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Deuterium oxide (D<jats:sub>2<\/jats:sub>O) is used for studies of protein turnover because of the advantages of long\u2010term labelling, but these methods introduce considerations that have been largely overlooked when studying conditions of protein gain or loss. The purpose of this study was to demonstrate the importance of accounting for a change in protein mass, a non\u2010steady state, during D<jats:sub>2<\/jats:sub>O labelling studies while also exploring the contribution of protein synthesis and breakdown to denervation\u2010induced muscle atrophy.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>Adult (6\u00a0months) male C57BL\/6 mice (<jats:italic>n<\/jats:italic>\u00a0=\u00a014) were labelled with D<jats:sub>2<\/jats:sub>O for a total of 7\u00a0days following unilateral sciatic nerve transection to induce denervation of hindlimb muscles. The contralateral sham limb and nonsurgical mice (<jats:italic>n<\/jats:italic>\u00a0=\u00a05) were used as two different controls to account for potential crossover effects of denervation. We calculated gastrocnemius myofibrillar and collagen protein synthesis and breakdown assuming steady\u2010state or using non\u2010steady\u2010state modelling. We measured RNA synthesis rates to further understand ribosomal turnover during atrophy.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Gastrocnemius mass was less in denervated muscle (137\u00a0\u00b1\u00a09\u00a0mg) compared with sham (174\u00a0\u00b1\u00a015\u00a0mg;<jats:italic>P<\/jats:italic>\u00a0&lt;\u00a00.0001) or nonsurgical control (162\u00a0\u00b1\u00a05\u00a0mg;<jats:italic>P<\/jats:italic>\u00a0&lt;\u00a00.0001). With steady\u2010state calculations, fractional synthesis and breakdown rates (FSR and FBR) were lower in the denervated muscle (1.49\u00a0\u00b1\u00a00.06%\/day) compared with sham (1.81\u00a0\u00b1\u00a00.09%\/day;<jats:italic>P<\/jats:italic>\u00a0&lt;\u00a00.0001) or nonsurgical control (2.27\u00a0\u00b1\u00a00.04%\/day;<jats:italic>P<\/jats:italic>\u00a0&lt;\u00a00.0001). When adjusting for change in protein mass, FSR was 4.21\u00a0\u00b1\u00a00.19%\/day in denervated limb, whereas FBR was 4.09\u00a0\u00b1\u00a00.22%\/day. When considering change in protein mass (k<jats:sub>syn<\/jats:sub>), myofibrillar synthesis was lower in denervated limb (2.44\u00a0\u00b1\u00a00.14\u00a0mg\/day) compared with sham (3.43\u00a0\u00b1\u00a00.22\u00a0mg\/day;<jats:italic>P<\/jats:italic>\u00a0&lt;\u00a00.0001) and non\u2010surgical control (3.74\u00a0\u00b1\u00a00.12\u00a0mg\/day;<jats:italic>P<\/jats:italic>\u00a0&lt;\u00a00.0001), whereas rate of protein breakdown (k<jats:sub>deg,<\/jats:sub>1\/t) was greater in denervated limb (0.050\u00a0\u00b1\u00a00.003) compared with sham (0.019\u00a0\u00b1\u00a00.001;<jats:italic>P<\/jats:italic>\u00a0&lt;\u00a00.0001) and nonsurgical control (0.023\u00a0\u00b1\u00a00.000;<jats:italic>P<\/jats:italic>\u00a0&lt;\u00a00.0001). Muscle collagen breakdown was completely inhibited during denervation. There was a strong correlation (<jats:italic>r<\/jats:italic>\u00a0=\u00a00.83,<jats:italic>P<\/jats:italic>\u00a0&lt;\u00a00.001) between RNA and myofibrillar protein synthesis in sham but not denervated muscle.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions<\/jats:title><jats:p>We show conflicting results between steady\u2010 and non\u2010steady\u2010state calculations on myofibrillar protein synthesis and breakdown during periods of muscle loss. We also found that collagen accumulation was largely from a decrease in collagen breakdown. Comparison between sham and non\u2010surgical control demonstrated a crossover effect of denervation on myofibrillar protein synthesis and ribosomal biogenesis, which impacts study design for unilateral atrophy studies. These considerations are important because not accounting for them can mislead therapeutic attempts to maintain muscle mass.<\/jats:p><\/jats:sec>","DOI":"10.1002\/jcsm.12772","type":"journal-article","created":{"date-parts":[[2021,8,21]],"date-time":"2021-08-21T19:32:43Z","timestamp":1629574363000},"page":"1764-1775","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":28,"title":["Determining the contributions of protein synthesis and breakdown to muscle atrophy requires non\u2010steady\u2010state equations"],"prefix":"10.1002","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1804-6333","authenticated-orcid":false,"given":"Kamil A.","family":"Kobak","sequence":"first","affiliation":[{"name":"Aging and Metabolism Research Program Oklahoma Medical Research Foundation Oklahoma City OK USA"},{"name":"Laboratory for Applied Research on Cardiovascular System, Department of Heart Diseases Wroclaw Medical University 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