Zinc chelation therapy mitigates muscle fibrosis and improves functional outcomes in a murine model of crush syndrome.

Abstract

The survivors from crush syndrome are often disabled due to the affected limbs, with excessive fibrosis changes at damaged muscle tissue. We previously reported that a zinc chelator (N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine: TPEN) administration to the crush syndrome mice model inhibited neutrophil activation induced by ischemia-reperfusion, alleviating the inflammatory response. In the present study, we investigated the effects of TPEN administration on fibrosis in affected limb muscles. Eight-week-old C57BL/6J mice were subjected to bilateral hindlimb compression using a rubber tourniquet for 2 h to create a crush syndrome model. Compression was then released to allow reperfusion. Gait evaluations and muscle tissue analyses were performed up to 4 weeks post-reperfusion. We investigated the effects of TPEN administration on functional recovery and histopathology of the affected muscles. In the chronic phase of the crush syndrome model, the gait function was impaired owing to muscle tissue fibrosis. The affected muscle tissue sustained sarcolemma hyperpermeability caused by basement membrane disruption and mitochondrial fission, resulting from elevated neutrophil elastase expression. In muscle tissue, PECAM-1-positive vascular endothelial cells co-expressed Notch1 and αSMA, indicating induction of endothelial-to-mesenchymal transition (EndMT) was occurring following persistent mitochondrial fission. TPEN administration reduced neutrophil elastase expression, improving the sarcolemma integrity due to the reduced infiltration of neutrophils. This led to the inhibition of EndMT, which minimized fibrosis and accelerated muscle regeneration, allowing for better functional recovery. This study demonstrates that zinc chelator administration not only alleviates fibrosis in muscle tissues damaged by crush syndrome but also stimulates tissue regeneration in the chronic phase. Our findings provide insight into the mechanism of muscle fibrosis and may represent a breakthrough in the treatment of crush syndrome, potentially minimizing long-term disability in the affected limbs.