Indexed on: 27 May '17Published on: 27 May '17Published in: Disease models & mechanisms
Loss of neuronal stimulation enhances protein breakdown and reduces protein synthesis, causing rapid muscle mass loss. To elucidate the pathophysiological adaptations that occur in atrophying muscles, we used stable isotope labelling and mass spectrometry to accurately quantify protein expression changes during denervation-induced atrophy after sciatic nerve section in the mouse gastrocnemius muscle (GAST). Additionally, mice were fed a SILAC diet containing (13)C6 lysine for four, seven, or eleven days to calculate relative levels of protein synthesis in denervated and control muscles. Ubiquitin remnant peptides (K-ϵ-GG) were profiled by immunoaffinity enrichment to identify potential substrates of the ubiquitin proteasomal pathway. Of the 4279 skeletal muscle proteins quantified, 850 were significantly differentially expressed within two weeks after denervation compared to control muscles. Moreover, pulse-labelling identified Lys6 incorporation in 4786 proteins of which 43 had differential Lys6 incorporation between control and denervated muscle. Enrichment of diglycine remnants identified 2100 endogenous ubiquitination sites and revealed a metabolic and myofibrillar protein diglycine signature, including myosin heavy chains (MyHC), myomesins and titin, during denervation. Comparative analysis of these proteomic datasets with known atrogenes using a random forest approach identified 92 proteins subject to atrogene-like regulation that have not previously been directly associated with denervation-induced atrophy. Comparison of protein synthesis and proteomic data indicated upregulation of specific proteins in response to denervation is mainly achieved by protein stabilization. This study provides the first integrated analysis of protein expression, synthesis and ubiquitin signatures during muscular atrophy in a living animal.