Studying arrhythmogenic right ventricular dysplasia with patient-specific iPSCs

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 screens. 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 ventricle, 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 years. We used previously published methods to generate iPSC lines from fibroblasts of two patients with ARVD/C and PKP2 mutations. Mutant PKP2 iPSC-CMs demonstrate abnormal plakoglobin nuclear translocation and decreased β-catenin activity 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.

Figure 1. Generation of c.2484C>T PKP2 mutant iPSCs

a, The JK#2 clone of mutant iPSCs expressed significant levels of pluripotent proteins. b, Representative images of cells of three germ layers from teratoma sections. c, Bisulphite sequencing analysis of the CpG methylation patterns of NANOG promoter regions in mutant PKP2 fibroblasts (FB), hESCs, and JK#2 clone of iPSCs. d, Normal karyotype of JK#2 iPSCs. e, Genomic DNA sequencing showed the homozygous c.2484C>T (asterisk) PKP2 mutations in JK#2 iPSCs. f, Analysis of genome-wide mRNA expression profiles (Heat map) of all iPSC lines, normal human FB (CF-FB)-derived CF-iPSC, and H9 hESC lines (see Methods and Supplementary Figs.1–2 for detailed description).

Figure 2. Induction of pathognomonic features of ARVD/C using mutant PKP2-iPSCs

a, H9 hESC-CMs showed sarcolemmal connexin 43 (Cx43) and Pkg distribution (red) at both cell membrane and nucleus (DAPI); yet (b) Pkg staining in mutant iPSC-CMs is restricted to the nuclei. c, At baseline, minimal TUNEL nuclear staining (apoptosis) or Nile-Red labelling (lipogenesis) was found in mutant iPSC-CMs. Pictures with low magnification are used to show the lack of pathologies. d, Protocols for lipogenic (3F) and pathogenic (5F) induction. e, Minimal apoptosis and (f) mild increase of lipid-laden (red) CMs were found after 3F treatment. g, Summary of expression levels of PPARα and PPARγ (relative to GAPDH) in beating EBs. PPARα: 4.3±0.3E-3 & 4.7±1.2E-3 at control condition (Con), 5.0±1.3E-3 & 1.1±0.2E-2 after 3F and 3.9±0.5E-3 & 7.0±1.3E-3 after 5F for H9 hESC- and mutant iPSC-CMs, respectively. PPARγ: 6.1±2.1E-6 & 2.9±1.7E-6 at Con, 1.6±0.4E-5 & 5.6±1.8E-6 after 3F and 2.0±0.5E-5 & 1.5±0.6E-4 after 5F. Pictures with higher magnification are used to show significant increase of (h) TUNEL-positive and (i) lipid-containing CMs after 5F. Summary of (j) degrees of apoptosis and (k) lipid-laden CMs in beating EBs. The number in each column represents the number of biological replicates tested. Asterisks indicate p<0.05 and NS, no significant difference by ANOVA. All data are shown as mean±s.e.m.

Figure 3. Rescue of pathological features of mutant PKP2-iPSC-CMs

a, Diagram of lentiviral vectors used to stably integrate control-GFP or WT-PKP2-GFP into mutant iPSC-CMs. b, Top panels: Pkg (red) remained restricted to nuclei with control-GFP vectors; yet, bottom panels show Pkg distributed at both cell membrane and nucleus (Hoechst) with WT-PKP2-GFP of purified mutant iPSC-CMs. c, After 5F and WT PKP2-GFP lentiviral infection, TUNEL nuclear staining could only be found in GFP-negative CMs. d, Large number of lipid droplets (red) could only be found in WT PKP2-GFP-negative (left, 22.5±3.8) or control-GFP-positive CMs (right, 30.3±12.2 lipid droplets per CM), but not in WT PKP2-GFP-positive CMs (5.5±2.0 small lipid droplets per CM). e, 13-HODE (20 μg/ml) with 3F could replace 5F for pathogenic induction. f, 3 μM GW and 0.5 μM T007 (not shown) prevent pathogenic induction by 13-HODE+3F. g, Summary of CM apoptotic index and (h) degrees of lipogenesis. The percentage of CMs that have TUNEL staining and lipid droplets in mutant iPSC-CMs after treatment with 3F, 13-HODE+3F, 13-HODE+3F+GW, 13-HODE+3F+T007 and 5F (re-plot from Fig. 2j–k) are: 3.9±1.8 & 4.8±1.0%, 22.4±4.0 & 37.5±6.0%, 2.5±2.0 & 3.9±2.5%, 1.5±0.9 & 4.0±1.0% and 39.6±8.5 & 31.6±3.3% respectively. i, N-acetyl-cysteine (NAC) prevented CM apoptosis induced by 5F. j, The apoptotic index for mutant iPSC-CMs after 5F with 1 mM NAC or ascorbic acid (AA) is 11.4±2.3% and 3.8±1.2%, respectively (versus 2.1±0.6% in control and 39.6±8.5 after 5F). The number in each column represents the number of biological replicates tested. Asterisks indicate p<0.05 and NS, no significant difference by ANOVA.

Figure 4. Metabolic and qRT-PCR assays of glucose and fatty acid utilization of mutant (JK#11 & #2) and normal hS-iPSC-CMs

The Etomoxir (ETO, 100 μM)-blocked component of oxygen consumption rate (OCR) and 2-deoxyglucose (2-DG, 50 mM)-blocked component of extracellular acidification rate (ECAR) represent FAO and glycolysis, respectively. Absolute values of OCR and ECAR are expressed as pmol/min/10⁶ cells and mpH/min/10⁶ cells, respectively. a, Real-time measurement of OCR showed that ETO blocked 14.4±10.2 (0F), 53.9±8.2 (3F) and 15.3±8.5% (5F) of baseline OCR (red arrow) for mutant iPSC-CMs. b, ETO-blocked absolute OCR (white boxes) for mutant iPSC-CMs are 85.2±196.6 (0F), 1865.3±383.5 (3F) and 254.5±115.4 (5F). c, ECAR measurement after ETO inhibition of β-oxidation showed a rapid ~21% compensatory increase of glycolysis only in the 3F condition (a switch in energy substrates); yet, after 5F, ETO transiently decreased ECAR by ~45% (green arrow) followed by ~28% compensatory increase in ECAR (glycolysis). d, 2DG-blocked absolute ECAR (glycolysis) for mutant iPSC-CMs after 0F, 3F or 5F are 1353.0±313.6, 1766.0±579.7 and 457.2±211.0, respectively. Comparable patterns in absolute and relative OCR or ECAR in normal hS-iPSC-CMs (WS#4) are shown in (e–h). For normal iPSC-CMs, ETO-blocked FAO after 0F, 3F or 5F are (e) (relative) 21.8±24.8%, 42.6±6.6% and 50.5±4.06%; or (f) (absolute) 329.4±235.8, 1114.5±316.6 and 807.8±196.4, respectively. 2DG-blocked glycolysis after 0F, 3F or 5F are (g) (relative) 65.5±5.3%, 54.9±3.8% and 58.8±4.8%; or (h) (absolute) 915.3±270.6, 929.4±314.3 and 556.8±217.9, respectively. Combining data shown in a–h, these results support that both normal and mutant iPSC-CMs 1) display embryonic metabolism at the baseline, and 2) show significantly increases FAO after 3F with ability to switch between FAO and glucose utilization (an adult-like metabolic pattern). Mutant iPSC-CMs after 5F behave like failing cardiomyocytes (also see Supplementary Fig. 8) with pathological glucose-dominant metabolism. i, A simple diagram to illustrate substrate utilization pathways in cardiomyocytes. qRT-PCR analysis of Puromycin-purified mutant iPSC-CMs showed that mutant iPSC-CMs displayed significantly decreased expression of (j) CPT-1b and (k) PDK4 mRNA transcripts when compared to control H9 hESC-CMs after 5F induction, leading to enhanced pyruvate oxidation and decreased FAO, respectively. Please see Supplementary Information for detailed legend of Fig. 4. Single asterisk indicates p<0.05 and NS, no significant difference by ANOVA. P values are shown when unpaired t-test was performed.