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Zebrafish genetic model of accelerated muscle atrophy associated with Fbxo32/Atrogin-1 expression

ABSTRACT: The degenerative loss of muscle associated with aging leading to muscular atrophy is called sarcopenia. Currently, practicing regular physical exercise is the only efficient way to delay the onset of sarcopenia. To identify therapeutic targets to suppress the symptoms of aging, there is a need for in vivo model organisms of accelerated muscle degeneration and atrophy. The zebrafish harbors hallmarks of aging, notably mitochondrial dysfunction, telomeres shortening and accumulation of senescent cells. Unfortunately, zebrafish aged slowly, and no specific zebrafish models of accelerated muscle atrophy associated with aging or sarcopenia are currently available. We have developed a new genetic tool to efficiently accelerate muscle fibers degeneration and muscle tissue atrophy in zebrafish larvae and adults. We used a gain-of-function strategy with a molecule that has been shown to be necessary and sufficient to induce muscle atrophy and a sarcopenia phenotype in mammals: Atrogin1 (also named Fbxo32). We report the generation, validation, and characterization of a zebrafish genetic model of accelerated muscle atrophy, the sarcofish. We demonstrated that specific expression of Atrogin1 in skeletal muscle tissue induces a muscle atrophic phenotype associated with locomotion dysfunctions in both larvae and adult fish. We identified that degradation of the myosin light chain as an event occurring before the degeneration of muscle fibers. Biological processes associated with muscle aging such as inflammation, proteolysis, apoptosis and stress response are upregulated in the sarcofish. Surprisingly, we observed a strong correlation between muscle fibers degeneration and a reduction of neuromuscular junctions in the peripheral nervous system, as well as neuronal cell bodies in the spinal cord, suggesting that muscle atrophy could underly a neurodegenerative phenotype in the central nervous system. Finally, while sarcofish larvae harbor regeneration of muscle fibers, spinal neurons and recover locomotive functions, adult sarcofish have impaired regenerative capacities as observed in mammals during muscle aging. In the future, the sarcofish could open new strategies in the fight against the sarcopenia disease to test therapeutic molecules aiming to treat or alleviate the symptoms of muscle aging.

ORGANISM(S): Danio rerio

PROVIDER: GSE274353 | GEO | 2024/08/09

REPOSITORIES: GEO

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Project description:Mitochondrial dysfunction caused by mitochondrial (mtDNA) deletions have been associated with skeletal muscle atrophy and myofiber loss. However, whether such defects occurring in myofibers cause sarcopenia is unclear. Also, the contribution of mtDNA alterations in muscle stem cells (MuSCs) to sarcopenia remain to be investigated. We expressed a dominant-negative variant of the mitochondrial helicase, which induces mtDNA alterations, specifically in differentiated myofibers (K320Eskm mice) and MuSCs (K320Emsc mice), respectively, and investigated their impact on muscle structure and function by immunohistochemistry, analysis of mtDNA and respiratory chain content, muscle transcriptome and functional tests. K320Eskm mice at 24 months of age had higher levels of mtDNA deletions compared to controls in soleus (SOL, 0.07673 % vs 0.00015 %, p=0.0167), extensor digitorium longi (EDL, 0.0649 vs 0.000925, p= 0.0015) and gastrocnemius (GAS, 0.09353 vs 0.000425, p=0.0004). K320Eskm mice revealed a progressive increase in the proportion of cytochrome c oxidase deficient fibers (COX-) in cross sections, reaching a maximum of 3.03%, 4.36%, 13.58% and 17.08% in EDL, SOL, tibialis anterior (TA) and GAS, respectively. However, mice did not show accelerated loss of muscle mass, muscle strength or physical performance. Histological analyses revealed ragged red fibers but also stimulated regeneration, indicating activation of MuSCs. RNAseq demonstrated enhanced expression of genes associated with protein synthesis, but also degradation, as well as muscle fiber differentiation and cell proliferation. In contrast, 7 days after destruction by cardiotoxin, regenerating TA of K320Emsc mice showed 30% of COX- fibers. Notably, regenerated muscle showed dystrophic changes, increased fibrosis (2.5% vs 1.6%, p=0.0003), increased abundance of fat cells (2.76% vs 0.23%, p=0.0144) and reduced muscle mass (regenerated TA: 40.0mg vs 60.2mg, p=0.0171). In contrast to muscles from K320Eskm mice, freshly isolated MuSCs from aged K320Emsc mice were completely devoid of mtDNA alterations. However, after passaging, mtDNA copy number as well as respiratory chain subunits and p62 levels gradually decreased.Taken together, accumulation of large-scale mtDNA alterations in myofibers alone is not sufficient to cause sarcopenia. Expression of K320E is tolerated in quiescent MuSCs, but progressively leads to mtDNA and respiratory chain depletion upon activation, in vivo and in vitro, possibly caused by an increased mitochondrial removal. Altogether, our results suggest that the accumulation of mtDNA alterations in myofibers activates regeneration during aging, which leads to sarcopenia if such alterations have expanded in MuSCs as well.

Project description:Background: Sarcopenia is a common and progressive skeletal muscle disorder characterized by atrophic muscle fibers and contractile dysfunction. Accumulating evidence shows that the number and function of satellite cells (SCs) decline and become impaired during aging, which may contribute to impaired regenerative capacity. A series of myokines/ small extracellular vesicles (sEVs) released from muscle fibers regulate metabolism in muscle and extramuscular tissues in an autocrine/paracrine/endocrine manner during muscle atrophy. It is still unclear whether myokines/sEVs derived from muscle fibers can affect satellite cell function during aging. Methods: Aged mice were used to investigate changes in the myogenic capacity of SCs during aging-induced muscle atrophy. The effects of atrophic myotube-derived sEVs on satellite cell differentiation were investigated by biochemical methods and immunofluorescence staining. Small RNA sequencing was performed to identify differentially expressed sEV microRNAs (miRNAs) between the control myotubes and atrophic myotubes. The target genes of the miRNA were predicted by bioinformatics analysis and verified by luciferase activity assays. The effects of identified miRNA on the myogenic capacity of SCs in vivo were investigated by intramuscular injection of adeno-associated virus (AAV) to overexpress or silence miRNA in skeletal muscle. Results: Our study showed that the myogenic capacity of SCs was significantly decreased (50%, n = 6, P < 0.001) in the tibialis anterior muscle of aged mice. We showed that atrophic myotube-derived sEVs inhibited satellite cell differentiation in vitro (n = 3, P < 0.001) and in vivo (35%, n = 6, P < 0.05). We also found that miR-690 was the most highly enriched miRNA among all the screened sEV miRNAs in atrophic myotubes [Log2 (Fold Change) = 7, P<0.001], which was verified in the atrophic muscle of aged mice (3-fold, n = 6, P < 0.001) and aged humans (2.8-fold, n = 10, P < 0.001). miR-690 can inhibit myogenic capacity of SCs by targeting myocyte enhancer factor 2, including Mef2a, Mef2c, and Mef2d, in vitro (n=3, P < 0.05) and in vivo (n=6, P < 0.05). Specific silencing of miR-690 in the muscle can promote satellite cell differentiation (n = 6, P < 0.001) and alleviate muscle atrophy in aged mice (n = 6, P < 0.001). Conclusions: Our study demonstrated that atrophic muscle fiber-derived sEV miR-690 may inhibit satellite cell differentiation by targeting myocyte enhancer factor 2 during aging.

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Zebrafish genetic model of accelerated muscle atrophy associated with Fbxo32/Atrogin-1 expression

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