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 < 0.0001) or nonsurgical control (162 +/- 5 mg; P < 0.0001). With steady-state calculations, fractional synthesis and breakdown rates (FSR and FBR) were lower in the denervated muscle (1.49 +/- 0.06%/day) compared with sham (1.81 +/- 0.09%/day; P < 0.0001) or nonsurgical control (2.27 +/- 0.04%/day; P < 0.0001). When adjusting for change in protein mass, FSR was 4.21 +/- 0.19%/day in denervated limb, whereas FBR was 4.09 +/- 0.22%/day. When considering change in protein mass (k(syn)), myofibrillar synthesis was lower in denervated limb (2.44 +/- 0.14 mg/day) compared with sham (3.43 +/- 0.22 mg/day; P < 0.0001) and non-surgical control (3.74 +/- 0.12 mg/day; P < 0.0001), whereas rate of protein breakdown (k(deg,) 1/t) was greater in denervated limb (0.050 +/- 0.003) compared with sham (0.019 +/- 0.001; P < 0.0001) and nonsurgical control (0.023 +/- 0.000; P < 0.0001). Muscle collagen breakdown was completely inhibited during denervation. There was a strong correlation (r = 0.83, P < 0.001) between RNA and myofibrillar protein synthesis in sham but not denervated muscle. Conclusions We show conflicting results between steady- and non-steady-state 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-surgical 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.

Automated summary

This study demonstrated the importance of accounting for a change in protein mass during D2O labelling studies, while also exploring the contribution of protein synthesis and breakdown to denervation-induced muscle atrophy. Conflicting results were found between steady- and non-steady-state calculations on myofibrillar protein synthesis and breakdown during periods of muscle loss. Additionally, collagen accumulation was largely from a decrease in collagen breakdown during denervation.