The MuSK-BMP pathway maintains myofiber size in slow muscle through regulation of Akt- mTOR signaling

Myofiber size regulation is critical in health, disease, and aging. MuSK (muscle-specific kinase) is a BMP (bone morphogenetic protein) co-receptor that promotes and shapes BMP signaling. MuSK is expressed at all neuromuscular junctions and is also present extrasynaptically in the slow soleus muscle. To investigate the role of the MuSK-BMP pathway in vivo we generated mice lacking the BMP-binding MuSK Ig3 domain. These ΔIg3-MuSKmice are viable and fertile with innervation levels comparable to wild type. In 3-month-old mice myofibers are smaller in the slow soleus, but not in the fast tibialis anterior (TA). Transcriptomic analysis revealed soleus-selective decreases in RNA metabolism and protein synthesis pathways as well as dysregulation of IGF1-Akt-mTOR pathway components. Biochemical analysis showed that Akt-mTOR signaling is reduced in soleus but not TA. We propose that the MuSK-BMP pathway acts extrasynaptically to maintain myofiber size in slow muscle by promoting protein synthetic pathways including IGF1-Akt-mTOR signaling. These results reveal a novel mechanism for regulating myofiber size in slow muscle and introduce the MuSK-BMP pathway as a target for promoting muscle growth and combatting atrophy.


Introduction
Maintaining myo ber size is essential for proper muscle function. Muscle atrophy characterizes aging, disuse, cancer cachexia and disease [1][2][3] . Notably, individual muscles and myo ber types are differentially affected in many of these settings 4 . For example, in sarcopenia upper leg muscles atrophy while the soleus muscle in the lower leg is spared 5 . In Duchenne muscular dystrophy limb-girdle muscles are affected in the rst years of life while upper limb muscles are spared until several years later 6 . In contrast, muscle weakness preferentially affects muscles in the anterior compartments of the face and leg in FSHD 4 . However, the molecular basis for such muscle-selective vulnerability to atrophy is largely unknown.
Members of the TGF-β (transforming growth factor-beta) superfamily, including myostatin and BMPs, are potent regulators of muscle size. Myostatin is a negative regulator of muscle mass, and its genetic deletion or pharmacological inhibition results in muscle hypertrophy 7 . In contrast, BMP signaling promotes muscle growth. Overexpression of BMP7 or a constitutively active BMP receptor BMPR1a (ALK3) in skeletal muscle results in increased muscle mass, ber size, and elevated canonical BMP and Akt/mTOR signaling 8 . Inhibiting BMP signaling by overexpressing the BMP sequestering protein noggin abolishes the hypertrophic phenotype observed in myostatin-de cient mice 9 . Either increasing BMP or reducing myostatin signaling can restore myo ber size in cancer cachexia 2,10 . These studies suggest that the BMP and myostatin/activin pathways antagonize each other and tipping the balance can result in either hypertrophy or atrophy. They also implicate BMP signaling as an attractive target for combatting atrophy. However, targeting BMPs therapeutically is challenging since unlike myostatin, BMPs are expressed ubiquitously and serve a wide range of functions throughout the body 11,12 . ( Fig. 1A, B). PCR ampli cation of the intronic regions of exon 5-6 and 5-8 borders yielded amplicons of the predicted size for the WT MuSK and MuSK ∆Ig3 alleles, respectively (Fig. 1C). MuSK ∆Ig3/∆Ig3 mice are viable and fertile with normal weights (Fig. 1D), grip strength (Fig. 1E), and innervation levels in both the fast sternomastoid (Fig. 1F) and the slow soleus (Fig. 1G). In this study we will term the MuSK ∆Ig3/∆Ig3 animals '∆Ig3-MuSK'.
BMP4 signaling is perturbed in ∆Ig3-MuSK cells We rst generated stable ΔIg3-MuSK myogenic cell lines to probe MuSK expression and BMP signaling in cells lacking the MuSK Ig3 domain (see Methods). Immunostaining of unpermeabilized myoblasts with a monoclonal antibody directed against the MuSK Ig2 domain showed that MuSK is expressed at the cell surface and at a comparable level and distribution in both WT and ∆Ig3-MuSK myoblasts ( Fig. 2A).
MuSK transcript expression is also comparable in WT and ∆Ig3-MuSK cells (Fig. 2B). To probe the role of the MuSK Ig3 domain in the BMP4 response, we serum-starved WT and ∆Ig3-MuSK myoblasts and examined the nuclear localization of pSmad1/5 in the absence or presence of added BMP4. As expected, nuclear pSmad1/5 levels were low at baseline in both genotypes. However, following BMP4 stimulation the level of nuclear pSmad1/5 was higher in WT compared to ΔIg3-MuSK cells (Fig. 2C). We then performed Western blots to quantify the pSmad1/5 response to BMP4 treatment (Fig. 2D). The ∆Ig3-MuSK cells showed a reduction in BMP4-stimulated pSmad1/5 levels compared to WT (Fig. 2D, E). We next assessed the role of the MuSK Ig3 domain in modulating the expression of MuSK-regulated BMP4induced transcripts Car3 and Wnt11 13 . Figure 2F shows that levels of BMP4-induced Car3 transcripts are reduced in ∆Ig3-MuSK myoblasts compared to WT. Similarly, BMP signaling is also perturbed in primary ∆Ig3-MuSK myotubes. As shown in Fig. 2G BMP4-induced Wnt11 transcript levels were reduced compared to WT myotubes. Finally, to establish that agrin signaling is preserved in ΔIg3-MuSK cells, we assessed agrin-induced AChR clustering in WT and ∆Ig3-MuSK primary myotubes. Figure 2H shows that the level of agrin-induced AChR clustering was comparable in both genotypes. This normal agrin response in cultured cells is in agreement with the comparable innervation levels observed in WT and ΔIg3-MuSK muscle (Fig. 1G, H). Taken together, these data show that in cultured cells the MuSK Ig3 domain regulates BMP4 pSmad1/5 and transcriptional responses and that agrin-and MuSK-BMPdependent signaling can be clearly distinguished.
Unique transcriptional pro les in ΔIg3-MuSK fast and slow muscles The results from cultured ∆Ig3-MuSK cells indicated that the MuSK Ig3 domain regulates BMP4 signaling and transcriptional response. We next explored the role of the MuSK-BMP pathway in vivo. The soleus muscle expresses several fold higher levels of MuSK transcript compared to the TA 13,16 , suggesting that MuSK may play a particularly important role in this muscle. We compared the transcriptional pro les in 3month-old WT and ∆Ig3-MuSK TA and soleus using RNA-seq. We isolated RNA from 36 muscles (n = 9 TA and soleus muscles per genotype) and sequenced at a depth of ~ 50 million reads/sample. The results revealed striking differences in the transcriptional pro les between these muscles. Figures 3A and 3B show heatmaps of the transcriptomic pro le differences in the 1000 most variable genes in ∆Ig3-MuSK compared to WT in TA and soleus, respectively. Principal component analysis of gene expression pro les showed that both the ∆Ig3-MuSK TA and soleus muscles clustered separately from WT (Fig. 3C, D).
Analysis of differentially expressed genes (DEGs) revealed signi cant changes in both muscles (Tables  S1, S2). The TA showed 485 upregulated and 287 downregulated genes compared to WT (Fig. 3E), while the soleus exhibited 531 upregulated and 253 downregulated genes (Fig. 3F). Notably, although the absolute number of DEGs was similar in both muscles, the changes in soleus showed broader ranges of log2 fold change and log10 FDR values compared to TA (Fig. 3G, H), indicating that the magnitude and signi cance of the DEG changes were larger in the soleus compared to the TA. Finally, the identity of DEGs in TA and soleus were strikingly different: only 19/799 of the combined downregulated genes and 34/704 of the combined upregulated genes were shared between TA and soleus (Fig. 3l). Taken together, these results show that the transcriptional pro les of TA and soleus WT and ∆Ig3-MuSK muscles are qualitatively and quantitatively distinct, with the soleus transcriptome being more affected by the downregulation of MuSK-BMP signaling.
To gain insight into cellular mechanisms impacted in ∆Ig3-MuSK muscles, we performed gene set enrichment analysis (GSEA) using GO terms for both TA and soleus. The speci c pathways and the extent of change within pathways differed markedly in TA and SOL ( Fig. 4A and B). Multiple upregulated pathways in the TA were related to regulation of GTPase activity and Ras/Rho signal transduction ( Fig. 4A, Table S3). In contrast, in the soleus the major upregulated pathways were related to ECM (extracellular matrix) structure, ECM organization, and in ammation (Fig. 4B, Table S3). In addition, we found upregulated pathways related to ERK1/2 signaling regulation as well as collagen bril organization ( Fig. 4B). Pathways downregulated in the TA included those related to translation, peptide biosynthesis, protein folding and ribosome biogenesis (Fig. 4A). Additionally, there were multiple processes related to mitochondrial energy metabolism such as mitochondrial organization, respiratory transport chain, mitochondrial ATP synthesis, and proton transport (Fig. 4A). The soleus showed a striking downregulation of biological process pathways related to RNA metabolism including mRNA and ncRNA processing, RNA splicing, and ribonucleoprotein complex biogenesis and subunit organization (all adjusted p-values between 10 − 6 and 10 − 11 ). These changes suggest that the ∆Ig3-MuSK soleus exhibits dysfunctional post-transcriptional RNA metabolism and protein synthesis (Fig. 4B).
Our analysis also revealed a small number (< 5%) of shared GO pathways in TA and soleus, including downregulation of mitochondrion organization and ribosome biogenesis (Fig. 4C). Shared upregulated pathways included synaptic signaling, trans-synaptic signaling, chemical synaptic transmission and anterograde trans-synaptic signaling (Fig. 4C), suggesting that although both muscles were fully innervated (Fig. 1F, G), the MuSK-BMP pathway may also play a role at the neuromuscular junction. Some pathways showed opposite directionality, with chromatin organization and histone modi cation downregulated in soleus while upregulated in the TA (Fig. 4C). Taken together, these analyses suggest that the MuSK-Ig3 domain regulates distinct pathways in ∆Ig3-MuSK TA and soleus.
Myo ber size is reduced in ∆Ig3-MuSK soleus The muscle-selective reductions in RNA metabolism pathways and ribosome biogenesis raised the possibility that myo ber size is reduced in ΔIg3-MuSK soleus. The overall structure of both the TA and the soleus as revealed by H&E staining was similar in both genotypes (Fig. 5A, B), with no evidence of degeneration observed. We did detect transcriptomic structural signatures related to increased ECM and in ammatory pathways (Fig. 4). While WGA staining in the TA was comparable in both genotypes ( Fig. 5C), we observed a marked increase in WGA signal in ∆Ig3-MuSK soleus (Fig. 5D). The staining levels for dystrophin were comparable in both muscles across genotypes (Fig. 5E, F). Muscle ber Feret diameter analysis in TA revealed that the myo ber sizes were comparable in ∆Ig3-MuSK and WT (Fig. 5G). In contrast, myo ber sizes were reduced in the ∆Ig3-MuSK soleus (Fig. 5H). This myo ber atrophy was observed when all myo bers were scored (Fig. 5H) and when either type I (Fig. 5I) or type IIa Type I and Type IIa myo bers comprise approximately 80% of the myo bers in this muscle 21 . Thus, the large majority of myo ber types in the soleus are atrophied, while the TA myo bers are unaffected in ∆Ig3-MuSK mice at this age.

MuSK expression in WT and ∆Ig3-MuSK muscle
We next examined MuSK mRNA expression and protein localization in WT and ∆Ig3-MuSK muscles. We rst assessed the levels of MuSK transcripts, which in cultured muscle cells is regulated by the MuSK-BMP pathway 13 . We and others have previously reported that MuSK transcripts are expressed at four to ve-fold higher levels in the soleus compared to fast muscles such as EDL and TA 13,16 . We con rmed these ndings in WT EDL and soleus (Fig. 6A). MuSK transcript levels were also higher in soleus as compared to EDL in ∆Ig3-MuSK animals (Fig. 6B). MuSK expression in EDL was comparable in ∆Ig3-MuSK and WT (Fig. 6C). However, MuSK transcript levels in soleus were reduced by ~ 1/2 in ∆Ig3-MuSK compared to WT (Fig. 6D).
We next assessed the localization of MuSK protein. MuSK was localized at all NMJs examined in TA and soleus in both WT and ∆Ig3-MuSK muscle ( Fig. 6E-H). In agreement with previous reports 16 , in WT muscles MuSK is localized at extrajunctional domains in the soleus but not in TA (Fig. 6E,G). However, MuSK was not detected in extrajunctional domains in the ∆Ig3-MuSK soleus (Fig. 6H). Taken together, these data indicate that MuSK-BMP signaling is important for regulating the expression and localization of extrajunctional MuSK in the soleus.
Reduced Akt-mTOR signaling in ∆Ig3-MuSK soleus We next investigated potential signaling pathways that may mediate the selective atrophy of soleus bers in ∆Ig3-MuSK mice. The transcriptomic analysis (Fig. 4) showed that multiple RNA metabolism pathways were selectively downregulated in the ∆Ig3-MuSK soleus. These observations suggested that reduced protein synthesis could underlie the atrophy observed in the soleus. One candidate pathway is IGF1-Akt-mTOR, which plays a central role in regulating muscle growth 22,23 . Moreover, several members of this pathway are selectively dysregulated in the ∆Ig3-MuSK soleus including Igf1, Igf2bp2, Igfbp2, IRS1, Txnip and its antisense lncRNA Gm15441 (Supplemental Table 6). To directly assess the activity of the Akt-mTOR pathway in ∆Ig3-MuSK mice, we measured the phosphorylation of 4EBP1, a major downstream target of mTOR (Fig. 7). Translation is promoted by phosphorylated 4EBP1 (p4EBP1) and is inhibited by the unphosphorylated form (4EBP1). We probed Western blots of muscle extracts from soleus and TA muscles of each genotype with antibodies against 4EBP1 and p4EBP1 ( Fig. 7A and B,). Quanti cation showed that p4EBP1 levels were reduced by > 40% in the ∆Ig3-MuSK soleus (1 ± 0.15 and 0.55 ± 0.06, ± SEM in WT and ∆Ig3-MuSK, respectively; p = 0.02; n = 5 muscles/genotype) (Fig. 7C). In contrast, p4EBP1 levels were comparable in WT and ∆Ig3-MuSK TA (Fig. 7D). Notably, pSmad 1/5 levels did not differ in ∆Ig3-MuSK compared to WT for both the TA and soleus ( Fig. 7E and F). Taken together, these results indicate that the Akt-mTOR pathway is selectively reduced in ∆Ig3-MuSK soleus.

Discussion
In this report we introduce the MuSK-BMP pathway as a novel regulator of myo ber size in slow muscle. This pathway is selective for the slow soleus muscle as compared to the predominantly fast TA. In ∆Ig3-MuSK mice, the soleus is atrophied, RNA metabolic pathways are downregulated and Akt-mTOR signaling is reduced compared to TA. Our ndings indicate that the locus of MuSK-BMP action is extrasynaptic throughout the soleus, rather than secondary to changes at the NMJ. These results also reveal a novel in vivo function for MuSK that is independent of its role in agrin-LRP4 signaling at the synapse. The results shed light on the mechanism for the muscle-selective regulation of myo ber size and reveal a new pathway for promoting muscle growth and combatting atrophy.
Our results demonstrate that deletion of the MuSK Ig3 domain selectively perturbs MuSK function as a BMP co-receptor. First, MuSK protein lacking the Ig3 domain is localized at the cell surface in cultured ∆Ig3-MuSK myogenic cells and the levels of MuSK mRNA are comparable in cultured ∆Ig3-MuSK and WT cells (Fig. 2). ∆Ig3-MuSK is also localized at all NMJs examined in vivo ( Fig. 6; see further discussion below). This high-delity expression and localization are consistent with the fact that the ∆Ig3-MuSK allele created by gene editing mimics a natural MuSK splice isoform 20,24 . Second, cultured ∆Ig3-MuSK myogenic cells show reduced levels of pSmad 1/5 signaling and target gene expression in response to BMP treatment (Fig. 2). Notably, the MuSK-regulated BMP induced transcripts include Wnt11 and Car3, which were previously identi ed in a study using cultured MuSK −/− myogenic cells 13 . In contrast, the agrin-LRP4 mediated functions of MuSK, which require its Ig1 domain, are spared: agrin-induced AChR clustering is robust in cultured ∆Ig3-MuSK myotubes; in vivo, NMJ innervation levels and grip strength are comparable to WT in these 3 month-old mice (Fig. 1).
Our transcriptomic, morphological, and biochemical results show that the MuSK-BMP pathway plays a selective role in slow as compared to fast muscle. The MuSK-BMP pathway regulates myo ber size in slow muscle. In soleus, muscle atrophy was observed in both type I and IIa bers (Fig. 5), which together comprise ~ 80% of ber types in soleus. In contrast, no signi cant differences were observed in the diameter of TA myo bers, which are predominantly Type IIb, in the 3-month age animals examined here (Fig. 5). The sets of both the up-and down-regulated genes in soleus and TA were also remarkably distinct. Out of a total of 1503 DEGs, only 19 downregulated and 34 upregulated genes were shared between soleus and TA, respectively (Fig. 3). GO pathway analysis also revealed distinct functions for the MuSK-BMP pathway in soleus and TA ( Fig. 4; Table S3). As discussed below, a large number of downregulated GO pathways involved in RNA metabolism were unique to soleus. However, it is noteworthy that the two shared downregulated GO pathways were related to mitochondria organization and ribosome biogenesis, which raises the possibility that the MuSK-BMP pathway may regulate energy metabolism and some aspects of protein synthesis in both fast and slow muscle. Finally, we observed a number of pathways related to synaptic signaling and organization, raising the possibility that the MuSK-BMP pathway, while not essential for synapse formation, may play a role at the NMJ.
Several lines of evidence indicate that the MuSK-BMP pathway maintains soleus myo ber size through the regulation of the IGF1-Akt-mTOR pathway, the primary anabolic regulator of muscle cell size (Fig. 8) 22,[25][26][27][28][29][30] . This pathway increases protein synthesis through mTOR-mediated phosphorylation of key elements regulating translation, notably 4EBP1. Our transcriptomic analysis revealed a host of downregulated GO pathways in RNA metabolism as well as dysregulation of members of the IGF1-Akt-mTOR pathway that were selective for the soleus. Importantly, biochemical analysis showed that p4EBP1, a direct target of mTOR, is downregulated in ∆Ig3-MuSK soleus but not TA (Fig. 7). Notably, others have observed muscle-selective effects of the mTOR inhibitor rapamycin on regulating myo ber size 29 . We saw no evidence that this atrophy was due to denervation, since the NMJs in both muscles were fully innervated (Fig. 1). Further, our transcriptomic analysis detected few signatures of upregulated protein degradation, such as the atrogenes, which are markedly upregulated following denervation 31 (Table S4,   S5). Taken together, our results support a model where the MuSK-BMP pathway maintains muscle mass by regulating protein translation through modulation of the Akt-mTOR pathway (Fig. 8).
The striking selectivity of the MuSK-BMP pathway in the soleus as compared to the TA is likely to re ect the distinct expression, localization and regulation of MuSK in this muscle. MuSK transcript levels are ~ 4-5-fold higher in the WT soleus compared to the fast EDL (Fig. 6). MuSK is present at NMJs in all muscles 29,32 , including TA and soleus (Fig. 6). However, in WT soleus MuSK is also localized at extrasynaptic domains along the extent of the myo ber (Fig. 6), which is in agreement with earlier reports 13,16 . Further, snRNAseq analysis shows robust MuSK expression in soleus as compared to TA myonuclei 17 . Importantly, both MuSK transcript levels and the localization of MuSK at extrasynaptic domains are selectively reduced in ∆Ig3-MuSK soleus. This reduction seems likely to be the result of perturbed autoregulation since MuSK itself is a MuSK-BMP dependent transcript 13 . On the organismal level, our results suggest that MuSK expression in the sarcolemma may be one mechanism conferring muscle-selective regulation of myo ber size in health and disease.
Our results add a novel dimension to our understanding of the role of BMP signaling in regulating muscle size. Previous studies have shown that increasing BMP signaling by overexpression of BMP7 or constitutively active BMPR1a (ALK 3) causes hypertrophy. Notably, the hypertrophy is blocked by the mTOR inhibitor rapamycin, establishing a link between BMP signaling and Akt-mTOR-mediated muscle growth 8 . These results also align with our observation that this pathway is an important output of MuSK-BMP signaling. On the other hand, studies of denervation atrophy have demonstrated a prominent role for ubiquitin ligases and protein degradation in this model of acute loss of muscle mass. We did not observe notable changes in atrogenes in our RNA-seq analysis (Table S4, S5), further supporting the hypothesis that the MuSK-BMP pathway works predominantly via anabolic protein synthesis pathways.
The role of MuSK in maintaining muscle size has potential implications for myasthenia gravis (MG) caused by autoantibodies to MuSK ('MuSK-MG') 33 . This form of MG is distinct from the more common anti-AChR MG, can be more severe and does not respond to cholinesterase inhibitors. The pathogenesis of MuSK-MG is mediated at least in part by IgG4 antibodies directed against the MuSK Ig1 domain that disrupt agrin-LRP4 binding and signaling 15,[34][35][36][37] . However, some clinical features of MuSK-MG suggest that non-synaptic pathology mediated by the MuSK autoantibodies may also contribute to the disease. MuSK-MG pathology is often more pronounced in restricted muscle groups, including bulbar and respiratory muscles. Moreover, muscle atrophy is observed in MuSK-MG where it is associated with nonuctuating weakness, fatty tissue in ltration and myopathic changes in electrophysiology recording. It is therefore plausible that antibodies targeting the MuSK expressed in the sarcolemma could contribute to MuSK MG pathology.
The MuSK-BMP pathway could also be a target for promoting muscle growth and treating conditions associated with muscle atrophy such as sarcopenia, immobilization, and cachexia. Maintenance of muscle mass is a balance between the homeostatic mechanisms regulating protein synthesis and degradation. Although the role for IGF1 as an anabolic pathway is well established, circulating IGF1 levels correlate incompletely with muscle status. Rather, muscle-derived IGF1 is likely to be the dominant mediator of growth 30 . The MuSK-BMP pathway represents an attractive target for developing speci c agents to modulate muscle growth. This pathway also offers prospects for the precise manipulation of BMP signaling in muscle. BMPs and their canonical receptors are ubiquitous and manipulating them leads to unwanted side effects; in contrast, MuSK expression is highly enriched in muscle. Moreover, the MuSK ectodomain would be accessible to manipulation by therapeutic antibodies, while antisense oligonucleotides could promote MuSK-BMP signaling without affecting the role of MuSK in synapse formation. Finally, MuSK is expressed in myonuclei in both fast and slow myo bers and its level increases with age in humans and rats 17,38,39 . The MuSK-BMP pathway thus emerges as an attractive target for selectively modulating muscle growth and combatting atrophy.

Animals
To target the MuSK Ig3 domain, we deleted exons 6 and 7 using CRISPR-Cas9 (Exon numbering according to ENSMUST00000081919; Fig. 1 Images were acquired on a Nikon Ti2-E inverted microscope equipped with a Photometrics Prime 95B sCMOS camera for uorescence imaging and a 16-megapixel Nikon DS-Ri2 color camera for imaging histology slides. Confocal z-stack images were obtained using a Zeiss 800 LSM laser scanning microscope equipped with a USRB laser module and GaAsP detectors. When comparing uorescence levels, all images were acquired on the same session and imaging parameters. Images were processed using ImageJ (NIH).
For visualization of NMJs in whole mount preparations muscles were collected and pinned at resting length for removal of connective tissue, xed in 4% PFA for 20 min, leted into bundles, washed with PBS 3 x 10 min, and labeled with tetramethylrhodamine-conjugated -bungarotoxin (1:40, Invitrogen T1175) for 15 min at room temperature. After washing, muscles were incubated in methanol at -20° C for 5 min.
Quantitative RT-PCR Total RNA from snap frozen muscles and cells stored in RNAlater (Invitrogen) was isolated using the RNeasy Fibrous and RNeasy mini kit (Qiagen), respectively, and DNase I treated according to manufacturer's protocol. cDNA from total RNA was reverse transcribed using SuperScript III cDNA synthesis kit (Invitrogen) and analyzed by quantitative RT-PCR using TaqMan assays (Applied and Id1 (Mm001281795_m1). Data analyzed by ∆∆Ct method using β-2 microglobulin (Mm00437762_m1) and 18S (Hs99999901_s1) reference genes.

RNA-seq Analysis
Total RNA from WT and ∆Ig3-MuSK mouse soleus and TA was extracted using a RNeasy Fibrous Tissue mini kit (Qiagen) for non-stranded RNA library preparation and sequencing (Genewiz). Samples were sequenced at a depth of approximately 50 million reads/ sample. Reads were assessed for quality and trimmed of adapter sequences using Trimmomatic then aligned with GSNAP to the Ensembl mouse reference genome (mm10) and read count matrices were generated using htseq-count. WT and ∆Ig3-MuSK read count matrices were uploaded to the integrated Differential Expression and Pathway (iDEP) analysis tool for exploratory data analysis, differential gene expression using DEseq2, and gene set enrichment pathway GO analysis (Ge et al., 2018). The raw datasets will be available in GEO at time of publication.

Statistical analysis
The average of biological replicates is shown as mean ± SEM. Experiments were replicated two to three times as indicated. Statistical comparisons between groups were performed using two-way ANOVA and unpaired Student's t-test when comparing multiple groups or two groups respectively. ANOVA analyses were corrected by post-hoc test as indicated. Signi cance was determined as P < 0.05 (*** P < 0.001, ** P < 0.01, *P < 0.05).

Declarations Ethical Approval
All protocols were conducted under accordance and approval of the Brown University Institutional Animal Care and Use Committee.      Table S3). Note the number of highly signi cant pathways in soleus (adjusted p values between 10 -7 and 10 -12 ) compared to TA. Selective enrichment in soleus down pathways were related to translation including those involving ribosome biogenesis as well as mRNA and ncRNA metabolism.
Soleus up pathways included those involved in ECM and in ammation. (C) Venn diagram showing the number of shared and unique downregulated and upregulated pathways between ∆Ig3-MuSK TA and soleus (see Table S3). Note that ≤5% of pathways are shared in any of the comparisons. No shared pathways were observed when comparing TA down and soleus up.   Akt-mTOR signaling is reduced in ∆Ig3-MuSK soleus but not TA. (A) WT and ∆Ig3-MuSK soleus muscle protein extracts were isolated for Western blotting of pSmad1/5, total Smad1, p-4EBP1, and total 4EBP1.
Total protein stain was used as loading control and for protein normalization. Levels of pSmad1/5 and p4EBP1 were determined as a ratio to total Smad1 and total 4EBP1, respectively. Note that p4EBP1 levels were reduced in ∆Ig3-MuSK soleus compared to WT (C), but unchanged in TA (D). pSmad 1/5 levels in ∆Ig3-MuSK soleus and TA were comparable to WT (E and F). Data are means ± SEM from ve biological replicates (*p < 0.05, unpaired two tailed Student's t test).

Figure 8
A model for MuSK-BMP regulation of muscle ber size in slow muscle. (A) MuSK containing the Ig3 domain binds BMP and promotes BMP signaling. (B) In soleus MuSK is expressed at the sarcolemma and regulates myo ber size via the AktmTOR pathway. C. MuSK is not detected in the sarcolemma in fast muscles (e.g. TA, EDL, STM). Note that MuSK is expressed at the NMJ of all muscles. We propose that MuSK-BMP signaling in the sarcolemma selectively regulates myo ber size in slow muscle by controlling the IGF1-Akt-mTOR pathway.

Supplementary Files
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