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Studying arrhythmogenic right ventricular dysplasia with patient-specific iPSCs

Nature volume 494, pages 105–110 (2013)Cite this article

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Abstract

Cellular reprogramming of somatic cells to patient-specific induced pluripotent stem cells (iPSCs) enables in vitro modelling of human genetic disorders for pathogenic investigations and therapeutic screens1,2,3,4,5,6,7. However, using iPSC-derived cardiomyocytes (iPSC-CMs) to model an adult-onset heart disease remains challenging owing to the uncertainty regarding the ability of relatively immature iPSC-CMs to fully recapitulate adult disease phenotypes. Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is an inherited heart disease characterized by pathological fatty infiltration and cardiomyocyte loss predominantly in the right ventricle8, which is associated with life-threatening ventricular arrhythmias. Over 50% of affected individuals have desmosome gene mutations, most commonly in PKP2, encoding plakophilin-2 (ref. 9). The median age at presentation of ARVD/C is 26 years8. We used previously published methods1,10 to generate iPSC lines from fibroblasts of two patients with ARVD/C and PKP2 mutations11,12. Mutant PKP2 iPSC-CMs demonstrate abnormal plakoglobin nuclear translocation and decreased β-catenin activity13 in cardiogenic conditions; yet, these abnormal features are insufficient to reproduce the pathological phenotypes of ARVD/C in standard cardiogenic conditions. Here we show that induction of adult-like metabolic energetics from an embryonic/glycolytic state and abnormal peroxisome proliferator-activated receptor gamma (PPAR-γ) activation underlie the pathogenesis of ARVD/C. By co-activating normal PPAR-alpha-dependent metabolism and abnormal PPAR-γ pathway in beating embryoid bodies (EBs) with defined media, we established an efficient ARVD/C in vitro model within 2 months. This model manifests exaggerated lipogenesis and apoptosis in mutant PKP2 iPSC-CMs. iPSC-CMs with a homozygous PKP2 mutation also had calcium-handling deficits. Our study is the first to demonstrate that induction of adult-like metabolism has a critical role in establishing an adult-onset disease model using patient-specific iPSCs. Using this model, we revealed crucial pathogenic insights that metabolic derangement in adult-like metabolic milieu underlies ARVD/C pathologies, enabling us to propose novel disease-modifying therapeutic strategies.

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Figure 1: Generation of c.2484C>T PKP2 mutant iPSCs.
Figure 2: Induction of pathognomonic features of ARVD/C using mutant PKP2-iPSCs.
Figure 3: Rescue of pathological features of mutant PKP2-iPSC-CMs.
Figure 4: Metabolic and qRT–PCR assays of glucose and fatty acid utilization of mutant (JK#11 and JK#2) and normal hS-iPSC-CMs.

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Acknowledgements

We thank the patients for their participation, microarray core facilities at SBMRI and Scripps Research Institute for their support, T. Yi for technical assistance, and G. W. Rogers for assistance in metabolic assays. This work was supported by NIH grants (RO1 HL058493 and RO1 HL101189) (to D.P.K.); NIH grants (RO1 AR056712 and RO1 AR052779) (to P.L.P); California Institute of Regenerative Medicine (CIRM) grants (RS1-00171-1, RB2-01512 and RB4-06276) and NIH grant (RO1 HL105194) (to H.-S.V.C.).

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Authors and Affiliations

  1. Del E. Webb Neuroscience, Aging & Stem Cell Research Center, Sanford-Burnham Medical Research Institute, La Jolla, 92037, California, USA

    Changsung Kim, Johnson Wong, Jianyan Wen, Shirong Wang, Cheng Wang & Huei-Sheng Vincent Chen

  2. Department of Cardiovascular Surgery, China-Japan Friendship Hospital, Beijing, 100029, China

    Jianyan Wen

  3. Muscle Regeneration and Development Program, Sanford-Burnham Medical Research Institute, La Jolla, 92037, California, USA

    Sean Spiering, Natalia G. Kan, Sonia Forcales & Pier Lorenzo Puri

  4. Dulbecco Telethon Institute, IRCCS Fondazione Santa Lucia, 00179 Rome, Italy ,

    Pier Lorenzo Puri

  5. Diabetes and Obesity Research Center, Sanford-Burnham Medical Research Institute, Orlando, 32827, Florida, USA

    Teresa C. Leone & Daniel P. Kelly

  6. Department of Medicine/Cardiology, Johns Hopkins University School of Medicine, Baltimore, 21287, Maryland, USA

    Joseph E. Marine, Hugh Calkins & Daniel P. Judge

  7. Department of Medicine/Cardiology, University of California-San Diego, San Diego, 92103, California, USA

    Huei-Sheng Vincent Chen

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  1. Changsung Kim
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Contributions

C.K. and H.-S.V.C. designed experiments and wrote the manuscript; J.E.M., H.C. and D.P.J. provided clinical assessment and patient’s fibroblasts as well as analysis of data and assistance with preparation of the manuscript; C.K., J.We., S.W., C.W., S.S., N.G.K. and H.-S.V.C. performed the experiments; S.F. and P.L.P. helped C.K. in performing bisulphide sequencing; J.Wo. analysed microarray, Seahorse and immunocytochemical data; D.P.K. and T.C.L. provided scientific advice and primers for metabolic assays; C.K., J.Wo. and H.-S.V.C performed and interpreted the calcium imaging data; all authors read and approved the manuscript, and H.-S.V.C. supervised the entire research project.

Corresponding author

Correspondence to Huei-Sheng Vincent Chen.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This fie contains a more detailed legend for Figure 4, Supplementary Figures 1-16, Supplementary Tables 1-4, Supplementary Methods and Supplementary References. (PDF 21705 kb)

Supplementary Data

This file contains the microarray data used to construct figure 1f. (XLS 6324 kb)

A beating 70 day-old H9 hESC-EB

A beating embryoid body derived from H9 human embryonic stem cells after it has been cultured for 70 days in cardiogenic media. (AVI 3116 kb)

A beating 70 day-old c.2484C>T mutant PKP2 iPSC-EB

A beating embryoid body derived from the homozygous c.2484C>T mutant PKP2-induced pluripotent stem cells of patient #1 after it has been cultured for 70 days in cardiogenic media. (AVI 2958 kb)

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Kim, C., Wong, J., Wen, J. et al. Studying arrhythmogenic right ventricular dysplasia with patient-specific iPSCs. Nature 494, 105–110 (2013). https://doi.org/10.1038/nature11799

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