Hui Chang

Remarkable preservation of Ca2+ 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 Ca2+ homeostasis and inhibition of apoptosis would be responsible for preventing muscle atrophy in hibernating Daurian ground squirrels. The results showed that intracellular Ca2+ homeostasis was maintained in soleus and extensor digitorum longus (EDL) in hibernation and post-hibernation, while cytosolic Ca2+ was overloaded in gastrocnemius (GAS) in hibernation with a recovery in post-hibernation. The Ca2+ 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 Ca2+ homeostasis in hibernation might be regulated by the cytosolic Ca2+ 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.

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.

Identification of the optimal dose and calpain system regulation of tetramethylpyrazine on the prevention of skeletal muscle atrophy in hindlimb unloading rats

Previous studies in our lab have shown that tetramethylpyrazine (TMP) could effectively attenuate disuse induced muscle atrophy. In order to screening out the optimal dose of tetramethylpyrazine (TMP) for protection against disuse induced muscle atrophy in hindlimb unloading (HLU) rats, in this study, we compared effects of 4 TMP doses on muscle wet weight (MWW), the ratios of muscle wet weight/body weight (MWW/BW) and muscle wet weight/dry weight (MWW/DW), fiber type composition, as well as cross-sectional area (CSA) in soleus (SOL) muscle. Consequently, we quantified optimal dose effects on both functional properties and protein expression (calpain-1, calpain-2, calpastatin and MuRF1) in SOL and extensor digitorum longus (EDL) muscles. Data indicated that the protective potential of TMP was dose-dependent: 60mg/kg TMP was most effective in terms of atrophy prevention. This dose reduced SOL MWW, MWW/BW and CSA muscle loss by 60, 60 and 54% (P<0.001), respectively. HLU-induced slow-to-fast fiber transition was reduced by 17% (P<0.01). 60mg/kg TMP also significantly lessened the decrease of contractile force, the increase of shorting velocity and fatigability induced by HLU. Besides, it also attenuated expressions of calpain-1 (SOL -8.6%, P<0.05; EDL -10.9%, P<0.05), calpain-2 (SOL -60%, P<0.001; EDL -32%, P<0.01) and MuRF1 expression (SOL -21%, P<0.001; EDL -10%, P<0.01), promoted the expression of calpastatin by 18% (P<0.05) in SOL muscle. Taken together, present study demonstrated that 60mg/kg body weight was the optimal dose of TMP against disuse induced muscle atrophy which effectively protected muscle function by inhibiting calpain-1, calpain-2 and MuRF1 expression, promoted calpastatin expression, especially in slow-twitch muscle.

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

BackgroundDaurian ground squirrels (Spermophilus dauricus) deviate from significant increase of protein catabolism and loss of myofibrillar contents during long period of hibernation inactivity.MethodsHere 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.ResultsThe 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.ConclusionThese 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.

Remarkable plasticity of Na + , K + -ATPase, Ca 2+ -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.

Unexpected regulation pattern of the IKKβ/NF-κB/MuRF1 pathway with remarkable muscle plasticity in the Daurian ground squirrel (Spermophilus dauricus)

As a typical hibernator, the Daurian ground squirrel (Spermophilus dauricus) spends considerable time in a state of reduced activity with prolonged fasting. Despite this, they experience little muscle atrophy and have thus become an interesting anti‐disuse muscle atrophy model. The IKKβ/NF‐κB signaling pathway is significant to muscle atrophy due to the protein degradation resulting from the upregulation of the E3 ubiquitin ligase MuRF1. The current study showed that the IKKβ/NF‐κB signaling pathway and MuRF1 maintained relatively steady mRNA and protein expression levels, with little muscle atrophy observed in the soleus (slow‐twitch, SOL) or extensor digitorum longus (fast‐twitch, EDL) during hibernation (HIB); however, mRNA expression significantly increased in the SOL and EDL muscle during interbout arousal (IBA), as did the MuRF1 mRNA level in the SOL and MuRF1 protein level in the EDL. Interestingly, the expressions of p50 and MuRF1 significantly increased during HIB in the gastrocnemius (mixed muscle, GAS) and showed moderate atrophy, but dramatically decreased during IBA. Elevated IKKβ and p50 mRNA and protein expression in the cardiac muscle (CM) during HIB did not accompany increased MuRF1 expression or muscle wasting. Importantly, almost all increased or decreased indicators in the tested tissues recovered to pre‐hibernation levels after HIB. This is the first study to report on the unexpected regulation of the IKKβ/NF‐κB/MuRF1 pathway with remarkable muscle plasticity in Daurian ground squirrels during hibernation. Furthermore, we found that different types of muscles exhibited different strategies to cope with prolonged hibernation‐induced disuse muscle atrophy.

Proteomic analysis reveals the distinct energy and protein metabolism characteristics involved in myofiber type conversion and resistance of atrophy in the extensor digitorum longus muscle of hibernating Daurian ground squirrels

Previous hibernation studies demonstrated that such a natural model of skeletal muscle disuse causes limited muscle atrophy and a significant fast-to-slow fiber type shift. However, the underlying mechanism as defined in a large-scale analysis remains unclarified. Isobaric tags for relative and absolute quantification (iTRAQ) based quantitative analysis were used to examine proteomic changes in the fast extensor digitorum longus muscles (EDL) of Daurian ground squirrels (Spermophilus dauricus). Although the wet weights and fiber cross-sectional area of the EDL muscle showed no significant decrease, the percentage of slow type fiber was 61% greater (P < 0.01) in the hibernation group. Proteomics analysis identified 264 proteins that were significantly changed (ratio < 0.83 or >1.2-fold and P < 0.05) in the hibernation group, of which 23 proteins were categorized into energy production and conversion and translation and 22 proteins were categorized into ribosomal structure and biogenesis. Along with the validation by western blot, MAPKAP kinase 2, ATP5D, ACADSB, calcineurin, CSTB and EIF2S were up-regulated in the hibernation group, whereas PDK4, COX II and EIF3C were down-regulated in the hibernation group. MAPKAP kinase 2 and PDK4 were associated with glycolysis, COX II and ATP5D were associated with oxidative phosphorylation, ACADSB was associated with fatty acid metabolism, calcineurin and CSTB were associated with catabolism, and EIF2S and EIF3C were associated with anabolism. Moreover, the total proteolysis rate of EDL in the hibernation group was significantly inhibited compared with that in the pre-hibernation group. These distinct energy and protein metabolism characteristics may be involved in myofiber type conversion and resistance to atrophy in the EDL of hibernating Daurian ground 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.

Controllable oxidative stress and tissue specificity in major tissues during the torpor–arousal cycle in hibernating Daurian ground squirrels

Mammalian hibernators experience repeated hypoxic ischaemia and reperfusion during the torpor–arousal cycle. We investigated levels of oxidative stress, antioxidant capacity, and the underlying mechanism in heart, liver, brain and kidney tissue as well as plasma during different periods of hibernation in Daurian ground squirrels (Spermophilus dauricus). Our data showed that the levels of hydrogen peroxide significantly increased in the heart and brain during late torpor (LT) compared with levels during the summer active (SA) state. The content of malondialdehyde (MDA) was significantly lower during interbout arousal (IBA) and early torpor (ET) than that during SA or pre-hibernation (PRE), and MDA levels in the LT brain were significantly higher than the levels in other states. Superoxide dismutase 2 protein levels increased markedly in the heart throughout the entire torpor–arousal cycle. Catalase expression remained at an elevated level in the liver during the hibernation cycle. Superoxide dismutase 1 and glutathione peroxidase 1 (GPx1) expression increased considerably in all tissues during the IBA and ET states. In addition, the activities of the various antioxidant enzymes were higher in all tissues during IBA and ET than during LT; however, GPx activity in plasma decreased significantly during the hibernation season. The expression of p-Nrf2 decreased in all tissue types during IBA, but significantly increased during LT, especially in liver tissue. Interestingly, most changed indicators recovered to SA or PRE levels in post-hibernation (POST). These results suggest that increased reactive oxygen species during LT may activate the Nrf2/Keap1 antioxidant pathway and may contribute to the decreased MDA levels found during the IBA and ET states, thereby protecting organisms from oxidative damage over the torpor-arousal cycle of hibernation. This is the first report on the remarkable controllability of oxidative stress and tissue specificity in major oxidative tissues of a hibernator.

Muscle-specific activation of calpain system in hindlimb unloading rats and hibernating Daurian ground squirrels: a comparison between artificial and natural disuse

To determine whether the regulation of calpain system is involved in non-hibernators and hibernators in disused condition, the soleus (SOL) and extensor digitorum longus (EDL) muscles were used for investigating the muscle mass, the ratio of muscle wet weight/body weight (MWW/BW), fiber-type distribution, fiber cross-sectional area (CSA), and the protein expression of MuRF1, calpain-1, calpain-2, calpastatin, desmin, troponin T, and troponin C in hindlimb unloading rats and hibernating Daurian ground squirrels. The muscle mass, MWW/BW, and fiber CSA were found significantly decreased in SOL and EDL of hindlimb unloading rats, but unchanged in hibernating ground squirrels. The MuRF1 expression was increased in both SOL and EDL of unloading rats, while it was only increased in SOL, but maintained in EDL of hibernating ground squirrels. The expression levels of calpain-1 and calpain-2 were increased in different degrees in unloaded SOL and EDL in rats, while they were maintained in EDL and even reduced in SOL of hibernating ground squirrels. Besides, the expression of calpastatin was decreased in unloaded rats, but increased in hibernating ground squirrels. The desmin expression was decreased in unloaded rats, but maintained in hibernating squirrels. Interestingly, the levels of troponin T and troponin C were decreased in both SOL and EDL of unloaded rats, but increased in hibernating ground squirrels with muscle-type specificity. In conclusion, differential calpain activation and substrate-selective degradation in slow and fast muscles are involved in the mechanisms of muscle atrophy of unloaded rats and remarkable ability of muscle maintenance of hibernating ground squirrels.