Muscle degeneration, characterized by atrophy, fat infiltration, fibrosis, and impaired regeneration, underlies numerous human pathologies and significantly reduces mobility. The ubiquitin proteasome system (UPS) plays a pivotal role in regulating these processes. Muscle-specific E3 ligases, such as Atrogin-1 and MuRF1, ubiquitinate proteins, marking them for degradation. Deubiquitinating enzymes (DUBs), conversely, remove ubiquitin molecules, acting as regulatory checkpoints in the UPS. Studies have linked sarcopenic muscles to ubiquitin accumulation and altered DUB expression and activity. Several DUBs, including USP19 and USP1, have been implicated in muscle atrophy and myogenesis. Myogenesis, the formation of new muscle fibers, depends on the timely turnover of muscle-specific regulatory factors, and DUBs like USP7 and USP10 have been shown to regulate myogenic factors. Despite the significant impact of muscle degeneration, the roles of the over 100 known DUBs in muscle biology remain largely unexplored. USP18, a cysteine protease DUB, is known for removing ISG15 from protein substrates (deISGylation) and regulating the ISG response. ISG15 modification can affect protein stability and localization, influencing processes like protein translation, DNA repair, innate immunity, and antiviral responses. USP18 can also inhibit type 1 interferon signaling independently of its enzymatic activity. While USP18 deficiency causes interferonopathies, its role in muscle pathologies is unclear. This study unexpectedly uncovered a crucial role for USP18 in muscle differentiation independent of the ISG response.

The existing literature highlights the importance of the ubiquitin proteasome system (UPS) in muscle physiology and pathology. Studies have shown the involvement of specific E3 ubiquitin ligases (Atrogin-1 and MuRF1) in muscle atrophy by ubiquitinating proteins and targeting them for degradation. Conversely, deubiquitinating enzymes (DUBs) counteract this process by removing ubiquitin molecules. Sarcopenia is associated with ubiquitin accumulation and altered DUB expression and activity. Some DUBs, such as USP19 and USP1, have been linked to muscle atrophy and myogenesis. The myogenic process relies on the precise regulation of muscle-specific factors, with DUBs like USP7 and USP10 influencing this regulation. However, the extensive roles of DUBs, particularly the over 100 identified DUBs, in muscle biology remain largely uncharted. USP18, a well-studied DUB in immune responses, removes ISG15 from proteins and regulates the ISG response. While USP18 deficiency leads to interferonopathies, its connection to muscle diseases is unknown. This study addresses this gap in knowledge by exploring USP18's role in muscle differentiation, specifically whether it operates independently of the ISG response.

The study employed a multifaceted approach, integrating genetic screening, RNA sequencing, proteomics, and functional assays. Initially, a DUB siRNA library was transfected into immortalized human skeletal muscle cells. After differentiation induction, high-throughput imaging quantified the fusion index (percentage of nuclei in fused cells). Knockdown of USP18 significantly reduced the fusion index. Subsequent experiments used serum-free medium to eliminate potential confounding factors from serum. To investigate the relationship between USP18 and cell proliferation, USP18 knockdown was performed both in proliferating and differentiating conditions. The effects of USP18 knockdown on cell proliferation were assessed by cell counting and confluency measurements. To determine whether USP18's role in differentiation is ISG15-dependent, ISG15 knockdown was performed alone and in combination with USP18 knockdown. RNA sequencing was conducted to analyze transcriptome-wide changes in USP18-depleted cells under both proliferating and differentiating conditions. Label-free liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to analyze the proteome of differentiated cells following USP18 knockdown. Finally, functional assays assessed calcium flux in multinucleated cells and contractile force in engineered 3D muscle bundles.

The study's key findings include: 1. **USP18 knockdown accelerates muscle cell differentiation initially:** USP18 depletion led to an earlier onset of differentiation, indicated by increased MyHC expression at 24 hours post-starvation. However, this was followed by reduced fusion index at 72 hours due to enhanced cell detachment. 2. **USP18's effect on differentiation is independent of its role in ISG15 deconjugation:** Knockdown of ISG15 did not affect differentiation, nor did it prevent USP18-knockdown-mediated differentiation. This suggests that USP18's impact on myogenesis is independent of its known role in regulating the ISG response. Furthermore, other ISGs were not upregulated during differentiation. 3. **USP18 depletion causes widespread transcriptomic changes:** RNA sequencing revealed significant changes in gene expression following USP18 depletion. Cell cycle genes were downregulated, while myogenic genes were upregulated, consistent with the observed shift towards differentiation. 4. **USP18 depletion impacts the myogenic differentiation program:** USP18 depletion altered the expression of various myogenic regulatory factors. While some genes followed the expected pattern during differentiation, others showed opposite expression patterns to the control cells. This suggests that USP18's regulatory role on the myogenic program involves not only the expression of muscle structural proteins but also that of the corresponding transcription factors. 5. **USP18 depletion has a minor effect on the proteome but significant impact on myofiber integrity:** Proteomic analysis revealed only minor changes in protein abundance after USP18 knockdown in differentiated cells. However, muscle structural proteins crucial for sarcomere integrity were downregulated. This correlated with reduced contractile force in engineered muscle bundles, highlighting USP18's importance in myofiber maintenance. 6. **Nuclear localization of USP18 isoform in differentiating muscle cells:** A truncated isoform of USP18 was found to be predominantly localized in the nucleus of differentiating cells. 7. **USP18 depletion affects calcium flux and contractile force:** Calcium flux measurements showed reduced calcium flux in USP18-depleted multinucleated cells formed under differentiating conditions. In 3D muscle bundles, USP18 depletion resulted in significantly reduced twitch and tetanic contractile forces, alongside disrupted sarcomere integrity.

This study unveils a novel, non-canonical role for USP18 in muscle cell differentiation and maturation, independent of its well-established function in ISG15 deconjugation and regulation of the ISG response. The observed increase in USP18 expression during differentiation contrasts with the lack of significant changes in other ISGs, underscoring USP18's distinct function in myogenesis. The profound transcriptomic changes following USP18 depletion, specifically the downregulation of cell cycle genes and upregulation of myogenic genes, suggest that USP18 acts as a gatekeeper, preventing premature entry into differentiation. The subsequent impact on sarcomere protein expression and contractile force highlights USP18's role in maintaining the structural and functional integrity of mature myofibers. Future studies focusing on the mechanism of USP18’s induction and its interaction with myogenic regulatory factors are necessary to completely elucidate the molecular underpinnings of this novel role. This finding opens new avenues for therapeutic interventions aimed at accelerating muscle regeneration and addressing muscle pathologies.

This research demonstrates a previously unknown role for USP18 in myogenesis that is independent of its ISG15 deconjugation activity. USP18 depletion triggers a switch from proliferation to differentiation, regardless of serum starvation. USP18 also maintains myofiber integrity and functionality. Future research should investigate the precise molecular mechanisms underlying USP18 induction and regulation of muscle cell differentiation.

The study's limitations include the use of immortalized muscle cells, which may not fully represent the complexity of primary muscle cells. The inability to generate USP18 knockout or catalytically dead mutant cells restricted the mechanistic studies. Future research using primary muscle cells and the development of a selective USP18 inhibitor would help to further investigate the role of USP18 in myogenesis. Also, the small number of altered proteins in response to USP18 knockdown may underrepresent the impact of USP18 on the proteome.