Rapsyn overexpression improves neuromuscular junction and skeletal muscle function after tourniquet-induced mouse hindlimb ischemia-reperfusion injuries

Tourniquet-induced hindlimb ischemia-reperfusion (tourniquet/IR) results in the fragmentation of nicotinic acetylcholine receptors (nAChR) clusters in the neuromuscular junction (NMJ) and impairs neurally controlled skeletal muscle contraction. Rapsyn is a cytoplasmic nAChR-associated protein and participates in the nAChR assembly at the NMJ. Here, we observed whether the down-expression of rapsyn mediated the structural damage and functional impairment of the NMJ and skeletal muscle from tourniquet/IR injuries in mouse hindlimb. Unilateral hindlimb of C57BL/6 mice was subjected to 3 h of ischemia by placing a rubber band and followed by 6 weeks of reperfusion (tourniquet/IR). Ad-m-RAPSN was injected into the gastrocnemius muscle to increase rapsyn levels. LV-m-Rapsyn shRNA was used to knockdown rapsyn levels. After 6 weeks of sham treatment or tourniquet/IR, skeletal muscle contraction, endplate potentials (EPPs), and morphology of NMJs were assessed. Compared to sham mice, NMJs in the gastrocnemius muscle were significantly fragmented with low rapsyn expression, and the amplitude of EPPs and muscle contraction were also decreased in tourniquet/IR mice. Ad-m-RAPSN transfection into tourniquet mice increased rapsyn expression, reduced fragmented NMJs (38.2% ± 4.0%, compared to 74.7% in tourniquet/IR alone, P < 0.05), and raised EPP amplitudes and skeletal muscle contraction. Conversely, rapsyn shRNA-downregulated rapsyn expression in normal mice resulted in the NMJ fragmentation and neuromuscular dysfunction. These data suggest that tourniquet/IR decreases rapsyn expression in the skeletal muscle, which results in the nAChR cluster fragmentation and impairs skeletal muscle contractile function. Rapsyn overexpression could be a therapeutic strategy to improve the morphological and functional recovery of the NMJ as well as skeletal muscle contraction from tourniquet/IR injuries.