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. 2016 Jan 21:6:2.
doi: 10.1186/s13395-016-0077-7. eCollection 2016.

Decrease of myofiber branching via muscle-specific expression of the olfactory receptor mOR23 in dystrophic muscle leads to protection against mechanical stress

Christophe Pichavant  1 , Thomas J Burkholder  2 , Grace K Pavlath  3
Affiliations

Decrease of myofiber branching via muscle-specific expression of the olfactory receptor mOR23 in dystrophic muscle leads to protection against mechanical stress

Christophe Pichavant et al. Skelet Muscle. .

Abstract

Background: Abnormal branched myofibers within skeletal muscles are commonly found in diverse animal models of muscular dystrophy as well as in patients. Branched myofibers from dystrophic mice are more susceptible to break than unbranched myofibers suggesting that muscles containing a high percentage of these myofibers are more prone to injury. Previous studies showed ubiquitous over-expression of mouse olfactory receptor 23 (mOR23), a G protein-coupled receptor, in wild type mice decreased myofiber branching. Whether mOR23 over-expression specifically in skeletal muscle cells is sufficient to mitigate myofiber branching in dystrophic muscle is unknown.

Methods: We created a novel transgenic mouse over-expressing mOR23 specifically in muscle cells and then bred with dystrophic (mdx) mice. Myofiber branching was analyzed in these two transgenic mice and membrane integrity was assessed by Evans blue dye fluorescence.

Results: mOR23 over-expression in muscle led to a decrease of myofiber branching after muscle regeneration in non-dystrophic mouse muscles and reduced the severity of myofiber branching in mdx mouse muscles. Muscles from mdx mouse over-expressing mOR23 significantly exhibited less damage to eccentric contractions than control mdx muscles.

Conclusions: The decrease of myofiber branching in mdx mouse muscles over-expressing mOR23 reduced the amount of membrane damage induced by mechanical stress. These results suggest that modifying myofiber branching in dystrophic patients, while not preventing degeneration, could be beneficial for mitigating some of the effects of the disease process.

Keywords: Mechanical stress; Muscle regeneration; Muscular dystrophy; Myofiber branching; mOR23.

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Figures

Fig. 1
Fig. 1
mOR23 expression in transgenic mouse muscles. a Schematic representation of the construct used to drive muscle-specific expression of mOR23. mOR23 was fused with rhodopsin (RHO) to increase mOR23 cell surface expression. HSA human α-skeletal actin. b mOR23 RNA levels were greatly increased in the gastrocnemius muscles of two different transgenic (TG) mouse lines compared to wild type (WT): TG1 (3302 ± 1141), TG2 (218 ± 21). Data are mean ± SEM with n = 4–6 for each genotype
Fig. 2
Fig. 2
Phase-contrast image of branched myofibers. a–c Phase-contrast images of the three branch types studied: bifurcated (a), split (b), and process (c). Bar = 100 μm. d Phase-contrast image of a highly branched myofiber from a 15-week-old mdx mouse. Bar = 250 μm
Fig. 3
Fig. 3
mOR23 over-expression decreases myofiber branching after muscle regeneration. a Myofiber cross-sectional areas (CSA) of WT and TG1 gastrocnemius muscles were comparable before injury and after 2 weeks of regeneration. b Representative images of injured WT and TG1 muscles after 2 weeks of regeneration. Bar = 100 μm. c The percentage of regenerated myofibers was not significantly different in WT and TG1 muscles 3 weeks after muscle injury but TG1 muscles contained significantly less branched regenerated myofibers than WT muscles (d). e The number of branches per branched myofiber was also significantly decreased in injured TG1 muscles compared to WT (chi-square = 21.6, df = 3). n = 94–196 myofibers isolated per genotype and mouse. Data are mean ± SEM and n = 4–6 mice for each genotype with *p < 0.05
Fig. 4
Fig. 4
Myofiber branching is decreased in mdx muscles over-expressing mOR23. a Representative images of 8-week-old mdx and Tg-mdx gastrocnemius muscles. Bar = 100 μm. b The percentage of regenerated myofibers was not significantly different between mdx and Tg-mdx muscles at the three ages (5, 8, and 15 weeks) studied. c Tg-mdx muscles had significantly less branched regenerated myofibers than mdx muscles at 5 and 8 weeks of age. d The number of branches per branched myofiber was also significantly decreased in Tg-mdx muscles compared to mdx at both 8 weeks (chi-square = 12.3, df = 3) and 15 weeks of age (chi-square = 13.9, df = 3). n = 32–103 myofibers isolated per age and mouse. Data are mean ± SEM and n = 3–4 for each genotype at each age with *p < 0.05
Fig. 5
Fig. 5
Over-expression of mOR23 in mdx muscles protects against membrane injury after eccentric muscle contractions. a Representative images of gastrocnemius muscles from mdx and Tg-mdx mice which underwent eccentric contraction (EC) or not (CTL, contralateral muscle). Evans blue dye (EBD) fluorescence identified myofibers with increased membrane permeability. Bar = 100 μm. b mdx muscles were significantly more permeable to EBD after EC than Tg-mdx muscles. Each line represents the CTL and EC muscle of one mouse, n = 7 for each genotype with *p < 0.05
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