Research ArticleMYOPATHY

Targeting protein homeostasis in sporadic inclusion body myositis

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Science Translational Medicine  23 Mar 2016:
Vol. 8, Issue 331, pp. 331ra41
DOI: 10.1126/scitranslmed.aad4583
  • Fig. 1. β-APP overexpression or exposure to inflammatory mediators induces sIBM-like pathology in cultured rat myocytes that is abrogated by arimoclomol.

    (A and B) Cytoplasmic inclusion bodies (white arrows) in rat myocytes transfected with full-length human β-APP that are immunoreactive for β-APP and ubiquitin, and TDP-43 and ubiquitin. (C) The number of rat myocytes containing ubiquitinated inclusion bodies as a percentage of the total number of myocytes present [n = 3; *P < 0.05, one-way analysis of variance (ANOVA)]. Ari, arimoclomol. (D and E) Expression of TDP-43 (green) after empty vector (EV) or β-APP transfection and arimoclomol treatment and the number of rat myocytes with cytoplasmic mislocalization of TDP-43 (n = 3; *P < 0.001, unpaired t test). (F and G) TDP-43 expression (green) after exposure to inflammatory mediators and arimoclomol and quantification of TDP-43 mislocalization in cytokine-treated cultures (n = 3; *P < 0.05, unpaired t test). (H) Western blot analysis of TDP-43 expression in rat myocyte cultures exposed to cytokines in the presence and absence of arimoclomol. (I and J) Images show the expression of NF-κB subunit p65 (green) in β-APP–transfected cultures [4′,6-diamidino-2-phenylindole (DAPI)–labeled nuclei in blue] and cultures exposed to cytokines in the presence and absence of arimoclomol. (K) The number of rat myocytes with the nuclear subunit of NF-κB, p65 as a percentage of the total number of myocytes present (n = 3; *P < 0.05, one-way ANOVA). Error bars represent SEM. Scale bars, 10 μm (A and B); 20 μm (D, F, I, and J).

  • Fig. 2. Arimoclomol augments HSP70 expression and improves survival of cultured rat myocytes.

    (A) HSP70 expression (red) in cultured rat myocytes after empty vector (EV) transfection (left-hand panel), β-APP overexpression (middle panel), or β-APP overexpression plus treatment with arimoclomol (right-hand panel). (B) HSP70 expression (red) in cultured rat myocytes that were untreated (control; left-hand panel) or exposed to IL-1β alone (middle panel) or IL-1β plus arimoclomol (right-hand panel). (C) Western blot analysis of HSP70 expression in rat myocyte cultures exposed to inflammatory mediators or after β-APP overexpression in the presence or absence of arimoclomol. (D) Cytotoxicity in β-APP–overexpressing rat myocyte cultures as a percentage of that in control cultures (n = 3; *P < 0.02, one-way ANOVA), as assessed by a lactate dehydrogenase (LDH) assay. (E) Cytotoxicity after exposure to inflammatory mediators in the presence or absence of arimoclomol (n = 3; *P < 0.05, one-way ANOVA), as assessed by an MTT [3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide] assay. Error bars represent SEM. Scale bars, 10 μm.

  • Fig. 3. Disruption of protein homeostasis in rat myocyte cultures is prevented by arimoclomol.

    (A) The cytosolic calcium ion response induced by the ER stressor thapsigargin (an indicator of ER stress) in β-APP–overexpressing rat myocyte cultures and in cell cultures exposed to inflammatory mediators (n = 3; *P < 0.05, one-way ANOVA). (B) Expression of the ER stress mediator CHOP determined by Western blot analysis in rat myocyte cultures overexpressing β-APP or exposed to inflammatory mediators, in the presence or absence of arimoclomol. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) The images show the presence of a cytoplasmic aggregate immunoreactive for p62 (red; white arrow) in a desmin-positive rat myocyte after β-APP transfection. (D) The images show the expression of the autophagic protein LC3-II (red) in β-APP–transfected desmin-positive rat myocytes after empty vector transfection (left-hand panel), β-APP overexpression (middle panel), or β-APP overexpression plus treatment with arimoclomol (right-hand panel). The white arrow indicates punctate LC3-II staining of autophagosomes. (E) The chymotrypsin-like proteasome activity assessed 48 hours after β-APP transfection in the presence or absence of arimoclomol (n = 3; *P < 0.05, one-way ANOVA). (F) The expression of the autophagosome marker LC3-II in β-APP–transfected rat myocytes with or without arimoclomol (n = 3; *P < 0.05, one-way ANOVA). EV, empty vector. Error bars represent SEM. Scale bars, 10 μm.

  • Fig. 4. Arimoclomol treatment improves muscle strength, muscle contractile characteristics, and IBM-like pathology in mutant VCP mice.

    (A) The change in grip strength in wild-type VCP (WT-VCP), mutant VCP (mVCP), and arimoclomol-treated mutant VCP (mVCP + Ari) mice at 4 and 14 months (M) of age (n = 10; *P < 0.0001, unpaired t test). (B) Typical traces of muscle twitch and maximum tetanic force of extensor digitorum longus (EDL) muscles in untreated and arimoclomol-treated mutant VCP mice. (C) Mean maximum force of extensor digitorum longus muscles of WT-VCP, mutant VCP, and arimoclomol-treated mutant VCP mice (n = 10; *P < 0.04, one-way ANOVA). (D) Hematoxylin and eosin (H&E) staining of tibialis anterior (TA) muscles of mice in each experimental group (white arrow, atrophied fiber; black arrowheads, hypertrophic fibers). (E) H&E staining showing clear inflammatory cell infiltration in mutant VCP tibialis anterior muscle. (F and G) Western blots show MHC-I and phospho-IκBα expression in tibialis anterior muscles of mice in each experimental group. (H) Transmission electron microscopy of tibialis anterior muscles of mice in each experimental group. (I) Muscle fiber diameter in tibialis anterior muscles from untreated and arimoclomol-treated mutant VCP mice compared to WT-VCP mice (n = 3; *P < 0.0001, one-way ANOVA). (J) Western blot analysis of HSP70 expression in tibialis anterior muscles from mice in each experimental group. Bar chart shows mean relative optical density (n = 3; *P = 0.01, unpaired t test). Error bars represent SEM. Scale bars, 50 μm (D and E); 2 μm (H).

  • Fig. 5. Arimoclomol abrogates IBM-like pathology in mutant VCP mouse muscle.

    (A to F) Cross sections of tibialis anterior muscle from WT-VCP, mutant VCP, and arimoclomol-treated mutant VCP mice immunostained for ubiquitin (red) and TDP-43 (green); nuclei stained with DAPI (blue). Scale bars, 50 μm; 25 μm (insets).

  • Fig. 6. Clinical trial secondary outcomes (efficacy measures).

    The change from baseline to end point on three different clinical scales assessed at 4, 8, and 12 months (means ± SEM) in sIBM patients treated with arimoclomol for 4 months. (A to C) IBMFRS score, MMT average score, and right-hand grip (HGR) MVICT score. The IBMFRS is a disease-specific functional questionnaire for patients with sIBM and measures physical function/disability. MMT is a measure of muscle strength scored by the physician on the basis of the clinical assessment. MVICT is a measure of muscle strength performed using a quantitative muscle assessment (QMA) system that uses an adjustable cuff to attach the patient’s arm or leg to an inelastic strap that is connected to a force transducer. Error bars represent SEM. No statistically significant clinical efficacy measures were observed.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/331/331ra41/DC1

    Materials and Methods

    Fig. S1. Overexpression of β-APP and exposure to inflammatory mediators induce sIBM-like pathology in cultured myocytes.

    Fig. S2. Mutant VCP mice show further signs of pathology.

    Fig. S3. Consort diagram of the patients participating in the clinical trial.

    Table S1. Baseline characteristics of the study population.

    Table S2. Summary of all adverse events over the course of 1 year.

    Table S3. Mean changes (±SD) in secondary outcome measures throughout the study period.

    Movie S1. Low-magnification serial block-face scanning electron microscopy of arimoclomol-treated and untreated mutant VCP mouse muscle.

    Movie S2. High-magnification serial block-face scanning electron microscopy of wild-type VCP mouse muscle.

    Movie S3. High-magnification serial block-face scanning electron microscopy of untreated mutant VCP mouse muscle.

    Movie S4. High-magnification serial block-face scanning electron microscopy of arimoclomol-treated mutant VCP mouse muscle.

    References (59, 60)

  • Supplementary Material for:

    Targeting protein homeostasis in sporadic inclusion body myositis

    Mhoriam Ahmed, Pedro M. Machado, Adrian Miller, Charlotte Spicer, Laura Herbelin, Jianghua He, Janelle Noel, Yunxia Wang, April L. McVey, Mamatha Pasnoor, Philip Gallagher, Jeffrey Statland, Ching-Hua Lu, Bernadett Kalmar, Stefen Brady, Huma Sethi, George Samandouras, Matt Parton, Janice L. Holton, Anne Weston, Lucy Collinson, J. Paul Taylor, Giampietro Schiavo, Michael G. Hanna, Richard J. Barohn, Mazen M. Dimachkie,* Linda Greensmith*

    *Corresponding author. E-mail: l.greensmith{at}ucl.ac.uk (L.G.); mdimachkie{at}kumc.edu (M.M.D.)

    Published 23 March 2016, Sci. Transl. Med. 8, 331ra41 (2016)
    DOI: 10.1126/scitranslmed.aad4583

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Overexpression of β-APP and exposure to inflammatory mediators induce sIBM-like pathology in cultured myocytes.
    • Fig. S2. Mutant VCP mice show further signs of pathology.
    • Fig. S3. Consort diagram of the patients participating in the clinical trial.
    • Table S1. Baseline characteristics of the study population.
    • Table S2. Summary of all adverse events over the course of 1 year.
    • Table S3. Mean changes (±SD) in secondary outcome measures throughout the study period.
    • Legends for movies S1 to S4
    • References (59, 60)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). Low-magnification serial block-face scanning electron microscopy of arimoclomol-treated and untreated mutant VCP mouse muscle.
    • Movie S2 (.mov format). High-magnification serial block-face scanning electron microscopy of wild-type VCP mouse muscle.
    • Movie S3 (.mov format). High-magnification serial block-face scanning electron microscopy of untreated mutant VCP mouse muscle.
    • Movie S4 (.mov format). High-magnification serial block-face scanning electron microscopy of arimoclomol-treated mutant VCP mouse muscle.

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