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

Figures

• 

  Figure 1. Generation of the ΔIg3-MuSK mouse. (A) Schematic representation of full length and ΔIg3-MuSK isoforms. The extracellular full-length isoform contains three immunoglobulin (Ig)-like domains (Ig1, Ig2, and Ig3) as well as a CRD/Fz and an intracellular tyrosine kinase domain. ΔIg3-MuSK lacks the BMP-binding Ig3 domain. (B) CRISPR-Cas9 was used to delete exon 6 and 7, which encode the Ig3 domain, along with the intervening intronic sequence to generate the ΔIg3-MuSK allele. (C) PCR amplification of WT and ΔIg3-MuSK alleles in WT and ΔIg3-MuSK heterozygous and homozygous mice using allele-specific primers (depicted in B). The animal weights (D) and grip strength (E) of 3-month-old WT and ΔIg3-MuSK were comparable. Innervation in the Ig3-MuSK animals, as assessed by overlap of pre- and post- synaptic structures, was equivalent to that in WT in both the fast EDL (F) and slow soleus (G) (EDL: n= 3 animals per genotype, SOL: n=6 animals per genotype, t-test n.s. in both muscles).

• 

  Figure 2. BMP signaling is selectively perturbed in ΔIg3-MuSK myogenic cells. (A, B), MuSK expression: (A) Unpermeabilized WT and ΔIg3-MuSK myoblasts cells were fixed, and cell surface MuSK was visualized by immunostaining with an antibody directed against the MuSK Ig2 domain (red). Note that comparable levels and distribution of immunostaining were observed in both genotypes. (B) WT and ΔIg3MuSK myoblast cell lines express comparable levels of MuSK transcript as assessed by qRT-PCR. Data are means ± SEM from five biological replicates and two independent experiments. (C-G), BMP signaling: (C) WT and ΔIg3-MuSK myoblasts were treated with 20 ng/ml BMP4 for 15 min and immunostained for pSmad1/5. Note the increased intensity of pSmad staining in nuclei of BMP4-treated WT compared to ΔIg3-MuSK cells. Data are from three independent experiments with three biological replicates per group. (D) pSmad1/5 and total Smad1 in BMP4-treated cells (as in C) were assessed by Western blotting. (E) The level of BMP4-induced pSmad1/5, normalized to total protein, was reduced ΔIg3-MuSK as compared to WT cells. Data are means ± SEM of three biological replicates and independent Western blots (**** p < 0.0001,…

• 

  Figure 3. Distinct transcriptomic profiles in ΔIg3-MuSK TA and soleus. WT and ΔIg3-MuSK soleus and TA muscles were subjected to RNA-sequencing at a depth of ~50 million reads per sample (n = 9 muscles/genotype; one muscle/animal). Heat map of the top 1000 variable genes in WT and ΔIg3-MuSK TA (A) and soleus (B). Each column represents one muscle sample. Principal component analysis of WT and ΔIg3MuSK TA (C) and soleus (D) shows clustering of samples with similar gene expression profiles (▲, WT; •, ΔIg3-MuSK; individual muscles). (E, F) Differentially expressed genes (DEGs) with 1.2 minimum fold-change and 0.1 false discovery rate (FDR) were determined using DEseq2 (See also Table. S1, S2). (G, H) Volcano plot of −log10 FDR versus gene log2 fold change (red dots up DEGs; blue dots down DEGs). (I) Venn diagram depicting the shared and unique upregulated and downregulated DEGs between ΔIg3-MuSK TA and soleus.

• 

  Figure 4. Distinct pathways are dysregulated in ΔIg3-MuSK TA and soleus. (A, B) Gene ontology pathway analysis of top 100 upregulated and downregulated biological process pathways in ΔIg3-MuSK TA (A) and soleus (B) (0.1 FDR; See Table S3). Note the number of highly significant 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 inflammation. (C) Venn diagram showing the number of shared and unique downregulated and upregulated pathways between ΔIg3-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.

• 

  Figure 5. Reduced myofiber size in ΔIg3-MuSK soleus but not TA. WT and ΔIg3MuSK TA and soleus sections were stained with H&E (A, B), WGA (C, D), or antidystrophin (E, F). In the TA, the structure (A), WGA staining (C), and mean Feret diameters (G), were indistinguishable in both genotypes. Data are means ± SEM from three animals of each genotype. Average number of muscle fibers counted per animal 963 ± 77. Soleus WT and ΔIg3-MuSK stained with H&E (B), WGA (D), or dystrophin (F). Note that the overall structure of the muscle was comparable, but there is increased interstitial WGA signal in ΔIg3-MuSK soleus. Soleus myofiber Feret diameters of all fibers (H), Type I fibers (I), or Type IIa fibers (J), were reduced in the ΔIg3-MuSK compared to WT. All fibers: 40.6 μm ± 0.2 WT and 38.4 μm ± 0.2 ΔIg3-MuSK (697 fibers/animal ± 72, n=3 animals); Type I fibers: 36.6 μm ± 0.2 WT and 34.4 μm ± 0.2 ΔIg3-MuSK (300 ± 33 fibers/animal, n=4 animals); Type IIa: 36.7 μm ± 0.2 WT and 33.9 μm ± 0.2 ΔIg3-MuSK (487 ± 50, fibers/animal n=4 animals; **** P < 0.0001, MannWhitney test).

• 

  Figure 6. MuSK extrasynaptic localization and transcript expression and reduced in ΔIg3-MuSK soleus. (A-D): MuSK transcript levels in WT and ΔIg3-MuSK EDL and soleus muscle were quantified by qRT-PCR. Note that MuSK transcript levels are selectively reduced in ΔIg3-MuSK soleus compared to WT while the levels in the EDL are comparable in both genotypes. Data are means ±SEM from 3 to 6 different biological replicates (*p < 0.05, unpaired two-tail Student’s t test). (E, F): MuSK localization in TA. TA sections from WT and ΔIg3-MuSK animals were immunolabeled with anti-MuSK (IgG4 serum fraction from MuSK myasthenia gravis patients45; 1:1000; red), and α-bungarotoxin (green; to visualize NMJs). In the TA, MuSK is restricted to the NMJ (arrows) in both WT and ΔIg3-MuSK muscle. No extrajunctional MuSK localization is detected in TA of either genotype. (G, H): MuSK localization in soleus. In WT soleus (G) MuSK is localized at both the NMJ (arrows) and extrajunctionally (arrowheads). In contrast, in ΔIg3-MuSK soleus (H) MuSK is present at NMJs at levels comparable to WT, but extrajunctional MuSK was not detected.

• 

  Figure 7. Akt-mTOR signaling is reduced in ΔIg3-MuSK soleus but not TA. (A) WT and ΔIg3-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 ΔIg3-MuSK soleus compared to WT (C), but unchanged in TA (D). pSmad 1/5 levels in ΔIg3-MuSK soleus and TA were comparable to WT (E and F). Data are means ± SEM from five biological replicates (*p < 0.05, unpaired two tailed Student’s t test).

• 

  Figure 8. A model for MuSK-BMP regulation of muscle fiber 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 myofiber 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 myofiber size in slow muscle by controlling the IGF1-Akt-mTOR pathway.