β‐hydroxy‐β‐methylbutyrate improves fast‐twitch muscle function, histopathology and mitochondrial respiration in the D2.mdx dystrophic mouse

Abstract Duchenne muscular dystrophy (DMD) is a severe inherited muscle disorder caused by mutations that eliminate the protein, dystrophin, resulting in disrupted protein homeostasis, mitochondrial dysfunction, chronic inflammation and progressive muscle degeneration. The leucine metabolite, β‐hydroxy‐β‐methylbutyrate (HMB), has shown therapeutic potential in dystrophic muscle. We previously reported that HMB supplementation improves fast‐twitch muscle histopathology and function in juvenile mdx mice during their peak damage phase (3–6 weeks). However, the mdx model exhibits a relatively mild pathology compared to human DMD, limiting clinical relevance. Here, we investigated 8 weeks of HMB supplementation (1 mg g −1  day −1 via drinking water) in the more severe D2.mdx mouse model at 3 and 6 months of age, representing skeletal muscle pathologies consistent with chronic inflammation and advanced muscle fibrosis, respectively. HMB‐treated D2.mdx mice had improved grip strength compared to controls, whereas isolated fast‐twitch extensor digitorum longus (EDL) muscles displayed increased fibre size, reduced tissue infiltrate and enhanced force production ex vivo . Mechanistically, HMB increased the phospho‐to‐total ratio of direct downstream mammalian target of rapamycin complex 1 (mTORC1) targets p70S6K1 and 4EBP1, consistent with enhanced anabolic signalling. Mitochondria assessed from treated flexor digitorum brevis muscles exhibited improved respiration and ATP production, with functional improvements aligning with elevated complex II succinate dehydrogenase activity in HMB‐treated EDL muscles. By contrast, no significant HMB‐induced effects were observed in the slow‐twitch soleus muscle. This study is the first to demonstrate that HMB enhances in vivo and ex vivo muscle function, downstream mTORC1 signalling, and mitochondrial performance in the severe D2.mdx model, supporting its potential as a therapeutic strategy for DMD. image Key points β‐Hydroxy‐β‐methylbutyrate (HMB) supplementation improves skeletal muscle function in the D2.mdx mouse model of Duchenne muscular dystrophy. HMB treatment increased voluntary grip strength and enhanced force production in isolated extensor digitorum longus extensor digitorum longus muscles in 3‐ and 6‐month‐old D2.mdx mice. Functional improvements were accompanied by reduced tissue infiltrate, increased mean muscle fibre size, and elevated succinate dehydrogenase activity. Τηεδδ adaptations coincided with increased markers of mammalian target of rapamycin complex 1 signalling, mitochondrial respiration and ATP production, suggesting improved metabolic capacity in dystrophic muscle.