Plasticity changes in iron homeostasis in hibernating Daurian ground squirrels (Spermophilus dauricus) may counteract chronically inactive skeletal muscle atrophy.

Disuse-induced muscular atrophy is frequently accompanied by iron overload. Hibernating animals are a natural animal model for resistance to disuse muscle atrophy. In this paper, we explored changes in skeletal muscle iron content of Daurian ground squirrels (Spermophilus dauricus) during different periods of hibernation as well as the regulatory mechanisms involved. The results revealed that compared with the summer active group (SA), iron content in the soleus muscle (SOL) decreased (- 65%) in the torpor group (TOR), but returned to normal levels in the inter-bout arousal (IBA); splenic iron content increased in the TOR group (vs. SA, + 67%), decreased in the IBA group (vs. TOR, - 37%). Expression of serum hepcidin decreased in the TOR group (vs. SA, - 22%) and returned to normal levels in the IBA groups; serum ferritin increased in the TOR group (vs. SA, + 31%), then recovered in the IBA groups. Soleus muscle transferrin receptor 1 (TfR1) expression increased in the TOR group (vs. SA, + 83%), decreased in the IBA group (vs. TOR, - 30%); ferroportin 1 increased in the IBA group (vs. SA, + 55%); ferritin increased in the IBA group (vs. SA, + 42%). No significant differences in extensor digitorum longus in iron content or iron metabolism-related protein expression were observed among the groups. Significantly, all increased or decreased indicators in this study returned to normal levels after the post-hibernation group, showing remarkable plasticity. In summary, avoiding iron overload may be a potential mechanism for hibernating Daurian ground squirrels to avoid disuse induced muscular atrophy. In addition, the different skeletal muscle types exhibited unique strategies for regulating iron homeostasis.

Remarkable Plasticity of Bone Iron Homeostasis in Hibernating Daurian Ground Squirrels (Spermophilus dauricus) May Be Involved in Bone Maintenance.

Iron overload is an independent risk factor for disuse osteoporosis. Hibernating animals are natural models of anti-disuse osteoporosis; however, whether iron metabolism is involved in bone adaptation and maintenance during hibernation is unclear. To investigate this question, Daurian ground squirrels (Spermophilus dauricus) (n = 5-6/group) were used to study changes in bone iron metabolism and its possible role in anti-disuse osteoporosis during hibernation. Iron content in the femur and liver first decreased in the torpor group (vs. summer group, -66.8% and -25.8%, respectively), then recovered in the post-hibernation group, suggesting remarkable plasticity of bone iron content. The expression of ferritin in the femur and hepcidin in the liver also initially decreased in the torpor group (vs. summer group, -28.5% and -38.8%, respectively), then increased in the inter-bout arousal (vs. torpor group, 126.2% and 58.4%, respectively) and post-hibernation groups (vs. torpor group, 153.1% and 27.1%, respectively). In conclusion, bone iron metabolism in hibernating Daurian ground squirrels showed remarkable plasticity, which may be a potential mechanism to avoid disuse bone loss during extended periods of inactivity. However, the specific location of iron during low-iron hibernation and the source of iron in post-hibernation recovery need to be further explored.

Resistance to disuse-induced iron overload in Daurian ground squirrels (Spermophilus dauricus) during extended hibernation inactivity.

Iron overload occurs in disuse-induced osteoporosis. Hibernators are a natural animal model of resistance to disuse osteoporosis. We hypothesized that hibernators avoid iron overload to resist disuse-induced osteoporosis. Here, the role of iron metabolism in resistance to disuse osteoporosis was investigated by studying differences in iron content and iron metabolism in the femurs and livers of Daurian ground squirrels (Spermophilus dauricus) between the summer active and torpid states. Results showed that the femurs were generally well-maintained during torpor, with no significant differences observed in most bone microstructural parameters, except for a significantly lower (by 40%) trabecular bone connection density. Femur and liver iron concentrations were significantly lower during torpor (by 59% and 49%, respectively). Based on histological staining, livers were iron-negative and femurs showed a reduction in iron-positive area (by 83%) during torpor; The number of osteoblasts and osteoclasts showed no significant differences between the two groups. Most iron metabolism/homeostasis proteins expression levels in the femur and liver showed no significant differences between the two groups, with their stable expression likely preventing iron overload during inactivity. Higher femoral transferrin receptor 1 (TfR1) expression (by 108%) and lower liver ferritin expression (by 45%) were found in torpid squirrels. Lower liver ferritin may be related to the lower iron content, with the elevation in femoral TfR1 potentially related to restoration of bone iron levels. In conclusion, despite long periods of inactivity, iron levels in the femur and liver of squirrels were lower, bone formation and resorption were balanced and no iron overload was observed, as is found under disuse conditions in non-hibernators. Therefore, avoiding iron overload may be a potential mechanism for hibernators to avoid disuse-induced bone loss.

Autophagy and Akt-mTOR signaling display periodic oscillations during torpor-arousal cycles in oxidative skeletal muscle of Daurian ground squirrels (Spermophilus dauricus).

Whether hibernation accelerates or suppresses autophagy is still unknown. In the current study, we examined changes in autophagy in oxidative soleus (SOL) muscle in summer active (SA), pre-hibernation (PRE), torpor (TOR), interbout arousal (IBA), and post-hibernation groups of Daurian ground squirrels (Spermophilus dauricus). Here, the SOL muscle showed no significant atrophy during hibernation in regard to muscle wet weight, fiber cross-sectional area, or MuRF1 protein level. Autophagy-related proteins beclin1 and Atg7 increased significantly, whereas LC3-II decreased significantly in the PRE group compared with the SA group. However, neither the expression nor activity of cathepsin L showed any differences between the SA and PRE groups. In addition, beclin1, LC3-II, and the LC3-II/LC3-I ratio increased, p62 decreased, LC3 puncta increased, p62 puncta decreased, and cathepsin L activity increased in the TOR group compared with the PRE group. In contrast, beclin1, LC3-II, and the LC3-II/LC3-I ratio decreased, p62 increased, LC3 puncta decreased, p62 puncta increased, and cathepsin L activity declined in the IBA group compared with the TOR group. Moreover, the phosphorylation of Akt (Ser473) and mTOR (Ser2448) changed significantly during hibernation and showed an inverse relationship with autophagy changes. In conclusion, autophagy proteins displayed periodic oscillation in the torpor-arousal cycle, which may be advantageous in maintaining SOL muscle mass during the entire hibernation period. Furthermore, the Akt-mTOR signaling was decreased in TOR and increased in IBA group in the SOL muscle of Daurian ground squirrels during hibernation.

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.

Remarkable plasticity of Na+, K+-ATPase, Ca2+-ATPase and SERCA contributes to muscle disuse atrophy resistance in hibernating Daurian ground squirrels.

We investigated cytosolic calcium (Ca2+) and sarcoplasmic reticulum Ca2+ regulation in skeletal muscle fibers of hibernating Daurian ground squirrels (Spermophilus dauricus), non-hibernating hindlimb-unloaded (HLU) squirrels, and HLU rats to clarify the molecular mechanisms involved in preventing muscle atrophy in hibernators. The Na+, K+-ATPase and Ca2+-ATPase activities in the soleus muscle (SOL) of squirrels were maintained in hibernation, decreased during interbout arousal (IB-A), and increased to autumn/pre-hibernation (AUT/Pre-H) levels in torpor after interbout arousal (Post-IBA), whereas activities in the extensor digitorum longus muscle (EDL) were stable during hibernation, but increased during post-hibernation (Post-H). Activities increased in the SOL of HLU rats, but were stable in HLU squirrels. Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) activity in the SOL decreased in IB-A squirrels, but returned to AUT/Pre-H levels in the Post-IBA group; no significant changes were found in the EDL. SERCA activity increased in the EDL of HLU squirrels and SOL of HLU rats. Compared with AUT/Pre-H, SERCA type 2 protein expression increased in the SOL and EDL of IB-A and Post-IBA squirrels, but increased in the SOL only in HLU animals. We also describe the protein kinase A changes in this paper. Thus, hibernating ground squirrels displayed remarkable Na+, K+-ATPase, Ca2+-ATPase, and SERCA plasticity.

Tetramethylpyrazine ameliorated disuse-induced gastrocnemius muscle atrophy in hindlimb unloading rats through suppression of Ca2+/ROS-mediated apoptosis.

The purpose of this study was to examine the possible mechanism underlying the protective effect of tetramethylpyrazine (TMP) against disuse-induced muscle atrophy. Sprague-Dawley rats were randomly assigned to receive 14 days of hindlimb unloading (HLU, a model of disuse atrophy) or cage controls. The rats were given TMP (60 mg/kg body mass) or vehicle (water) by gavage. Compared with vehicle treatment, TMP significantly attenuated the loss of gastrocnemius muscle mass (-33.56%, P < 0.01), the decrease of cross-sectional area of slow fiber (-10.99%, P < 0.05) and fast fiber (-15.78%, P < 0.01) during HLU. Although TMP failed to further improve recovery of muscle function or fatigability compared with vehicle treatment, it can suppress the higher level of lactate (-22.71%, P < 0.01) induced by HLU. Besides, TMP could effectually reduce the increased protein expression of muscle RING-finger protein 1 induced by HLU (-14.52%, P < 0.01). Furthermore, TMP can ameliorate the calcium overload (-54.39%, P < 0.05), the increase of malondialdehyde content (-19.82%, P < 0.05), the decrease of superoxide dismutase activity (21.34%, P < 0.05), and myonuclear apoptosis (-78.22%, P < 0.01) induced by HLU. Moreover, TMP significantly reduced HLU-induced increase of Bax to B-cell lymphoma 2 (-36.36%, P < 0.01) and cytochrome c release (-36.16%, P < 0.05). In conclusion, TMP attenuated HLU-induced gastrocnemius muscle atrophy through suppression of Ca2+/reactive oxygen species increase and consequent proteolysis and apoptosis. Therefore, TMP might exhibit therapeutic effect against oxidative stress, cytosolic calcium overload, and mitochondrial damage in disuse-induced muscle atrophy.

Remarkable preservation of Ca(2+) homeostasis and inhibition of apoptosis contribute to anti-muscle atrophy effect in hibernating Daurian ground squirrels.

The underlying mechanisms that hibernators deviated from muscle atrophy during prolonged hibernating inactivity remain elusive. This study tested the hypothesis that the maintenance of intracellular Ca(2+) homeostasis and inhibition of apoptosis would be responsible for preventing muscle atrophy in hibernating Daurian ground squirrels. The results showed that intracellular Ca(2+) homeostasis was maintained in soleus and extensor digitorum longus (EDL) in hibernation and post-hibernation, while cytosolic Ca(2+) was overloaded in gastrocnemius (GAS) in hibernation with a recovery in post-hibernation. The Ca(2+) overload was also observed in interbout arousals in all three type muscles. Besides, the Bax/Bcl-2 ratio was unchanged in transcriptional level among pre-hibernation, hibernation and interbout arousals, and reduced to a minimum in post-hibernation. Furthermore, the Bax/Bcl-2 ratio in protein level was reduced in hibernation but recovered in interbout arousals. Although cytochrome C was increased in GAS and EDL in post-hibernation, no apoptosis was observed by TUNEL assay. These findings suggested that the intracellular Ca(2+) homeostasis in hibernation might be regulated by the cytosolic Ca(2+) overload during interbout arousals, which were likely responsible for preventing muscle atrophy via inhibition of apoptosis. Moreover, the muscle-specificity indicated that the different mechanisms against disuse-induced atrophy might be involved in different muscles in hibernation.

Muscle composition after 14-day hindlimb unloading in rats: effects of two herbal compounds.

AIM: We studied the potential of two herbal compounds (HC-1 and HC-2) in different dosages (HC-1, containing ligustrazine; HC-2, containing radix astragali) as a countermeasure against muscle atrophy in a hindlimb unloading rat model. Soleus muscle weight, fiber type distribution, cross-sectional area (CSA), and myosin ATPase activity were measured. METHODS: Six rats each were assigned to a nine-group design: Control (CON); hindlimb unloading only (HLU); hindlimb unloading plus intragastric water instillation (HLU-W); and hindlimb unloading plus different dosages of instilled HC-1 (HLU-HC-1l, HLU-HC-1m, HLU-HC-1h) or HC-2 (HLU-HC-2l, HLU-HC-2m, HLU-HC-2h). RESULTS: As expected, in HLU and HLU-W, soleus muscle-to-body weight ratio and CSA of type I and II fiber went down, and myosin ATPase activity and the percentage of type II fibers went up, all compared with CON. Compared to untreated rats, high-dose HC-1 enhanced type I and II fiber CSA by 77% and 55%, respectively; myosin ATPase activity (type II fiber percentage) were lower, but soleus muscle-to-body weight ratio did not change. High-dose HC-2 enhanced soleus muscle-to-body weight ratio by 33%; and type I / type II fiber CSA by 143% and 83% above HLU-W values, respectively, and resulted in a slower myosin ATPase activity (corresponding to a lower type II fiber percentage). Low- to mid-dose HC-1 and HC-2 showed some preventive effects as well. CONCLUSION: HC-1 and HC-2 in proper dosages diminished muscle atrophy that otherwise occurs in simulated weightlessness.

[Effects of Ligustrazine and Radix Astragali on activities of myosin adenosine triphosphatase of soleus muscle and muscle atrophy in tail-suspended rats].

OBJECTIVE: To study the effects of Ligustrazine (Lig) and Radix Astragali (Rad) on activities of myosin adenosine triphosphatase (mATPase) of soleus muscle and atrophy in tail-suspended rat. METHOD: Weightlessness was simulated by tail suspension in female rats. The activities of mATPase of intrafusal and extrafusal fibres in soleus muscle were detected by method of Ca2(+)-ATPase. RESULT: 1) Compared with tail-suspended (TS) group, the percentage of type II fibres of SOL in both Ligustrazine (Lig) group and Rad group decreased distinctly. Furthermore, the percentage of type I and type II fibres of SOL in Rad group showed no difference with control (CON) group. 2) The type I CSA of Lig group was markedly larger than that in TS group, and there was no difference as compared with that of CON group. CSA of type I, II and average CSA of Rad group were remarkably larger than those in TS group, and there were no difference as compared with those of CON group. 3) mATPase activities of intrafusal fibres in both Lig group and Rad group were approximate to that of CON group. CONCLUSION: Lig and Rad are both able to effectively prevent muscle atrophy caused by tail suspension, restrain the slow-twitch muscle transform to fast-twitch muscle and control the increase of mATPase activities caused by weightlessness.