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 1

Atrogin-1 deficiency results in contraction-dependent muscle fiber detachment.

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Atrogin-1 deficiency results in contraction-dependent muscle fiber detac...
Schematic of wild-type atrogin-1 (atrogin-1+/+) and mutant atrogin-1 (atrogin-1–/–) protein structure and mRNA sequence, with the mutant predicted to incorporate a premature stop in exon 1. The mutant was generated using CRISPR/Cas9 genome editing, which resulted in a 34 bp insertion (red). Numbers in the protein box are amino acids, and numbers in the mRNA box are base pairs. (B) qRT-PCR analysis showing significant reduction in atrogin-1 levels in atrogin-1–/– mutants compared with atrogin-1+/+ wild-type larvae. Error bars represent mean ± SEM for 3 replicate experiments, with each experiment comprising a pooled sample of at least 5 fish. *P < 0.05 determined using a 1-way ANOVA with Tukey’s multiple correction post hoc test. Muscle fibers span the entire length of the somite in the 3 dpf atrogin-1+/+ (C), atrogin-1 heterozygous (atrogin-1+/–) (D), and atrogin-1–/– mutant (E) larvae, as seen by F-Actin labeling. (F) Quantification of the muscle phenotype, with atrogin-1+/+, atrogin-1+/–, and atrogin-1–/– displaying indistinguishable muscle structure, as determined using a χ2 test. Incubation of 3 dpf atrogin-1+/– (H) and atrogin-1–/– (I) in methyl cellulose results in muscle fiber detachment, which is not evident in atrogin-1+/+ larvae (G). (J) Percentage of affected atrogin-1+/+, atrogin-1+/–, and atrogin-1–/– larvae, with the latter 2 genotypes having a significant increase in the proportion of fish displaying the muscle fiber detachment, as determined using a χ2 test. At 6 dpf, atrogin-1+/– (L) and atrogin-1–/– (M) display sporadic muscle fiber detachment but not in atrogin-1+/+ larvae (K). (N) Percentage of affected atrogin-1+/+, atrogin-1+/–, and atrogin-1–/– larvae, with the latter 2 genotypes having a significant increase in the proportion of fish displaying the muscle fiber detachment, as determined using a χ2 test. Methyl cellulose incubation of 6 dpf atrogin-1+/– (P) and atrogin-1–/– (Q) results in muscle fiber detachment, which is not evident in atrogin-1+/+ larvae (O). (R) Percentage of affected atrogin-1+/+, atrogin-1+/–, and atrogin-1–/– larvae, with the latter 2 genotypes having a significant increase in the proportion of fish displaying the muscle fiber detachment, as determined using a χ2 test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. All experiments were performed in triplicate, with the total number of fish examined in each replicate documented in Supplemental Table 2.