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

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) and α-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.


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 is inherited in an autosomal dominant pattern. 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, progressive heart failure, and even SCD, but on the other hand the mutation carrier can be completely asymptomatic.
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 -myosin heavy chain (MYH7) or in the myosin-binding protein C (MYBPC3) genes [1]. In Finland, two founder mutations located in MYBPC3 and -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 disease is directed toward the symptom relief and there is no specific therapy to prevent the onset or progression of the 2 Stem 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 and to study the underlying disease mechanisms [5]. To date, hiPSCs have already been used to model a variety of cardiac diseases: electrical defects, for example, long-QT syndrome [6][7][8] and catecholaminergic polymorphic ventricular tachycardia (CPVT) [9,10] as well as cardiomyopathies including dilated cardiomyopathy (DCM) [11] and HCM [12][13][14].
Here we have derived hiPSCs from patients carrying two of the Finnish HCM founder mutations either in MYBPC3 (MYBPC3-Gln1061X) or in TPM1 (TPM1-Asp175Asn) gene. We have differentiated the patient-specific hiPSCs into CMs and compared the phenotypes of the diseased and control CMs.

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).

RT-PCR.
The RNA was extracted from the hiPSC lines by NucleoSpin RNA II Kit (Macherey-Nagel GmbH & Co., Düren, Germany) and 500-1000 ng of RNA was transcribed to cDNA by High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Life Technologies Ltd.). The presence of pluripotency genes Nanog, SOX2, REX1, OCT4, and c-MYC and the absence of virally imported exogenes (OCT4, SOX2, c-MYC, and KLF4) 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.).

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). 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.

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. Hamden, USA). The perfusion was controlled by a gravity driven VC 3 8 application system (ALA Scientific Instruments Inc., NY, USA). Coverslip was perfused for 15 minutes for Fura-2 AM deesterification before experimental recordings. Ca 2+ 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 DPS control unit. TILLvisION or Live Acquisition software (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.

Characterization of hiPSC-Derived
For Ca 2+ 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 Ca 2+ 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 the end, or in between two Ca 2+ spikes (low/middle peaks); two or three peaks which do not return to the baseline (double peaks); Ca 2+ 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 Ca 2+ 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.

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 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 (APD 50 and APD 90 ), 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 APD 90 /APD 50 < 1.3 and APA > 90 mV.

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 ∘ C until the DNase I digestion and reverse transcription-specific target amplification (RT-STA) by using CellsDirect One-Step qRT-PCR 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 Technologies Ltd.) used in the qRT-PCR are collected in Table 1. All samples were analyzed in duplicate and the fold changes were calculated by the 2 −ΔΔCT method [21]. EEF1A1 and GAPDH genes were used as endogenous control genes and UTA.04511.WT cell line was used as a calibrator.

Western
Blot. hiPSC-derived CMs were lysed in M-PER 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 g of protein was run 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, sc-166081, Santa Cruz Biotechnology), cTnT (1 : 2000, ab64623, Abcam), TPM1 (1 : 200, sc-73225, Santa Cruz Biotechnology), or -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 -actin expression from the same cell line. Mann-Whitney test 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 Ca 2+ 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. < 0.05 was considered statistically significant. All error bars are presented as standard error of the mean (SEM).

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 -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 -blocker medication. Due to bradycardia and frequent nonsustained ventricular tachycardia episodes, an implantable cardioverter defibrillator (ICD) 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).

Mutation-Specific HCM Phenotypes
Were Observed in hiPSC-Derived Cardiomyocytes. All the cell lines used in the present study differentiated into cardiomyocytes similarly. Beating aggregates were formed 14-20 days after the initiation 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 Ca 2+ 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), = 96-100, < 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 ( < 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     Figure 2(c)).
The Ca 2+ handling properties of hiPSC-derived CMs were studied by Ca 2+ 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), = 6 in statistical analysis, < 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 Ca 2+ signals (Figures 2(e) and 2(f)). HCMT-CMs had significantly higher number of double peaks than HCMM-CMs ( < 0.05, = 6 in statistical analysis) when both cell lines and all time points were combined for each group. 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%), HCMT-CM (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 number of APs. We found that the DAD rate in HCMM-CMs was significantly higher than in WT-CMs (Figure 3  The average APD at 50% repolarization (APD 50 ) and 90% repolarization (APD 90 ) of HCMT-CMs was significantly longer than those of the WT-CMs (APD 50 ( < 0.005) and APD 90 ( < 0.005)) and HCMM-CMs (APD 50 ( < 0.05) and APD 90 ( < 0.05)) ( Table 3). APD 90 of HCMM-CMs was significantly longer than that of the WT-CMs ( < 0.05) ( Table 3). Furthermore, the beating rates of both HCMT-CMs and HCMM-CMs were significantly lower than in WT-CMs (WT-CM versus HCMT-CM ( < 0.005) and WT-CM versus HCMM-CM ( < 0.005)). In addition, the APA of HCMM-CMs was significantly higher than in the WT-CMs ( < 0.05). However, no significant differences were found for the MDP between any groups (Table 3).

Differences in the Gene Expression Profiles of hiPSC-Derived
Cardiomyocytes. Dissociated hiPSC-derived CMs were cultured for one week before qRT-PCR analysis was performed. The results are presented in Figure 4. The expression of sarcomeric genes MYBPC3, TNNT2, ACTN2, TTN, MYL7, and MYL9 was significantly higher in both HCMT-CMs and HCMM-CMs than in the WT-CMs ( < 0.005 in all cases). The expression of TPM1 and TNNC1 was significantly increased only in the HCMM-CMs when compared to WT-CMs ( < 0.005 in both cases). On the other hand, the expression of MYH6 was on the same level in all hiPSCderived CMs. Moreover, the expression of some sarcomeric genes (TNNT2, ACTN2, TNNC1, TTN, MYL7, and MYL9) was significantly higher in the HCMM-CMs than in the HCMT-CMs ( < 0.005 in other than TNNT2 and MYL9 < 0.05). The expression of natriuretic peptide A (NPPA) was similar in all hiPSC-derived CMs while the expression of natriuretic peptide B (NPPB) was increased in HCMT-CMs and HCMM-CMs when compared to WT-CMs ( < 0.005 in both cases). Nodal marker HCN4 was also significantly increased in both HCM-CMs when compared to WT-CMs ( < 0.05 for HCMT-CMs versus WT-CMs and < 0.005 for HCMM-CMs versus WT-CMs). Further, the highest expression of potassium channel KCNQ1 and sodium channel SCN5A as well as sodium calcium exchanger SLC8A1 was observed in HCMM-CMs ( < 0.005 when compared to WT-CMs in all cases).
We found differences also in the expression of genes related to the Ca 2+ handling. The expression of CACNA1C and PLN was increased in both HCMT-CMs ( < 0.05 for CACNA1C and < 0.005 for PLN) and HCMM-CMs ( < 0.005 in both cases) when compared to WT-CMs while the expression of ATP2A2 and ITPR2 was on the same level in all hiPSC-derived CMs. However, the expression of CASQ2 and RYR2 was significantly higher in HCMT-CMs and HCMM-CMs when compared to WT-CMs ( < 0.005 in all cases). Moreover, the expression of RYR2 was almost six times higher in the HCMM-CMs than in the WT-CMs ( < 0.005) and around three times higher than in HCMT-CMs ( < 0.005).

Truncated MYBPC Protein Was Not Detected in hiPSC-
Derived Cardiomyocytes Carrying MYBPC3-Gln1061X Mutation. Both wild type and the mutant TPM1 mRNA were present in HCM-CMs carrying the TPM1-Asp175Asn mutation while hiPSC-derived control CMs expressed only wild type mRNA. However, in hiPSC-CMs carrying the MYBPC3-Gln1061X mutation the mutant mRNA was not clearly detected ( Supplementary Figure 7). At a protein level all hiPSC-derived CMs expressed MYBPC, cTnT, and TPM1 (Figures 5(a) and 5(b)). The truncated MYBPC protein (predicted size: 116 kDa) was not detected in HCMM-CMs with western blot analysis ( Figure 5(b)). However, the expression level of total MYBPC was slightly reduced in HCMM-CMs when compared to WT-CMs ( Figure 5(c)). In addition, the expression of cTnT and TPM1 was elevated in both HCM-CMs ( Figure 5(c)).

Discussion
Here we have analyzed characteristics of hiPSC-derived HCM-CMs carrying either TPM1-Asp175Asn or MYBPC3-Gln1061X gene mutation. The size of the MYBPC3-Gln1061X CMs was significantly larger than that of TPM1-Asp175Asn CMs, while the CMs carrying the latter mutation had significantly more abnormal Ca 2+ transients. Additionally, CMs with TPM1-Asp175Asn mutation had significantly more prolonged action potentials. However, both types of HCM-CMs had increased amount of arrhythmogenic events (DADs and EADs) in electrophysiological recordings when compared to control CMs. In addition to morphological and functional differences, also gene expression profiles were different between CMs carrying either TPM1-Asp175Asn or MYBPC3-Gln1061X gene mutation.
Genetic HCM is primarily due to mutations in sarcomeric genes, while changes at the cellular level include disturbed Ca 2+ metabolism and decreased contraction force generation in addition to enlarged cell size [23]. To our knowledge,  total of three reports studying the characteristics as well as the pathophysiological mechanisms of the HCM by using the patient-specific hiPSCs have been published [12][13][14]. In two of these publications, the mutation is located in the MYH7 (MYH7-R663H or MYH7-R442G) [12,13], whereas, in the most recent publication, the hiPSCs were derived from three HCM patients, from whom one carried the MYBPC3-999-1004del2, while the other mutations were unknown [14].
In addition to these three publications, hiPSC-derived CMs carrying MYBPC3 mutations have been used in one study where the effects of serum on the phenotype of neonatal rat CMs as well as hPSC-derived CMs have been explored [24]. hiPSC-derived CMs carrying MYBPC3 mutation were used only when studying the effects of serum on the cellular enlargement [24]. In this current study, we obtained hiPSCs from HCM patients carrying either the MYBPC3-Gln1061X or TPM1-Asp175Asn mutation. We believe that this is the first report where hiPSC-derived CMs carrying different gene mutations have been compared in the same study with similar experimental settings. Our HCM-CMs demonstrated cellular enlargement similarly to previous HCM studies with hiPSCs [12][13][14]. However, we observed a significant difference in the cellular enlargement between the two HCM mutations. CMs carrying the MYBPC3-Gln1061X mutation presented more pronounced and earlier cellular enlargement than CMs carrying the TPM1-Asp175Asn mutation. In a previous study, serum has been shown to mask hypertrophic phenotype of the CMs with mutations in the MYBPC3 [24]. The CMs from the HCM patients were larger in serum-free conditions without any external stimuli, while the serum seemed to increase the cellular enlargement in WT-CMs but not in CMs with HCM mutations [24]. We used 20% serum in our CM culture medium that did not seem to mask the cellular enlargement with our CMs. Already after one week of culture, CMs carrying MYBPC3-Gln1061X mutation were significantly larger than the WT-CMs. The enlargement of CMs carrying TPM1-Asp175Asn mutation was detectable only after three weeks of culture. In the previous clinical studies, as well as in the patient data analyzed in this current study, the hypertrophy has been in the same range in patients carrying either of these two mutations [2]. Therefore, these differences in CM size between the two mutations do not correlate with the extent of clinical hypertrophy.
The higher Ca 2+ sensitivity, observed in animal models and myectomy samples, has been suggested to be a common feature for all HCM mutations [25,26]. In the previous studies with hiPSC-derived CMs, irregularities in Ca 2+ transients have been observed in the MYH7-R663H and the MYH7-R442G mutations [12,13]. The higher Ca 2+ sensitivity has been related to lower phosphorylation levels of the MYBPC and the Troponin I proteins and the difference could at least partly be explained by hypophosphorylation of the sarcomeric proteins compared to the actual mutations [26]. In our study, the amount of abnormalities in Ca 2+ transients was significantly increased only in the hiPSC-derived CMs with the TPM1-Asp175Asn mutation. Indeed, the amount of irregularities in Ca 2+ handling properties was similar in the MYBPC3-Gln1061X compared to that in the WT-CMs. The phosphorylation of the proteins was not analyzed in this current study. However, we analyzed the expression of genes related to Ca 2+ handling and they were at the highest level in hiPSC-derived CMs carrying the MYBPC3-Gln1061X mutation. This might be at least partly due to the larger cell size of these CMs. Han and coworkers found decreased level of RYR2 expression in HCM-CMs carrying MYH7-R442G mutation [13], while in our study the expression of RYR2 was significantly higher in both mutations and almost six times higher in the hiPSC-derived CMs carrying the MYBPC3-Gln1061X mutation than in the WT-CMs. These observations suggest that abnormal Ca 2+ transients in HCM-CMs carrying different mutations may be caused by distinct mechanisms.
One of the fundamental features of the HCM is its association with ventricular arrhythmias responsible for severe cardiac malfunctions including sudden cardiac death [27,28]. We found increased amount of arrhythmogenic events (DADs and EADs) in both HCM-CMs. Furthermore, decreased beating rate was observed in both types of HCM-CMs, which could be due to higher occurrence of DADs between the two consecutive APs. In addition, the APD 90 of hiPSC-derived CMs carrying either TPM1-Asp175Asn or MYBPC3-Gln1061X mutation was longer than in WT-CMs, which is in line with previous findings with different mutations [12,13,29]. The mechanism of arrhythmias in HCM is not yet fully understood; however, imbalances in Ca 2+ homeostasis are considered as a main cause of arrhythmias shown in the previous studies [12,13]. Clinically, patients carrying TPM1-Asp175Asn mutation have been reported to 14 Stem Cells International be more prone to arrhythmias than those carrying MYBPC3-Gln1061X mutation [30]. Our data with hiPSC-derived CMs support this finding by demonstrating more abnormal Ca 2+ transients and longer APD 90 in TPM1-Asp175Asn CMs than in HCM-CMs carrying the MYBPC3-Gln1061X mutation. Like most of the HCM mutations located in the MYBPC3, also MYBPC3-Gln1061X is a nonsense mutation that leads to premature stop-codon [31]. Nonsense mutations are suggested to act through haploinsufficiency in which the mutated protein is either degraded or not produced at all. The truncated form of MYBPC has not been found in human cardiac samples while the total expression level of MYBPC has been reported to vary from being decreased to even increased [32][33][34][35][36]. Interestingly, when studying myectomy samples from HCM hearts with MYBPC3 mutation, Helms et al. observed that the total amount of MYBPC3 mRNA was increased, while the total amount of MYBPC protein was on the same level compared to that in the control hearts. They hypothesized that the upregulation of the MYBPC3 compensates the degraded truncated MYBPC protein [35]. We could not detect mutant allele on mRNA expression level or truncated MYBPC protein in hiPSC-derived CMs carrying the MYBPC3-Gln1061X mutation. Similar absence of truncated protein has been reported earlier with a different MYBPC mutation in hiPSC-derived CMs [14]. These data suggest that the mutant mRNA might be degraded. However, further research is still needed to confirm the results and to discover the actual degradation mechanism.
The two HCM mutations analyzed in this study are the most frequent mutations in Finland accounting for about 18% of all Finnish HCM patients [3]. In our study, we found differences in the morphological and biochemical properties, as well as in Ca 2+ cycling and electrophysiological properties between the CMs carrying either TPM1-Asp175Asn or MYBPC3-Gln1061X mutation. However, we have not analyzed the possible effects of other gene mutations and epigenetic factors on the phenotype differences observed in our study. Additionally, we used only two hiPSC lines from two different patients in each mutation in our experiments. In the future, we need to extend our studies including further patients and studying the effects of additional gene mutations and epigenetic factors. The immature nature of hiPSCderived CMs is a general limitation when using these cells in disease modeling. Further development in the differentiation and maturation protocols will increase the reliability of studies conducted with hiPSC-derived CMs. Finally, the exact pathophysiology in abnormal Ca 2+ transients or electrical abnormalities is not known and further research with combined patch clamp and Ca 2+ imaging is required in the future to reveal the significance of the cellular findings for clinical phenotypes as well as for treatment options.

Conclusions
In conclusion, both HCM hiPSC-derived CMs either carrying the TPM1-Asp175Asn or MYBPC3-Gln1061X mutation exhibited pathological changes related to HCM. However, significant differences between the two mutations were observed. The hiPSC-derived cell models, established in this study, can be exploited to study further the pathophysiological mechanisms of HCM as well as to screen drugs and potentially optimize treatments in mutation-specific way.