Beta-catenin knockdown improves recovery of the muscle function from tourniquet-induced ischemia-reperfusion injury in mouse hindlimb

The neuromuscular junction (NMJ) is a synaptic connection between a motor nerve terminal and a skeletal muscle and influences the muscle contractile function. Tourniquet-induced hindlimb ischemia-reperfusion (IR) results in the fragmentation of nicotinic acetylcholine receptors (nAChR) clusters in the NMJ and impairs neural-controlled skeletal muscle contraction. β-Catenin, a crucial downstream component of the Wnt signaling pathway, participates in synaptic assembly and plays an important part in muscle contraction. We found that the protein of β-catenin was increased in the skeletal muscle with tourniquet-induced IR. Here, we observed the effect of β-catenin knockdown on morphological and functional recovery of the NMJ from tourniquet-induced IR injuries in mouse hindlimb. Unilateral hindlimb of C57/BL6 mice were subjected to 3 hours of ischemia by placing a rubber band and followed 6 weeks of reperfusion. LV-m-β-catenin shRNA (1.4 x 10 7 TU/ml) was injected into gastrocnemius muscle (50 µl) by Hamilton microliter syringe after 2 weeks of tourniquet-induced IR. After 6 weeks of IR, rotarod test was done to measure the latency before falling off, and then the sciatic nerve-stimulated gastrocnemius muscle contraction and endplate potential (EPP) were recorded in in-situ muscle samples. The NMJs were labelled with alpha-bungarotoxin (BTX) and nerofilment-200 (NF-200) + synaptophysin (Syn) antibodies, and NMJ images were captured with Zeiss LSM 800. The mice with 6 weeks of IR displayed a weak skeletal muscle contraction, demonstrated by a short latency to fall in rotarod test and nerve-stimulated muscle contraction (98.8 ± 10.7 sec and 89.3 ± 7.3 g, respectively), compared to sham mice (146.3 ± 10.7 sec and 107.5 ± 6.3 g). Additionally, NMJs in the gastrocnemius muscle were impaired in mice with 6 weeks of IR, including significantly fragmented nAChR clusters (8.5 ± 0.6 fragments in IR mice vs. 1.9 ± 0.1 fragments in sham mice) and small fragment area (91.7 ± 12.2 µm 2 in IR mice vs. 269.7 ± 14.2 µm 2 in sham mice). The amplitude of the EPP was lower in mice with 6 weeks of IR (21.9 ± 0.8 mV) than that in sham mice (26.8 ± 0.8 mV). After LV- m-β-catenin shRNA was injected into the skeletal muscle of IR mice, it reduced β-catenin expression and improved the rotarod performance (135.7 ± 8.7 sec), nerve-stimulated muscle contraction (101.0 ± 2.5 g), and EPP amplitude (24.7 ± 0.8 mV). Catenin shRNA also mitigated fragmentation of nAChR clusters (4.2 ± 0.3 fragments) and enlarged fragment area (200.4 ± 14.5 µm 2 ). The data suggest that β-catenin overexpression is involved in IR-induced NMJ and skeletal muscle damage, and β-catenin knockdown improves the recovery of the NMJ and skeletal muscle from tourniquet-induced hindlimb IR injuries. This research is supported by the NIH/NIGMS 1R01GM145736 to Y-LL. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.