Myostatin Function during In Vitro Myogenesis: Considerations for Knockout-Based Mechanistic Analysis.

BACKGROUND: Myostatin (MSTN) is a well-known negative regulator of skeletal muscle growth, and its pharmacological blockade, such as with follistatin (FST), an endogenous MSTN inhibitor, is under active investigation as a treatment for muscle-wasting conditions. However, the dynamics of MSTN signaling during in vitro myogenesis and its modulation by culture conditions remain incompletely understood. METHODS: Primary myoblasts were isolated from wild-type (WT), Mstn-/-, and FST transgenic (F66) mice to evaluate the effects of MSTN inhibition on myotube formation. Myoblasts were differentiated on Matrigel-coated surfaces in the presence of horse serum. Myotube maturation was evaluated by confocal microscopy and reverse transcription-polymerase chain reaction; protein synthesis was assessed using a puromycin incorporation assay; and MSTN concentrations in serum were quantified by enzyme-linked immunosorbent assay. RESULTS: Mstn expression peaked at 24 hr of differentiation, coinciding with early myotube formation, and progressively declined as myotubes matured. Both Mstn-/- and F66 myotubes showed increased thickness and protein synthesis compared to WT controls, with more pronounced effects observed in F66 myotubes. Consistent with this, F66 myotubes also displayed higher expression of myogenic maturation markers. Notably, horse serum used in the culture medium contained detectable levels of MSTN, which may have partially restored MSTN signaling in Mstn-/- cultures and masked the full knockout phenotype. CONCLUSIONS: FST overexpression induces greater myotube hypertrophy and protein synthesis than Mstn deletion, likely due to its broader inhibition of both endogenous and serum-derived MSTN. These findings highlight the importance of serum composition in interpreting phenotypes from in vitro knockout models targeting secreted factors such as MSTN.