Mustn1 in Skeletal Muscle: A Novel Regulator?

Abstract

Skeletal muscle is a complex organ essential for locomotion, posture, and metabolic health. This review explores our current knowledge of Mustn1, particularly in the development and function of skeletal muscle. Mustn1 expression originates from Pax7-positive satellite cells in skeletal muscle, peaks during around the third postnatal month, and is crucial for muscle fiber differentiation, fusion, growth, and regeneration. Clinically, Mustn1 expression is potentially linked to muscle-wasting conditions such as muscular dystrophies. Studies have illustrated that Mustn1 responds dynamically to injury and exercise. Notably, ablation of Mustn1 in skeletal muscle affects a broad spectrum of physiological aspects, including glucose metabolism, grip strength, gait, peak contractile strength, and myofiber composition. This review summarizes our current knowledge of Mustn1's role in skeletal muscle and proposes future research directions, with a goal of elucidating the molecular function of this regulatory gene.

Keywords: Mustn1; development; knockout; mouse; repair; skeletal muscle.

Figure 1

The diagram delineates the progression from stem cells in the paraxial mesoderm to mature myofibers, encapsulating stages of myogenesis during embryonic development in mice. Stem cells expressing Pax3 and Pax7 proceed to asymmetric division and myogenic commitment via MyoD and Myf5 expression. Myoblasts, upon MyoG influence, transition into committed myocytes, expressing Des and myosin. Myocytes undergo fusion, creating myotubes which subsequently differentiate and mature into primary myofibers through the incorporation of myofibrils during primary myogenesis. Secondary myogenesis guides the development of secondary myofibers, influenced by further innervation and myoblast fusion to mature fiber types (slow Type I or fast Type II).

Figure 2

Molecular modeling of Mustn1 in 3D AlphaFold. Protein structure predictions for Mustn1 in various vertebrate organisms: (A) H. sapiens, (B) P. troglodytes, (C) B. taurus, (D) C. lupus familiaris, (E) R. norvegicus, (F) M. musculus, (G) X. laevis, and (H) X. tropicalis. All images are aligned to xy-axis. AlphaFold produces a per-residue model confidence score (pLDDT) between 0 and 100 and colored regions indicate pLDDT with Dark blue = Very high (pLDDT > 90); Light blue = High (90 > pLDDT > 70); and Yellow = Low (70 > pLDDT > 50). Predictions obtained from the AlphaFold Protein Structure Database (https://alphafold.ebi.ac.uk/ (accessed on June 1, 2024).

Figure 3

(A) Mustn1 ^PRO-driven GFP expression at various stages of embryonic development in transgenic mice in (a) somites (white arrows), (b) trapezius muscle (what arrows), (c) intercostal muscles (white arrowheads), and (d) forelimb muscles (white arrowheads). (B) The study extended its observations to the context of skeletal muscle regeneration and repair following injury, showing a clear surge in Mustn1-GFP expression 3 days post injury, (a,d) with some areas overlaying with desmin (denoted by *) but numerous examples of mononuclear cells expressing only GFP (denoted by arrow). By 5 days post-injury (b,e) expression gradually subsided as newly formed muscle fibers (*) became more prominent, as shown by co-staining with GFP (red, arrow), DAPI (blue), and desmin (green). Finally, Desmin expression is most robust at 10 days post-injury (c,f), and only regions of GFP are evident overlay with desmin (*) and located at the periphery of newly formed muscle fibers. Tm, Trapezius muscle; IcM, Intercostal muscle; Dg, Digit; Cc, Costal cartilage. Modified from [14].

Figure 4

Mustn1 expression in smooth muscle and as a secretory protein. (A) Immunofluorescence images of Mustn1 co-expression with α-smooth muscle actin (α-SMA) in smooth muscle of blood vessels in tibialis anterior muscle cross sections from Mustn1 knockout mice and wild-type littermates, scale bars: 100 μm (n = 3), (B) immunoblotting of cell supernatant and 5% cell lysate of cultured smooth muscle cells transduced with Mustn1-expressing adenovirus (Ad-Mustn1) and control adenovirus (Ad-CON), collected 3, 6, or 9 h after medium change, which shows increased Mustn1 expression in cell supernatant. Representative blots (left panel) and quantification (right panel, n = 3). Data represented by mean ± SEM. Modified from [39].

Figure 5

Mustn1 in Golden Retriever Muscular Dystrophy (GRMD) dogs treated with stem cells from the hindlimb muscle and assessed weekly with (A) clinical score as a percentage of a theoretical healthy dog score, (B) stem-cell treated dogs (GRMD^MuStem) show a plateau in their score, (C) untreated dogs (GRMD) show a continued decline in their score. PCR performed on the bicep femoris showing that (D) Mustn1 is one of the regulatory genes that were upregulated in GRMD^MuStem dogs and (E) one of the top 5 upregulated genes, (F) compared to healthy and GRMD dogs, Mustn1 was significantly upregulated in GRMD^MuStem dogs. Modified from [64].

Figure 6

Mustn1 perturbation in Xenopus laevis. Antisense Mustn1 MO injected at the 4-cell stage targeting the start codon of Mustn1 mRNA led to distinct malformations of the eye, body axis length and tail curvature (A,B,D,E). Rescue group with Mustn1-MO embryos co-injected with a modified, MO-resistant Mustn1 mRNA, significantly reduced or corrected the developmental defects observed (C,F). Quantitative analysis of (G) eye, (H) body axis length, and (I) tail curvature. Modified from [18].