Aquaporin‐4 Orchestrates Muscle Cell Differentiation and Satellite Cell Preservation: A Novel Target in Age‐Related Muscle Decline
María D. Girón, Manuel Manzano, Suzette L. Pereira, Antonio Reinoso, Chiara Burgio, Nora Montero‐Martin, Guillermo Jiménez‐Jiménez, Josep M. Argilés, Ricardo Rueda, José M. López‐Pedrosa, Rafael SaltoABSTRACT
Background
Impaired muscle regeneration is a key contributor to age‐related muscle decline, yet the molecular mechanisms linking muscle water homeostasis to regenerative failure remain incompletely understood. Aquaporin‐4 (AQP4), a major skeletal muscle water channel, may have functions beyond water transport that are relevant to muscle maintenance and repair. We investigated whether altered AQP4 expression contributes to age‐related defects in skeletal muscle regeneration and whether AQP4 may represent a targetable pathway for muscle dysfunction associated with ageing.
Methods
Skeletal muscle from young adult (25‐week; n = 9) and aged (85‐week; n = 8) rats was analysed for extracellular and intracellular water content by bioimpedance. AQP4, AQP1 and myogenic markers were measured by Western blot. Satellite cells isolated from skeletal muscle were assessed for proliferation, differentiation and expression of differentiation markers. To define the mechanistic role of AQP4 in myogenesis, CRISPR‐generated AQP4‐silenced L6 rat myoblasts were evaluated for proliferation, differentiation, senescence and Akt/PKB signalling. Rescue experiments were performed by restoring AQP4 expression in silenced cells. Data are reported as mean ± SEM.
Results
Compared with young rats, aged rats showed an altered intracellular water‐to‐lean mass ratio (0.94 vs. 0.62, p < 0.05), a 33% reduction in AQP4 expression ( p < 0.05) and reduced muscle strength (3.1 vs. 2.6, p < 0.05), with AQP4 levels correlating with muscle performance. Satellite cells from aged muscle exhibited 29% lower AQP4 expression and 39% lower Pax7 expression (both p < 0.05), together with reduced myogenic differentiation (30% decrease, p < 0.05). In L6 myoblasts, AQP4 silencing reduced proliferation (20% decrease, p < 0.05), impaired myotube differentiation (50% decrease, p < 0.05), increased senescence (30% increase, p < 0.05) and decreased Akt/PKB signalling by 42% ( p < 0.05). AQP4 silencing was also associated with decreased dystrophin localization at the plasma membrane (50%, p < 0.05). Re‐expression of AQP4 reversed these abnormalities and restored the phenotype toward wild‐type levels (all p < 0.05).
Conclusions
Reduced AQP4 expression is associated with impaired muscle regeneration and functional decline in aged skeletal muscle. Experimental AQP4 loss disrupts myogenic signalling, membrane organization and increases progression to senescence, whereas AQP4 restoration rescues these defects. These findings identify AQP4 as a biologically plausible therapeutic target for strategies aimed at preserving or restoring muscle health and regenerative capacity in ageing‐associated muscle dysfunction.