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Adult Disease Model from iPSC Progeny

“Studying arrhythmogenic right ventricular dysplasia with patient-specific iPSCs”

Modelling genetic disorders using induced pluripotent stem cells (iPSCs) is an emerging tool for researchers;  however cells derived from iPSCs, such as cardiomyocytes (CMs), have not yet been qualified as useful models of adult disease phenotypes. Now researchers from the group of Huei-Sheng Vincent Chen at the Sanford-Burnham Medical Research Institute, California, USA have studied the inherited heart disease arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) (Calkins and Marcus) an inherited heart disease characterized by pathological fatty infiltration and cardiomyocyte loss in the right ventricle. From patient-specific mutation bearing fibroblasts they generated iPSCs and subsequently iPSC-CMs; finding that induction of adult-like metabolism has a critical role in establishing an adult-onset disease model (Kim et al).

Using fibroblasts taken from a patient with clinical ARVD/C and a homozygous c.2484C.T mutation in PKP2, three iPSC lines were generated and extensively characterised, demonstrating their similarity to ESCs, silencing of exogenous retroviral transgenes and the endurance of the PKP2 mutation. Using a standard cardiogenic protocol (Kim et al), beating embryoid bodies (EBs) were produced using the disease-specific iPSCs, hESCs and wild type iPSCs. Subsequent analysis found, as expected (Garcia-Gras et al), abnormal nuclear translocation of junction plakoglobin proteins (PKG) and very low b-catenin activity and expression in the mutant iPSC-CMs as compared to the hESC and normal iPSCs. However, the lack of exaggerated lipogenesis or apoptosis was consistent with the delayed, adult-onset clinical course of ARVD/C.

Detailed analysis of cardiomyocytes in EBs (positive for cardiac a-actinin or troponin I, or CTNI) found no significant difference between lipogenesis in hESC-CMS and mutant iPSC-CMs. The induction of an adult-like energy metabolism (fatty acid metabolism as compared to glycolysis in embryonic cardiomyocytes) using 3 factors (3F); insulin, dexamethasone and 3-isobutyl-1-methilxanthine (IBMX), to accelerate pathogenesis found mildly increased lipogenesis with minimal apoptosis and induced the expression of PPAR-alpha (PPAR-a), the major transcriptional regulator of fatty acid metabolism in adult cardiomyocytes (Lopaschuk et al), in beating mutant iPSC-EBs only.   PPAR-g, a pathway which is over-activated in right ventricle tissue samples of ARVD/C hearts (Djouadi et al) and whose overexpression in cardiomyocytes leads to dilated cardiomyopathy (Son et al), was only slightly activated in any CMs. Boosting over-activation of PPAR-g by the addition of rosiglitazone and indomethacin (5F) (and later by the endogenous PPAR-g activator 13-hydroxyocta-decadienoic acid (13-HODE)), triggered increased lipogenesis and apoptosis in the mutant iPSC-CMs only, with PPAR-g nuclear staining observed at 2 weeks. However, rosiglitazone and indomethacin treatment alone (without 3F) did not induce exaggerated lipogenesis or apoptosis suggesting that activation of both normal PPAR-a and abnormal PPAR-g pathways in mutant iPSC-CMs is required for eliciting pathologies of ARVD/C. The specificity of mutations in PKP2 to the disease pathogenesis was verified through the rescue of overt phenotypes in mutant iPSC-CMs by the re-introduction of wild-type (WT) PKP2.

Analysis of fatty acid oxidation (FAO) and glycolysis found that both normal and mutant iPSC-CMs had dominant glycolytic energetics (an embryonic-stage pattern (Onay-Besikci)) at the baseline, with FAO being boosted by PPAR-g activation. 5F induction led to a depressed metabolic state, with huge reductions in FAO and glycolysis, similar to that observed in failing hearts (Neubauer), with glycolysis usage being favoured in this state. The authors therefore claim that the reported protocol recapitulates the metabolic and pathological signatures of failing ARVD/C hearts and also went onto repeat and confirm these findings in genome integration-free iPSCs generated from a different mutant (heterozygous c.2013delC in exon 10 of PKP2).  

Moving to analysis of cardiomyocyte properties, mutant iPSC-CMs demonstrated slower intracellular calcium flux at baseline as compare to normal iPSC-CMs, which was further exacerbated after 5F pathogenic induction. Additionally, mRNA levels of sarcoplasmic reticulum Ca2+-ATPase (SERCA, for Ca2+ re-uptake) were reduced and Na+Ca2+ exchanger 1 (NCX1, for Ca2+ extrusion)  remained similar at baseline, while under 5F conditions, both genes mRNAs were significantly reduced. The differences in Ca2+ also led to detectable electrophysiological differences in mutant cells.

Finally, altering the levels of Islet l-positive (Isl1+) cardiac progenitor cells, and thereby altering numbers of right-ventricle-like cardiomyocytes was assessed. Increasing their number in EBs through treatment with 6-bromoindirubin-39-oxime (BIO) (Qyang et al) followed by 5F treatment significantly increased lipogenesis and apoptosis in cardiomyocytes, while a reduction in progenitor number through Dickkopf-1 (Dkk1) (Qyang et al) treatment led to lower lipogenesis and apoptosis, suggesting that  Isl1+ cells confer the dominant pathologies in the right ventricle.

Overall, this paper successfully recapitulates an adult-onset disease in cells derived from patient-specific and disease-specific iPSCs and demonstrates the vital importance of PPAR activation and the role of Isl1+ cells in the pathogenesis of ARVD/C thus providing an excellent model system for further research into the disease and hopefully for therapeutic screens.



  • Calkins, H. & Marcus, F. Arrhythmogenic right ventricular cardiomyopathy/dysplasia: an update. Curr. Cardiol. Rep. 10, 367–375 (2008).
  • Djouadi, F. et al. A potential link between peroxisome proliferator-activated receptor signalling and the pathogenesis of arrhythmogenic right ventricular cardiomyopathy. Cardiovasc. Res. 84, 83–90 (2009).
  • Garcia-Gras, E. et al. Suppression of canonical Wnt/b-catenin signaling by nuclear plakoglobin recapitulates phenotype of arrhythmogenic right ventricular cardiomyopathy. J. Clin. Invest. 116, 2012–2021 (2006).
  • Kim, C. et al. Non-cardiomyocytes influence the electrophysiological maturation of human embryonic stem cell-derived cardiomyocytes during differentiation. Stem Cells Dev. 19, 783–795 (2010).
  • Lopaschuk, G. D. et al. Myocardial fatty acid metabolism in health and disease. Physiol. Rev. 90, 207–258 (2010).
  • Neubauer, S. The failing heart—an engine out of fuel. N. Engl. J. Med. 356, 1140–1151 (2007).
  • Onay-Besikci, A. Regulation of cardiac energy metabolism in newborn. Mol. Cell. Biochem. 287, 1–11 (2006).
  • Qyang, Y. et al. The renewal and differentiation of Isl1+ cardiovascular progenitors are controlled by a Wnt/b-catenin pathway. Cell Stem Cell 1, 165–179 (2007).
  • Son, N.-H. et al. Cardiomyocyte expression of PPARg leads to cardiac dysfunction in mice. J. Clin. Invest. 117, 2791–2801 (2007).

Study originally appeared in Nature.

Stem Cell Correspondent Stuart P. Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.