Iron Deficiency Impairs Muscle Stem Cell Proliferation and Skeletal Muscle Regeneration via HIF-2α Stabilization.

BACKGROUND: Functional iron deficiency affects a large proportion of patients with chronic diseases and is increasingly observed in older adults. Clinical evidence links iron deficiency to sarcopenia, yet the mechanistic relationship between iron status and muscle regeneration remains poorly defined. This study investigates how iron depletion alters muscle stem cell (MuSC) proliferation and skeletal muscle regeneration, focusing on HIF-2α signalling. METHODS: Male and female C57BL/6 J mice (4 week old, n > 20 per group in total) were fed iron-sufficient (IS) or iron-deficient (ID) chow for 4 weeks before cardiotoxin-induced tibialis anterior (TA) muscle injury. Muscle mass, MuSC proliferation and histological changes in regenerating TA muscles were evaluated at 10 and 30 days after injury (dpi). Pharmacological HIF-2α inhibition (PT2385) was used to determine causal mechanisms. Data were analyzed by t tests and one-way ANOVA. RESULTS: Iron deficiency significantly reduced MuSC proliferation (-10.2% Ki67(+) MuSC at 10 dpi, p < 0.01, n = 5) and myoblast EdU incorporation (-18.1%, p < 0.001), leading to smaller regenerating myofibres (-22.7% median cross-sectional area at 30 dpi, p < 0.01, n = 3) and impaired muscle mass recovery (males: -13.9% p < 0.001, females: -9.4% p < 0.05, n = 6). HIF-2α inhibition with PT2385 in ID mice increased MuSC proliferation (+7.1% Ki67(+) MuSC at 10 dpi, p < 0.01, n = 5) and restored muscle mass (males: +10.3% p < 0.001, females: +5.5% p < 0.05, n = 6). Mechanistically, iron deficiency stabilized HIF-2α in proliferating MuSC, which upregulated retinoblastoma protein (Rb1), repressed E2F target RNA levels and induced G0/G1 cell cycle arrest. This impaired myoblast expansion and delayed muscle regeneration in vitro and in vivo. In ID mice, PT2385 restored MuSC proliferation, accelerated myofibre maturation and enhanced muscle mass recovery without compromising MuSC self-renewal. Chromatin immunoprecipitation demonstrated HIF-2α binding at the Rb1 promoter, increasing Rb transcription and reducing H3K27 acetylation at E2F target loci. CONCLUSIONS: Iron deficiency impairs skeletal muscle regeneration by stabilizing HIF-2α in MuSC, inducing Rb1 RNA expression, and repressing E2F-dependent proliferation. Transient HIF-2α inhibition rescues MuSC proliferation and muscle repair under iron-deficient conditions, highlighting HIF-2α as a potential therapeutic target to counteract sarcopenia in aging and chronic diseases.