{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,11]],"date-time":"2026-10-11T10:46:12Z","timestamp":1791715572007,"version":"4.3.4"},"reference-count":43,"publisher":"Wiley","license":[{"start":{"date-parts":[[2026,10,11]],"date-time":"2026-10-11T00:00:00Z","timestamp":1791676800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,10,11]],"date-time":"2026-10-11T00:00:00Z","timestamp":1791676800000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/501100000038","name":"Natural Sciences and Engineering Research Council of Canada","doi-asserted-by":"publisher","award":["RGPIN\u20102023\u201005382"],"award-info":[{"award-number":["RGPIN\u20102023\u201005382"]}],"id":[{"id":"10.13039\/501100000038","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001804","name":"Canada Research Chairs","doi-asserted-by":"publisher","award":["950\u2010231201"],"award-info":[{"award-number":["950\u2010231201"]}],"id":[{"id":"10.13039\/501100001804","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["physoc.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Experimental Physiology"],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    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\u2010characterized. Stable isotope\u2010labelled 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\u2010induced 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\u00a0h of AA withdrawal microtubes and myotubes underwent 2\u00a0h of acute AA and\/or ketone treatment (\u00b1contraction in microtissues). In microtissues, sarcoplasmic FSR increased significantly during amino acid re\u2010addition (\n                    <jats:italic>P\u00a0<\/jats:italic>\n                    &lt;\u00a00.0001), whereas myofibrillar FSR displayed a main effect of condition (\n                    <jats:italic>P\u00a0=\u00a0<\/jats:italic>\n                    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\n                    <jats:italic>P<\/jats:italic>\n                    \u00a0\u2265\u00a00.985). 2D myotubes showed significant increases in sarcoplasmic and myofibrillar FSR in response to amino acid treatment (both\n                    <jats:italic>P<\/jats:italic>\n                    \u00a0\u2264\u00a00.0003). Notably, microtissues demonstrated a more stable myofibrillar protein fraction, with a sarcoplasmic\u2010to\u2010myofibrillar FSR ratio of \u223c2:1 which closely resembled that of native human muscle. The puromycin\u2010based approach only detected subtle differences in 2D myotubes (main effect\n                    <jats:italic>P\u00a0=\u00a0<\/jats:italic>\n                    0.031) with AA provision being greater than ketones (\n                    <jats:italic>P\u00a0=\u00a0<\/jats:italic>\n                    0.0196) with no other\n                    <jats:italic>post hoc<\/jats:italic>\n                    differences (all\n                    <jats:italic>P<\/jats:italic>\n                    \u00a0\u2265\u00a00.221). Puromycin failed to detect MPS responses to any stimulus in microtissues (all\n                    <jats:italic>P<\/jats:italic>\n                    \u00a0\u2265\u00a00.834), highlighting the greater sensitivity of tracer\u2010based measurements, particularly where longer time scales are needed to capture an effect. These findings support the use of engineered muscle and isotope\u2010derived measurements of MPS in future studies of stimuli regulating skeletal muscle mass.\n                  <\/jats:p>","DOI":"10.1113\/ep094100","type":"journal-article","created":{"date-parts":[[2026,10,11]],"date-time":"2026-10-11T09:49:32Z","timestamp":1791712172000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Stable isotope\u2010derived measurements of protein synthesis detect amino acid\u2010induced anabolism in skeletal muscle microtissues not captured by puromycin labelling"],"prefix":"10.1113","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-6783-7652","authenticated-orcid":false,"given":"Yekaterina","family":"Tiper","sequence":"first","affiliation":[{"name":"Institute of Biomedical Engineering University of Toronto  Toronto Ontario Canada"},{"name":"Donnelly Centre University of Toronto  Toronto Ontario Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9367-1852","authenticated-orcid":false,"given":"Cassidy T.","family":"Tinline\u2010Goodfellow","sequence":"additional","affiliation":[{"name":"Faculty of Kinesiology and Physical Education University of Toronto  Toronto Ontario Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5509-9616","authenticated-orcid":false,"given":"Penney M.","family":"Gilbert","sequence":"additional","affiliation":[{"name":"Institute of Biomedical Engineering University of Toronto  Toronto Ontario Canada"},{"name":"Donnelly Centre University of Toronto  Toronto Ontario Canada"},{"name":"Department of Cell and Systems Biology University of Toronto  Toronto Ontario Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5865-4398","authenticated-orcid":false,"given":"Daniel R.","family":"Moore","sequence":"additional","affiliation":[{"name":"Faculty of Kinesiology and Physical Education University of Toronto  Toronto Ontario Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,10,11]]},"reference":[{"issue":"11","key":"e_1_2_9_2_1","doi-asserted-by":"crossref","first-page":"3350","DOI":"10.1093\/jn\/nxab261","article-title":"Incorporation of Dietary Amino Acids Into Myofibrillar and Sarcoplasmic Proteins in Free\u2010Living Adults Is Influenced by Sex, Resistance Exercise, and Training Status","volume":"151","author":"Abou Sawan S.","year":"2021","journal-title":"Journal of Nutrition"},{"issue":"9","key":"e_1_2_9_3_1","doi-asserted-by":"crossref","first-page":"2381","DOI":"10.1074\/mcp.E113.031658","article-title":"Western blots versus selected reaction monitoring assays: Time to turn the tables?","volume":"12","author":"Aebersold R.","year":"2013","journal-title":"Molecular & Cellular Proteomics"},{"issue":"1","key":"e_1_2_9_4_1","doi-asserted-by":"crossref","first-page":"6918","DOI":"10.1038\/s41598-020-62837-8","article-title":"A 96\u2010well culture platform enables longitudinal analyses of engineered human skeletal muscle microtissue strength","volume":"10","author":"Afshar M. 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R.","year":"2009","journal-title":"The Journal of Physiology"},{"issue":"11","key":"e_1_2_9_36_1","doi-asserted-by":"crossref","first-page":"1009","DOI":"10.1038\/ncb1101-1009","article-title":"Mediation of IGF\u20101\u2010induced skeletal myotube hypertrophy by PI(3)K\/Akt\/mTOR and PI(3)K\/Akt\/GSK3 pathways","volume":"3","author":"Rommel C.","year":"2001","journal-title":"Nature Cell Biology"},{"issue":"4","key":"e_1_2_9_37_1","doi-asserted-by":"crossref","first-page":"275","DOI":"10.1038\/nmeth.1314","article-title":"SUnSET, a nonradioactive method to monitor protein synthesis","volume":"6","author":"Schmidt E. K.","year":"2009","journal-title":"Nature Methods"},{"issue":"11","key":"e_1_2_9_38_1","doi-asserted-by":"crossref","first-page":"1405","DOI":"10.1113\/EP086581","article-title":"Electrical pulse stimulation: An in vitro exercise model for the induction of human skeletal muscle cell hypertrophy. 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