Research ArticleCARDIOMYOPATHY

Severe dystrophic cardiomyopathy caused by the enteroviral protease 2A–mediated C-terminal dystrophin cleavage fragment

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Science Translational Medicine  01 Jul 2015:
Vol. 7, Issue 294, pp. 294ra106
DOI: 10.1126/scitranslmed.aaa4804
  • Fig. 1. Generation of transgenic mice and expression of dystrophin and DGC proteins.

    (A) (Top) Schematic of dystrophin structure. Hinge domains are indicated by blue boxes. Spectrin-like repeats are indicated by red boxes, with yellow boxes constituting the rod domain actin binding region of dystrophin. Arrow at hinge 3 indicates 2Apro cleavage site. (Bottom) Schematics of the NtermDys and CtermDys transgenes with N-terminal epitope tags. NtermDys contains an N-terminal FLAG epitope tag, the N-terminal domain of dystrophin, and the N-terminal portion of the central rod domain (including spectrin-like repeats 1 to 19, and hinges 1, 2, and part of hinge 3). CtermDys contains an N-terminal MYC epitope tag, dystrophin’s cysteine (Cys)–rich domain, C-terminal domain, and C-terminal portion of the rod domain (containing part of hinge 3, hinge 4, and spectrin-like repeats 20 to 24). (B) Representative Western blot for dystrophin expression in membrane proteins of NTg, CtermDys, and NtermDys transgenic mice. Quantification of dystrophin expression shown as expression relative to loading normalized to NTg. (C) Immunofluorescence detection of CtermDys in a heart section of CtermDys mice. Scale bar, 100 μm. Inset: Merged laminin (green) and CtermDys (red), indicating the basal lamina. (D to H) Quantification of β-dystroglycan (D), γ-sarcoglycan (E), α-sarcoglycan (F), α-dystroglycan (G), and utrophin (H) expression in membranes isolated from NTg and transgenic mice. Quantification of protein expression shown as expression relative to loading normalized to NTg. In (B) and (D) to (H), data are means ± SEM (n = 4 to 7 per group); *P < 0.05 versus NTg, analysis of variance (ANOVA) and Dunnett’s multiple comparison test.

  • Fig. 2. Heart morphology, fibrosis, and membrane instability in hearts of CtermDys mice.

    (A) Whole-heart images (scale bar, 2 mm) and transverse sections [hematoxylin and eosin (H&E)], and heart-to-tibia length ratio. Data (expressed as ratio of heart weight to tibia length) are means ± SEM (n = 4). P value was determined by t test. The representative H&E-stained heart transverse section indicates inflammation in fibrotic area [scale bars, 1 and 0.5 mm (inset)]. (B) Sirius Red–Fast Green staining of representative NTg, NtermDys, CtermDys, and mdx mouse hearts (age 6 months). Scale bar, 200 μm. Fibrotic area was quantified from the stained images. Data are means ± SEM (n = 8 to 15). P values were determined by ANOVA. (C) Representative mosaic images of EBD uptake (18 hours after dye injection) in hearts of NTg, mdx, NtermDys, CtermDys, NtermDys(mdx), CtermDys(mdx), and DTg mice. Scale bars, 1 mm. The dye uptake percentage out of whole-heart area was quantified from the images. Data are means ± SEM (n = 7 to 11 per group). *P < 0.05; **P < 0.01; ***P < 0.001 versus NTg, ANOVA.

  • Fig. 3. Ischemic injury in isolated hearts and adrenergic stress testing of transgenic mice in vivo.

    (A and B) LVDP and recovery of LVDP in hearts of NTg and CtermDys mice during ischemia/reperfusion injury. *P < 0.05 versus CtermDys by two-way ANOVA. Data are means ± SEM (n = 7 per group). (C) LVEDP of NTg and CtermDys hearts during ischemia/reperfusion injury. Data are means ± SEM (n = 7 per group). P value was determined by t test. (D) Survival study by cardiac stress testing in vivo. Animals were administered with isoproterenol (three times per day, every 8 hours) starting at time 0 (arrows), and survival of NTg, mdx, and CtermDys mice was evaluated over the course of 5 days. n = 10 to 15 animals per group. P values were determined by Mantel-Cox test.

  • Fig. 4. Expression of full-length functional dystrophin corrects dystrophic cardiomyopathy in CtermDys transgenic mice.

    (A) Representative Western blots of heart total protein extracts from NTg and transgenic animals. Dystrophin expression was detected using an antibody that detects both mouse and human dystrophin. Desmin served as the loading control. (B) Quantification of CtermDys protein expression in (A) is relative to desmin and normalized to CtermDys mice, and is expressed as means ± SEM (n = 4 to 5). P value was determined by ANOVA. (C) Quantification of EBD uptake in NTg, CtermDys, and DTg(mdx) hearts. Data are means ± SEM (n = 4 to 5 per group). P values were determined by ANOVA. (D) Survival of NTg, CtermDys, CtermDys-hDys649(mdx), and CtermDys-hDys600(mdx) mice during prolonged adrenergic stress. n = 11 per group. P value comparing CtermDys to NTg and CtermDys-hDys649(mdx) was determined by Mantel-Cox test. (E) Representative images of EBD uptake in NTg, CtermDys, and DTg(mdx) hearts. Scale bars, 1 mm.

  • Fig. 5. Survival during physiological stress testing and gene-dose analysis by in vivo cardiac titration of the CtermDys peptide.

    (A) CtermDys mice were crossed with mice expressing a cardiac-specific tamoxifen-inducible Cre recombinase (MCM). Tamoxifen was administered to the resulting DTg CtermDys × MCM mice to study the decay of the CtermDys protein in the heart after gene excision in vivo. (B) Representative Western blot showing CtermDys expression level decay and full-length endogenous dystrophin increase at the sarcolemma in CtermDys × MCM mice after tamoxifen treatment. Dystrophin and CtermDys were both detected with the same C-terminal antibody. Desmin was used as loading control. Quantification of full-length dystrophin and CtermDys expression in CtermDys × MCM mice after tamoxifen treatment. Data are means ± SEM (n = 6 per time point). (C) Survival curve of transgenic and NTg mice (n = 8 to 12 per group) starting 5 days after tamoxifen treatment, during isoproterenol stress test. (D) Survival curves for transgenic and NTg mice during isoproterenol stress test (no tamoxifen injected) (n = 6 to 8 per group). P value was determined by Mantel-Cox test.

  • Fig. 6. Two-hit model of CtermDys as a loss of dystrophin function peptide.

    (A) Normal DGC network in human cardiac muscle. (B) Proposed two-hit model of EV infection–based CtermDys cardiomyopathy showing severed link to actin and, because CtermDys is anchored to the DGC, prevention of utrophin compensatory binding. (C) Dystrophin-deficient myocyte with capacity for utrophin to bind and compensate, in part, for loss of dystrophin.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/294/294ra106/DC1

    Fig. S1. Transgene expression in hearts of NtermDys and CtermDys transgenic mice.

    Fig. S2. DGC protein expression in the hearts of transgenic mice.

    Fig. S3. Subcellular localization of NtermDys protein in the heart.

    Fig. S4. Subcellular localization of CtermDys protein in the heart.

    Fig. S5. Evidence of normal membrane integrity in the hearts of NtermDys transgenic lines.

  • Supplementary Material for:

    Severe dystrophic cardiomyopathy caused by the enteroviral protease 2A–mediated C-terminal dystrophin cleavage fragment

    Matthew S. Barnabei, Frances V. Sjaastad, DeWayne Townsend, Fikru B. Bedada, Joseph M. Metzger*

    *Corresponding author. E-mail: metzgerj{at}umn.edu

    Published 1 July 2015, Sci. Transl. Med. 7, 294ra106 (2015)
    DOI: 10.1126/scitranslmed.aaa4804

    This PDF file includes:

    • Fig. S1. Transgene expression in hearts of NtermDys and CtermDys transgenic mice.
    • Fig. S2. DGC protein expression in the hearts of transgenic mice.
    • Fig. S3. Subcellular localization of NtermDys protein in the heart.
    • Fig. S4. Subcellular localization of CtermDys protein in the heart.
    • Fig. S5. Evidence of normal membrane integrity in the hearts of NtermDys transgenic lines.

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