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

Abstract

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, Ca(2+) 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.

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. Scale bars: 200 μm. (b) The virally transferred Sendai exogenes, exo-OCT4 (483 bp), exo-KLF4 (410 bp), exo-SOX2 (451 bp), and exo-c-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).

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 WT- and HCMT-CMs (^$$ 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 n numbers 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 n numbers 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.

Figure 3

Arrhythmogenic events (DADs and EADs) were observed in HCM-CMs. (a)–(e) Representative recordings of control hiPSC-derived 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 WT-CMs (^∗∗ p < 0.005).

Figure 4

Gene expression profiles in control hiPSC-derived CMs (WT) and in hiPSC-derived CMs carrying TPM1-Asp175Asn (HCMT) or MYBPC3-Gln1061X (HCMM) mutations. (^∗ represents HCMT or HCMM versus WT, and ^$ represents HCMT versus HCMM. ^∗∗ or ^$$ p < 0.005, ^∗ or ^$ p < 0.05  n = 16, except for NPPA n = 8).

Figure 5

Cardiac-specific protein expression in hiPSC-derived CMs. (a) Representative images of hiPSC-derived CMs carrying TPM1-Asp175Asn mutation (HCMT) or MYBPC3-Gln1061X mutation (HCMM) and hiPSC-derived control CMs (WT) stained with cTnT, MYBPC, and TPM1. These images are not to quantify the protein expression but to demonstrate the presence of cTnT, MYBPC, and TPM1 proteins in the hiPSC-derived CMs. (b) hiPSC-derived CMs from all cell lines expressed MYBPC, cTnT, and TPM1 proteins. The truncated MYBPC was not detected in HCMM cells (size of the wild type protein 141 kDa and the predicted size of the truncated protein 117 kDa). (c) The expression of MYBPC, cTnT, and TPM1 in WT-CMs, HCMT-CMs, and HCMM-CMs normalized to the expression of β-actin. Protein expressions were quantified from western blots using ImageJ software. Quantitation data show the averages of MYBPC/β-actin, cTnT/β-actin, and TPM1/β-actin relations from hiPSC-derived CMs from two different hiPSC lines in each group. Because of the lack of replicates, statistical analysis was not performed.