Copper Promotes Myogenic Differentiation of Bovine Skeletal Muscle-Derived Cells by Activating the HIF-1α/β-catenin Signaling Axis

Copper is an essential trace element for beef cattle, yet its muscle developmental regulatory mechanism remains unclear. This study investigated the effects of copper sulfate (CuSO₄) on myogenic differentiation of bovine skeletal muscle-derived cells (BSMC) and the underlying molecular mechanisms. BSMC were isolated from the longissimus thoracis muscle of three 30-month-old crossbred steers, with each steer defined as an independent experimental unit corresponding to a separate biological replicate ( n  = 3). Based on cell viability, CuSO₄ was used at 0, 0.1, 1, and 10 µM. All cell samples were harvested at differentiation day 6 (D6). The 0.1 µM CuSO₄ treatment during cell growth significantly promoted D6 myotube formation ( P  = 0.049), and upregulated mRNA and protein expression of myogenic differentiation 1 (MyoD) ( P  = 0.044, P  = 0.030), as well as protein expression of hypoxia-inducible factor-1α (HIF-1α) ( P  = 0.037) and β-catenin ( P  = 0.050). During differentiation, 1 µM CuSO₄ significantly facilitated D6 myotube formation ( P  = 0.0004), increased myogenic factor 5(Myf5) mRNA and protein expression ( P  = 0.030, P  = 0.006), and elevated HIF-1α ( P  = 0.025) and β-catenin ( P  = 0.004) protein levels, whereas Wnt10b protein expression remained unaltered ( P  = 0.930). Single or combined pharmacological inhibition of HIF-1α and β-catenin markedly reversed copper’s pro-myogenic effect on BSMC. In conclusion, copper modulates BSMC myogenic differentiation via the HIF-1α/β-catenin axis by enhancing β-catenin protein stability independent of Wnt10b transcription. HIF-1α acts upstream to drive β-catenin, and a reciprocal regulatory relationship may exist in which β-catenin in turn helps sustain HIF-1α abundance, thereby supporting cascade signaling and downstream myogenic transcription. This study elucidates copper’s molecular mechanism governing muscle development and provides theoretical support for precise copper supplementation to improve beef production performance.