Modeling and study of the mechanism of dilated cardiomyopathy using induced pluripotent stem cells derived from individuals with Duchenne muscular dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene (DMD), and is characterized by progressive weakness in skeletal and cardiac muscles. Currently, dilated cardiomyopathy due to cardiac muscle loss is one of the major causes of lethality in late-stage DMD patients. To study the molecular mechanisms underlying dilated cardiomyopathy in DMD heart, we generated cardiomyocytes (CMs) from DMD and healthy control induced pluripotent stem cells (iPSCs). DMD iPSC-derived CMs (iPSC-CMs) displayed dystrophin deficiency, as well as the elevated levels of resting Ca(2+), mitochondrial damage and cell apoptosis. Additionally, we found an activated mitochondria-mediated signaling network underlying the enhanced apoptosis in DMD iPSC-CMs. Furthermore, when we treated DMD iPSC-CMs with the membrane sealant Poloxamer 188, it significantly decreased the resting cytosolic Ca(2+) level, repressed caspase-3 (CASP3) activation and consequently suppressed apoptosis in DMD iPSC-CMs. Taken together, using DMD patient-derived iPSC-CMs, we established an in vitro model that manifests the major phenotypes of dilated cardiomyopathy in DMD patients, and uncovered a potential new disease mechanism. Our model could be used for the mechanistic study of human muscular dystrophy, as well as future preclinical testing of novel therapeutic compounds for dilated cardiomyopathy in DMD patients.

Keywords: Dilated cardiomyopathy; Duchenne muscular dystrophy; Induced pluripotent stem cells.

Conflict of interest statement

Competing interests

The authors declare no competing or financial interests.

© 2015. Published by The Company of Biologists Ltd.

Figures

Fig. 1.

CM differentiation from control and DMD iPSCs. (A) Representative images showing morphologies of control Y1, S3 iPS4 and DMD iPSCs. DMD-iPSC1 cells were positive for OCT4 immunostaining (green) and DMD15 iPSCs were positive for live staining of pluripotency surface marker, alkaline phosphatase (red). Scale bars: 100 μm. (B) Schematic showing dystrophin mutations in DMD-iPS1 and DMD15 iPSCs. Both DMD-iPS1 and DMD15 iPSCs have the same DMD mutation. (C) Schematic showing the Dp427m DMD isoform expressed in iPSC-CMs and the locations where anti-DMD antibodies bind to the N-terminal (NT), Rod domain and C-terminal (CT) of dystrophin. (D) Immunostaining of dystrophin (green) with the anti-DMD antibodies described in C in control S3 and DMD-iPS1-cell-derived CMs (CTNT, red). Scale bars: 10 µm.

Fig. 2.

Detection of apoptosis. (A) Representative images showing iPSC-EBs at day 22 of differentiation. Red arrows indicate the cell debris on iPSC-EBs. Scale bars: 200 μm. (B) Representative immunostaining images of cleaved caspase-3 (C-CASP3, red) in iPSC-CMs (CTNT, green). Red arrowheads indicate the C-CASP3+ CMs. Scale bars: 50 µm. (C) Quantification of C-CASP3-positive CMs in control and DMD iPSC-CMs (Y1, n=657; S3, n=1129; DMD-iPS1, n=860; DMD15, n=53). (D) CMs with PI staining followed by FACS quantification of DNA fragmentation. All results are mean±s.d. of four independent experiments. **P<0.01, *P<0.05 (two-tailed Student's t-test).

Fig. 3.

Whole-transcriptome sequencing of DMD iPSC-CMs. (A) Whole transcriptome sequencing of iPSC-EBs at day 22 of differentiation. Fragment per kilobase of exon per million reads (FPKM) of genes from DMD iPS1 CMs (n=3 each) and control S3 iPSC-CMs (n=3) were averaged and compared. The FPKM value indicates the relative expression level of a sequenced gene. Heat maps were drawn with log2 of FPKM (mean±s.d. of triplicate experiments) for representative genes in control versus DMD CMs. (B) Validation of gene expression by q-RT-PCR. Error bars show s.d. of triplicate experiments. *P<0.05, **P<0.01 (two-tailed Student's t-test). (C) IPA analysis of the differentially expressed genes in DMD-iPS1 CMs versus control S3 iPSC-CMs. Green indicates the upregulated, and red indicates the downregulated bio-function categories in DMD iPSC-CMs when compared with control iPSC-CMs.

Fig. 4.

Mitochondria-mediated apoptosis network in DMD iPSC-CMs. (A) TEM of mitochondria in iPSC-CMs. Yellow arrowheads indicate the healthy mitochondria and blue arrows indicate the swollen mitochondria. Scale bars: 2 μm. (B) Green JC-1 staining indicates damaged mitochondria with disrupted membrane potential. Quantification of green JC-1 was conducted in all day 22 control and DMD iPSC-CMs. The y-axis indicates the relative change of the ratio of cells showing JC-1 green. Error bars show s.d. of five independent experiments. *P<0.05, **P<0.01 (two tailed Student's t-test). (C) Western blot analysis of day 22 control and DMD iPSC-EBs (left and middle panels). Mitochondria-free cytosol and mitochondria were fractionated from iPSC-EBs, followed by western blotting to detect the expressions of DIABLO and CYCS (right panel). The same amounts of samples were loaded based on their GAPDH expression levels before fractioning. UC-CASP3, uncleaved CASP3. (D) iPSC-CMs were stained with MitoTracker dye (red) to stain mitochondria and anti-DIABLO antibody (green). White arrows indicate DIABLO in mitochondria. Pink arrows indicate the cytosolic DIABLO released from mitochondria. Scale bars: 10 μm. (E) A schematic depicting the mitochondria-mediated apoptosis network in DMD iPSC-CMs. Red stars indicate the increased, and green stars indicate the decreased protein levels in DMD iPSC-CMs compared with control iPSC-CMs.

Fig. 5.

Ca2+ handling of control and DMD iPSC-CMs. (A) Measurement of L-type Ca2+ currents in single iPSC-CMs using patch clamp. (Control, n=12; DMD-iPS1, n=12). (B) Quantification of resting [Ca2+]i. (Control, n=80; DMD-iPS1, n=62.) All error bars show s.d. **P<0.01 (two-tailed Student's t-test).

Fig. 6.

P188 suppresses CASP3 cleavage in DMD iPSC-CMs. (A) DMD iPSC-CMs were treated with P188 (1 mg/ml) for 7 days, followed by Ca2+ imaging. P188 suppressed the resting [Ca2+]i (DMD-iPS1 CMs, n=62; DMD-iPS1 CMs+P188, n=36). *P<0.05. (B) P188 suppressed cleavage of CASP3 in DMD iPSC-CMs. Western blot analysis of DMD iPSC-CMs with and without P188 treatment (1 mg/ml) for 7 days. Representative images of western blots are shown in the upper panel and statistical analysis of band intensity is shown in the lower panel. *P<0.05 (n=3). (C) Representative images of C-CASP3 immunostaining (left panel) and quantification of C-CASP3+ CTNT+ CM ratios (right panel) in DMD-iPS1 CMs with and without P188 treatment (DMD-iPS1 CMs, n=546; DMD-iPS1 CMs+P188, n=332). **P<0.01. Scale bars: 10 µm. (D) A schematic summary of the possible mechanism, as well as potential therapeutic strategies for dilated cardiomyopathy in DMD patients. In all graphs, error bars show the s.d., and the P values were calculated with a two-tailed Student's t-test.