Delayed Reinnervation During Muscle Regeneration After NO‐Donor Pre‐Treatment

Abstract

Activation of satellite cells (SCs) toward myogenesis leads to structural regeneration of damaged myofibers and reinnervation is essential to muscle function. SC activation is mediated by NO (a signaling and neurotransmitter molecule) and manipulation of the NO signal is shown to enhance muscle repair and promote growth in older mice in combination with exercise. SCs up‐regulate mRNA expression of a neural chemorepellent, semaphorin 3A (Sema3A) that peaks 6–8 days after muscle injury. Since Sema3A expression is implicated in regulating the timing of axon growth toward regenerating fibers, we hypothesized that premature activation of SC before an injury would advance Sema3A synthesis and disrupt the process of reinnervation. The time‐ course of reinnervation in regenerating tibialis anterior muscle (TA) of 6–10‐week‐old C57BL6 mice was examined after injury by crush (a large traumatic injury) or injection of cardiotoxin (CTX, which damages only fibers). We tested whether early activation of SC before injury using a 2‐day daily pre‐treatment with isosorbide dinitrate (ISDN, an NO‐donor drug) affects the process of new‐fiber innervation post‐injury. Muscle was collected 0, 4, 6, 8, 10, and 14 days after unilateral TA injury (n=4–5 per time point in crush, CTX, and respective control groups). The position of the nuclei and the length of the newly‐formed myotubes were used to track muscle regeneration on coded slides. CTX‐injured muscles had noticeably more advanced formation of neuromuscular junctions (NMJs) and faster fiber regeneration compared to crush‐ injured muscles at the same time point post‐injury. Combined staining of NMJs for acetylcholinesterase (AchE, silver staining) and acetylcholine receptors (AchRs, α‐bungarotoxin epifluorescence) on coded slides, revealed immature or mature NMJs (positive for AchE and AchRs) by day 14 in untreated crush‐ or CTX‐injured muscles, respectively. ISDN pre‐treatment in both crush‐ and CTX‐injured muscles delayed NMJ development on myotubes up to day 14 post‐injury. Results are consistent with the action of Sema3A as a neural chemorepellent, since SC activation before injury resulted in delayed myotube reinnervation. Therapeutic manipulation of Sema3A during regeneration, in time and/or concentration, may have potential to improve reinnervation and muscle function in conditions where nerve‐muscle interactions are disrupted by injury or disease.Support or Funding InformationSupported by a NSERC Discovery grant (to JEA) and a University of Manitoba Graduate Fellowship (to ND)