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

Research Article Mutation-Specific Phenotypes in hiPSC-Derived Cardiomyocytes Carrying Either Myosin-Binding Protein C Or 𝛼-Tropomyosin Mutation for Hypertrophic Cardiomyopathy Marisa Ojala,1Chandra Prajapati,1Risto-Pekka Pölönen,1Kristiina Rajala,1 Mari Pekkanen-Mattila,1Jyrki Rasku,2Kim Larsson,1and Katriina Aalto-Setälä1,3,4 1BioMediTech, University of Tampere, 33014 Tampere, Finland 2School of Information Sciences, University of Tampere, 33014 Tampere, Finland 3Medical School, University of Tampere, 33014 Tampere, Finland 4Heart Hospital, Tampere University Hospital, 33521 Tampere, Finland Correspondence should be addressed to Katriina Aalto-Set¨ al¨ a; kat[email protected] Received 21 May 2015; Revised 22 July 2015; Accepted 20 September 2015 Academic Editor: Farah Sheikh Copyright © 2016 Marisa Ojala et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Hypertrophic cardiomyopathy (HCM) is a genetic cardiac disease, which affects the structure of heart muscle tissue. The clinical symptoms include arrhythmias, progressive heart failure, and even sudden cardiac death but the mutation carrier can also be totally asymptomatic. To date, over 1400 mutations have been linked to HCM, mostly in genes encoding for sarcomeric proteins. However, the pathophysiological mechanisms of the disease are still largely unknown. Two founder mutations for HCM in Finland are located in myosin-binding protein C (MYBPC3-Gln1061X)and𝛼-tropomyosin (TPM1-Asp175Asn) genes. We studied the properties of HCM cardiomyocytes (CMs) derived from patient-specific human induced pluripotent stem cells (hiPSCs) carrying either MYBPC3-Gln1061X or TPM1-Asp175Asn mutation. Both types of HCM-CMs displayed pathological phenotype of HCM but, more importantly, we found differences between CMs carrying either MYBPC3-Gln1061X or TPM1-Asp175Asn gene mutation in their cellular size, Ca2+ handling, and electrophysiological properties, as well as their gene expression profiles. These findings suggest that even though the clinical phenotypes of the patients carrying either MYBPC3-Gln1061X or TPM1-Asp175Asn gene mutation are similar, the genetic background as well as the functional properties on the cellular level might be different, indicating that the pathophysiological mechanisms behind the two mutations would be divergent as well. 1. Introduction Hypertrophic cardiomyopathy (HCM) is one of the most common genetic cardiac diseases with worldwide prevalence of 1 : 500, as well as the most common cause of sudden cardiac death (SCD) among young competing athletes. HCM isinheritedinanautosomaldominantpattern.Nevertheless, a large clinical diversity and age-related penetrance are typical for HCM. On the tissue level, HCM is characterized by the disarray of cardiomyocytes (CMs) and fibrosis of cardiac tissue, as well as thickened interventricular septum or free left ventricular wall. Clinical symptoms include arrhythmias, progressiveheartfailure,andevenSCD,butontheother handthemutationcarriercanbecompletelyasymptomatic. Altogether more than 1400 mutations in 11 genes encoding for the sarcomeric proteins have been identified and related to HCM. The majority of the mutations are found either in the 𝛽-myosin heavy chain (MYH7) or in the myosin-binding protein C (MYBPC3) genes [1]. In Finland, two founder mutations located in MYBPC3 and 𝛼-tropomyosin (TPM1) genes and one common mutation in MYH7 gene together account around 24% of all Finnish HCM cases [2, 3]. Although the genetic information related to HCM has been growing in the recent years due to the development of sequencing technologies, exact information of the disease mechanisms remains unclear. Thus, current medication of the diseaseisdirectedtowardthesymptomreliefandthereisno specific therapy to prevent the onset or progression of the Hindawi Publishing Corporation Stem Cells International Volume 2016, Article ID 1684792, 16 pages http://dx.doi.org/10.1155/2016/1684792 2Stem Cells International disease [1]. Most of the HCM studies have been conducted with model systems, mainly either with transgenic mice or by studying human tissues obtained from surgical myectomy from end-stage HCM patients [4]. However, animal models carry only the mutated gene lacking the rest of the genome and myectomy samples are obtained from patients in the late stage of HCM development. Therefore, the discovery of the human induced pluripotent stem cells (hiPSCs) has offered a new valuable tool to model HCM and other cardiac diseases andtostudytheunderlyingdiseasemechanisms[5].Todate, hiPSCs have already been used to model a variety of cardiac diseases: electrical defects, for example, long-QT syndrome [6–8] and catecholaminergic polymorphic ventricular tachycardia (CPVT) [9, 10] as well as cardiomyopathies including dilated cardiomyopathy (DCM) [11] and HCM [12–14]. Here we have derived hiPSCs from patients carrying two of the Finnish HCM founder mutations either in MYBPC3 (MYBPC3-Gln1061X)orinTPM1 (TPM1-Asp175Asn)gene. We have differentiated the patient-specific hiPSCs into CMs and compared the phenotypes of the diseased and control CMs. 2. Materials and Methods 2.1. Ethical Issues. This study was conducted in accordance with the Ethics Committee of Pirkanmaa Hospital District to establish, culture, and differentiate hiPSC lines (R08070). Skin biopsies for hiPSC establishment were received from the Heart Hospital, Tampere University Hospital, Tampere, Finland. Patients donating skin biopsies signed an informed consent after receiving both oral and written descriptions of the study. The teratoma assay, described in Section 2.3.6, was approved by ELLA-Animal Experiment Board of Regional State Administrative Agency for Southern Finland (ESAVI/6543/04.10.03/2011). 2.2. Generation and Culture of Patient-Specific hiPSC Lines. hiPSC lines were generated from skin’s fibroblasts either with Sendai reprogramming vectors OCT4,KLF4,c-MYC,and SOX2 using CytoTune-iPS Reprogramming Kit (Life Technologies Ltd., Paisley, UK) according to the manufacturer’s instructions or by using pMX retroviral vectors OCT4,KLF4, c-MYC,andSOX2 with or without Cre-LoxP site as described earlier [6, 15]. UTA.13602.HCMT, UTA.02912.HCMT, and UTA.04511.WT hiPSC lines were generated by using Sendai vectors and UTA.07801.HCMM and UTA.06108.HCMM by using pMX retroviral vectors with Cre-LoxP site and UTA.04602.WT was generated by using pMX retroviral vectors without Cre-LoxP site. In the present study, one line of each patient was used. hiPSC lines were derived and cultured on mouse embryonic fibroblast (MEF) feeder cell layers (26000 cells/cm2, CellSystems Biotechnologie Vertrieb GmbH, Troisdorf, Germany) in human pluripotent stem cell (hPSC) culture medium consisting of knockout-DMEM (koDMEM,Gibco,LifeTechnologiesLtd.)supplementedwith 20% knockout serum replacement (ko-SR, Gibco, Life Technologies Ltd.), 1% nonessential amino acids (NEAA, Lonza Group Ltd., Basel, Switzerland), 2 mM GlutaMax (Gibco, Life Technologies Ltd.), 50 U/mL penicillin/streptomycin (Lonza Group Ltd.), 0.1 mM 2-mercaptoethanol (Gibco, Life Technologies Ltd.), and 4 ng/mL basic fibroblast growth factor (bFGF, PeproTech, Rocky Hill, NJ, USA). 2.3. Characterization of hiPSC Lines 2.3.1. Mutation Analysis by Genotyping. DNA samples from thehiPSClineswerepreparedwithTaqManSample-toSNP Kit (Applied Biosystems, Life Technologies Ltd.) and the presence of MYBPC3-Gln1061X and TPM1-Asp175Asn mutation in the patient-specific hiPSC lines was confirmed by custom TaqMan SNP Genotyping Assays (Applied Biosystems, Life Technologies Ltd.) according to the manufacturer’s instructions. In the genotyping assays, MYBPC3-gene as well as TPM1-gene was amplified with specific primers. Furthermore, the presence of the mutations was assessed with mutation-specific FAM labeled probes. VIC labeled probes were used to assess the presence of the wild type allele. Sequencesfortheprimersandprobesusedintheassayare listed in Supplementary Table 1 (see Supplementary Material available online at http://dx.doi.org/10.1155/2016/1684792). 2.3.2. The Expression of Mutant and Wild Type Alleles in hiPSC-Derived CMs. RNA samples were collected and extracted from hiPSC-derived CMs (UTA.04511.WT, UTA.02912.HCMT, UTA.07801.HCMM, and UTA.06108. HCMM) with Norgen’s Total RNA Purification Plus Kit (Norgen Biotek Corp., Ontario, Canada) according to manufacturer’s instructions. 50–100 ng of RNA was transcribed to cDNA by High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Life Technologies Ltd.). The expression of TPM1-Asp175Asn or MYBPC3-Gln1061X mutation on mRNA level in the hiPSC-derived CMs was studied by Custom TaqMan SNP Genotyping Assays (Applied Biosystems, Life Technologies Ltd.) similarly as that for genotyping described above. Sequences for the primers and probesusedintheassayarelistedinSupplementaryTable1. 2.3.3. Immunocytochemistry. Undifferentiated hiPSC colonies were fixed with 4% paraformaldehyde (PFA, SigmaAldrich, Saint Louis, USA), stained with primary antibodies for Nanog (R&D systems Inc., Minneapolis, MN, USA), OCT4 (R&D systems Inc.), SOX2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), TRA-1-60 (Millipore, Billerica, MA, USA), and TRA-1-81 (Millipore), and visualized with secondary antibodies as described before [16]. Finally, the cells were mounted with Vectashield (Vector Laboratories Inc., Burlingame, CA, USA) containing 40,6-diamidino-2phenylindole (DAPI) for the nuclei staining and imaged with an Olympus IX51 phase contrast microscope equipped with fluorescence optics and Olympus DP30BW camera (Olympus Corporation, Hamburg, Germany). 2.3.4. RT-PCR. The RNA was extracted from the hiPSC lines by NucleoSpin RNA II Kit (Macherey-Nagel GmbH & Co., D¨ uren, Germany) and 500–1000 ng of RNA was transcribed to cDNA by High-Capacity cDNA Reverse Transcription Kit Stem Cells International 3 (Applied Biosystems, Life Technologies Ltd.). The presence of pluripotency genes Nanog,SOX2,REX1,OCT4,andc-MYC andtheabsenceofvirallyimportedexogenes(OCT4,SOX2, c-MYC,andKLF4) were confirmed by RT-PCR. GAPDH was used as an endogenous control. The primer sequences for pluripotency genes and virally imported exogenes have been published earlier [5]. The primer sequences used for detection of Sendai transgenes are described in CytoTune-iPS Reprogramming Kit’s manual (Life Technologies Ltd.). 2.3.5. Karyotype Analysis. The karyotypes of hiPSC lines were studied by G-banding (Medix Laboratories, Espoo, Finland) or by KaryoLite assay [17] (Turku Centre for Biotechnology, University of Turku, Turku, Finland). 2.3.6. Pluripotency Analysis. The pluripotency of hiPSC lines was confirmed in vitro by embryoid body (EB) formation and in vivo by teratoma assay. hiPSCs were removed from feeder cell layer and cultured in suspension to form EBs. The EBs were cultured in EB medium consisting of ko-DMEM supplemented with 20% fetal bovine serum (FBS, Biosera, Boussens,France),1%NEAA(LonzaGroupLtd.),2mM GlutaMax (Invitrogen, Life Technologies Ltd.), and 50 U/mL penicillin/streptomycin (Lonza Group Ltd) for 4–6 weeks before RNA extraction. 200 ng of RNA was transcribed to cDNAfortheRT-PCRanalysis.Thepresenceofallthreegerm layers, endoderm (AFP, SOX17), ectoderm (SOX1, NESTIN, and Musashi), and mesoderm (KDR, alpha cardiac actin), was studied with RT-PCR. For in vivo pluripotency assay, hiPSCs were injected under the testis capsule of nude mice and the formed teratomas were collected and fixed with 4% PFA 8 weeks after the injection. Teratomas were embedded in paraffin, cut in sections, and stained with haematoxylin and eosin. 2.4. Differentiation of Cardiomyocytes. hiPSCs were differentiated into CMs by coculturing with mouse visceral endodermal-like cells (END-2) (Hubrecht Institute, Utrecht, Netherlands) as described before [18]. After 15–30 days beating areas were cut from cocultures and dissociated into single cells in EB medium by Collagenase A (Roche Diagnostics, Mannheim, Germany) as described earlier [18] and plated to 0.1% gelatin-coated cover slips or well plates for further analysis. 2.5. Characterization of hiPSC-Derived Cardiomyocytes 2.5.1. Immunocytochemistry and Image Analysis. Dissociated CMs were fixed with 4% PFA and stained with Troponin T(cTnT,1:2000,ab64623,Abcam,Cambridge,MA,USA), MYBPC (1 : 400, sc-166081, Santa Cruz Biotechnology), and TPM1 (1 : 200, sc-73225, Santa Cruz Biotechnology) primary antibodies, followed by labeling with secondary antibodies. Images were obtained with Olympus IX51 phase contrast microscope equipped with fluorescence optics and Olympus DP308W camera (Olympus Corporation) or with Zeiss AxioScope A1 fluorescent microscope and Zeiss AxioCam MRc5 camera (Carl Zeiss, Jena, Germany). Size of the Troponin T stainedCMswasanalyzedfrom46to50CMsineachcellline by in-house made software (unpublished method). CMs were analyzed from pictures obtained with Olympus IX51 phase contrast microscope. The proportion of multinucleated CMs was determined from the same images (46–50 CMs/cell line). 2.5.2. Ca2+ Imaging. The clusters of CMs were cut, dissociated, plated on 0.1% gelatin-coated coverslips, and cultured for1,3,and6weeks.TostudytheCa 2+ handling properties of hiPSC-derived CMs, cells were loaded with 4 𝜇M Fura-2 AM (Molecular Probes, Life Technologies Ltd.) for 30 minutes in perfusate medium. The perfusate medium consisted of (in mM) 137 NaCl, 5 KCl, 0.44 KH2PO4, 20 HEPES, 4.2 NaHCO3, 5 D-glucose, 2 CaCl2,1.2MgCl 2,and1Na-pyruvate dissolvedinH 2O. pH of the perfusate medium was adjusted to 7.4 with NaOH. The coverslip, containing the dissociated hiPSC-derived CMs, was mounted to an RC-25 recording chamber and continuously perfused with perfusate medium preheated to 35-36∘C by an SH-27B inline-heater controlled byaTC-324Bunit(allfromWarnerInstrumentsInc., Hamden, USA). The perfusion was controlled by a gravity driven VC38 application system (ALA Scientific Instruments Inc., NY, USA). Coverslip was perfused for 15 minutes for Fura-2 AM deesterification before experimental recordings. Ca2+ handling of spontaneously beating CMs was imaged with an inverted IX70 microscope using UApo/340 x20 air objective (Olympus Corporation) and ANDOR iXon 885 CCD camera (Andor Technology, Belfast, Northern Ireland) synchronized with a Polychrome V light source by a real time DPScontrolunit.TILLvisIONorLiveAcquisitionsoftware (TILL Photonics, Munich, Germany) was used for recording. Fura-2 AM was excited at 340 nm and 380 nm light and the emission was recorded for 10–30 seconds at 505 nm. For Ca2+ imaging analysis, single beating CMs were selected as regions of interests and background noise, recorded from a cell-free area in the same coverslip, was subtracted before further processing. Data is presented as ratios of 340/380 nm (F340/F380). The spontaneously beating CMs were divided into five different rhythm categories based on the abnormalities observed in their Ca2+signals: normal beating with regular peaks (normal); more than three peaks which do not return to the baseline (oscillation); signals with small or middle sized amplitude events in the beginning, in theend,orinbetweentwoCa 2+ spikes (low/middle peaks); two or three peaks which do not return to the baseline (double peaks); Ca2+ spikes with prolonged rise or decay time (plateau abnormality). In the low/middle peaks category, the small amplitude was at least 10% from the preceding Ca2+ spike amplitude. Full-length, 10–30 seconds long recordings were analyzed, while most of the analyzed recordings were 12 seconds long. The distribution of CMs in different categories is presented for each cell lines separately. 2.5.3. Electrophysiological Measurements: Recording and Analysis of Action Potentials. The action potentials (APs) were recorded by conventional patch clamp [19] in perforated patch configuration using Amphotericin B [20] in final concentration of 0.24 mg/mL [8]. Data acquisition was 4Stem Cells International Table1:TaqManassaysusedinqRT-PCRexperiments. Gene Description/alias Function TaqMan assay ID EEF1A1 Eukaryotic translation elongation factor 1 alpha 1 Housekeeping gene Hs00265885 g1 GAPDH Glyceraldehyde-3-phosphate dehydrogenase Housekeeping gene Hs02758991 g1 TNNT2 Troponin T Sarcomeric gene Hs00165960 m1 MYH6 Myosin heavy chain 6 Sarcomeric gene Hs01101425 m1 ACTN2 𝛼-actinin 2 Sarcomeric gene Hs00153809 m1 TPM1 𝛼-tropomyosin Sarcomeric gene Hs00165966 m1 MYBPC3 Myosin-binding protein C Sarcomeric gene Hs00165232 m1 TTN Titin Sarcomeric gene Hs00399225 m1 TNNC1 Troponin C type 1 Sarcomeric gene Hs00896999 g1 MYL9 Myosin, light chain 2/MLC2 Sarcomeric gene Hs00697086 m1 MYL7 Myosin, light chain 7 Sarcomeric gene Hs01085598 g1 NPPA Natriuretic peptide A Hypertrophy marker Hs01081097 m1 NPPB Natriuretic peptide B Hypertrophy marker Hs01057466 g1 HCN4 Hyperpolarization activated cyclic nucleotide-gated potassium channel 4 Potassium channel Hs00975492 m1 KCNQ1 Voltage-gated potassium channel, KQT-like subfamily, member 1 Potassium channel Hs00923522 m1 CACNA1C Voltage-dependent calcium channel, L type, alpha 1C subunit/CaCNA1.2 Calcium channel Hs00167681 m1 SCN5A Voltage-gated sodium channel, V type, alpha subunit Sodium channel Hs00165693 m1 SLC8A1 Solute carrier family 8, member 1/NCX1 Na+/Ca2+ exchanger Hs01062258 m1 PLN Phospholamban/PLB Protein kinase substrate Hs01848144 s1 ATP2A2 ATPase, Ca2+ transporting, cardiac muscle, slow twitch 2/SERCA2a Ca2+-ATPase Hs00544877 m1 CASQ2 Calsequestrin Ca2+ binding protein in SR Hs00154286 m1 ITPR2 Inositol 1,4,5-trisphosphate receptor, type 2/IP3R2 Ca2+ receptor Hs00181916 m1 RYR2 Ryanodine receptor 2 (cardiac) Ryanodine receptor Hs00892883 m1 conducted using Axon Series 200B patch-clamp amplifier connected to Digidata 1440a AD/DA converter driven by pCLAMP 10.2 software (all from Molecular devices LLC). On the day of use, the coverslips containing dissociated hiPSC-derived CMs were transferred to RC-24N recording chamber (Warner Instruments Inc.) and mounted on an inverted Olympus IX71 microscope (Olympus Corporation). The patch electrodes had tip resistance of 3.0–3.5 MΩand contained the following intracellular solution (in mM): 132 KMeSO4,20KCl,1MgCl 2, and 1 CaCl2(pH was adjusted to 7.2 with KOH). The extracellular solution contained (in mM) 143 NaCl, 4.8 KCl, 1.8 CaCl2, 1.2 MgCl2,5glucose,and10 HEPES (pH was adjusted to 7.4 with NaOH). The preheated extracellular solution was continuously perfused with similar setupcomparedtowhatispresentedinSection2.5.2.Patch pipettes (Harvard Apparatus Ltd., Holliston, MA, USA) were freshly prepared using PC-10 micropipette puller and then flame-polished with MF-830 microforge (both from Narishige Int., Tokyo, Japan). APs were recorded in the gap-free mode in the current clamp from the spontaneously beating hiPSC-derived CMs. Current-clamp recordings were digitally sampled at 20 kHz and filtered at 2 kHz using low pass Bessel filter on recording amplifier. Beats per minute (BPM), AP duration (APD50 and APD90), AP amplitude (APA), and maximum diastolic potential (MDP) were analyzed from the recorded APs by using Origin 9.1 (OriginLab Corp., Northampton, USA). Only ventricular-like waveforms are presented here to avoid any biasness among different hiPSC lines. The ventricularlike CMs were characterized by APD90/APD50 <1.3 and APA >90 mV. 2.5.4. Real-Time qRT-PCR Analysis. After one week of culture, dissociated CMs were collected into a lysis solution buffer of CellsDirect One-Step qRT-PCR Kit (Life Technologies Ltd.) according to the manufacturer’s instructions. Two replicate samples were collected and stored at −70∘Cuntilthe DNase I digestion and reverse transcription-specific target amplification (RT-STA) by using CellsDirect One-Step qRTPCR Kit. Real-Time qPCR was performed with Biomark HD system (Fluidigm Corp., San Francisco, USA) according to the manufacturer’s instructions. The TaqMan assays (Life TechnologiesLtd.)usedintheqRT-PCRarecollectedin Table1.Allsampleswereanalyzedinduplicateandthefold changeswerecalculatedbythe2−ΔΔCT method [21]. EEF1A1 and GAPDH genes were used as endogenous control genes and UTA.04511.WT cell line was used as a calibrator. 2.5.5. Western Blot. hiPSC-derived CMs were lysed in MPER protein extraction reagent (Thermo Scientific, Life Technologies Ltd.), supplemented with complete protease inhibitor cocktail (Roche Diagnostics). The protein concentration was quantified with BCA protein assay kit (Thermo Scientific, Life Technologies Ltd.). 10 𝜇gofproteinwasrun to 4–15% mini-PROTEAN TGX precast polyacrylamide gel Stem Cells International 5 (Bio-Rad, Hercules, CA, USA) and transferred to PVDF membrane (Amersham Hybond-P, GE Healthcare, Little Chalfont, UK). Membranes were blocked with 5% milk for 1 h at RT and proteins were stained with MYBPC (1 : 1500, sc166081, Santa Cruz Biotechnology), cTnT (1 : 2000, ab64623, Abcam), TPM1 (1 : 200, sc-73225, Santa Cruz Biotechnology), or 𝛽-actin (1 : 1000, sc-47778, Santa Cruz Biotechnology) primary antibodies over night at +4∘C. Horseradish peroxidase- (HRP-) conjugated polyclonal rabbit anti-mouse (DAKO, P0260) and rabbit anti-goat IgG (Santa Cruz Biotechnology, sc-2922) were used as secondary antibodies. Stained proteins were detected by using Clarity ECL substrate (Bio-Rad) and visualized by Molecular Imager ChemiDOc XRS+ (Bio-Rad). ImageJ software (National Institutes of Health, USA) was used to compare the expression of MYBPC, cTnT, and TPM1 with the 𝛽-actin expression from the same cell line. 2.6. Statistical Analysis. For statistical analysis, control cell lines and cell lines in each mutation were combined in groups: UTA.04602.WT and UTA.04511.WT hiPSC lines in WT-CM group, UTA.02912.HCMT and UTA.13602.HCMT in HCMTCM group, and UTA.07801.HCMM and UTA.06108.HCMM in HCMM-CM group. Mann-Whitney 𝑈test with Bonferroni’s correction was used to analyze the differences between WT-, HCMT-, and HCMM-CMs in cell size analysis, proportion of multinucleated CMs, and Ca2+ imaging experiments as well as in gene expression analysis. For the statistical comparison between the three groups, one-way ANOVA followed by Tukey test was used for the patch-clamp result analysis. 𝑝 < 0.05 was considered statistically significant. All error bars are presented as standard error of the mean (SEM). 3. Results 3.1. hiPSCs Were Derived from HCM Patients with Different Backgrounds. We derived hiPSCs from four patients carrying a HCM causing mutation either in TPM1 (TPM1-Asp175Asn) or in MYBPC3 (MYBPC3-Gln1061X). UTA.13602.HCMT and UTA.02912.HCMTs carry TPM1-Asp175Asn and UTA.07801. HCMM and UTA.06108.HCMM MYBPC3-Gln1061X mutation. The hiPSC lines and their mutations and abbreviations, used below, are presented in Table 2. UTA.13602.HCMT (46, XX) is derived from a 48-year-old female, whose mother died suddenly at the age of 51. Our patient has had one collapse at the age of 20 with normal heart structure, but later slight thickening of septum (16 mm) has been observed. Currently she is not on medication due to low blood pressure. UTA.02912.HCMT (46, XY) is derived from a 33-year-old male whose family member has died suddenly at the age of 21. The maximal septal thickness of our patient has been measured to be 26 mm on echocardiogram. The patient has been asymptomatic but is currently on 𝛽-blocker medication. UTA.07801.HCMM (46, XY) is derived from a 61-year-old male with no SCDs in the family. On echocardiogram, his myocardial septum has been observed to be 25 mm. He has atrial fibrillation and he is on 𝛽-blocker medication. Due to bradycardia and frequent nonsustained ventricular tachycardia episodes, an implantable cardioverter defibrillator (ICD) Table 2: The hiPSC lines and their mutations and abbreviations used in the study. Group Cell line Mutation Name in Figure 2(f) 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 implanted. UTA.06108.HCMM (46, XY) is derived from a 55-year-old male whose father died suddenly at the age of 36 and uncle at the age of 38. Our patient has been asymptomatic with maximal septal thickness of 22 mm on echocardiogram. He is not on medication due to low blood pressure. Control hiPSC lines were derived from healthy individuals: UTA.04602.WT (46, XX) from a 56-year-old female and UTA.04511.WT (46, XY) from a 34-year-old male. The pluripotent characteristics of the hiPSC-lines used were assessed (Figure 1 and Supplementary Figures 1–5). UTA.04602.WT cell line has been characterized earlier [22]. All the lines formed colonies, which expressed proteins and genes typical for hPSCs. The virally transferred exogenous genes were silenced and karyotypes of the hiPSC lines were normal. The pluripotency of hiPSC lines was proven in vitro by EB formation or in vivo by teratoma formation. The presence of TPM1-Asp175Asn and MYBPC3-Gln1061X mutations in the patient-specific hiPSC lines was confirmed by custom TaqMan SNP Genotyping Assays (Supplementary Figure 6). 3.2. Mutation-Specific HCM Phenotypes Were Observed in hiPSC-Derived Cardiomyocytes. All the cell lines used in the present study differentiated into cardiomyocytes similarly. Beatingaggregateswereformed14–20daysaftertheinitiation of coculturing with END-2 cells and there was no difference between the lines when the beating areas appeared. After cardiac differentiation, beating clusters were dissociated into single cells and cultured for 1, 3, and 6 weeks. The differences in the cell sizes and Ca2+ handling properties between two different mutations and control cells were analyzed in each time point. When comparing different types of CMs, HCMM-CMs were significantly larger than HCMT-CMs and WT-CMs in all three time points (Figures 2(a) and 2(b), 𝑛=96–100, 𝑝 < 0.005 in all cases). The enlargement of HCMT-CMs was detected after three weeks of culture when they were significantly larger than WT-CMs (𝑝< 0.005). Generally, the size of the hiPSC-derived CMs in all groups increased when the cells cultured for three weeks. Within each group, there were no differences in cell sizes between three and six weeks, except with HCMT-CMs, which seemed to be smaller in size 6-week time point. However, during 6 weeks of culture, cell types other than CMs had the tendency to overgrow the CM culture, which might have affected the cellular enlargement. This phenomenon was 6Stem Cells International 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) Mesoderm Endoderm Ectoderm (d) Figure 1: Characterization of UTA.13602.HCMT cell line. (a) The hiPSCs formed colonies expressing Nanog, OCT4, SOX2, TRA-1-60, and TRA-1-81.Scalebars:200𝜇m. (b) The virally transferred Sendai exogenes, exo-OCT4 (483 bp), exo-KLF4 (410 bp), exo-SOX2 (451 bp), and exoc-MYC (532 bp), were silenced in the hiPSCs. + indicates positive controls, for which RNA was extracted from cells 1 week after transduction. hiPSCs expressed 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 control. (c) The hiPSC line was karyotypically normal, 46 XX. (d) The pluripotency of hiPSCs was confirmed by in vivo teratoma assay, in which hiPSCs formed all three germ layers (mesoderm, endoderm, and ectoderm). Stem Cells International 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 $$ && (b) ∗∗ 50 40 30 20 10 (%) WT 1w WT 3w WT 6w HCMT 1w HCMT 3w HCMT 6w HCMM 1w HCMM 3w HCMM 6w (c) ∗∗ 50 40 30 20 10 (%) WT 1w WT 3w WT 6w HCMT 1w HCMT 3w HCMT 6w HCMM 1w HCMM 3w HCMM 6w (d) Normal 246810 Time (s) F340/F380 Oscillation 246810 Time (s) F340/F380 Low/middle peaks 246810 Time (s) F340/F380 Plateau abnormality 246810 Time (s) F340/F380 Double peaks 246810 Time (s) F340/F380 (e) Figure 2: Continued. 8Stem Cells International 50 60 70 80 90 40 30 20 10 (%) n= WT11w WT13w WT16w WT21w WT23w WT26w HCMT11w HCMT13w HCMT16w HCMT21w HCMT23w HCMT26w HCMM11w HCMM13w HCMM16w HCMM21w HCMM23w HCMM26w 89 24 20 99 66 50 99 104 19 145 24 97 78 141 83 90 29 42 (f) Figure 2: The cell size and Ca2+ handling of hiPSC-derived CMs after 1-, 3-, and 6-week culture as single cells. (a) Representative images of WT-CMs (WT), HCMT-CMs (HCMT), and HCMM-CMs (HCMM) stained with antibodies for cTnT (red) and MYBPC (green) proteins. Scale bars are 100 𝜇m. (b) The size of the HCMM-CMs was significantly larger in all three time points when compared to WTand HCMTCMs ($$𝑝< 0.005 when compared to WT-CMs or HCMT-CMs in the 1-week time point, ∗∗𝑝< 0.005 when compared to WT-CMs or HCMT-CMs in 3-week time point, and ##𝑝< 0.005 when compared to WT-CMs or HCMT-CMs in 6-week time point). HCMT-CMs were significantly larger than WT-CMs in 3-week time point (&&𝑝< 0.005 when compared to WT-CMs. 𝑛 = 100, except in HCMT 6 w 𝑛=96.) (c) The proportion of the multinucleated CMs was significantly higher in HCMT-CMs than in WT-CMs and HCMM-CMs when both cell lines and all time points were combined for each group (in statistical analysis 𝑛=6,∗𝑝< 0.05). The averages of multinucleated CMs were determined from the same cells, whose sizes and 𝑛numbers are presented in (b). (d) Significantly more CMs with Ca2+ handling abnormalities were observed in HCMT-CMs than in WT-CMs and HCMM-CMs when both cell lines and all time points were combined for each group (in statistical analysis 𝑛=6,∗𝑝< 0.05). The proportions of CMs with abnormalities in their Ca2+ handling were determined from the same Ca2+ imaging results presented in (f). The total 𝑛numbers of the analyzed CMs are presented in (f). (e) Representative images of Ca2+ rhythm categories. (f) Distributions of hiPSC-derived CMs in different Ca2+ rhythm categories (e) in each time point. 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. observed especially with UTA.13602.HCMT cell line (data not shown). When all time points in each group (WT-CM, HCMT-CM, and HCMM-CM) were combined, the number of multinucleated CMs was significantly higher in HCMTCMs than in WT-CMs and HCMM-CMs (𝑛=6in statistical analysis, 𝑝 < 0.05, Figure 2(c)). The Ca2+handling properties of hiPSC-derived CMs were studied by Ca2+ imaging. HCMT-CMs had significantly more abnormalities than WT-CMs and HCMM-CMs, when all time points in each group were combined (Figure 2(d), 𝑛= 6in statistical analysis, 𝑝 < 0.05). Spontaneously beating CMs were divided into five different rhythm categories (normal, oscillation, low/middle peaks, double peaks, and plateau abnormality) based on the abnormalities observed in their Ca2+ signals (Figures 2(e) and 2(f)). HCMT-CMs had significantly higher number of double peaks than HCMMCMs (𝑝 < 0.05,𝑛=6in statistical analysis) when both cell lines and all time points were combined for each group. 3.3. Action Potential Characteristics of WT and HCM hiPSCDerived Cardiomyocytes. The spontaneous action potentials were recorded from the beating hiPSC-derived CMs to establish the electrophysiological baselines. Most of the cells (>80%) were ventricular-like CMs in all the hiPSC lines studied. For this reason, only ventricular-like waveforms are presented here. We first analyzed the percentage of the arrhythmias in each cell line (Figures 3(a)–3(f)) and found similar percentage in both cell lines within the groups (UTA.04602.WT (13%) versus UTA.04511.WT (15%), UTA.02912.HCMT (42%) versus UTA.13602.HCMT (47%), and UTA.07801.HCMM (50%) versus UTA.06108.HCMM (50%)). Based on the percentage of the arrhythmias, we combined hiPSC-derived CMs into groups (WT-CM, HCMTCM, and HCMM-CM) for further analysis. Both HCMT-CMs and HCMM-CMs had more arrhythmic events including delayed after depolarizations (DADs) and early after depolarizations (EADs) when compared to the WT-CMs (WT-CM (14%), HCMTCM (45%), and HCMM-CM (50%)). We quantified the occurrence of DADs in hiPSC-derived CMs as a rate (DADs/min) calculated as total number of DADs/total numberofAPs.WefoundthattheDADrateinHCMMCMs was significantly higher than in WT-CMs (Figure 3(g), 𝑝 < 0.005). Stem Cells International 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 Normal UTA.04602.WT UTA.04511.WT UTA.02912.HCMT UTA.13602.HCMT UTA.07801.HCMM UTA.06108.HCMM (f) 2 4 6 8 DADs/min n=43 WT n=71 HCMT n=54 HCMM ∗∗ (g) Figure 3: Arrhythmogenic events (DADs and EADs) were observed in HCM-CMs. (a)–(e) Representative recordings of control hiPSCderived CMs (WT) and hiPSC-derived CMs carrying TPM1-Asp175Asn (HCMT) or MYBPC3-Gln1061X (HCMM) mutations. Typical DADs (arrows) are presented in (b) and (c) and EADs (arrows) in (d) and (e) for HCMT-CMs and HCMM-CMs, respectively. Scale bars represent 40 mv and 5 seconds, respectively. Scale bars in (a) are representative for (b) and (c), and scale bars in (d) are representative for (e). (f) Distribution of CMs exhibiting arrhythmogenic events in each cell line. (g) DAD rate was significantly higher in HCMM-CMs than in WTCMs (∗∗𝑝< 0.005). 16 Stem Cells International [28] B. J. Maron, “Hypertrophic cardiomyopathy: a systematic review,” The Journal of the American Medical Association,vol. 287, no. 10, pp. 1308–1320, 2002. [29] P. Liang, F. Lan, A. S. Lee et al., “Drug screening using a library of human induced pluripotent stem cell-derived cardiomyocytes reveals disease-specific patterns of cardiotoxicity,” Circulation, vol.127,no.16,pp.1677–1691,2013. [30] P. J¨ a¨ askel¨ ainen, R. Miettinen, P. K¨ arkk¨ ainen, L. Toivonen, M. Laakso, and J. Kuusisto, “Genetics of hypertrophic cardiomyopathy in eastern Finland: few founder mutations with benign or intermediary phenotypes,” Annals of Medicine,vol.36,no.1, pp.23–32,2004. [31] P. J¨ a¨ askel¨ ainen, J. Kuusisto, R. Miettinen et al., “Mutations in the cardiac myosin-binding protein C gene are the predominant cause of familial hypertrophic cardiomyopathy in eastern Finland,” Journal of Molecular Medicine,vol.80,no.7,pp.412–422, 2002. [32] S. Marston, O. Copeland, A. Jacques et al., “Evidence from human myectomy samples that MYBPC3 mutations cause hypertrophic cardiomyopathy through haploinsufficiency,” Circulation Research,vol.105,no.3,pp.219–222,2009. [33] W. Rottbauer, M. Gautel, J. Zehelein et al., “Novel splice donor site mutation in the cardiac myosin-binding protein-C gene in familial hypertrophic cardiomyopathy. Characterization of cardiac transcript and protein,” Journal of Clinical Investigation, vol. 100, no. 2, pp. 475–482, 1997. [34] S. J. Van Dijk, D. Dooijes, C. Dos Remedios et al., “Cardiac myosin-binding protein C mutations and hypertrophic ardiomyopathy haploinsufficiency, deranged phosphorylation, and cardiomyocyte dysfunction,” Circulation, vol. 119, no. 11, pp. 1473–1483, 2009. [35] A. S. Helms, F. M. Davis, D. Coleman et al., “Sarcomere mutation-specific expression patterns in human hypertrophic cardiomyopathy,” Circulation: Cardiovascular Genetics,vol.7, no. 4, pp. 434–443, 2014. [36]J.L.Theis,J.M.Bos,J.D.Theisetal.,“Expressionpatterns of cardiac myofilament proteins: genomic and protein analysis of surgical myectomy tissue from patients with obstructive hypertrophic cardiomyopathy,” Circulation: Heart Failure,vol. 2, no. 4, pp. 325–333, 2009. 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