Establishment and characterization of an equine skeletal muscle in vitro platform: gene expression validation and hypoxia-responsive signatures

Background In response to the increasing interest in equine regenerative medicine and translational nutrigenomics, we sought to establish an in vitro platform for studying skeletal muscle biology in horses. The study focused on generating a primary equine skeletal muscle cell line, confirming its myogenic identity through qPCR analysis of the markers MYOD1 , MYF5 , MYOG , and PAX7 , identifying and validating suitable endogenous reference genes for normalization, and assessing cellular responses to normoxia and hypoxia based on the expression profile of HIF1A , a master regulator reflecting oxygen availability in working muscle. Methods Primary equine skeletal muscle cells were isolated using collagenase and pronase treatment or the explant method and cultured under standard conditions. Gene expression was assessed by qPCR, including the evaluation of housekeeping genes for normalization. Hypoxic cultures were subsequently maintained at 3% O₂ in a HypoxyLab incubator, while normoxic controls were kept at 37°C with 5% CO₂. All cultures were handled in parallel under otherwise identical conditions. Results Collagenase digestion provided the most viable myogenic cells, whereas pronase and explant methods yielded less suitable populations. Among eight candidate reference genes evaluated with ΔCt, BestKeeper, NormFinder, and geNorm, ACTB emerged as the most stable, followed by RN18S , SDHA , and GAPDH . In contrast, B2M and TFRC showed the lowest stability and were deemed unsuitable for normalization. Conclusions Collagenase digestion is the most suitable method for establishing equine myogenic cell cultures, with ACTB as the optimal endogenous control. Furthermore, these findings suggest that hypoxia does not markedly affect myogenic progression, thus it may not be a favourable condition for modelling accelerated muscle regeneration or stress-induced differentiation in high-performance animals such as racing horses.