc i e hype ophic ca diomyopa hy,” Ci cula ion: Hea Failu e, ol. 2, no. 4, pp. 325–333, 2009. 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