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 4

Atrogin-1 deficiency also results in altered mitochondrial dynamics.

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Atrogin-1 deficiency also results in altered mitochondrial dynamics. (A)...
(A) Overrepresentation analyses on all differentially regulated proteins in atrogin-1–/– larvae revealed a significant enrichment of multiple Kyoto Encyclopedia of Genes and Genomes pathway terms, with oxidative phosphorylation (OXPHOS) being the most significant. (B) Heatmap of the relative abundance of OXPHOS proteins ndufb6, ndufa10, ndufs3, cox4i1, cox5aa, atp5pb, atp6v1e1b, atp6v1ab, atp5fa1, and atp5f1b in atrogin-1+/+ and atrogin-1–/– larvae. (C) Representative Western blot images for VDAC1, 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. (D) Quantification of VDAC1 levels normalized to total protein, with atrogin-1–/p4 larvae displaying a significant reduction compared with atrogin-1+/+ larvae, as determined using an unpaired t test. Data are shown as mean ± SD. (E–H) Live images of 6 dpf methyl cellulose–treated atrogin-1+/+ (E and F) and atrogin-1–/– (G and H) larvae showing mosaic expression of actc1b:mitoGFP labeling the mitochondria in green. While atrogin-1+/+ larvae display small mitochondria some of which form an intricate network, mitochondria in atrogin-1–/– larvae are large and rounded. Scale bar: 100 μm (left); 20 μm (right). F and H are zoomed in views of E and G, respectively. (I) The proportion of muscle fibers displaying altered mitochondrial morphology in methyl cellulose–treated atrogin-1+/+ or atrogin-1–/– larvae, as per Fisher’s exact test. ****P < 0.0001. (J–M) Electron micrographs of the muscle in 6 dpf methyl cellulose–treated atrogin-1+/+ and atrogin-1–/– larvae. While atrogin-1+/+ larvae display normal sarcomeric and mitochondrial structure (J and K), atrogin-1–/– mutants (L and M) display fiber disintegration, evident by the disorganized arrangement of sarcomeres (arrow), and abnormal mitochondria with large and swollen matrices (arrowheads). K and M are zoomed in views of J and L, respectively. (N and O) 3 dpf methyl cellulose–treated atrogin-1–/– larvae show a significant reduction in basal (N) and maximum respiration (O) compared with atrogin-1+/+ larvae, as determined using an unpaired t test. Data are shown as mean ± SD.*P < 0.05; ****P< 0.0001. All experiments performed in triplicate, with the total number of fish examined in each replicate being documented in Supplemental Table 2.