Atrogin-1 promotes muscle homeostasis by regulating levels of endoplasmic reticulum chaperone BiP
Avnika A. Ruparelia, Margo Montandon, Jo Merriner, Cheng Huang, Siew Fen Lisa Wong, Carmen Sonntag, Justin P. Hardee, Gordon S. Lynch, Lee B. Miles, Ashley Siegel, Thomas E. Hall, Ralf B. Schittenhelm, Peter D. Currie
Avnika A. Ruparelia, Margo Montandon, Jo Merriner, Cheng Huang, Siew Fen Lisa Wong, Carmen Sonntag, Justin P. Hardee, Gordon S. Lynch, Lee B. Miles, Ashley Siegel, Thomas E. Hall, Ralf B. Schittenhelm, Peter D. Currie
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Research Article Muscle biology

Atrogin-1 promotes muscle homeostasis by regulating levels of endoplasmic reticulum chaperone BiP

Abstract

Skeletal muscle wasting results from numerous pathological conditions affecting both the musculoskeletal and nervous systems. A unifying feature of these pathologies is the upregulation of members of the E3 ubiquitin ligase family, resulting in increased proteolytic degradation of target proteins. Despite the critical role of E3 ubiquitin ligases in regulating muscle mass, the specific proteins they target for degradation and the mechanisms by which they regulate skeletal muscle homeostasis remain ill-defined. Here, using zebrafish loss-of-function models combined with in vivo cell biology and proteomic approaches, we reveal a role of atrogin-1 in regulating the levels of the endoplasmic reticulum chaperone BiP. Loss of atrogin-1 resulted in an accumulation of BiP, leading to impaired mitochondrial dynamics and a subsequent loss in muscle fiber integrity. We further implicated a disruption in atrogin-1–mediated BiP regulation in the pathogenesis of Duchenne muscular dystrophy. We revealed that BiP was not only upregulated in Duchenne muscular dystrophy, but its inhibition using pharmacological strategies, or by upregulating atrogin-1, significantly ameliorated pathology in a zebrafish model of Duchenne muscular dystrophy. Collectively, our data implicate atrogin-1 and BiP in the pathogenesis of Duchenne muscular dystrophy and highlight atrogin-1’s essential role in maintaining muscle homeostasis.

Authors

Avnika A. Ruparelia, Margo Montandon, Jo Merriner, Cheng Huang, Siew Fen Lisa Wong, Carmen Sonntag, Justin P. Hardee, Gordon S. Lynch, Lee B. Miles, Ashley Siegel, Thomas E. Hall, Ralf B. Schittenhelm, Peter D. Currie

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Figure 3

Atrogin-1 mutants display increased levels of BiP, which is sufficient to cause muscle fiber detachment.

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Atrogin-1 mutants display increased levels of BiP, which is sufficient t...
(A) Volcano plot highlighting differentially regulated proteins in atrogin-1–/– larvae compared with atrogin-1+/+ wild-type larvae – identified from untargeted proteomics. Proteins significantly (q < 0.05) upregulated and downregulated are shown in red and blue, respectively, as determined using an unpaired t test. (B) Representative Western blot images for BiP, and total protein direct blue stain, on whole cell protein lysates obtained from 3 independent biological replicates, each containing multiple atrogin-1+/+ or atrogin-1–/– larvae. (C) Quantification of BiP levels normalized to total protein with atrogin-1–/– larvae displaying a significant reduction compared with atrogin-1+/+, as determined using an unpaired t test. Data are shown as mean ± SD. (D–F) 6 dpf tunicamycin- (Tm-) or thapsigargin-treated (Tg-treated) larvae display muscle fiber detachment following incubation in methyl cellulose. (G) The percentage of affected larvae, with Tm or Tg treatment resulting in a significant increase in the proportion of fish displaying the muscle fiber detachment, as determined using a χ2 test. (H and I) Confocal images of F-actin–stained, methyl cellulose–treated, 6 dpf atrogin-1–/– mutants on the Tg(actc1b:KalTA4;cryaa:GFPpc54Tg) only [labeled as Control (KaltA4)] or Tg(actc1b:KalTA4;cryaa:GFPpc54Tg) and Tg(4XUAS:NLSCas9;cmlc2:RFP gl37Tg) (labeled as BiP KO) background. While control atrogin-1–/– mutants display fiber detachment, atrogin-1–/– mutants with BiP deficiency specifically in the muscle show normal muscle structure. (J) The percentage of affected atrogin-1–/– control larvae and BiP-KO larvae, with the latter having a significant decrease in the proportion of fish displaying the muscle fiber detachment, as determined using Fisher’s exact test. *P < 0.05, **P < 0.01. All experiments performed in triplicate with the total number of fish examined in each replicate being documented in Supplemental Table 2. Scale bar: 200 μm.