Stable isotope‐derived measurements of protein synthesis detect amino acid‐induced anabolism in skeletal muscle microtissues not captured by puromycin labelling

Abstract Skeletal muscle microtissues are valuable in vitro models for studying the stimuli regulating muscle protein synthesis (MPS), the key determinant of changes in muscle mass. Differentiated between opposing posts, microtissues contain aligned, contractile myotubes, providing a controlled system for investigating the responses of skeletal muscle to nutrient and contractile stimulation. However, microtissue MPS responses to these stimuli remain under‐characterized. Stable isotope‐labelled amino acid tracers deliver sarcoplasmic and myofibrillar fractional synthetic rates (FSR) for MPS in human studies but have not been implemented in engineered skeletal muscle. We close these gaps by characterizing amino acid (AA), ketone and contraction‐induced MPS in 3D microtissues and 2D myotubes (without contraction) derived from the same primary myoblast line, using stable isotope tracers and puromycin incorporation. Following 4 h of AA withdrawal microtubes and myotubes underwent 2 h of acute AA and/or ketone treatment (±contraction in microtissues). In microtissues, sarcoplasmic FSR increased significantly during amino acid re‐addition ( P  P =  0.0018), with an overall lesser magnitude of change compared to the sarcoplasmic fraction. However, both fractions were unresponsive to ketone stimulation, and the selected contraction regime (when combined with ketones and/or AA; all P  ≥ 0.985). 2D myotubes showed significant increases in sarcoplasmic and myofibrillar FSR in response to amino acid treatment (both P  ≤ 0.0003). Notably, microtissues demonstrated a more stable myofibrillar protein fraction, with a sarcoplasmic‐to‐myofibrillar FSR ratio of ∼2:1 which closely resembled that of native human muscle. The puromycin‐based approach only detected subtle differences in 2D myotubes (main effect P =  0.031) with AA provision being greater than ketones ( P =  0.0196) with no other post hoc differences (all P  ≥ 0.221). Puromycin failed to detect MPS responses to any stimulus in microtissues (all P  ≥ 0.834), highlighting the greater sensitivity of tracer‐based measurements, particularly where longer time scales are needed to capture an effect. These findings support the use of engineered muscle and isotope‐derived measurements of MPS in future studies of stimuli regulating skeletal muscle mass.