Determining the contributions of protein synthesis and breakdown to muscle atrophy requires non‐steady‐state equations

Background Ageing and cachexia cause a loss of muscle mass over time, indicating that protein breakdown exceeds protein synthesis. Deuterium oxide (D2O) is used for studies of protein turnover because of the advantages of long‐term 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‐steady state, during D2O labelling studies while also exploring the contribution of protein synthesis and breakdown to denervation‐induced muscle atrophy. Methods Adult (6 months) male C57BL/6 mice (n = 14) were labelled with D2O for a total of 7 days following unilateral sciatic nerve transection to induce denervation of hindlimb muscles. The contralateral sham limb and nonsurgical mice (n = 5) 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‐state or using non‐steady‐state modelling. We measured RNA synthesis rates to further understand ribosomal turnover during atrophy. Results Gastrocnemius mass was less in denervated muscle (137 ± 9 mg) compared with sham (174 ± 15 mg; P