Dynamic changes in the skeletal muscle proteome during denervation-induced atrophy

Abstract

Loss of neuronal stimulation enhances protein breakdown and reduces protein synthesis, causing rapid loss of muscle mass. To elucidate the pathophysiological adaptations that occur in atrophying muscles, we used stable isotope labelling and mass spectrometry to quantify protein expression changes accurately during denervation-induced atrophy after sciatic nerve section in the mouse gastrocnemius muscle. Additionally, mice were fed a stable isotope labelling of amino acids in cell culture (SILAC) diet containing 13C6-lysine for 4, 7 or 11 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 differentially expressed significantly within 2 weeks after denervation compared with 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, myomesins and titin, during denervation. Comparative analysis of these proteomic data sets with known atrogenes using a random forest approach identified 92 proteins subject to atrogene-like regulation that have not previously been associated directly with denervation-induced atrophy. Comparison of protein synthesis and proteomic data indicated that 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.

Skeletal Muscle-Specific Methyltransferase METTL21C Trimethylates p97 and Regulates Autophagy-Associated Protein Breakdown

“…Preparation of complex muscle lysates for proteomic analysis was performed as described previously (Shevchenko et al, 1996;Lang et al, 2017). Briefly, proteins were separated using 4%-12% Bis-Tris gels (Invitrogen).…”

“…Although activation of the UPS is primarily responsible for the degradation of 80%-90% of all proteins (Rock et al, 1994), it is not entirely clear how the autophagy system interacts with the UPS and contributes to overall remodeling during catabolic conditions (Lilienbaum, 2013). Previous studies showed that not all muscles are equally affected by atrophy and respond very differently, depending on their fiber type composition (Ciciliot et al, 2013;Lang et al, 2017;Schiaffino and Reggiani, 2011).…”

A mini review: Proteomics approaches to understand disused vs. exercised human skeletal muscle

“…Therefore, elucidating the mechanisms that 1) determine the molecular pathways altered by disuse, 2) determine basal muscle function and fitness (and thereby enable physical activity), and 3) mediate activityinduced skeletal muscle responses is important for finding new ways to target disuse muscle atrophy and metabolic deregulation. To understand molecular pathways regulated by disuse and exercise, multi-omics approaches have been employed in the field, such as genomics, transcriptomics, metabolomics, and proteomics (39,40,53,59,61). Given that protein expression and function are modulated by translation and posttrans-lational modifications, proteomics analysis provide a global snapshot of how the proteome is changed or altered in physiological and pathophysiological conditions.…”

Identification and Characterization of Fbxl22, a novel skeletal muscle atrophy-promoting E3 ubiquitin ligase

“…In comparison, MuRF1 and MAFbx expression remain elevated for a longer time period in neurogenic muscle atrophy models, returning to baseline after 14 days. (9,12,16,22,35). Thus, we hypothesized that Fbxl22 could be involved in the early initiation of the muscle atrophy process.…”