Plasticity changes in neuromuscular junction morphology and related regulatory proteins in the hibernating ground squirrel.

Skeletal muscle disuse atrophy can cause degenerative changes in neuromuscular junction morphology. Although Daurian ground squirrels (Spermophilus dauricus) are a natural anti-disuse animal model for studying muscle atrophy during hibernation, little is known about the morphological and regulatory mechanisms of their neuromuscular junctions. Here, we found that morphological indices of the soleus muscle were significantly lower during hibernation (torpor and interbout arousal) compared with pre-hibernation but recovered during post-hibernation. In the extensor digitorum longus muscle, neuromuscular junction morphology did not change significantly during hibernation. Agrin-Lrp4-MuSK is a key pathway for the formation and maintenance of the neuromuscular junction. Our results showed that low-density lipoprotein receptor-associated protein 4 (Lrp4) expression in the soleus (slow muscle) decreased by 46.2% in the interbout arousal group compared with the pre-hibernation group (P = 0.019), with recovery in the post-hibernation group. Compared with the pre-hibernation group, agrin expression in the extensor digitorum longus (fast muscle) increased by 67.0% in the interbout arousal group (P = 0.016). In conclusion, periodic up-regulation in agrin expression during interbout arousal may be involved in the maintenance of neuromuscular junction morphology in the extensor digitorum longus muscle during hibernation. The degenerative changes in neuromuscular junction morphology and the periodic decrease in Lrp4 protein expression in the soleus during hibernation, these changes recovered to the pre-hibernation levels in the post-hibernation group, exhibiting significant plasticity. This plasticity may be one of the important mechanisms for resisting disuse atrophy in hibernating animals.NEW & NOTEWORTHY This study is the first to explore the neuromuscular junction morphology of slow- and fast-twitch muscles in Daurian ground squirrels during different periods of hibernation. Results showed that the neuromuscular junction maintained stable morphology in the extensor digitorum longus muscle. The degenerative changes in neuromuscular junction morphology and the periodic decrease in Lrp4 protein expression in the soleus muscle during hibernation recovered in post-hibernation, exhibiting significant plasticity.

Different fuel regulation in two types of myofiber results in different antioxidant strategies in Daurian ground squirrels (Spermophilus dauricus) during hibernation.

We previously showed that different skeletal muscles in Daurian ground squirrels (Spermophilus dauricus) possess different antioxidant strategies during hibernation; however, the reason for these varied strategies remains unclear. To clarify this issue, we studied REDD1, FOXO4, PGC-1α, FOXO1 and atrogin-1 proteins to determine the potential cause of the different antioxidant strategies in Daurian ground squirrels during hibernation, and to clarify whether different strategies affect atrophy-related signals. Results showed that the soleus (SOL) muscle experienced intracellular hypoxia during interbout arousal, but no oxidative stress. This may be due to increased PGC-1α expression enhancing antioxidant capacity in the SOL under hypoxic conditions. Extensor digitorum longus (EDL) muscle showed no change in oxidative stress, hypoxia or antioxidant capacity during hibernation. The FOXO1 and PGC-1α results strongly suggested differentially regulated fuel metabolism in the SOL and EDL muscles during hibernation, i.e. enhanced lipid oxidation and maintained anaerobic glycolysis, respectively. Atrogin-1 expression did not increase during hibernation in either the SOL or EDL, indicating that protein synthesis was not inhibited by atrogin-1. Thus, our results suggest that different fuel regulation may be one mechanism related to antioxidant defense strategy formation in different kinds of skeletal muscle fibers of Daurian ground squirrels during hibernation.

A Temporal Examination of Cytoplasmic Ca2 + Levels, Sarcoplasmic Reticulum Ca2 + Levels, and Ca2 + -Handling-Related Proteins in Different Skeletal Muscles of Hibernating Daurian Ground Squirrels.

To explore the possible mechanism of the sarcoplasmic reticulum (SR) in the maintenance of cytoplasmic calcium (Ca2+) homeostasis, we studied changes in cytoplasmic Ca2+, SR Ca2+, and Ca2+-handling proteins of slow-twitch muscle (soleus, SOL), fast-twitch muscle (extensor digitorum longus, EDL), and mixed muscle (gastrocnemius, GAS) in different stages in hibernating Daurian ground squirrels (Spermophilus dauricus). Results showed that the level of cytoplasmic Ca2+ increased and SR Ca2+ decreased in skeletal muscle fiber during late torpor (LT) and inter-bout arousal (IBA), but both returned to summer active levels when the animals aroused from and re-entered into torpor (early torpor, ET), suggesting that intracellular Ca2+ is dynamic during hibernation. The protein expression of ryanodine receptor1 (RyR1) increased in the LT, IBA, and ET groups, whereas the co-localization of calsequestrin1 (CSQ1) and RyR1 in GAS muscle decreased in the LT and ET groups, which may increase the possibility of RyR1 channel-mediated Ca2+ release. Furthermore, calcium pump (SR Ca2+-ATPase 1, SERCA1) protein expression increased in the LT, IBA, and ET groups, and the signaling pathway-related factors of SERCA activity [i.e., ß-adrenergic receptor2 protein expression (in GAS), phosphorylation levels of phospholamban (in GAS), and calmodulin kinase2 (in SOL)] all increased, suggesting that these factors may be involved in the up-regulation of SERCA1 activity in different groups. The increased protein expression of Ca2+-binding proteins CSQ1 and calmodulin (CaM) indicated that intracellular free Ca2+-binding ability also increased in the LT, IBA, ET, and POST groups. In brief, changes in cytoplasmic and SR Ca2+ concentrations, SR RyR1 and SERCA1 protein expression levels, and major RyR1 and SERCA1 signaling pathway-related factors were unexpectedly active in the torpor stage when metabolic functions were highly inhibited.

Remarkable Homeostasis of Protein Sialylation in Skeletal Muscles of Hibernating Daurian Ground Squirrels (Spermophilus dauricus).

As the most common post-translational protein modification, glycosylation is intimately linked to muscle atrophy. This study aimed to investigate the performance of protein glycosylation in the soleus muscle (SOL) in Daurian ground squirrels (Spermophilus dauricus) and to determine the potential role of protein glycosylation in the mechanism underlying disuse muscle atrophy prevention. The results showed that (1) seven glycan structures comprising sialic acid α2-3 galactose (SAα2-3Gal) were altered during hibernation; (2) alterations in the SAα2-3Gal structure during hibernation were based on changes in the expression levels of beta-galactoside alpha-2 and 3-sialyltransferases; and (3) α2-3-linked sialylated modifications of heat shock cognate 70 and pyruvate kinase and expression of 14-3-3 epsilon protein were oscillatorily changed during hibernation. Our findings indicate that the skeletal muscles of hibernating Daurian ground squirrels maintain protein sialylation homeostasis by restoring sialylation modification during periodic interbout arousal, which might protect the skeletal muscles against disuse atrophy.

Novel findings on ultrastructural protection of skeletal muscle fibers during hibernation of Daurian ground squirrels: Mitochondria, nuclei, cytoskeleton, glycogen.

We examined ultrastructure protective phenomena and mechanisms of slow and fast muscles in hibernating Daurian ground squirrels (Spermophilus dauricus). Some degenerative changes such as slightly decreased sarcomere length and vacuolization occurred in hibernation, but periaxonal capsular borders in intrafusal fibers remained distinct and the arrangement of extrafusal fibers and Z-lines unscathed. In soleus samples, the number of glycogenosomes more than tripled during hibernation. The expression of phosphorylated glycogen synthase remained unaltered while that of glycogen phosphorylase decreased during hibernation. The number of extensor digitorum longus glycogenosomes decreased and the expression of phosphorylated glycogen synthase decreased, while glycogen phosphorylase expression remained unaltered. The nuclei number remained unchanged. Kinesin and desmin, preventors of nuclear loss and damage, were maintained or just slightly reduced in hibernation. The single-fiber mitochondrial concentration and sub-sarcolemmal mitochondrial number increased in both muscle types. The expression of vimentin, which anchors mitochondria and maintains Z-line integrity, was increased during and after hibernation. Also, dynamin-related protein 1, mitochondrial fission factor, and adenosine triphosphate synthase were elevated in both muscle types. These findings confirm a remarkable ultrastructure preservation and show an unexpected increase in mitochondrial capacity in hibernating squirrels.

A dramatic blood plasticity in hibernating and 14-day hindlimb unloading Daurian ground squirrels (Spermophilus dauricus).

We compared the effects of hibernation inactivity and 14-day hindlimb unloading in non-hibernating period on biochemical, rheological, and hematological parameters of blood in Daurian ground squirrels (Spermophilus dauricus). Twenty-four squirrels were randomly divided into four groups: control (CON), hibernation (HIB), post-hibernation (POST), and 14-day hindlimb unloading (HU). The results showed that serum enzymes (L-lactate dehydrogenase, alanine aminotransferase, and aspartate aminotransferase) activities decreased in HIB, POST, and HU squirrels compared with CON. Total protein (including albumin and globulin) maintained in HIB but decreased in HU compared with CON. Total cholesterol and high-density lipoprotein-cholesterol increased in HIB but maintained in HU and POST compared with CON. Meanwhile, serum creatinine decreased and urea increased in HU compared with CON. All blood ions concentrations were unchanged in HIB, POST, and HU squirrels compared with CON except calcium which increased in HIB compared with CON, and phosphorus which increased in HIB and POST compared with CON. Most of detected serum biochemical analytes in POST recovered to the CON level. Blood viscosity, which was unchanged in all shear rates in HU, increased in HIB and recovered in POST in lower shear rates compared with CON. Erythrocyte and corpuscular volume decreased in HIB and HU but maintained in POST compared with CON. All the routine hematological parameters recovered in POST as compared with CON except platelet, which decreased in HIB and POST but maintained in HU compared with CON. In conclusion, our results suggested a remarkable ability to maintain blood homeostasis in hibernating squirrels.

iTRAQ-based proteomic analysis of myofibrillar contents and relevant synthesis and proteolytic proteins in soleus muscle of hibernating Daurian ground squirrels (Spermophilus dauricus).

BACKGROUND: Daurian ground squirrels (Spermophilus dauricus) deviate from significant increase of protein catabolism and loss of myofibrillar contents during long period of hibernation inactivity. METHODS: Here we use iTRAQ based quantitative analysis to examine proteomic changes in the soleus of squirrels in pre-hibernation, hibernation and post-hibernation states. The total proteolysis rate of soleus was measured by the release of the essential amino acid tyrosine from isolated muscles. Immunofluorescent analysis was used to determine muscle fiber cross-sectional area. Western blot was used for the validation of the quantitative proteomic analysis. RESULTS: The proteomic responses to hibernation had a 0.4- to 0.8-fold decrease in the myofibrillar contractile protein levels of myosin-3, myosin-13 and actin, but a 2.1-fold increase in myosin-2 compared to pre-hibernation group. Regulatory proteins such as troponin C and tropomodulin-1 were 1.4-fold up-regulated and 0.7-fold down-regulated, respectively, in hibernation compared to pre-hibernation group. Moreover, 10 proteins with proteolytic function in hibernation, which was less than 14 proteins in the post-hibernation group, were up-regulated relative to the pre-hibernation group. The total proteolysis rates of soleus in hibernation and post-hibernation groups were significantly inhibited as compared with pre-hibernation group. CONCLUSION: These findings suggest that the myofibrillar remodeling and partial suppression of myofibrillar proteolysis were likely responsible for preventing skeletal muscle atrophy during prolonged disuse in hibernation. This is the first study where the myofibrillar contents and relevant synthesis and proteolytic proteins in slow soleus was discussed based on proteomic investigation performed on wild Daurian ground squirrels. Our results lay the foundation for further research in preventing disuse-induced skeletal muscle atrophy in mammals.