Myomaker is a membrane activator of myoblast fusion and muscle formation

Abstract

Fusion of myoblasts is essential for the formation of multi-nucleated muscle fibres. However, the identity of muscle-specific proteins that directly govern this fusion process in mammals has remained elusive. Here we identify a muscle-specific membrane protein, named myomaker, that controls myoblast fusion. Myomaker is expressed on the cell surface of myoblasts during fusion and is downregulated thereafter. Overexpression of myomaker in myoblasts markedly enhances fusion, and genetic disruption of myomaker in mice causes perinatal death due to an absence of multi-nucleated muscle fibres. Remarkably, forced expression of myomaker in fibroblasts promotes fusion with myoblasts, demonstrating the direct participation of this protein in the fusion process. Pharmacological perturbation of the actin cytoskeleton abolishes the activity of myomaker, consistent with previous studies implicating actin dynamics in myoblast fusion. These findings reveal a long-sought myogenic fusion protein that controls mammalian myoblast fusion and provide new insights into the molecular underpinnings of muscle formation.

Figure 1. Muscle-specific expression of Myomaker

a, In situ hybridization for Myomaker in WT embryos illustrates muscle specificity. b, qPCR for Myomaker, Myogenin, and MyoD on tongues at the indicated ages shows down-regulation after myogenesis. c, Gene expression during differentiation of C2C12 myoblasts. d, X-gal staining on E16 and P2 Myomaker^+/− (Myomaker-LacZ) mice confirms expression in all skeletal muscles. e, Cardiotoxin-injured and X-gal stained tibialis anterior (TA) muscle from 6 week old Myomaker^+/− mice shows the re-activation of Myomaker. Serial H&E stained sections indicate muscle injury. Control represents uninjured contralateral TA. Scale bars: a, d, 2 mm; e 200 µm.

Figure 2. Myomaker is essential for skeletal muscle development

a, Full term WT (+/+) and Myomaker^−/− embryos were dissected and skinned to illustrate the lack of muscle surrounding Myomaker^−/− limbs. b, Paraffin sectioning and H&E staining on tongues reveal a lack of muscle fibers in E17.5 Myomaker^−/− embryos. c, Longitudinal sections of E14 hindlimb muscles stained with a myosin antibody to determine the multi-nucleation of the muscle cells. WT limbs exhibit myofibers containing multiple nuclei, which are absent in Myomaker^−/− sections. Scale bars: a, 2 mm; b, 100 µm; c, 40 µm.

Figure 3. Control of myoblast fusion by Myomaker

a, Myoblasts from WT (+/+) and Myomaker^−/− E17 embryos were differentiated for 3 days, and stained for myosin and a nuclear stain (Hoechst). Myomaker^−/− myoblasts failed to fuse. b, Quantitation of the number of nuclei present in a myosin⁺ cell indicates Myomaker^−/− myoblasts cannot form myotubes with three or more nuclei. c, Differentiation index, calculated as the percentage of nuclei in myosin⁺ cells, indicated null myoblasts can activate the myogenic program. d, C2C12 cells infected with a retrovirus encoding GFP or Myomaker were induced to differentiate for 4 days then stained with a myosin antibody and Hoechst (nuclei). e, Quantitation of the percentage of myosin⁺ cells that contained the indicated number of nuclei. Quantification was performed after 3 days of differentiation in (b), (c), and after 4 days in (e). Scale bars: a, 100 µm e, 200 µm. Data are presented as mean ± SEM from three independent experiments. * P < 0.05 compared to +/+ in b, c or GFP-infected cells in e. ns in c is not statistically significant.

Figure 4. Myomaker is expressed on the cell membrane of myoblasts

a, C2C12 cells were infected with Myomaker-Flag and live cells were stained 2 days after differentiation with Flag antibody on ice. After Flag staining, cells were then fixed and permeabilized and stained with Phalloidin (F-actin) and Hoechst (nuclei) to illustrate cell membrane localization of Myomaker-Flag. b, Cells were stained as in (a) to visualize Myomaker-Flag in fusing cultures. The red arrow depicts sites of cell-cell interaction. f, Myomaker-Flag infected C2C12 cells were fixed, permeabilized, and stained with Flag antibody, Phalloidin, and Hoechst showing the vesicle localization of the intracellular protein. Scale bars: 20 µm.

Figure 5. Myomaker participates in the myoblast membrane fusion reaction

a, Myomaker^+/− and Myomaker^−/− myoblasts express LacZ, and were either plated alone or mixed with WT myoblasts, induced to differentiate for 4 days, and stained with X-gal and nuclear fast red to determine the amount of fusion. Myomaker^+/− myoblasts, alone or in the presence of WT myoblasts fused normally, illustrated by myotubes with robust LacZ staining. Myomaker^−/− myoblasts alone exhibited an inability to fuse. Addition of WT myoblasts to Myomaker^−/− myoblasts resulted in chimeric myotubes (arrow) indicating fusion between the two cell populations. b, Quantitation of the percentage of LacZ⁺ myotubes containing ≥3 nuclei shows null myoblasts can only form myotubes with three or more nuclei in the presence of WT myoblasts. c, Phalloidin and Flag staining of C2C12 myoblasts after infection with Myomaker-Flag illustrates the lack of Flag staining in myotubes. d, 10T1/2 fibroblasts were infected with GFP-retrovirus and either Empty- or Myomaker-retrovirus and then mixed with C2C12 cells and differentiated. Myotube formation was monitored by myosin staining, and fusion of fibroblasts was determined by visualization of GFP in myosin⁺ myotubes. Myosin⁺ GFP⁺ myotubes (arrowheads) are evident in cultures containing Myomaker-infected fibroblasts, whereas myosin⁺ GFP⁻ myotubes (arrows) were observed in Empty-infected cultures. e, Quantitation of the percentage of GFP⁺ fibroblasts, infected with Empty- or Myomaker-retrovirus, that fused to myosin⁺ myoblasts. Scale bars: a, 100 µm; c, 20 µm; d, 200 µm. Data are presented as mean ± SEM from three independent experiments. * P < 0.05 compared to −/− in b and compared to empty in e.