| BackgroundSkeletal muscle comprises about 40% of the mass of the human body and is involved in many functions necessary for maintaining a healthy life including force generation and locomotion, heat production. Clinically, loss of skeletal muscle mass occurs under conditions of inactivity and unloading. A common cause of skeletal muscle atrophy is disuse, which means a combination of decreased loading and decreased neural activation. Hibernation is an important survival strategy for many animal species involving prolonged periods of inactivity, unloading and starvation; however, remarkably loss of skeletal muscle mass is minimal. The ability of hibernating animals to maintain muscle mass under conditions of prolonged disuse could provide valuable insights into strategies to prevent muscle atrophy in humans. Our previous studies have shown that anti-atrophy mechanisms of fast twitch and slow twitch muscles may be different rather than homogeneous. The specific ways explored for each muscle to avoid atrophy are unrevealed. And currently there are no researches done from this perspective, so in the present study daurian ground squirrels (Spermophilus dauricus), a common hibernating species in the north, were subjected to our investigation. Ultrastructure of two skeletal muscles were detected by transmission electron microscopy and high-throughput differential protein profiles of slow twitch muscles (soleus, SOL) and fast twitch muscle (extensor digitorum longus, EDL) in different periods were obtained by iTRAQ proteomics methods. Bioinformatics was explored to analyze the proteomics data and construction regulatory networks models for two muscles to maintain muscle mass.FocusThe subjects were divided into six groups including slow twitch and fast twitch muscles of pre-hibernation group, hibernating group and post-hibernation group of daurian ground squirrels. Soleus muscle was taken as slow twitch muscle and extensor digitorum longus for fast twitch muscle.Section 1Ultrastructural changes in skeletal muscles were investigated by transmission electron microscopy. During hibernation, it was found that the ultrastructure was kept intact for both muscles. And no degradation of myofibrils was detected in muscle fibers. Mitochondria kept their crista and membrane intact but the number of sub-sarcolemmal mitochondria increased. Number of myofibril glycogen increased in SOL and decreased in EDL. In the post-hibernation group, both muscles have degenerative changes with disordered muscle fibers. Besides mitochondria orientation was found deranged in EDL.Section 2iTRAQ LC-MS/MS was applied to investigate proteomics profiles of slow twitch muscle and fast twitch muscle for ground squirrels. A total of 2059 proteins were identified. Compared with pre-hibernation, proteins in hibernating group partly significantly changed. The slow twitch muscle has a total of 170 different proteins with 96 increased and74 decreased; the last twitch muscle has a total of 264 different proteins with 203 increased and 61 decreased. When compared with hibernating group, proteins in post-hibernation also partly changed. The slow twitch muscle has a total of 273 different proteins with 216 increased and57 decreased; the fast twitch muscle has a total of 188 different proteins with 121 increased and 67 decreased.355 proteins of metabolic pathway accounted for 19.7% with the highest proportion in all identified proteins. Analysis of Cluster of Orthofogous Groups of proteins indicates that proteins with function of post translation modification and protein turnover take the second place except general function only proteins.Section 3Bioinformatics was explored to analyze the proteomics data and construction regulatory networks models for two muscles to maintain muscle mass.Functional armotation clustering of gene ontology was implemented by using DAVID tools. And the results indicated that during hibernation, the most enriched terms are related to mitochondrial and proteasome in SOL and mitochondrial and protein synthesis in EDL respectively. In the post-hibernation group, the most enriched terms are related to cytoskeleton, mitochondrial, protein synthesis, cell membrane target proteins, oxidative stress in SOL and myofibrils and the mitochondria in EDL respectively.Pathway enrichment analysis showed that oxidative phosphorylation, proteasome and PPAR signaling pathways were significantly enriched in SOL while oxidative phosphorylation, protein synthesis and degradation, contractile protein were significantly enriched in EDL. In the post-hibernation group, Complement and coagulation cascades, ribosome, muscle contraction pathways were significantly enriched in SOL while oxidative phosphorylation and impairment of axonal transport were significantly enriched in EDL.Hierarchical analysis of significant enriched terms indicated that carbohydrate and branched chain fatty acid metabolism, positive regulation and negative regulation of ubiquitin protein ligase activity were most significant child terms in SOL while respiratory electron transfer chain, oxidative phosphorylation and positive regulation of ubiquitin protein ligase activity were most significant child terms in EDL during hibernation. In the post-hibernation groups, blood coagulation, extracellular matrix organization and hydrogen peroxide metabolism were most significant child terms in SOL while respiratory electron transfer chain, oxidative phosphorylation, hydrogen peroxide metabolism and glutathione metabolism were most significant child terms in EDL?Function evaluation of energy metabolism and protein metabolism was achieved IPA software. During hibernation, glycolysis and oxidative phosphorylation is constant but fatty acid metabolism, protein breakdown and synthesis decreased in SOL. Meanwhile, EDL was with decreased glycolysis, unchanged oxidative phosphorylation and increased ability of fatty acid metabolism, protein breakdown and synthesis. In the post-hibernation group, only glycolysis decreased, while oxidative phosphorylation, fatty acid metabolism, protein breakdown and synthesis were all increased in SOL. In EDL, glycolysis, protein breakdown and synthesis were increased while oxidative phosphorylation and fatty acid metabolism decreased.Reactome analysis showed overrepresented functions were signal transduction and DNA replication in SOL and respiratory electron transfer chain and DNA synthesis of cell cycle in EDL during hibernation. In the post-hibernation group, platelet aggregation induced by integrin alpha ?b beta 3 signal was overrepresented in SOL and gluconeogenesis pathways was overrepresented in EDL.The protein interaction network indicated that co-expression accounted for more than 50% among all the protein relationships in each period, and the direct physical interactions were different according to the network. During hibernation, network of SOL was composed with 57.32% co-expression and 37.64% physical interaction while EDL was composed of 73.03% co-expression and 6.29% physical interaction. In the post-hibernation group, network of SOL was composed with 61.43% co-expression and 19.66% physical interaction while EDL was composed with 64.71% co-expression and 10.04% physical interaction.Upstream regulators of protein networks were predicated and were used to construct models for muscles to avoid atrophy. The results showed that the TGFB1 signaling pathway was inhibited which increases protein degradation in SOL, and meanwhile the IGF1R signaling pathway was also inhibited which stimulates protein synthesis and inhibits protein degradation. In EDL, TGFB1 signaling pathway is activated and its inhibitors calcineurin and RICTOR pathway were suppressed. Besides, insulin signaling pathway was activated which could enhance protein synthesis.ConclusionsChanges of structure and proteome in post-hibernation are not simply reverse processes of hibernation. Remodeling of cytoskeleton occurred after hibernation while structure of myfilament maintains integrity during hibernation. Besides, biological processes related to exercise stress come up rather than the same biological process-in hibernation. Energy and protein metabolisms of post-hibernation are more similar to training rather than a reversal of hibernation.During hibernation, the stress responses of the slow twitch muscle and the fast twitch muscle are different. Although both the slow twitch and fast twitch muscle are avoided from disuse atrophy, they achieve the balance of protein metabolism through different regulating pathways. TGFB1 signaling pathway was inhibited and it causes a decrease of protein degradation in SOL, and meanwhile the IGF1R signaling pathway was also inhibited which induced a reduction of protein synthesis. In EDL, TGFB1 signaling pathway is activated and its inhibitors calcineurin and RICTOR pathway were suppressed. That would induce an increase of protein degradation. However, insulin signaling pathway was activated which could enhance protein synthesis. |
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