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. 2014 Nov 11;3(6):e001263.
doi: 10.1161/JAHA.114.001263.

Endothelin-1 induces myofibrillar disarray and contractile vector variability in hypertrophic cardiomyopathy-induced pluripotent stem cell-derived cardiomyocytes

Atsushi Tanaka  1 , Shinsuke Yuasa  2 , Giulia Mearini  3 , Toru Egashira  2 , Tomohisa Seki  2 , Masaki Kodaira  2 , Dai Kusumoto  2 , Yusuke Kuroda  2 , Shinichiro Okata  4 , Tomoyuki Suzuki  2 , Taku Inohara  2 , Takuro Arimura  5 , Shinji Makino  2 , Kensuke Kimura  2 , Akinori Kimura  5 , Tetsushi Furukawa  6 , Lucie Carrier  3 , Koichi Node  7 , Keiichi Fukuda  2
Affiliations

Endothelin-1 induces myofibrillar disarray and contractile vector variability in hypertrophic cardiomyopathy-induced pluripotent stem cell-derived cardiomyocytes

Atsushi Tanaka et al. J Am Heart Assoc. .

Abstract

Background: Despite the accumulating genetic and molecular investigations into hypertrophic cardiomyopathy (HCM), it remains unclear how this condition develops and worsens pathologically and clinically in terms of the genetic-environmental interactions. Establishing a human disease model for HCM would help to elucidate these disease mechanisms; however, cardiomyocytes from patients are not easily obtained for basic research. Patient-specific induced pluripotent stem cells (iPSCs) potentially hold much promise for deciphering the pathogenesis of HCM. The purpose of this study is to elucidate the interactions between genetic backgrounds and environmental factors involved in the disease progression of HCM.

Methods and results: We generated iPSCs from 3 patients with HCM and 3 healthy control subjects, and cardiomyocytes were differentiated. The HCM pathological phenotypes were characterized based on morphological properties and high-speed video imaging. The differences between control and HCM iPSC-derived cardiomyocytes were mild under baseline conditions in pathological features. To identify candidate disease-promoting environmental factors, the cardiomyocytes were stimulated by several cardiomyocyte hypertrophy-promoting factors. Interestingly, endothelin-1 strongly induced pathological phenotypes such as cardiomyocyte hypertrophy and intracellular myofibrillar disarray in the HCM iPSC-derived cardiomyocytes. We then reproduced these phenotypes in neonatal cardiomyocytes from the heterozygous Mybpc3-targeted knock in mice. High-speed video imaging with motion vector prediction depicted physiological contractile dynamics in the iPSC-derived cardiomyocytes, which revealed that self-beating HCM iPSC-derived single cardiomyocytes stimulated by endothelin-1 showed variable contractile directions.

Conclusions: Interactions between the patient's genetic backgrounds and the environmental factor endothelin-1 promote the HCM pathological phenotype and contractile variability in the HCM iPSC-derived cardiomyocytes.

Keywords: ET‐1; HCM; MYBPC3; disease modeling; iPS cells.

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Figures

Figure 1.
Figure 1.
Patients' clinical characteristics. Detailed clinical informations, electrocardiogram, and 4‐chamber images of echocardiogram or cardiac magnetic resonance imaging of each patient with HCM. *The age when the one's iPSCs were established. **Maron's classification. HCM indicates hypertrophic cardiomyopathy; iPSCs, induced pluripotent stem cells; LA, left atrium; LV, left ventricle; LVOTO, left ventricular outflow tract obstruction; RA, right atrium; RV, right ventricle.
Figure 2.
Figure 2.
iPSC generation from control and patients with HCM. A, Immunostaining for human pluripotent stem cell markers, Nanog, OCT 3/4, SSEA 3, SSEA 4, TRA‐1‐60, and TRA‐1‐81. B, Teratoma formation from control 1 , HCM 1 , HCM 2, and HCM3 iPSCs in the immunocompromised NOD‐SCID mice. Sections of teratomas were stained with hematoxylin–eosin, and tissues representative of all 3 germ layers were observed. HCM indicates hypertrophic cardiomyopathy; iPSCs, induced pluripotent stem cells.
Figure 3.
Figure 3.
Cardiomyocyte generation from control and HCM iPSCs. The single cardiomyocytes at 60 days after differentiation were cultured for 7 days under adherent culture conditions. A, Immunostaining for α‐actinin and MLC2a with DAPI nuclear staining in the single cardiomyocytes derived from each iPSC. B, Immunostaining for MLC2v and cTnT with DAPI nuclear staining in the single cardiomyocytes derived from each iPSCs. C, Immunostaining for α‐actinin and ANP with DAPI nuclear staining in the single cardiomyocytes derived from each iPSC. ANP indicates atrial natriuretic peptide; cTnT, cardiac troponin T; DAPI, 4′,6‐diamidino‐2‐phenylindole; HCM, hypertrophic cardiomyopathy; iPSCs, induced pluripotent stem cells; MLC2a, myosin light chain 2a; MLC2v, myosin light chain 2v.
Figure 4.
Figure 4.
Time course of drug stimulation protocols for beating single cardiomyocytes. EBs were formed under floating culture conditions at day 0 and then maintained until 30, 60, or 90 days. The floating EBs were then enzymatically dissociated and single cardiomyocytes were cultured under adherent conditions for 7 days. EBs indicates embryoid bodies; iPS, induced pluripotent stem.
Figure 5.
Figure 5.
In vitro pathological findings: cell surface area in the isolated iPSC‐derived cardiomyocytes. Embryoid bodies (EBs) were maintained for 30, 60, or 90 days in floating culture. After dissociation, cardiomyocytes were cultured 7 days under adherent culture conditions. A, Immunostaining for cardiac troponin T (cTnT) with DAPI nuclear staining in the single cardiomyocytes derived from each control and HCM iPSCs. B, Immunostaining for α‐actinin with DAPI nuclear staining in the single cardiomyocytes derived from each control and HCM iPSCs. C, Cell surface areas of 798 to 823 randomly chosen cTnT‐positive cardiomyocytes from each control (1 to 3) and HCM (1 to 3) group were measured at each time point. **P<0.01 vs control in the same time point (repeated‐measures ANOVA and Mann–Whitney U test).HCM indicates hypertrophic cardiomyopathy; iPSC, induced pluripotent stem cell.
Figure 6.
Figure 6.
Structural characterization of iPSC‐derived cardiomyocytes. A, Electron microscopic images showing well‐organized striated myofibrils derived from beating EBs from control and HCM iPSCs at 60 days after differentiation. Scale bars, 1 μm. B, Electron microscopic images showing myofibrillar disarray derived from beating EBs from HCM iPSC at 60 days after differentiation. Scale bars, 1 μm. C, Immunostaining for cTnT in the single cardiomyocytes at 60 days derived from each HCM iPSC, showing myofibrillar disarray. D, The percentages of cardiomyocytes with myofibrillar disarray were assessed by cTnT immunostaining of the single cardiomyocytes. N=851 to 995. *P<0.05, **P<0.01 vs control in the same time point (χ2 test). cTnT indicates cardiac troponin T; EBs, embryoid bodies; HCM, hypertrophic cardiomyopathy; iPSC, induced pluripotent stem cell.
Figure 7.
Figure 7.
Schema of myofibrillar disarray in single induced pluripotent stem cell (iPSC)‐derived cardiomyocytes. A, Cardiomyocyte with normal myofibrillar alignment. B, Cardiomyocyte with myofibrillar disarray.
Figure 8.
Figure 8.
Cardiac‐specific protein expression in control and HCM iPSC‐derived cardiomyocytes. A, Western blotting for α‐actinin, cTnT, ANP, and GAPDH in control and HCM iPSC‐derived beating EBs at 60 days after differentiation. B through D, Statistical analyses of α‐actinin, cTnT, and ANP protein levels. N=4 to 6. *P<0.05 vs control (Mann–Whitney U test). E, Western blotting for cMyBP‐C in control and HCM3 iPSC‐derived beating EBs at 60 days after differentiation. F, Statistical analysis of cMyBP‐C protein level. N=6. *P<0.05 vs control (Mann–Whitney U test). ANP indicates atrial natriuretic peptide; cMyBP‐C, cardiac myosin‐binding protein C; cTnT, cardiac troponin T; HCM, hypertrophic cardiomyopathy; iPSC, induced pluripotent stem cell; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase.
Figure 9.
Figure 9.
ET‐1 augments pathological features in HCM iPSC‐derived cardiomyocytes. A, Immunostaining for cardiac troponin‐T (cTnT) of the single cardiomyocytes derived from each control and HCM iPSC. The single cardiomyocytes at 60 days were cultured for 7 days with ET‐1, Ang II, IGF‐1, or PE. B and C, Cell surface areas of 788 to 819 randomly chosen cTnT‐positive cardiomyocytes in each group and condition were measured. The single cardiomyocytes were cultured for 7 days with ET‐1, Ang II, IGF‐1, or PE (1‐way ANOVA with Steel's multiple comparison post‐test). D and E. The percentages of cardiomyocytes with myofibrillar disarray were assessed by immunostaining for cTnT. N=844 to 995. *P<0.05, **P<0.01, †P<0.0001 vs free condition (χ2 test). Ang II indicates angiotensin II; ET‐1, endothelin‐1; HCM, hypertrophic cardiomyopathy; IGF‐1, insulin‐like growth factor‐1; iPSC, induced pluripotent stem cell; PE, phenylephrine.
Figure 10.
Figure 10.
Increased pathological features in HCM iPSC‐derived cardiomyocytes. A, The single cardiomyocytes at 60 days after differentiation were cultured for 7 days with ET‐1, Ang II, IGF‐1, or PE. Cell surface areas of cTnT‐positive cardiomyocytes were measured. N=788 to 819. †P<0.0001 vs control in the same condition (Mann–Whitney U test). B, The percentages of cardiomyocytes with myofibrillar disarray were assessed by immunostaining for cardiac troponin‐T. N=844 to 995. **P<0.01, †P<0.0001 vs control in the same condition (χ2 test). Ang II indicates angiotensin II; cTnT, cardiac troponin‐T; ET‐1, endothelin‐1; HCM, hypertrophic cardiomyopathy; IGF‐1, insulin‐like growth factor‐1; iPSC, induced pluripotent stem cell; PE, phenylephrine.
Figure 11.
Figure 11.
ET‐1 augments myofibrillar disarray in HCM iPSC‐derived cardiomyocytes. A, Electron microscopic observation of beating EBs at 60 days, which were cultured with ET‐1, Ang II, IGF‐1, or PE for 7 days. Scale bars, 1 μm. B and C, Relative gene expression of EDNRA and EDNRB in beating EBs at 30 days, normalized against TNNT2. N=3 (Control=3, HCM 1=3, HCM 1=3, HCM 3=3) (Kruskal–Wallis test). Ang II indicates angiotensin II; EBs, embryoid bodies; EDNRA, endothelin receptor type A; ET‐1, endothelin‐1; HCM, hypertrophic cardiomyopathy; IGF‐1, insulin‐like growth factor‐1; iPSC, induced pluripotent stem cell; PE, phenylephrine.
Figure 12.
Figure 12.
ET‐1 strengthens pathological features in HCM iPSC‐derived cardiomyocytes in a concentration‐dependent manner. A, Cell surface areas of 789 to 822 randomly chosen cardiac troponin‐T (cTnT)‐positive cardiomyocytes in each group and condition were measured. The cardiomyocytes were dissociated from EBs at 60 days and cultured for 7 days with various concentrations of ET‐1 (1, 10, 100, or 1000 nmol/L) (1‐way ANOVA with Steel's multiple comparison post‐test). B, The percentages of cardiomyocytes with myofibrillar disarray were assessed by immunostaining for cTnT of the single cardiomyocytes after 7 days of culture with ET‐1. N=875 to 995 (per each group and condition). *P<0.05, **P<0.01, †P<0.0001 vs ET‐1–free (0 nmol/L) condition in the same group (χ2 test). C and D, Nuclear translocation of NFATc4 was assessed by immunostaining for NFATc4 and cTnT. Isolated cardiomyocytes at 60 days were cultured for 7 days with or without ET‐1. The presented images were the iPSC‐derived cardiomyocytes cultured for 7 days with ET‐1. Scale bars, 20 μm. N=376 to 448 (per each group and condition). *P<0.01, **P<0.001 vs free condition in each group (χ2 test). EBs indicates0 embryoid bodies; ET‐1, endothelin‐1; HCM, hypertrophic cardiomyopathy; iPSC, induced pluripotent stem cell; NFAT, nuclear factor of activated T cells.
Figure 13.
Figure 13.
ET‐1–ETA axis has a pivotal role in pathological deterioration in HCM iPSC‐derived cardiomyocytes. A, Immunostaining for cardiac troponin‐T (cTnT) of the single cardiomyocytes derived from HCM 1, HCM 2, and HCM 3 iPSC‐derived cardiomyocytes. The single cardiomyocytes at 60 days were cultured for 7 days with ETA‐b and/or ETB‐b in the presence or absence of ET‐1. B, Cell surface areas of 809 to 825 randomly chosen cTnT‐positive cardiomyocytes in each condition were measured (1‐way ANOVA with Steel's multiple comparison post‐test). C, The percentages of cardiomyocytes with myofibrillar disarray were assessed by immunostaining for cTnT of the single cardiomyocytes. N=880 to 995 (per each condition). †P<0.0001 vs ET‐1 condition (χ2 test). D, Electron microscopic observation of beating EBs at 60 days cultured with ETA‐b or ETB‐b in the presence of ET‐1. Scale bars, 200 nm. E, Lower magnified electron microscopic images corresponding with (D). F and G, Nuclear translocation of NFATc4 was assessed by immunostaining for NFATc4 and cTnT. The single cardiomyocytes at 60 days were cultured for 7 days with ETA‐b and/or ETB‐b in the presence of ET‐1. Scale bars, 20 μm. N=391 to 751 (per each culture condition). †P<0.0001 vs ET‐1 condition (χ2 test). EBs indicates embryoid bodies; ET‐1, endothelin‐1; ETA‐b, endothelin receptor type A blocker; ETB‐b, endothelin receptor type B blocker; HCM, hypertrophic cardiomyopathy; iPSC, induced pluripotent stem cell; NFAT, nuclear factor of activated T cells.
Figure 14.
Figure 14.
ETA‐b regresses the HCM pathological phenotype. A, Time course of drug stimulation protocols for beating single cardiomyocytes. The single cardiomyocytes derived from each of the HCM iPSCs, which were dissociated from EBs at 60 days, were cultured for 7 days with or without ET‐1, and then subsequently cultured for 7 days with or without ETA‐b. Immunostainings for cTnT with DAPI nuclear staining were performed at 14 days. B, The isolated cardiomyocytes at 60 days were cultured for 7 days with or without ET‐1 and subsequently cultured for 7 days with or without ET‐1, or ETA‐b. C, Cell surface areas of total 300 randomly chosen cTnT‐positive cardiomyocytes were measured in each culture condition (1‐way ANOVA with Steel's multiple‐comparison post‐test). D, The percentages of cardiomyocytes with myofibrillar disarray were assessed by immunostaining for cTnT of the single cardiomyocytes. N=317 to 361 (per each culture condition). *P<0.001 (χ2 test). cTnT indicates cardiac troponin‐T; EBs, embryoid bodies; ET‐1, endothelin‐1; ETA‐b, endothelin receptor type A blocker; HCM, hypertrophic cardiomyopathy; iPSCs, induced pluripotent stem cells.
Figure 15.
Figure 15.
ET‐1 augments the incidence of myofibrillar disarray in cardiomyocytes isolated from heterozygous Mybpc3‐targeted knock‐in mice. A, Immunostaining for α‐actinin and cMyBP‐C with DRAQ5 nuclear staining of cardiomyocytes derived from neonatal wild‐type (WT) and Mybpc3‐targeted heterozygous knock‐in (HET) mice. Scale bars, 20 μm. B, Immunostaining for α‐actinin and cMyBP‐C with DRAQ5 nuclear staining of the single cardiomyocytes derived from WT and HET mice, which were cultured with ET‐1 for 48 hours. Scale bars, 20 μm. C, Cell surface areas of 173 to 178 randomly chosen α‐actinin–positive cardiomyocytes in each condition were measured. *P<0.05, **P<0.01 vs WT or as indicated on the figure (Mann–Whitney U test). D, The percentages of cardiomyocytes with myofibrillar disarray were assessed by immunostaining for α‐actinin of the single cardiomyocytes. N=215 to 236. *P<0.05, **P<0.01 vs WT or as indicated on the figure (χ2 test). E through G, Quantitative RT‐PCR analyses for Acta1, Nppa, and Nppb. N=3 to 5. *P<0.05, **P<0.01 (Kruskal–Wallis test). Acta1 indicates α‐skeletal muscle; cMyBP‐C, cardiac myosin‐binding protein C; ET‐1, endothelin‐1; Mybpc3, human/mouse gene encoding cardiac myosin‐binding protein C; Nppa, natriuretic peptide type A; Nppb, natriuretic peptide type B; RT‐PCR, reverse transcription‐polymerase chain reaction.
Figure 16.
Figure 16.
Schema of motion vector analysis for single beating cardiomyocytes. A, Normal cardiomyocytes, beating synchronously in 1 direction, show symmetrical twin peaks under 360° analyses. The summation data indicates a single peak with lower SD scores. B, Cardiomyocytes with myofibrillar disarray, beating nonsynchronously, show ≥2 morphologically irregular peaks under 360° analyses. The summation data indicate flattened peak with higher SD score (double‐headed red arrow).
Figure 17.
Figure 17.
Motion vector analysis of single beating cardiomyocytes derived from control and HCM iPSCs. A, The iPSC‐derived single cardiomyocytes at 60 days, which were cultured for 24 hours, were stimulated for 48 hours with or without ET‐1 and/or each endothelin receptor blocker. Phase contrast image (PC) showed the single beating cardiomyocytes obtained in this system. Vectors showed traced motion vectors overlaid in the cardiomyocyte image in PC. A histogram showed the frequency of the motion vector angles (angles between each vector and x‐axis [horizontal axis] in the image) in a single cardiomyocyte. The histograms and SD scores were calculated at 0, 24, and 48 hours after stimulation. B, The SD scores of the vector angle histograms in control and HCM iPSC‐derived beating cardiomyocytes before ET‐1 stimulation (0 hour). N=30 (control) or 90 (HCM). C, The SD scores of the vector angle histograms after 24‐hour stimulation. N=21 to 24 (control) or 79 to 87 (HCM). D, The SD scores of the vector angle histograms after 48‐hour stimulation. The SD scores of HCM iPSC‐derived cardiomyocytes with ETA‐b were significantly reduced, compared with those of ET‐1. N=27 to 28 (control) or 67 to 73 (HCM). *P<0.05, **P<0.01 vs as indicated on the figure (Student t test). ET‐1 indicates endothelin‐1; ETA‐b, endothelin receptor type A blocker; ETB‐b, endothelin receptor type B blocker; HCM, hypertrophic cardiomyopathy; iPSCs, induced pluripotent stem cells.
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