| Disuse muscle atrophy is one of the urgent clinical problems that remains unsolved. Even though substantial research has been conducted, few effective approaches to prevent and treat disuse muscle atrophy have been found up to date due to lack of understanding about the molecular mechanisms of the disease. Hibernation, often lasting for several months, is a unique survival strategy adopted by hibernators combating adverse factors like low temperature and limited food availability. Interestingly, after experiencing over-extended hibernation inactivity, there are no signs of atrophy of the skeletal muscles in hibernators. In this regard, hibernators can be used as a natural model for the study of the protective ability against disuse muscle atrophy. Elucidating the mechanisms underlying this unique adaptation in hibernators' skeletal muscles is one of the urgent important topics in the field of physiological ecology, and is enligntening in the fields of aerospace medicine, clinical medicine, sports medicine and rehabilitation medicine.Protein glycosylation is the enzymatic addition of sugars or oligosaccharides to proteins and one of the most common post-translational protein modifications. It is estimated that as many as about 70% of all human proteins are modified with a diversity of glycan structures. Increasing evidences suggest that a variety of pathological related muscle atrophies are intimately linked to protein glycosylation. As one of the glycosylational approaches, O-acetylglycosamine (O-GlcNAc) can regulate disuse muscle atrophy through generating myoprotein. The decrease of sialylation level is a critical factor causing degradation and weakening of myofiber, and loss of muscle mass. Additionally, a wide spectrum of muscular disorders (e.g. distal myopathy with rimmed vacuoles, DMRV, congenital muscular dystrophy, CMD) that cause muscle atrophy or dysfunction are closely correlated with abnormal protein glycosylation states in muscle. We used Daurian ground squirrels (Spermophilus dauricus) as the subjects concerned in this study, and chose the typical slow muscle soleus (SOL) as a representative. This is the first attempt to explore the glycoproteomics of the SOL of Daurian ground squirrels in different hibernation states, and to elucidate the potential mechanism underlying the anti-diuse muscle atrophy in hibernation animals from a novel perspective.Part 1:We applied lectin microarrays to investigate the precision alteration of protein glycosylationglycoyation of SOL muscles from ground squirrels in different periods of hibernation which is validated by lectin histochemistry using the lectin microarrays. The analysis of the lectin microarray revealed that the fluorescence intensities of eight lectins were significantly changed, including MAL-II, PTL-??WFA. GSL-??PTL-??DSA? EEL and PHA-E+L. The results showed that 1) compared with the Pre-hibernation (PRE) group, SA (sialic acid)a2-3Gal (galactose) recognized specifically by lectin MAL-II was significantly decreased in the Hibernation (HIB) group, but was significantly increased and recovered to the level of the Pre-hibernation state in both the Interbout arousal (IBA) and the Post-hibernation (POST) groups; 2) the change in the levels of aGalNAc and Gal recognized by PTL-I was similar to that of SAa2-3Gal, that is, decrease in the HIB group, and increase in the IBA and the POST groups; 3) compared with the HIB group, Terminal GalNAc recognized by WFA was significantly increased in the IBA and the POST groups; 4) compared with the other three groups, aGalNAc and Gal recognized by GSL-I and PTL-II were significantly increased in the IBA group; 5) compared with the PRE group, GlcNAc recognized by DSA was significantly increased in the HIB group, but was significantly increased and recovered to the level of the Pre-hibernation state in the IBA and the POST groups; 6) compared with the other three groups, Gala 1-3 (Fucal-2) Gal recognized by lectin EEL was increased in the HIB group; and 7) compared with the PRE group, Tri-and tetra-antennary complex-type N-glycan recognized by PHA-E+L were significantly increased in the in the HIB, the IBA and the POST groups. We selected four lectins (MAL-II, PTL-I, DSA and PHA-E+L) for lectin histochemistry. The results of lectin histochemistry were consistent with those from the lectin microarrays, validating the conclusion of the lectin microarrays. Overall, our results suggest that in the course of periodic interbout arousal of ground squirrels, marked rise in the level of SAa2-3Gal effectively offset its decrease in hibernation, which is critical for preventing their skeletal muscles disuse atrophy. Since O-glycans are the common sugar chains identified by the four lectins (PTL-I, WFA, GSL-I, PTL-?), we speculate that the increase of O-glycans at interbout arousal might be involved in the innate protective mechanisms of the hibernating ground squirrels. Moreover, the levels of tri-and tetra-antennary N-glycans were increased dramatically, which might be associated with the mechanism of physiological adaptation to hibernation disuse in ground squirrels skeletal muscles.Part2:Chapter2 indicates that in the course of periodic interbout arousal of ground squirrels, marked rise in the level of SA?2-3Gal effectively offset its decrease in hibernation, while normal content of SA plays an important role in maintaining morphology, structure and functions of skeletal muscles. Therefore, The purpose of this part is to analyze the mechanisms of the altered levels of SAa2-3Ga at the mRNA level of glycan-related genes in different periods of hibernation by determining the alterations of the levels of the mRNA expressions of sialyltransferasebeta-galactoside alpha-2,3-sialyltransferases (including ST3Gall, ST3Gal2, ST3Gal3, ST3Gal4, ST3Gal5, ST3Gal6) and sialidases (including NEU1, NEU2, NEU3, NEU4). Compared with the PRE group, ST3GallmRNA expression was significantly decreased in the HIB group (-55.00%; P<0.01); compared with the HIB group, ST3Gall mRNA expression was significantly increased in the IB A and the POST groups (+114.30% and+126.20%, respectively, P<0.01); there were no statistical differences among the PRE, the IBA and the POST groups (P>0.05). Compared with the PRE group, ST3Gal2 mRNA expression was significantly decreased in the HIB group (-42.70%; P<0.01); compared with the HIB group, ST3Gal2 mRNA expression was significantly increased in the IBA and the POST groups (+77.30% and +76.50%, respectively, P<0.01); there were no statistical difference among the PRE, the IBA and the POST groups (P>0.05). Compared with the PRE group, ST3Gal5 mRNA expression was significantly decreased in the HIB group (-60.91%; P<0.01); compared with the HIB group, ST3Gal5 mRNA expression was significantly increased in the IBA and the POST groups (+160.56% and +125.48% respectively, P<0.01); there were no statistical difference among the PRE, the IBA and the POST groups (P>0.05). There were no statistical differences in NEU1, NEU and ST3Gal6 mRNA expressions between four groups (PRE, HIB, IBA and POST groups) (P>0.05). Our results suggest that the changes in mRNA expressions of ST3Gall, ST3Gal2 and ST3Gal5 might be the major factor that leads to the change in SAa2-3Gal of ground squirrels SOL during hibernation.Part3:Chapter 2 indicates that during hibernation, the marked change of SAa2-3Gal, which is specifically identified by the Lectin MAL-II in soleus muscle of the ground squirrels, may play a key role in anti-disuse muscle atrophy. Accordingly, in this part, lectin MAL-II magnetic particles composites were synthesized and used to extract the glycoproteins with the terminal of SAa2-3Gal. The isolated glycoproteins were identified by LC-ESI-MS/MS and further analyzed by bio informatics methods.227 glycoproteins from the PRE group and 185 glycoproteins from the HIB group were identified.141 glycoproteins were exclusively identified from the PRE group, while 99 glycoproteins were exclusively identified from the HIB groups. There was an overlap of 86 between the two groups. Gene Ontology (GO) function analysis shows the following results:according to cellular component (CC) annotations, the identified glycoproteins were mainly intracellular proteins and macromolecular complexes, and mainly distributed in organelle and membrane; according to molecular function (MF) annotations, the major molecular functions of the glycoproteins included binding, catalytic activity and structural molecule activity; according to biological process (MP) annotations, the biological processes of the glycoproteins were mainly involved in cellular process, single-organism process, metabolic process and biological regulation. Enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway revealed that 40 pathways and 36 pathways were significantly enriched in the PRE and HIB groups, respectively; 14 pathways were only significantly enriched in the PRE group, while 10 pathways were only significantly enriched in the HIB group. Following the analysis of the protein-protein interaction using the IntAct database, the protein-protein interaction networks (PPI network) of the glycoproteins from the PRE and HIB groups were built by Cytoscape software. In addition, the nine core proteins with a degree greater than 30 were screened.Part4:We selected two glycoproteins for validation, including heat shock cognate 70 (HSC70) and pyruvate kinase (PK). We investigated the changes in the protein expressions and SAa2-3Gal structures of the two glycoproteins during hibernation, respectively. In addition, we measured the changes in the protein expression of 14-3-3 protein epsilon (?) in SOL of ground squirrels during hibernation to study its potential role in preventing disuse muscle atrophy. Our results showed that compared with the PRE group, the protein expression of HSC70 was significantly decreased in the HIB group (-37.39%, P<0.01); compared with HIB group, the protein expressions of HSC70 were significantly increased in the IBA and the POST groups (the IBA:+51.39%, P<0.01; the POST:+41.67%, P<0.01); there were no statistical differences among the PRE, the IBA and the POST groups (P>0.05). The protein expression of pyruvate kinase did not statistically differ between four groups (PRE, HIB, IBA and POST groups) (P>0.05). Compared with the PRE group, the protein expression of 14-3-3 epsilon was significantly decreased in the HIB group (-42.59%, P<0.01); compared with HIB group, the protein expressions of 14-3-3 epsilon were significantly increased in the IBA and the POST groups (the IBA:+51.61%, P<0.01; the POST:+58.06%, P<0.01); there were no statistical differences among the PRE, the IBA and the POST groups (P>0.05). Compared with the PRE group, the SAo2-3Gal structures of HSC70 was significantly decreased in the HIB group (-30.08%, P<0.01); compared with HIB group, the SAa2-3Gal structures of HSC70 were significantly increased in the IBA and the POST groups (the IBA:+30.91%, P<0.05; the POST:+34.41%, P<0.05); there were no statistical differences among the PRE, the IBA and the POST groups (P>0.05). The SA?2-3Gal structures in pyruvate kinase was significantly decreased in the HIB group (-30.08%, P<0.01); compared with HIB group, the SAa2-3Gal structures in pyruvate kinase were significantly increased in the IBA and the POST groups (the IBA:+30.91%, P<0.05; the POST:+34.41%, P<0.05); there were no statistical differences among the PRE, the IBA and the POST groups (P>0.05). The results suggest that the changes in HSC70, pyruvate kinase and 14-3-3 epsilon (protein expression and SAa2-3Gal structure) of SOL muscles of ground squirrels in different periods of hibernation might be associated with their innate mechanisms preventing disuse muscle atrophy. |
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