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iPSC-derived cardiomyocytes reveal abnormal TGF-β signalling in left ventricular non-compaction cardiomyopathy

Nature Cell Biology volume 18, pages 1031–1042 (2016)Cite this article

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Abstract

Left ventricular non-compaction (LVNC) is the third most prevalent cardiomyopathy in children and its pathogenesis has been associated with the developmental defect of the embryonic myocardium. We show that patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from LVNC patients carrying a mutation in the cardiac transcription factor TBX20 recapitulate a key aspect of the pathological phenotype at the single-cell level and this was associated with perturbed transforming growth factor beta (TGF-β) signalling. LVNC iPSC-CMs have decreased proliferative capacity due to abnormal activation of TGF-β signalling. TBX20 regulates the expression of TGF-β signalling modifiers including one known to be a genetic cause of LVNC, PRDM16, and genome editing of PRDM16 caused proliferation defects in iPSC-CMs. Inhibition of TGF-β signalling and genome correction of the TBX20 mutation were sufficient to reverse the disease phenotype. Our study demonstrates that iPSC-CMs are a useful tool for the exploration of pathological mechanisms underlying poorly understood cardiomyopathies including LVNC.

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Figure 1: Characterization of patient-specific LVNC iPSC-CMs carrying a TBX20 mutation.
Figure 2: Upregulation of TGF-β signalling in the LVNC phenotype.
Figure 3: Developmental arrest in cardiomyocyte-specific TGF-β1-overexpressing mouse embryo heart.
Figure 4: Disturbed expansion of embryonic cardiomyocytes and trabecular/compact layer ratio in the left ventricle of the TGF-β1-overexpressing mouse.
Figure 5: TBX20 regulates the expression of TGF-β signalling modifier genes in developing cardiomyocytes.
Figure 6: PRDM16 is a candidate target gene of TBX20 in cardiomyocytes.
Figure 7: Rescue of pathological features of LVNC iPSC-CMs.

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Acknowledgements

We thank N. Sun, S. Hu and J. Lee for their help with functional assessments; H. Yamagishi (Keio University, Japan) for providing plasmid; A. B. Glick (Pennsylvania State University, USA) for providing dominant-negative TGFBRII overexpression adeno virus; B. Huber, B. Patlolla and G. Berry for their help with analysing the in vivo study. We are grateful for the support provided by the Neuroscience Microscopy Service (NMS), and FACS Core at the Institute for Stem Cell Biology and Regenerative Medicine, Stanford University. This work is supported by the Uehara Memorial Foundation Research Fellowship, American Heart Association Postdoctoral Fellowship 15POST25160016 (K.K.), American Heart Association Postdoctoral Fellowship 15POST22940013 and NIH K99HL130416 (S.-G.O.), The Ministry of Education, Culture, Sports, Science and Technology in Japan (F.I.), National Institutes of Health P01 GM099130 (M.P.S.), NIH K18 HL11708301, Children’s Cardiomyopathy Foundation (D.B.), AHA Established Investigator Award, NIH R01 HL113006, NIH R01 HL130020, NIH R01 126527, NIH R01 128170, NIH R01 HL123968 and NIH R24 HL117756 (J.C.W.).

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Author notes

  1. Kazuki Kodo and Sang-Ging Ong: These authors contributed equally to this work.

Authors and Affiliations

  1. Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, California 94305, USA

    Kazuki Kodo, Sang-Ging Ong, Vittavat Termglinchan, Kolsoum InanlooRahatloo, Antje D. Ebert, Praveen Shukla, Oscar J. Abilez, Jared M. Churko, Ioannis Karakikes, Gwanghyun Jung, Sean M. Wu, Daniel Bernstein & Joseph C. Wu

  2. Department of Medicine, Division of Cardiology, Stanford University School of Medicine, Stanford, California 94305, USA

    Kazuki Kodo, Sang-Ging Ong, Vittavat Termglinchan, Kolsoum InanlooRahatloo, Antje D. Ebert, Praveen Shukla, Oscar J. Abilez, Jared M. Churko, Ioannis Karakikes, Sean M. Wu & Joseph C. Wu

  3. Department of Genetics, Stanford University School of Medicine, Stanford, California 94305, USA

    Fereshteh Jahanbani & Michael P. Snyder

  4. Institute of Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California 94305, USA

    Vittavat Termglinchan, Antje D. Ebert, Jared M. Churko, Ioannis Karakikes & Joseph C. Wu

  5. Department of Pediatrics, University of Toyama, Toyama-shi, Toyama 930-8555, Japan

    Keiichi Hirono & Fukiko Ichida

  6. Department of Pediatrics, Stanford University School of Medicine, Stanford, California 94305, USA

    Gwanghyun Jung & Daniel Bernstein

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Contributions

All authors have read and approved the manuscript. K.K., S.-G.O., D.B. and J.C.W. designed research, performed the experiments and wrote the manuscript; F.I., D.B. and J.C.W. recruited the patients; K.K., J.M.C. and S.-G.O. generated and performed experiments on iPSCs/ESCs with the help of A.D.E. and G.J.; K.K., F.J., K.H., K.I. and J.M.C. performed sequencing and bioinformatics analysis; S.M.W. generated NK-TGCK transgenic mouse; K.K., V.T. and I.K. generated genome-corrected iPSC lines; K.K., P.S. and O.J.A. performed and interpreted the patch clamping and calcium imaging data; M.P.S. provided scientific advice; and J.C.W. provided funding and supervised the entire research project.

Corresponding authors

Correspondence to Daniel Bernstein or Joseph C. Wu.

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Competing interests

J.C.W. is co-founder of Stem Cell Theranostics. The remaining authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Characterization of patient-specific iPSCs.

(a–b) Combined overexpression of cardiac transcription factors and wild-type, Y317∗, or T262M mutant TBX20 revealed lack of synergistic activity of mutant TBX20 protein with NKX2-5 and GATA4 (a), or TBX5 (b), on NPPA promoter in luciferase assay using HeLa cells. n = 6 independent experiments per each group. (c) TBX20 Y317∗ and T262M mutant protein could not repress the MSX2 promoter activity in luciferase assay using HeLa cells. n = 6 independent experiments per each group. (d) iPSCs derived from the family members could form teratomas when injected into a NOD/SCID mouse background, and could differentiate into all three germ layers (ectoderm, endoderm, and mesoderm) as shown by hematoxylin and eosin (HE) staining. (e) Validation of pluripotent gene expression profile using quantitative real-time PCR (qRT-PCR) in all cell lines n = 6 independent experiments per each group. (f) Immunofluorescence of representative iPSC colony derived from patient-specific fibroblasts or PBMCs staining with markers of pluripotency, including TRA-1-60 (green), OCT3/4 (red), SSEA-4 (green), and NANOG (red). Scale bars, 100 μm. (g) SNP-based analysis revealed a normal karyotype for all iPSC lines derived from the family members. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.005; ns, not significant in one-way ANOVA followed by Tukey post hoc test. The bar graphs show the mean and error bars represent s.e.m. Scale bars, 100 μm. Statistics source data can be found in Supplementary Table 12.

Supplementary Figure 2 Supplementary Figure 2. Characterization of patient-specific iPSC-CMs.

(a) Schematic of cardiac differentiation protocol. (b) Representative immunostaining for cardiac troponin T (TNNT2) and alpha-sarcomeric actin (αSA) demonstrating no significant differences in sarcomeric structures between iPSC-CMs from control and family members at 4 weeks after induction of cardiac differentiation. Scale bars, 20 μm. (c) Spontaneous action potentials in control, mild DCM, and LVNC iPSC-CMs at 4 weeks after induction of cardiac differentiation measured in current-clamp mode. (d–g) The results of Ca2+ imaging study using iPSC-CMs at 4 weeks. Time to peak (d), F/Frest (e), 50% decay (f), and 90% decay (g) were observed between control and patient-specific iPSC-CM lines with 1 Hz or 2 Hz electric stimulation. Data were recorded from n = 30 cells (control and mild DCM groups) or n = 60 cells (LVNC group). ∗P < 0.05; ns, not significant in one-way ANOVA. (h) Quantification of cell size for control and patient-specific iPSC-CMs (n = 60 cells per group) at 2, 4, 6, and 8 weeks after induction of cardiac differentiation. (i) Quantification of multinucleation in control and LVNC (III-4) iPSC-CMs at 2, 4, 6, and 8 weeks. n = 3 independent experiments per each group. (j) mRNA expression of sarcomere components in control and patient-specific iPSC-CMs were validated by qRT-PCR at 2 weeks after induction of cardiac differentiation. n = 6 independent experiments per each group. (k) mRNA expression profile of mesodermal transcription factors in differentiating iPSCs were validated by qRT-PCR at 2 days after induction of cardiac differentiation. n = 6 independent experiments per each group. CON, unrelated control. ∗P < 0.05; ns, not significant in one-way ANOVA followed by Tukey post hoc test. The bar graphs show the mean and error bars represent s.e.m. Statistics source data can be found in Supplementary Table 12.

Supplementary Figure 3 Gene expression profile and proliferative potential of differentiating iPSC-CMs.

(a) Relative mRNA expression profile of cardiac transcription factors including GATA4, TBX5, NKX2-5, MEF2C, TBX20, and ISL1 from day 0 to day 16 after induction of cardiac differentiation. The LVNC iPSCs showed significant decrease of mRNA expressions of cardiac transcription factors in day 6 and 9. n = 6 independent experiments per each group. ∗P < 0.05.∗∗∗P < 0.005; ns, not significant in one-way ANOVA followed by Tukey post hoc test. (b) The number (left) and population doubling level (right) of iPSC-CMs with or without serum or growth factors (IGF1, IGF2, FGF1 or NRG1) from day 14 to day 28 after induction of cardiac differentiation. n = 6 independent experiments per group. (c) The measurement of cumulative population doublings showed no significant difference in the growth speed between of control, mild DCM, and LVNC iPSC lines (n = 9, 3 and 7 independent experiments for control, mild DCM and LVNC per group respectively). The bar graphs show the mean and error bars represent s.e.m. Statistics source data can be found in Supplementary Table 12.

Supplementary Figure 4 Abnormal activation of TGFβ signaling in patient-specific LVNC iPSC-CMs.

(a) Upstream regulator analysis of growth factors comparing LVNC and control iPSC-CMs at 2 weeks. (b) Significant increase of TGFβ expression in patient-specific iPSC-CMs versus control iPSC-CMs in mRNA-sequencing. Fold-change of mRNA-sequencing data was obtained by FKPM (fragments per kilobase of exon per million) of LVNC (A-III-2, 3, 4; mean of four samples) or mild DCM (A-II-2; mean of two samples) iPSC-CMs per CON (unrelated controls; mean of two samples) iPSC-CMs. (c) mRNA expression of downstream target genes of TGFβ1 in control and patient-specific iPSC-CMs are validated by mRNA-sequencing at 2 weeks. LVNC iPSC-CMs showed increasing activity of TGFβ signaling compared with control iPSC-CMs (unrelated controls; mean of two samples, mild DCM; mean of two samples, LVNC; mean of four samples). (d–e) qRT-PCR analysis showed significant increase in TGFβ (d) and downstream target gene (e) mRNA expression in LVNC iPSC-CMs compared to control iPSC-CMs at 2 weeks. (f) Significant increase of CDKN1A expression in patient-specific iPSC-CMs versus control iPSC-CMs in mRNA-sequencing (unrelated controls; mean of two samples, mild DCM; mean of two samples, LVNC; mean of four samples). (g) Validation of mRNA expression of cyclin-dependent kinase inhibitors using qRT-PCR. †q < 0.05 in Benjamini-Hochberg correction. One-way ANOVA followed by Tukey post hoc test were performed for the validation of qRT-PCR. ∗P < 0.05.∗∗P < 0.01.∗∗∗P < 0.005; ns, not significant. The bar graphs show the mean and error bars represent s.e.m. Statistics source data can be found in Supplementary Table 12.

Supplementary Figure 5 Phenotype of cardiomyocyte-specific TGFβ1 overexpression mouse embryos.

(a) The schematic of the β1glo and αMHC-Cre transgene. (b) Activation of TGFB1 transgene in double transgenic (β1glo/αMHC-Cre) embryos at embryonic day (E) E10.5. Hearts of double transgenic embryos and wild-type littermates (-/-) were corrected and TGFβ1 and Gapdh mRNA expression were validated by RT-PCR. (c) Immunostaining of nuclear (blue), phosphor-histone H3 (phospho-HH3) (red), and αSA (green) in coronal sections of control and β1glo/αMHC-Cre double transgenic embryo hearts at E10.5. (d) Percentage of phospho-histone H3 positive (pHH3+) cardiomyocytes in compact layer of control (n = 7 hearts) and β1glo/αMHC-Cre double transgenic embryo hearts (n = 4 hearts) at E10.5. (e) The schematic of the β1glo and NK-TGCK transgene. NK enhancer/promoter-driven eGFP-Cre fusion proteins are expressed without doxycycline (DOX) and tTA proteins inhibit eGFP-Cre expression with existence of DOX. (f) Schematic of DOX-treatment protocol. (g) Cre activities of αMHC-Cre and NK-TGCK transgene with or without DOX treatment. αMHC-Cre and NK-TGCK transgenic mice were crossed with mT/mG reporter mice which express cell membrane-localized green fluorescence in Cre recombinase expressing cells. Immunostaining for nuclear (blue), αSA (red), and GFP (green) showed ∼90% Cre activity in αMHC-Cre transgenic mouse, ∼40% in DOX-untreated NK-TGCK transgenic mouse, and ∼25% in DOX-treated NK-TGCK transgenic mouse at E12.5. (h) mRNA expression of TGFB1 transgene in αMHC-Cre/β1glo (n = 6 hearts) and NK-TGCK/β1glo double transgenic mouse embryos with (n = 9 hearts) or without DOX treatment (n = 9 hearts) at E10.5. (i) mRNA expression of TGFB1 downstream target genes including Cdkn1a and Serpine1 in NK-TGCK/β1glo double transgenic mouse embryos with (n = 5 hearts) or without DOX treatment (n = 8 hearts) compared with wild-type littermates (CON; n = 5 hearts per group with or without DOX) at E10.5. (j) Immunostaining of nuclear (blue), phospho-histone H3 (phosphor-HH3) (red), and αSA (green) in coronal sections of wild-type (control) and β1glo/NE-TGCK double transgenic embryo hearts with (DOX+) or without (DOX−) doxycycline treatment at E12.5. (k) Percentage of phospho-histone H3 positive (pHH3+) cardiomyocytes in compact layer of control (n = 7 hearts) and β1glo/NE-TGCK double transgenic embryo hearts with (DTG+; n = 6 hearts) or without (DTG-; n = 7 hearts) doxycycline treatment at E12.5. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.005; ns, not significant in unpaired two-tailed t-test or one-way ANOVA followed by Tukey post hoc test. The bar graphs show the mean and error bars represent s.e.m. Scale bars, 100 μm. Statistics source data can be found in Supplementary Table 12. Unprocessed original scans of gels are shown in Supplementary Fig. 8.

Supplementary Figure 6 Modification of TGFβ signaling rescues proliferation in LVNC iPSC-CMs and TBX20 knockdown ESC-CMs.

(a) Representative immunostaining for nuclear (blue), TNNT2 (red), and EdU (green) in control and LVNC (III-3) iPSC-CMs at 2 weeks after induction of cardiac differentiation with or without treatment of TGFβ receptor-1 inhibitor (SD208 or RepSox) for 2 continuous days. (b) Representative immunostaining for nuclear (blue), TNNT2 (green), and EdU (red) in scramble and TBX20 knockdown ESC-CMs (TBX20KD ESC-CMs) at 2 weeks after induction of cardiac differentiation with or without treatment of TGFβ receptor-1 inhibitor (SD208 or RepSox) for 2 continuous days. (c) Representative immunostaining for nuclear (blue), TNNT2 (green), and EdU (red) in LVNC iPSC-CMs at 2 weeks after induction of cardiac differentiation with adenoviral mediated overexpression of GFP (GFP-Ad) or dominant negative form of TGFBRII (TGFBRIIDN-Ad). Scale bars, 100 μm.

Supplementary Figure 7 Generation of TBX20 mutation corrected patient-specific iPSC lines using TALEN.

(a) TALEN recognition sites on TBX20 exon 7. TALEN pair was designed to bind to the specific sequence of mutant allele including stop-gain mutation site (c.951C > A, red character) using the rapid TALEN assembly system. (b) Sequence of left and right arms of the targeting vector. To make a targeting vector, 500 bp fragments of left and right arms were designed to share homologies with TBX20 exon 7 carrying wild-type sequence (c.951C, blue character) and flanking introns. TTAA site for piggyBac excision was created by introducing silent mutations near the intended modification site (red character in left arm). To avoid re-cleavage of substituted genomic sequence, three silent mutations (red character) were created into the TALEN recognition sites on right arm (yellow box). Both arms were cloned into a vector carrying piggyBac transposon with PGK promoter puroΔtk selection cassette (PGK-puroΔtk) to make the targeting vector. (c) Overview of targeted correction of TBX20 Y317∗ mutation using TALEN and piggyBac transposon system. TALEN-mediated double strand DNA break promotes homologous recombination of the targeting vector into cleaved site, and clones with integrated PGK-puroΔtk cassette were selected by puromycin. Subsequently, piggyBac-PGK-puroΔtk cassette was excised by transient expression of piggyBac transposase. Mutation-corrected lines were obtained after negative selection using ganciclovir treatment. Genotyping primer positions are indicated as Seq Fw, Seq Rv, 5′ Fw, 5′ Rv, 3′ Fw and 3′ Rv. d, PCR analysis showing transposon removal. Pre, pre-transfection of TALEN pairs and targeting vector; PB, after puromycin selection; Ex, after gancyclovir selection; 5′ Fw-Rv, PCR using 5′ Fw and 5′ Rv primers; 3′ Fw-Rv, PCR using 3′ Fw and 5′ Rv primers; Seq Fw-Rv, PCR using Seq Fw and Seq Rv primers. Unprocessed original scans of gels are shown in Supplementary Fig. 8. (e) Precise repair of the stop-gain mutation site. Mutation-corrected lines showed the wild-type sequence (c.951C, yellow box) instead of stop-gain mutation (c.951C > M, red box) and possessed the designed synonymous mutation as indicated in the orange box. (f) Mechanistic schema of the pathology of LVNC caused by TBX20 loss of function mutation. During the myocardium development, Tbx20 interacts with other cardiac transcription factors, including NKX2-5, GATA4, and TBX5, and initiates cardiac chamber-specific gene expression and myocardium growth. On the other hand, TBX20 inhibits activation of TGFβ signaling partially via PRDM16 and promotes embryonic cardiomyocyte proliferation. TBX20 mutation disturbs synergistic regulation of cardiac chamber specification and myocardium growth. Furthermore, failed suppression of TGFβ leads to improper upregulation of CDKN1A and promotes early exit of cardiomyocyte cell cycle. Decreased ventricular cardiomyocyte specification and proliferation leads to failed formation of compact layer of developing myocardium and results in LVNC.

Supplementary Figure 8 Unprocessed original scans.

Uncropped images of scanned western blots and gels shown in the Figures are provided.

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Kodo, K., Ong, SG., Jahanbani, F. et al. iPSC-derived cardiomyocytes reveal abnormal TGF-β signalling in left ventricular non-compaction cardiomyopathy. Nat Cell Biol 18, 1031–1042 (2016). https://doi.org/10.1038/ncb3411

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