Effective Reinnervation of Skeletal Muscle is Impaired by Disrupting Microvascular Regeneration Following Acute Injury

Abstract

Acute injury of skeletal muscle damages myofibers, microvessels and neuromuscular junctions (NMJs). Although myofiber regeneration is well‐characterized, little is known of concurrent events in microvessels and motor nerves during skeletal muscle regeneration. Because growth and patterning are coordinated between nerves and microvessels during development, we tested the hypothesis that effective microvascular regeneration is required for restoring neuromuscular structure and function post injury in the adult. To impair angiogenesis, mice were bred for endothelial cell (EC)‐specific deletion of ephrin‐B2. Male mice (3–6 months old; n=2–3/group) were injected with tamoxifen to delete ephrin‐B2 in ECs (EfnB2CKO; confirmed with PCR); uninjected mice of the same genotype were used as controls. The gluteus maximus (GM) or tibialis anterior (TA) muscle was injured by local injection of 1.2% BaCl2 and muscles were evaluated at 10 days post injury, by which time morphological and functional myofiber regeneration will have occurred. Intravascular injection of fluorescent wheat germ agglutinin (WGA; stains only perfused vessels) or immunolabeling for CD31 (stains all ECs) revealed microvessels of the GM in EfnB2CKO to be vacuolated with disorganized ECs vs. intact microvascular networks with aligned ECs in control mice. Using indirect stimulation via the sciatic nerve, isometric force production in the TA was 19±6 N/cm2 in EfnB2CKO vs. 33±3 N/cm2 in control mice; force produced with direct electrical field stimulation was similar between groups. Immunostaining for neurofilament‐heavy revealed reduced presynaptic coverage of NMJs in EfnB2CKO vs. control [60±9% vs. 74±10% of acetylcholine receptors (AChRs) per 200 μm2 NMJ area]; postsynaptic staining with α‐bungarotoxin revealed dispersed AChRs [200±118 vs. 92±43 fragments/200 μm2, respectively]. These preliminary findings indicate that the loss of ephrin‐B2 in ECs results in functional deficits for neuromuscular transmission and NMJ morphology in association with impaired microvascular regeneration following acute injury. We conclude that regeneration of intact NMJs with functional reinnervation of myofibers requires effective revascularization and recovery of the microcirculation following skeletal muscle injury.Support or Funding InformationSupport: APS Postdoctoral Fellowship (ABM), R01AR067450 (DDWC), R37HL041026 (SSS)