USP18 is an essential regulator of muscle cell differentiation and maturation

Skeletal muscle degeneration involves atrophy, fibrosis, fat infiltration, and impaired regeneration, processes tightly controlled by the ubiquitin–proteasome system (UPS). While muscle E3 ligases (e.g., Atrogin-1/MAFbx, MuRF1) have established roles, the roles of deubiquitinating enzymes (DUBs) in myogenesis remain poorly defined despite over 100 DUBs in the human genome. Prior work implicated specific DUBs (USP7, USP10, USP19, USP1) in atrophy and regulatory factor turnover during myogenesis. USP18 is a cysteine protease best known for deISGylation of ISG15-modified proteins and negative regulation of type I interferon (IFN-I) signaling, with deficiencies causing interferonopathies and links to tumorigenesis. The study’s objective was to systematically identify DUBs regulating human muscle cell differentiation and to define the role and mechanism of USP18 in myogenic differentiation and maturation. The central hypothesis emerging from the screen is that USP18 is a critical regulator of myogenic differentiation acting independently of canonical IFN/ISG pathways.

- UPS components regulate muscle homeostasis; Atrogin-1 (MAFbx) and MuRF1 promote proteasomal degradation and are central to atrophy. - DUBs can reverse ubiquitination, serving as regulatory checkpoints. Altered DUB activity and ubiquitin accumulation are observed in sarcopenia. - Specific DUBs implicated in muscle: USP19 (inhibits myogenesis via UPR signaling), USP1 (in starvation signaling), USP7 and USP10 (stabilize myogenic regulators), SENP2 (SUMO protease) affects myogenesis. - Myogenesis requires timely turnover of myogenic regulators (MRFs/MEFs) to progress from myoblasts to myotubes. - USP18 biology: removes ISG15 from substrates (deISGylation), can inhibit IFN-I signaling via IFNAR2/STAT2 interactions, induced by cytokines (IFN-I/III, TNF-α, LPS). USP18 deficiency causes interferonopathies in mice and humans; also involved in tumorigenesis via effects on proliferation and signaling. Its role in muscle biology had been obscure prior to this study.

- DUB siRNA screen: Immortalized human skeletal muscle cells were transfected with an ON-TARGETplus siRNA library targeting DUBs (10 nM; Lipofectamine RNAiMAX) for 48 h at ~70% confluence, then induced to differentiate by serum starvation (DMEM + 2% horse serum) for 72 h. Differentiation was assessed by Myosin heavy chain (MyHC) immunostaining and calculating the fusion index via high-throughput imaging. - Follow-up differentiation protocol: To avoid exogenous cytokine effects from horse serum, most subsequent assays used serum- and antibiotic-free DMEM (DMEM-/-). Cells were seeded at ~50% confluence before transfection to minimize spontaneous differentiation, transfected with USP18 siRNAs (primarily siRNA-C; 10 nM), incubated 48 h in transfection medium, then switched to proliferation or differentiation media. Differentiation was quantified at 24 h and 72 h (MyHC staining, fusion index) and cell counts recorded. - Detachment/cell death assessment: After 72 h differentiation, proteins from attached cells and culture supernatants were collected separately and analyzed by Western blot for MyHC and GAPDH to assess detachment versus cell death. - Proliferation assays: Real-time confluency monitoring (IncuCyte Zoom) in proliferation medium following USP18 knockdown. Brightfield imaging to visualize spontaneous myotube formation under proliferation conditions. Western blots for MyHC; RT-qPCR to confirm USP18 KD where USP18 protein was below detection. - Primary/progenitor validation: Human muscle induced pluripotent stem cell-derived progenitors were subjected to USP18 KD and assessed for differentiation (24 h and 72 h) in proliferation medium (MyHC IF, fusion index, nuclei counts). - ISG15 dependence testing: siRNA KD of ISG15 alone and combined with USP18 KD; validation by IFN-β treatment (1,000 U/mL, 24 h) and Western blot for ISG15 conjugates. Differentiation quantified by MyHC IF and fusion index to test ISG15 dependence. - IFN/ISG pathway activation during differentiation: RNA-seq of control cells undergoing differentiation (proliferation vs 24 h differentiation in DMEM-/-) to evaluate ISG induction; analysis for IFN-α/β, ISG15, and broader ISG signatures. - Transcriptomics under USP18 KD: RNA-seq (N=3) of USP18 KD vs control in (i) proliferation medium (24 h after medium change), and (ii) differentiation medium (24 h). DESeq2 used for differential expression (adj. p<0.05); focus on FC>|2| sets and hierarchical clustering of top-affected genes (N=509, FC>4). Enrichment analyses (DAVID/STRING) identified networks (cell cycle, ECM, GPCR, Ca2+ channels, muscle contraction). Nuclear-enriched transcripts were specifically analyzed to assess transcriptional regulators (MRFs/MEFs) perturbed by USP18 KD. - Subcellular localization of USP18: Cellular fractionation followed by Western blot to distinguish cytosolic vs nuclear isoforms (36 kDa cytosol, 34 kDa nuclear) and immunofluorescence to localize USP18 in multinucleated myofibers. - Proteomics: Label-free LC-MS/MS (Orbitrap Fusion Lumos) on differentiated cultures with and without USP18 KD. Statistical thresholds set at FDR 0.05 (stringent) and 0.25 (lenient) to account for heterogeneity. STRING/Cytoscape PPI networks and hierarchical clustering identified USP18-dependent vs -independent protein clusters; comparison to RNA changes assessed. - Calcium flux: Fluo-4 live-cell imaging quantified KCl (10–20 mM)–induced Ca2+ transients in unfused and multinucleated cells under conditions: siControl (Diff.), siUSP18 (Prolif.), siUSP18 (Diff.). Time courses recorded pre- and post-stimulation and analyzed with CALIMA. - 3D engineered muscle bundles: hiPSC-derived muscle progenitors embedded in Matrigel/fibrin hydrogel, with siUSP18 or siControl complexes incorporated during casting. After 48 h in proliferation medium, switched to differentiation for 7 days with 6-ACA. Contractile function measured (twitch 1 Hz, tetanic 20 Hz) by pillar deflection; specific force normalized by cross-sectional area. USP18 KD verified by RT-qPCR. Structural integrity assessed by titin immunostaining. - Standard molecular techniques: Western blots (RIPA lysis; Odyssey CLx imaging), RT-qPCR (SYBR Green; TBP housekeeping), RNA-seq pipeline (BIOWDL v5.0.0; STAR/HTSeq-count; Ensembl v104), proteomics data processing (MaxQuant/Perseus), and statistical analyses (Student’s t-test, Benjamini–Hochberg FDR).

- DUB siRNA screen identified multiple DUBs affecting myogenic fusion; USP18 KD reduced fusion index at 72 h (validated by three distinct siRNAs), indicating a role in differentiation. - USP18 expression increased during differentiation and preceded MyHC expression; despite cytokine-free conditions (DMEM-/-), USP18 rose while other ISGs did not, suggesting non-canonical regulation. - USP18 KD accelerated early differentiation: at 24 h, MyHC+ cells appeared and fusion index increased versus controls even with fewer nuclei; by 72 h, fusion index decreased due to enhanced detachment, not cell death (≈90% of MyHC in supernatant vs ≈20% in controls). - Proliferation was halted by USP18 KD; spontaneous myotube formation and MyHC expression occurred under proliferation conditions in both immortalized cells and human muscle progenitors. - USP18-mediated differentiation is independent of ISG15 and IFN-I signaling: ISG15 conjugates remained low/unchanged during differentiation; ISG15 KD alone had no effect on differentiation and did not rescue or block USP18 KD-driven differentiation; RNA-seq showed USP18 upregulation with no upregulation of other ISGs (ISG15 unchanged or down) and no detectable IFN-α/β transcripts. - Transcriptomics (USP18 KD vs control, 24 h): Massive changes (1,445 genes with FC>|2|, p<0.05 FDR), with strong downregulation of cell-cycle genes and upregulation of myogenic genes. Clustering across conditions (N=509, FC>4) revealed: Cluster IV (276 genes) downregulated cell-cycle network (FDR ≈ 3×10^-68); Cluster I/III enriched for ECM, GPCR, and Ca2+ channel genes; muscle contraction genes enriched but downregulated in USP18 KD (Cluster II), indicating impaired maturation. - Nuclear programs affected: Among dysregulated nuclear-encoded genes (N=235, FC>2), 36 muscle-specific transcription regulators were enriched. Key MRF/MEF genes were perturbed: MYOG up; MYF5/MYF6 down in expected patterns, but MYOD1 and MEF2A showed opposite expression trends versus controls. - USP18 localization: A truncated 34 kDa USP18 isoform accumulated in nuclei of differentiated cells, while the 36 kDa isoform was cytosolic. - Proteomics: Despite transcriptome-wide effects, the USP18-dependent proteome shift was modest (FDR 0.05: 6 proteins; FDR 0.25: 63 proteins; ≈0.18% and 1.88% of quantified proteins, respectively). USP18 KD-dependent clusters showed increases in muscle proteins (e.g., MyHC, TNNI1, TNNT3, KLHL31) with parallel decreases in DNA replication proteins; however, a subset of mitochondrial and endosomal proteins (Cluster D) rose in controls but not in USP18 KD, and some sarcomere/contractile gene expression was reduced at RNA level in USP18 KD. - Calcium handling: KCl-induced Ca2+ flux was robust in control myotubes; USP18 KD myotubes formed in proliferation medium displayed strong Ca2+ transients, whereas those formed in differentiation medium failed to mount Ca2+ flux, indicating functional immaturity/defect dependent on context. - Engineered 3D muscle bundles: USP18 KD reduced both twitch and tetanic specific forces compared to controls; titin staining showed disrupted myofiber continuity and weak striation, evidencing sarcomeric defects. RT-qPCR confirmed USP18 KD in bundles. - Overall, USP18 acts as a gatekeeper preventing premature differentiation (via maintaining cell-cycle gene expression and restraining myogenic transcription programs) and later supports expression of sarcomeric genes and functional maturation/maintenance, independent of ISG15/ISG signaling.

The study demonstrates a previously unrecognized, ISG-independent role for USP18 in human myogenesis. USP18 expression increases during differentiation without activation of IFN-I or broader ISG programs, and USP18 depletion precipitates a switch from proliferation to differentiation even under growth conditions, accompanied by a profound transcriptomic reprogramming. Early events include suppression of cell-cycle networks and activation of myogenic and ECM/GPCR/Ca2+ channel gene sets, implicating USP18 as a safeguard against premature differentiation. The nuclear accumulation of a truncated USP18 isoform in differentiating cells suggests potential nuclear functions that influence transcriptional regulators (MRFs/MEFs), consistent with the observed dysregulation of MYOD1 and MEF2A and the enrichment of muscle-specific transcription regulators upon USP18 loss. At later stages, USP18 depletion compromises sarcomeric gene expression and function, leading to defective calcium handling and reduced contractile force, with structural disorganization of titin and myofibers in 3D muscle bundles. The limited changes in the proteome compared to widespread transcriptomic shifts indicate USP18 mainly influences gene expression programs rather than global protein stability in this context. Collectively, USP18 operates at two critical junctures: inhibiting entry into differentiation during proliferation and enabling sarcomeric maturation/maintenance during later differentiation, independent of ISG15/deISGylation. These insights expand USP18 biology beyond immune regulation, with implications for muscle regeneration and inflammatory myopathies such as dermatomyositis where USP18 is elevated.

This work identifies USP18 as an essential regulator of skeletal muscle cell differentiation and maturation, functioning independently of ISG15 and canonical IFN-I signaling. USP18 restrains premature differentiation by sustaining cell-cycle programs and limiting myogenic transcriptional activation, then supports sarcomeric gene expression and functional maturation to maintain myotube integrity. The discovery of a nuclear USP18 isoform during differentiation and the strong transcriptomic, but modest proteomic, changes highlight a principal role in regulating nuclear gene programs. Future studies should define the upstream cues inducing USP18 during myogenesis, delineate USP18’s nuclear partners and direct effects on transcriptional complexes, and employ selective USP18 inhibitors or catalytically inactive mutants to dissect catalytic versus scaffolding roles. Engineered muscle models incorporating immune components may further elucidate USP18’s role at the intersection of inflammation and regeneration and inform therapeutic strategies to modulate myogenesis.

USP18 depletion halted proliferation and induced rapid differentiation, preventing generation of stable USP18 knockout lines or introduction of catalytically inactive mutants in human muscle cells. A lack of selective USP18 inhibitors precluded direct separation of catalytic (deISGylating) versus non-catalytic functions in this system. Although knockdown and multi-omics analyses support an ISG15-independent mechanism, definitive pharmacological dissection will require specific USP18 inhibitors to validate therapeutic potential and mechanistic pathways.