Stable isotope tracer captures the anabolic response of human skeletal muscle microtissues undetected by puromycin labeling

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-labeled 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 stimulation-induced MPS, in microtissues and 2D myotubes derived from the same primary myoblast line, using stable isotope tracers and puromycin incorporation. In microtissues, sarcoplasmic FSR increased significantly during the two-hour period following amino acid treatment (p < 0.0001), whereas myofibrillar FSR remained unchanged (p = 0.159). However, both fractions were unresponsive to ketone stimulation and contraction (all p [&ge;] 0.703). 2D myotubes showed significant increases in sarcoplasmic and myofibrillar FSR in response to amino acid treatment (both p = 0.002). 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 failed to detect MPS responses to any stimulus (all p [&ge;] 0.677), highlighting the superior sensitivity of tracer-based measurements, particularly where longer timescales 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. New FindingsO_ST_ABSWhat is the central question of this study?C_ST_ABSStable isotope tracers are emerging as a powerful approach to measure fraction-specific protein synthesis. However, their efficacy relative to conventional puromycin labeling remains unreported, and they have not been applied to engineered skeletal muscle. What is the main finding and its importance?By implementing stable isotope tracers in engineered muscle, we showcase the ability to capture anabolic responses that are undetected by puromycin-based methods. We found that the myofibrillar protein fraction of microtissues is more stable than the sarcoplasmic fraction, a property of native muscle, absent in 2D myotubes. These findings demonstrate the physiological relevance of engineered muscle and support the adoption of isotope-derived measurements in future studies.