Fusogenic micropeptide Myomixer is essential for satellite cell fusion and muscle regeneration

Abstract

Regeneration of skeletal muscle in response to injury occurs through fusion of a population of stem cells, known as satellite cells, with injured myofibers. Myomixer, a muscle-specific membrane micropeptide, cooperates with the transmembrane protein Myomaker to regulate embryonic myoblast fusion and muscle formation. To investigate the role of Myomixer in muscle regeneration, we used CRISPR/Cas9-mediated genome editing to generate conditional knockout Myomixer alleles in mice. We show that genetic deletion of Myomixer in satellite cells using a tamoxifen-regulated Cre recombinase transgene under control of the Pax7 promoter abolishes satellite cell fusion and prevents muscle regeneration, resulting in severe muscle degeneration after injury. Satellite cells devoid of Myomixer maintain expression of Myomaker, demonstrating that Myomaker alone is insufficient to drive myoblast fusion. These findings, together with prior studies demonstrating the essentiality of Myomaker for muscle regeneration, highlight the obligatory partnership of Myomixer and Myomaker for myofiber formation throughout embryogenesis and adulthood.

Keywords: CRISPR/Cas9; Myomaker; fusion; satellite cells; skeletal muscle.

Fig. 1.

Generation of Myomixer^loxP alleles. (A) Schematic of experimental design to insert loxP1 and loxP2 sites into intron 2 and the 3′-UTR region of the Myomixer (Mymx) gene, respectively. Note that upon Cre-mediated DNA recombination, the short Myomixer isoform is removed. (B and C) PCR genotyping with primers that amplify the regions where the loxP1 and loxP2 sites were inserted. (D) PCR amplification of genomic DNA isolated from Mymx^loxp1,2/+ fibroblasts at 48 h postadenoviral infection. (E) Schematic of experimental design to insert loxP3 and loxP4 sites into the 5′- and 3′-UTR regions of the Myomixer gene, respectively. Note that upon Cre-mediated DNA recombination, the ORF encoding the long Myomixer isoform is removed. (F and G) PCR genotyping with primers that amplify the regions where the loxP3 and loxP4 sites were inserted. (H) PCR amplification of genomic DNA isolated from Mymx^loxp3,4/+ fibroblasts at 48 h postadenoviral infection. ssODNA, single-stranded oligodeoxynucleotide DNA. HDR, homology-directed repair.

Fig. 2.

Validation of Myomixer deletion in satellite cells. (A) Schematic outlining the strategy of tamoxifen and cardiotoxin treatment of mice. Control and Myomixer KO mice were treated with tamoxifen every 2 d over a period of 10 d before injury of skeletal muscle by cardiotoxin injection. Muscles were collected for analysis at 3 d postinjury. (B) PCR amplification of genomic DNA isolated from noninjured EDL muscles of Mymx^loxp1,2/1,2 (lane 1), Pax7^CreERT2/Mymx^loxp1,2/1,2 (lane 2), Mymx^loxp3,4/3,4 (lane 3), and Pax7^CreERT2/Mymx^loxp3,4/3,4 (lane 4) mice treated as shown in A. (C) Quantification by qPCR of Myomixer gene expression in gastrocnemius muscle 3 d postinjury. n = 3 for control (CTL) mice, n = 5 for Myomixer KO mice. (D) Western blot analysis of Myomixer and Gapdh in gastrocnemius muscle 3 d postinjury. *P < 0.05; ***P < 0.001. Student’s t test. Data are mean ± SEM.

Fig. 3.

Deletion of Myomixer in adult satellite cells does not affect the early response to muscle injury. (A) Quantification by qPCR of gene expression in gastrocnemius muscle 3 d postinjury. n = 3 for control (CTL) mice, n = 5 for Myomixer KO mice. (B) H&E staining of gastrocnemius muscle cross-sections at day 3 postinjury. (Scale bar: 50 µm.) (C) Immunohistochemistry of Laminin, Myogenin and Desmin in gastrocnemius muscle cross-sections at day 3 postinjury. (Scale bars: 20 µm.) *P < 0.05; **P < 0.01; ***P < 0.001. Student’s t test. Data are mean ± SEM.

Fig. 4.

Myomixer is essential for skeletal muscle regeneration after injury. (A and D) Schematic outlining strategy of tamoxifen and cardiotoxin treatment. (B and E) H&E staining of TA muscle cross-sections at (B) day 7 and (E) day 14 postinjury. (Scale bar: Top, 500 µm; Bottom, 50 µm.) (C and F) Immunohistochemistry of Laminin, Myosin, and Desmin on TA muscle cross-sections at (C) day 7 and (F) day 14 postinjury. (Scale bars: 20 µm.)