In this report we introduce the MuSK-BMP pathway as a novel regulator of myofiber 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 findings 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 myofiber 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-fidelity expression and localization are consistent with the fact that the ∆Ig3-MuSK allele created by gene editing mimics a natural MuSK splice isoform20,24. 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 identified in a study using cultured MuSK−/− myogenic cells13. 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 myofiber size in slow muscle. In soleus, muscle atrophy was observed in both type I and IIa fibers (Fig. 5), which together comprise ~ 80% of fiber types in soleus. In contrast, no significant differences were observed in the diameter of TA myofibers, 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 myofiber size through the regulation of the IGF1-Akt-mTOR pathway, the primary anabolic regulator of muscle cell size (Fig. 8)22, 25–30. 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 myofiber size29. 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 denervation31 (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 reflect 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 muscles29,32, including TA and soleus (Fig. 6). However, in WT soleus MuSK is also localized at extrasynaptic domains along the extent of the myofiber (Fig. 6), which is in agreement with earlier reports13,16. Further, snRNAseq analysis shows robust MuSK expression in soleus as compared to TA myonuclei17. 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 transcript13. On the organismal level, our results suggest that MuSK expression in the sarcolemma may be one mechanism conferring muscle-selective regulation of myofiber 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 growth8. 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 signaling15, 34–37. 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 non-fluctuating weakness, fatty tissue infiltration 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 growth30. The MuSK-BMP pathway represents an attractive target for developing specific 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 myofibers and its level increases with age in humans and rats17,38,39. The MuSK-BMP pathway thus emerges as an attractive target for selectively modulating muscle growth and combatting atrophy.