Inducible deletion of endothelial cell EfnB2 attenuates neuromuscular regeneration in mouse skeletal muscle
Authors: Morton Aaron , Jacobsen Nicole , Diller Alexandra , Cornelison DD , Segal Steven 
Published: May 2023

Background: Acute injury of skeletal muscle disrupts myofibers, microvessels and neuromuscular junctions (NMJs). While myofiber regeneration is well-characterized, little is known of concurrent events in microvessels and motor nerves following injury. Endothelial cell [EC] ephrin B2 [EfnB2] is required for sprouting angiogenesis during embryonic development, for promoting vascularization of tumors in the adult, and for microvascular-dependent guidance of axon regeneration following nerve resection. Hypothesis: We hypothesized that following acute injury to skeletal muscle 1) EC EfnB2 is required for restoring microvascular volume; and 2) loss of EC EfnB2 would impair recovery of neuromuscular structure and function. Methods: Mice (male and female, age 3-5 months) were bred for EC-specific conditional knockout [CKO] of EfnB2 following tamoxifen injection; noninjected CKO mice served as “wildtype” controls [WT]. Following injury by local injection of the myotoxin BaCl2, the gluteus maximus [GM] muscle was studied for microvascular analysis or the tibialis anterior [TA] muscle was evaluated for recovery of neuromuscular function. Mice were studied (n=4-6/group) at 3 timepoints for vascular studies (0, 5, and 10 days post injury [dpi]) and 2 timepoints for neuromuscular structure and function (0 and 10 dpi). Results: Intravascular staining with fluorescent wheat germ agglutinin revealed a diminished microvascular area in the GM of CKO mice at 5 dpi vs. 10 dpi (means ± SE; CKO 5 dpi 35.5% ± 2.5 vs. CKO 10 dpi 49.8% ± 3.9, P<0.01). No difference in vascular area was recorded in WT groups at 5 vs. 10 dpi (WT 5 dpi 45.5% ± 2.6 vs. WT 10 dpi 46.7% ± 2.2, P=0.99). Direct electrical stimulation of the TA resolved a reduction in maximal force at 10 dpi when compared to 0 dpi for both WT and CKO (WT 0 dpi 128.6 g ± 4.1 vs. WT 10 dpi 64.7 g ± 3.0, P<0.0001) (CKO 0 dpi 112.7 g ± 5.0 vs. CKO 10 dpi 65.9 g ± 5.0, P<0.0001). Indirect stimulation (sciatic nerve) resolved initial neuromuscular transmission failure (INMTF) reduced submaximal force in CKO but not WT (CKO 0 dpi 2.0 ± 4.3% vs. CKO 10 dpi -30.0 ± 6.5 %, P<0.001), which correlated with perturbed recovery of NMJ innervation; (R2=0.328, P=0.003), endplate area; (R2=0.479, P=0.001), and nicotinic receptor clusters; (R2=0.207, P=0.025). Conclusion: Following acute injury of skeletal muscle in the adult mouse, loss of EfnB2 in ECs delays microvessel regeneration in the GM and disrupts the recovery of NMJ structure and function in the TA. Support: NIH grant F32 HL-152558 (NLJ), APS Postdoctoral Fellowship (ABM), NIH LRP (ABM), Margaret Proctor Mulligan Fellowship, MU (NLJ, SSS), NIH R01 AR078045 (DDC), School of Medicine Development Award, MU (SSS), NIH MERIT Award R37 HL-041026 (SSS). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.