β-Hydroxy-β-methylbutyrate reduces myonuclear apoptosis during recovery from hind limb suspension-induced muscle fiber atrophy in aged rats

β-Hydroxy-β-methylbutyrate (HMB) is a leucine metabolite shown to reduce protein catabolism in disease states and promote skeletal muscle hypertrophy in response to loading exercise. In this study, we evaluated the efficacy of HMB to reduce muscle wasting and promote muscle recovery following disuse in aged animals. Fisher 344×Brown Norway rats, 34 mo of age, were randomly assigned to receive either Ca-HMB (340 mg/kg body wt) or the water vehicle by gavage (n = 32/group). The animals received either 14 days of hindlimb suspension (HS, n = 8/diet group) or 14 days of unloading followed by 14 days of reloading (R; n = 8/diet group). Nonsuspended control animals were compared with suspended animals after 14 days of HS (n = 8) or after R (n = 8). HMB treatment prevented the decline in maximal in vivo isometric force output after 2 wk of recovery from hindlimb unloading. The HMB-treated animals had significantly greater plantaris and soleus fiber cross-sectional area compared with the vehicle-treated animals. HMB decreased the amount of TUNEL-positive nuclei in reloaded plantaris muscles (5.1% vs. 1.6%, P < 0.05) and soleus muscles (3.9% vs. 1.8%, P < 0.05). Although HMB did not significantly alter Bcl-2 protein abundance compared with vehicle treatment, HMB decreased Bax protein abundance following R, by 40% and 14% (P < 0.05) in plantaris and soleus muscles, respectively. Cleaved caspase-3 was reduced by 12% and 9% (P < 0.05) in HMB-treated reloaded plantaris and soleus muscles, compared with vehicle-treated animals. HMB reduced cleaved caspase-9 by 14% and 30% (P < 0.05) in reloaded plantaris and soleus muscles, respectively, compared with vehicle-treated animals. Although, HMB was unable to prevent unloading-induced atrophy, it attenuated the decrease in fiber area in fast and slow muscles after HS and R. HMB's ability to protect against muscle loss may be due in part to putative inhibition of myonuclear apoptosis via regulation of mitochondrial-associated caspase signaling.     

Skeletal muscle atrophy leads to loss and dysfunction of muscle precursor cells

Atrophy of skeletal muscle leads to decreases in myofiber size and nuclear number; however, the effects of atrophic conditions on muscle precursor cells (MPC) are largely unknown. MPC lie outside myofibers and represent the main source of additional myonuclei necessary for muscle growth and repair. In the present study, we examined the properties of MPC after hindlimb suspension (HS)-induced atrophy and subsequent recovery of the mouse hindlimb muscles. We demonstrated that the number of MPC in atrophied muscles was decreased. RT-PCR analysis of cells isolated from atrophied muscles indicated that several mRNA characteristic of the myogenic program in MPC were absent. Cells isolated from atrophied muscles failed to properly proliferate and undergo differentiation into multinucleated myotubes. Thus atrophy led to a decrease in MPC and caused dysfunction in those MPC that remained. Upon regrowth of the atrophied muscles, these deleterious effects were reversed. Our data suggest that preventing loss or dysfunction of MPC may be a new pharmacological target during muscle atrophy. satellite cells; hindlimb suspension; proliferation; differentiation; myotubes     

Analysis of altered gene expression in rat soleus muscle atrophied by disuse

The present study involved a global analysis of genes whose expression was modified in rat soleus muscle atrophied after hindlimb suspension (HS). HS muscle unloading is a common model for muscle disuse that especially affects antigravity slow-twitch muscles such as the soleus muscle. A cDNA cloning strategy, based on suppression subtractive hybridization technology, led to the construction of two normalized soleus muscle cDNA libraries that were subtracted in opposite directions, i.e., atrophied soleus muscle cDNAs subtracted by control cDNAs and vice versa. Differential screening of the two libraries revealed 34 genes with altered expression in HS soleus muscle, including 11 novel cDNAs, in addition to the 2X and 2B myosin heavy chain genes expressed only in soleus muscles after HS. Gene up- and down-regulations were quantified by reverse Northern blot and classical Northern blot analysis. The 25 genes with known functions fell into seven important functional categories. The homogeneity of gene alterations within each category gave several clues for unraveling the interplay of cellular events implied in the muscle atrophy phenotype. In particular, our results indicate that modulations in slow- and fast-twitch-muscle component balance, the protein synthesis/secretion pathway, and the extracellular matrix/cytoskeleton axis are likely to be key molecular mechanisms of muscle atrophy. In addition, the cloning of novel cDNAs underlined the efficiency of the chosen technical approach and gave novel possibilities to further decipher the molecular mechanisms of muscle atrophy.     

PGC-1α and FOXO1 mRNA levels and fiber characteristics of the soleus and plantaris muscles in rats after hindlimb unloading

Fifteen-week-old rats were subjected to unloading induced by hindlimb suspension for 3 weeks. The peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and forkhead box-containing protein O1 (FOXO1) mRNA levels and fiber profiles of the soleus and plantaris muscles in rats subjected to unloading (unloaded group) were determined and compared with those of age-matched control rats (control group). The body weight and both the soleus and plantaris muscle weights were lower in the unloaded group than in the control group. The PGC-1α mRNA was downregulated in the soleus, but not in the plantaris muscle of the unloaded group. The FOXO1 mRNA was upregulated in both the soleus and plantaris muscles of the unloaded group. The oxidative enzyme activity was reduced in the soleus, but not in the plantaris muscle of the unloaded group. The percentage of type I fibers was decreased and the percentages of type IIA and IIC fibers were increased in the soleus muscle of the unloaded group, whereas there was no change in fiber type distribution in the plantaris muscle of the unloaded group. Atrophy of all types of fibers was observed in both the soleus and plantaris muscles of the unloaded group. We conclude that decreased oxidative capacity and fiber atrophy in unloaded skeletal muscles are associated with decreased PGC-1α and increased FOXO1 mRNA levels.     

Ca2+ movements in sarcoplasmic reticulum of rat soleus fibers after hindlimb suspension.

The functional capacity of skeletal muscle sarcoplasmic reticulum (SR) was examined in the slow soleus of rats submitted to 15 days of disuse produced by hindlimb suspension (HS). By using caffeine-induced contractions of single skinned fibers, Ca2+ uptake, Ca2+ release, and passive Ca2+ leakage through the SR membrane were investigated. In the SR of atrophied muscles, the amounts of Ca2+ uptake and Ca2+ release were significantly higher than in the control muscles and were close to those found for a fast muscle, the plantaris. Moreover, the study of the Ca2+ leakage showed that the time required to empty the SR previously loaded with Ca2+ was reduced by a factor of two after HS. Such disturbances of the Ca2+ movements in the SR suggested that alterations of the SR membrane occurred after HS. The results supported the idea that after hindlimb unweighting the slow soleus muscle acquired SR properties that were very much like those of a faster muscle.     

Proteomic analysis of rat soleus muscle undergoing hindlimb suspension-induced atrophy and reweighting hypertrophy

A proteomic analysis was performed comparing normal rat soleus muscle to soleus muscle that had undergone either 0.5, 1, 2, 4, 7, 10 and 14 days of hindlimb suspension-induced atrophy or hindlimb suspension-induced atrophied soleus muscle that had undergone 1 hour, 8 hour, 1 day, 2 day, 4 day and 7 days of reweighting-induced hypertrophy. Muscle mass measurements demonstrated continual loss of soleus mass occurred throughout the 21 days of hindlimb suspension; following reweighting, atrophied soleus muscle mass increased dramatically between 8 hours and 1 day post reweighting. Proteomic analysis of normal and atrophied soleus muscle demonstrated statistically significant changes in the relative levels of 29 soleus proteins. Reweighting following atrophy demonstrated statistically significant changes in the relative levels of 15 soleus proteins. Protein identification using mass spectrometry was attempted for all differentially regulated proteins from both atrophied and hypertrophied soleus muscle. Five differentially regulated proteins from the hindlimb suspended atrophied soleus muscle were identified while five proteins were identified in the reweighting-induced hypertrophied soleus muscles. The identified proteins could be generally grouped together as metabolic proteins, chaperone proteins and contractile apparatus proteins. Together these data demonstrate that coordinated temporally regulated changes in the skeletal muscle proteome occur during disuse-induced soleus muscle atrophy and reweighting hypertrophy.     

Interleukin-15 responses to aging and unloading-induced skeletal muscle atrophy

Interleukin-15 (IL-15) mRNA is constitutively expressed in skeletal muscle. Although IL-15 has proposed hypertrophic and anti-apoptotic roles in vitro, its role in skeletal muscle cells in vivo is less clear. The purpose of this study was to determine if skeletal muscle aging and unloading, two conditions known to promote muscle atrophy, would alter basal IL-15 expression in skeletal muscle. We hypothesized that IL-15 mRNA expression would increase as a result of both aging and muscle unloading and that muscle would express the mRNA for a functional trimeric IL-15 receptor (IL-15R). Two models of unloading were used in this study: hindlimb suspension (HS) in rats and wing unloading in quail. The absolute muscle wet weight of plantaris and soleus muscles from aged rats was significantly less when compared with muscles from young adult rats. Although 14 days of HS resulted in reduced muscle mass of plantaris and soleus muscles from young adult animals, this effect was not observed in muscles from aged animals. A significant aging times unloading interaction was observed for IL-15 mRNA in both rat soleus and plantaris muscles. Patagialis (PAT) muscles from aged quail retained a significant 12 and 6% of stretch-induced hypertrophy after 7 and 14 days of unloading, respectively. PAT muscles from young quail retained 15% hypertrophy at 7 days of unloading but regressed to control levels following 14 days of unloading. A main effect of age was observed on IL-15 mRNA expression in PAT muscles at 14 days of overload, 7 days of unloading, and 14 days of unloading. Skeletal muscle also expressed the mRNAs for a functional IL-15R composed of IL-15R, IL-2/15R-, and -c. Based on these data, we speculate that increases in IL-15 mRNA in response to atrophic stimuli may be an attempt to counteract muscle mass loss in skeletal muscles of old animals. Additional research is warranted to determine the importance of the IL-15/IL-15R system to counter muscle wasting. atrophy; interleukins; sarcopenia; gene signaling     

Early changes of alpha B-crystallin mRNA in rat skeletal muscle to mechanical tension and denervation.

alpha B-Crystallin specifically decreases in atrophied rat soleus muscle with hindlimb suspension (HS). alpha B-Crystallin cDNA was cloned from rat heart cDNA library using oligonucleotide probe, and its complete coding and partial non-coding regions were sequenced. Northern blot analysis revealed that alpha B-crystallin mRNA in slow muscle decreases at 36 hour after HS but recovered at 24 hour after HS stopped. Denervation decreased the expression of alpha B-crystallin mRNA in slow muscle but increased it in fast muscles, which hardly expressed in normal condition. Passive tension increased the expression of alpha B-crystallin mRNA in both muscle types. Based upon these Northern blot analysis of alpha B-crystallin, nerve innervation and external load on muscle are essential regulatory factors on the expression of the mRNA of alpha B-crystallin in rat skeletal muscle.     

Quantitative assessment of degenerative changes in soleus muscle after hindlimb suspension and recovery

The aim of this study was to quantify the degenerative and regenerative changes in rat soleus muscle resulting from 3-week hindlimb suspension at 45° tilt (HS group, n = 8) and 4-week normal cage recovery (HS-R group, n = 7). Degenerative changes were quantified by microscope examination of muscle cross sections, and the myosin heavy chain (MHC) composition of soleus muscles was studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis. At the end of 3-week hindlimb suspension, histological signs of muscle degenerative changes were detected in soleus muscles. There was a significant variability in the percentage of fibres referred to as degenerating (%dg) in individual animals in the HS group [%dg = 8.41 (SEM 0.5)%, range 4.66%–14.08%]. Moreover, %dg varied significantly along the length of the soleus muscle. The percentage of fibres with internal nuclei was less than %dg in HS-soleus muscles [4.12 (SEM 0.3)%, range 1.24%–8.86%]. In 4-week recovery rats, the greater part of the fibres that were not referred to as normal, retained central nuclei [15.8 (SEM 2.2)%, range 6.2%–21.1%]. A significant increase in the slow isoform of MHC was recorded in the HS-R rats, compared to muscles from age-matched rats (P < 0.01). These results would suggest that a cycle of myofibre degeneration-regeneration occurred during HS and passive recovery, and that the increased accumulation of slow MHC observed in soleus muscles after recovery from HS could be related to the prevalence of newly formed fibres. Accepted: 14 October 1996     

Conditioned medium derived from umbilical cord mesenchymal stem cells regenerates atrophied muscles

We investigated the regenerative effects and regulatory mechanisms of human umbilical cord mesenchymal stem cells (UC-MSCs)-derived conditioned medium (CM) in atrophied muscles using an in vivo model. To determine the appropriate harvest point of UC-CM, active factor content was analyzed in the secretome over time. A muscle atrophy model was induced in rats by hindlimb suspension (HS) for 2 weeks. Next, UC-CM was injected directly into the soleus muscle of both hind legs to assess its regenerative efficacy on atrophy-related factors after 1 week of HS. During HS, muscle mass and muscle fiber size were significantly reduced by over 2-fold relative to untreated controls. Lactate accumulation within the muscles was similarly increased. By contrast, all of the above analytical factors were significantly improved in HS-induced rats by UC-CM injection compared with saline injection. Furthermore, the expression levels of desmin and skeletal muscle actin were significantly elevated by UC-CM treatment. Importantly, UC-CM effectively suppressed expression of the atrophy-related ubiquitin E3-ligases, muscle ring finger 1 and muscle atrophy F-box by 2.3- and 2.1-fold, respectively. UC-CM exerted its actions by stimulating the phosphoinositol-3-kinase (PI3K)/Akt signaling cascade. These findings suggest that UC-CM provides an effective stimulus to recover muscle status and function in atrophied muscles.     

Myogenic differentiation during regrowth of atrophied skeletal muscle is associated with inactivation of GSK-3beta

Muscle atrophy contributes to morbidity and mortality in aging and chronic disease, emphasizing the need to gain understanding of the mechanisms involved in muscle atrophy and (re)growth. We hypothesized that the magnitude of muscle regrowth during recovery from atrophy determines whether myonuclear accretion and myogenic differentiation are required and that insulin-like growth factor (IGF)-I/Akt/glycogen synthase kinase (GSK)-3 signaling differs between regrowth responses. To address this hypothesis we subjected mice to hindlimb suspension (HS) to induce atrophy of soleus (–40%) and plantaris (–27%) muscle. Reloading-induced muscle regrowth was complete after 14 days and involved an increase in IGF-IEa mRNA expression that coincided with Akt phosphorylation in both muscles. In contrast, phosphorylation and inactivation of GSK-3 were observed during soleus regrowth only. Furthermore, soleus but not plantaris regrowth involved muscle regeneration based on a transient increase in expression of histone 3.2 and myosin heavy chain-perinatal, which are markers of myoblast proliferation and differentiation, and a strong induction of muscle regulatory factor (MRF) expression. Experiments in cultured muscle cells showed that IGF-I-induced MRF expression is facilitated by inactivation of GSK-3 and selectively occurs in the myoblast population. This study suggests that induction of IGF-I expression and Akt phosphorylation during recovery from muscle atrophy is independent of the magnitude of muscle regrowth. Moreover, our data demonstrate for the first time that the regenerative response characterized by myoblast proliferation, differentiation, and increased MRF expression in recovering muscle is associated with the magnitude of regrowth and may be regulated by inactivation of GSK-3. glycogen synthase kinase-3; Akt; muscle growth; muscle atrophy     

Effects of hindlimb suspension and androgen treatment on testosterone receptors in rat skeletal muscles

This study tested the specific and combined effects of testosterone treatment and hindlimb suspension (HS) on the properties of steroid receptors in skeletal muscle. Male rats were either administered weekly high doses of testosterone heptylate (10 mg x kg(-1)) or olive oil placebo, and were either tail-suspended or acted as controls. After 3 weeks of treatment, three muscles were excised from each animal, soleus (SOL), extensor digitorum longus (EDL), and plantaris. The results showed that the testosterone treatment was unable to minimise the HS-induced atrophy of skeletal muscle. As expected, HS altered the fibre-type composition of SOL muscles (-33% of type I, +188% and +161% of type IIa and intermediate fibres respectively, P < 0.01). No overall effect of treatment was detected on the fibre-type composition of either slow or fast-twitch muscles. Binding capacity determined by a radiocompetition technique was increased by HS, especially in SOL and EDL muscles (P < 0.01), while HS or steroid treatment decreased the affinity of the steroid receptors. The combination of HS and testosterone administration resulted in a decrease in binding capacity and affinity of steroid receptors in skeletal muscles. Steroid receptors in fast-twitch muscles exhibited a higher affinity than those in slow-twitch muscles, and it is suggested that it is likely that testosterone treatment is more effective in fast-twitch than in slow-twitch muscles. It was concluded that the lack of preventive effect of testosterone treatment on HS-induced SOL muscle atrophy could be explained by both a decrease in steroid sensitivity and the removal of mechanical factors.     

mRNA levels for alpha-subunit of prolyl 4-hydroxylase and fibrillar collagens in immobilized rat skeletal muscle.

There is evidence that immobilization causes a decrease in total collagen synthesis in skeletal muscle within a few days. In this study, early immobilization effects on the expression of prolyl 4-hydroxylase (PH) and the main fibrillar collagens at mRNA and protein levels were investigated in rat skeletal muscle. The right hindlimb was immobilized in full plantar flexion for 1, 3, and 7 days. Steady-state mRNAs for alpha- and beta-subunits of PH and type I and III procollagen, PH activity, and collagen content were measured in gastrocnemius and plantaris muscles. Type I and III procollagen mRNAs were also measured in soleus and tibialis anterior muscles. The mRNA level for the PH alpha-subunit decreased by 49 and 55% (P < 0.01) in gastrocnemius muscle and by 41 and 39% (P < 0.05) in plantaris muscle after immobilization for 1 and 3 days, respectively. PH activity was decreased (P < 0.05-0.01) in both muscles at days 3 and 7. The mRNA levels for type I and III procollagen were decreased by 26-56% (P < 0.05-0.001) in soleus, tibialis anterior, and plantaris muscles at day 3. The present results thus suggest that pretranslational downregulation plays a key role in fibrillar collagen synthesis in the early phase of immobilization-induced muscle atrophy.     

Denervation and reinnervation of fast and slow muscles. A histochemical study in rats.

A histochemical study, using myosin-adenosine triphosphatase activity at pH 9.4, was conducted in soleus and plantaris muscles of adult rats, after bilateral crushing of the sciatic nerve at the sciatic notch. The changes in fiber diameter and per cent composition of type I and type II fibers plus muscle weights were evaluated along the course of denervation-reinnervation curve at 1, 2, 3, 4 and 6 weeks postnerve crush. The study revealed that in the early denervation phase (up to 2 weeks postcrush) both the slow and fast muscles, soleus and plantaris, resepctively, atrophied similarly in muscle mass. Soleus increased in the number of type II fibers, which may be attributed to "disuse" effect. During the same period, the type I fibers of soleus atrophied as much or slightly more than the type II fibers; whereas the type II fibers of plantaris atrophied significantly more than the type I fibers, reflecting that the process of denervation, in its early stages, may affect the two fiber types differentially in the slow and fast muscles. It was deduced that the type I fibers of plantaris may be essentially different in the slow (soleus) and fast (plantaris) muscles under study. The onset of reinnervation, as determined by the increase in muscle weight and fiber diameter of the major fiber type, occurred in soleus and plantaris at 2 and 3 weeks postcrush, respectively, which confirms the earlier hypotheses that the slow muscles are reinnervated sooner than the fast muscles. It is suggested that the reinnervation of muscle after crush injury may be specific to the muscle type or its predominant fiber type.     

A physiological level of clenbuterol does not prevent atrophy or loss of force in skeletal muscle of old rats.

Supraphysiological levels of clenbuterol (CL) reduce muscle degradation in both young and old animals; however, these pharmacological levels induce side effects that are unacceptable in the elderly. In this study, we tested the hypothesis that a "physiological" dose of CL (10 microg. kg(-1). day(-1)) would attenuate the loss of in situ isometric force and mass in muscles of senescent rats during hindlimb suspension (HS). Adult (3 mo) and senescent (38 mo) Fischer 344 x Brown Norway rats received CL or a placebo during 21 days of normal-weight-bearing or HS conditions (8 rats/age group). HS reduced soleus muscle weight-to-body weight ratio by 31%, muscle cross-sectional area by 37%, and maximal isometric tetanic force (P(o)) by 76% in senescent rats. CL attenuated the loss of P(o) and muscle weight by 17 and 8%, respectively, in the soleus of senescent rats relative to HS+placebo conditions, but it did not improve muscle weight normalized for body weight. CL did not reduce the decrease in soleus P(o) or mass after HS in adult rats. CL failed to reduce the loss of plantaris weight (-20%) and P(o) (-46%) in senescent rats after HS. Our data support the conclusion that physiological levels of CL do not improve fast muscle atrophy and only modestly reduce slow muscle atrophy, and, therefore, it is largely an ineffective countermeasure for preventing muscle wasting from HS in senescent rats.     

Identification of cold-shock protein RBM3 as a possible regulator of skeletal muscle size through expression profiling

Changes in gene expression associated with skeletal muscle atrophy due to aging are distinct from those due to disuse, suggesting that the response of old muscle to inactivity may be altered. The goal of this study was to identify changes in muscle gene expression that may contribute to loss of adaptability of old muscle. Muscle atrophy was induced in young adult (6-mo) and old (32-mo) male Brown Norway/F344 rats by 2 wk of hindlimb suspension (HS), and soleus muscles were analyzed by cDNA microarrays. Overall, similar changes in gene expression with HS were observed in young and old muscles for genes encoding proteins involved in protein folding (heat shock proteins), muscle structure, and contraction, extracellular matrix, and nucleic acid binding. More genes encoding transport and receptor proteins were differentially expressed in the soleus muscle from young rats, while in soleus muscle from old rats more genes that encoded ribosomal proteins were upregulated. The gene encoding the cold-shock protein RNA-binding motif protein-3 (RBM3) was induced most highly with HS in muscle from old rats, verified by real-time RT-PCR, while no difference with age was observed. The cold-inducible RNA-binding protein (Cirp) gene was also overexpressed with HS, whereas cold-shock protein Y-box-binding protein-1 was not. A time course analysis of RBM3 mRNA abundance during HS showed that upregulation occurred after apoptotic nuclei and markers of protein degradation increased. We conclude that a cold-shock response may be part of a compensatory mechanism in muscles undergoing atrophy to preserve remaining muscle mass and that RBM3 may be a therapeutic target to prevent muscle loss.     

Age effects on rat hindlimb muscle atrophy during suspension unloading.

Disuse can induce numerous adaptive alterations in skeletal muscle. In the present study the effects of hindlimb unloading on muscle mass and biochemical responses were examined and compared in adult (450 g) and juvenile (200 g) rats after 1, 7, or 14 days of whole body suspension. Quantitatively and qualitatively the soleus (S), gastrocnemius (G), plantaris (P), and extensor digitorum longus (EDL) muscles of the hindlimb exhibited a differential sensitivity to suspension and weightlessness unloading in both adults and juveniles. The red slow-twitch soleus exhibited the most pronounced atrophy under both conditions, with juvenile responses being greater than adult. In contrast, the fast-twitch EDL hypertrophied during suspension and atrophied during weightlessness, with no significant difference between adults and juveniles. Determination of biochemical parameters (total protein, RNA, and DNA) indicated a less rapid rate of response in adult muscles. This was corroborated by assessment of muscle alpha-actin mRNA levels, which indicated a rapid (within 1 day) and significant (P less than 0.05) effect in juveniles but not in adults. The results of this investigation indicate 1) a qualitatively similar differential effect of unloading on muscles of adults and juveniles, 2) a quantitatively reduced and less rapid effect of suspension on adult muscles, and 3) a close similarity of adult and juvenile muscle responses during suspension and spaceflight, suggesting that this ground-based model simulates many of the unloading effects of weightlessness.     

Changes in αB-crystallin,tubulin, and MHC isoforms by hindlimb unloading show different expression patterns in various hindlimb muscles

[Purpose]

αB-crystallin is a small heat shock protein that acts as a molecular chaperone under various stress conditions. Microtubules, which consist of tubulin, are related to maintain the intracellular organelles and cellular morphology. These two proteins have been shown to be related to the properties of different types of myofibers based on their contractile properties. The response of these proteins during muscular atrophy, which induces a myofibril component change, is not clearly understood.

[Methods]

We performed 15 days of hindlimb unloading on rats to investigate the transitions of these proteins by analyzing their absolute quantities. Protein contents were analyzed in the soleus, plantaris, and gastrocnemius muscles of the unloading and control groups (N = 6).

[Results]

All three muscles were significantly atrophied by hindlimb unloading (P < 0.01): soleus (47.5%), plantaris (16.3%), and gastrocnemius (21.3%) compared to each control group. αB-crystallin was significantly reduced in all three examined unloaded hindlimb muscles compared to controls (P < 0.01) during the transition of the myosin heavy chain to fast twitch muscles. α-Tubulin responded only in the unloaded soleus muscle. Muscle atrophy induced the reduction of αB-crystallin and α-tubulin expressions in plantar flexor muscles with a shift to the fast muscle fiber compared to the control.

[Conclusion]

The novel finding of this study is that both proteins, αB-crystallin and α-tubulin, were downregulated in slow muscles (P < 0.01); However, α-tubulin was not significantly reduced compared to the control in fast muscles (P < 0.01).     

肌卫星细胞在失重肌萎缩中的可塑性变化及机制

肌卫星细胞在骨骼肌生长发育和出生后骨骼肌损伤修复中起着重要的作用,但是有关肌萎缩中肌卫星细胞的可塑性变化、作用及其机制尚不清楚.本研究采用小鼠尾悬吊模拟失重效应诱导失重肌萎缩,动态分析了失重肌萎缩发生过程中不同类型肌纤维的肌卫星细胞数量和增殖、分化潜能可塑性的改变,发现在失重肌萎缩过程中,处于安静状态的肌卫星细胞显著增多、激活增殖的肌卫星细胞显著减少,而具有成肌分化潜能的肌卫星细胞有持续减少趋势.此外,在失重肌萎缩比目鱼肌单根肌纤维移出的体外培养中,证明了失重肌萎缩肌纤维肌卫星细胞可塑性降低的特征性变化.进一步,通过对比分析Smad3基因敲除及其同窝野生型小鼠,在失重肌萎缩中肌卫星细胞可塑性的差异性变化,揭示了Smad3在调控失重肌萎缩肌卫星细胞可塑性变化中的关键作用.     

Passive stretch reduces calpain activity through nitric oxide pathway in unloaded soleus muscles

Unloading in spaceflight or long-term bed rest induces to pronounced atrophy of anti-gravity skeletal muscles. Passive stretch partially resists unloading-induced atrophy of skeletal muscle, but the mechanism remains elusive. The aims of this study were to investigate the hypotheses that stretch tension might increase protein level of neuronal nitric oxide synthase (nNOS) in unloaded skeletal muscle, and then nNOS-derived NO alleviated atrophy of skeletal muscle by inhibiting calpain activity. The tail-suspended rats were used to unload rat hindlimbs for 2 weeks, at the same time, left soleus muscle was stretched by applying a plaster cast to fix the ankle at 35° dorsiflexion. Stretch partially resisted atrophy and inhibited the decreased protein level and activity of nNOS in unloaded soleus muscles. Unloading increased frequency of calcium sparks and elevated intracellular resting and caffeine-induced Ca(2+) concentration ([Ca(2+)]i) in unloaded soleus muscle fibers. Stretch reduced frequency of calcium sparks and restored intracellular resting and caffeine-induced Ca(2+) concentration to control levels in unloaded soleus muscle fibers. The increased protein level and activity of calpain as well as the higher degradation of desmin induced by unloading were inhibited by stretch in soleus muscles. In conclusion, these results suggest that stretch can preserve the stability of sarcoplasmic reticulum Ca(2+) release channels which prevents the elevated [Ca(2+)]i by means of keeping nNOS activity, and then the enhanced protein level and activity of calpain return to control levels in unloaded soleus muscles. Therefore, stretch can resist in part atrophy of unloaded soleus muscles.