Experimental investigations on hypokinesis of skeletal muscles with different functions, III. Changes in protein fractions of subcellular components

Changes occurring in the protein fractions of rabbits' immobilized skeletal muscles with different functions were studied. Disuse of the muscles resulted in a gradual reduction in the contractile proteins. The specific proteins of the tonic muscle (m. soleus) were degraded to a greater extent than those of the tetanic (white) muscles (m. gastrocnemius). Parallel with the decrease in the structural proteins the sarcoplasmic protein exhibited a relative increase. The tonic muscles underwent greater damage than the tetanic muscles, indicating that the dedifferentiation was more marked in the tonic muscle. The results are explained by the biological importance of the function and activity of the cell: disuse leads to changes in the physiological and biochemical characteristics of the muscle, and to dedifferentiation of the cells.     

Mycobacterium ulcerans infections cause progressive muscle atrophy and dysfunction, and mycolactone impairs satellite cell proliferation

Clinical observations from Buruli ulcer (BU) patients in West Africa suggest that severe Mycobacterium ulcerans infections can cause skeletal muscle contracture and atrophy leading to significant impairment in function. In the present study, male mice C57BL/6 were subcutaneously injected with M. ulcerans in proximity to the right biceps muscle, avoiding direct physical contact between the infectious agent and the skeletal muscle. The histological, morphological, and functional properties of the muscles were assessed at different times after the injection. On day 42 postinjection, the isometric tetanic force and the cross-sectional area of the myofibers were reduced by 31% and 29%, respectively, in the proximate-infected muscles relative to the control muscles. The necrotic areas of the proximate-infected muscles had spread to 7% of the total area by day 42 postinjection. However, the number of central nucleated fibers and myogenic regulatory factors (MyoD and myogenin) remained stable and low. Furthermore, Pax-7 expression did not increase significantly in mycolactone-injected muscles, indicating that the satellite cell proliferation is abrogated by the toxin. In addition, the fibrotic area increased progressively during the infection. Lastly, muscle-specific RING finger protein 1 (MuRF-1) and atrogin-1/muscle atrophy F-box protein (atrogin-1/MAFbx), two muscle-specific E3 ubiquitin ligases, were upregulated in the presence of M. ulcerans. These findings confirmed that skeletal muscle is affected in our model of subcutaneous infection with M. ulcerans and that a better understanding of muscle contractures and weakness is essential to develop a therapy to minimize loss of function and promote the autonomy of BU patients.     

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.     

Of bears, frogs, meat, mice and men: complexity of factors affecting skeletal muscle mass and fat

Extreme loss of skeletal muscle mass (atrophy) occurs in human muscles that are not used. In striking contrast, skeletal muscles do not rapidly waste away in hibernating mammals such as bears, or aestivating frogs, subjected to many months of inactivity and starvation. What factors regulate skeletal muscle mass and what mechanisms protect against muscle atrophy in some species? Severe atrophy also occurs with ageing and there is much clinical interest in reducing such loss of muscle mass and strength (sarcopenia). In the meat industry, a key aim is optimizing the control of skeletal muscle growth and meat quality. The impaired response of muscle to insulin resulting in diabetes, that is a consequence of the metabolic impact of increasing obesity and fat deposition in humans, is also of increasing clinical concern. Intensive research in these fields, combined with mouse models, is reviewed with respect to the molecular control of muscle growth (myogenesis) and atrophy/hypertrophy and fat deposition (adipogenesis) in skeletal muscle, with a focus on IGF‐1/insulin signaling. BioEssays 28: 994–1009, 2006. © 2006 Wiley Periodicals, Inc.     

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.     

Muscle-specific overexpression of NCOATGK, splice variant of O-GlcNAcase, induces skeletal muscle atrophy

The protein O-linked β-N-acetylglucosamine (O-GlcNAc) modification plays an important role in skeletal muscle development and physiological function. In this study, bitransgenic mice were generated that overexpressed NCOAT(GK), an O-GlcNAcase-inactive spliced variant of the O-GlcNAcase gene, specifically in skeletal muscle using the muscle creatine kinase promoter. Expression of the chimeric enhanced green fluorescent protein-NCOAT(GK) transgene caused an increase of cellular O-GlcNAc levels, along with the accumulation and activation of proapoptotic factors in muscles of bitransgenic mice. The consequence of overexpressing the transgene for a 2-wk period was muscle atrophy and, in some cases, resulted in the death of male mice. Muscle atrophy is a common complication of many diseases, some of which correlate markedly with high cellular O-GlcNAc levels, such as diabetes. Our study provides direct evidence linking muscle atrophy and the disruption of O-GlcNAcase activity.     

Physiology of a microgravity environment invited review: microgravity and skeletal muscle.

Spaceflight (SF) has been shown to cause skeletal muscle atrophy; a loss in force and power; and, in the first few weeks, a preferential atrophy of extensors over flexors. The atrophy primarily results from a reduced protein synthesis that is likely triggered by the removal of the antigravity load. Contractile proteins are lost out of proportion to other cellular proteins, and the actin thin filament is lost disproportionately to the myosin thick filament. The decline in contractile protein explains the decrease in force per cross-sectional area, whereas the thin-filament loss may explain the observed postflight increase in the maximal velocity of shortening in the type I and IIa fiber types. Importantly, the microgravity-induced decline in peak power is partially offset by the increased fiber velocity. Muscle velocity is further increased by the microgravity-induced expression of fast-type myosin isozymes in slow fibers (hybrid I/II fibers) and by the increased expression of fast type II fiber types. SF increases the susceptibility of skeletal muscle to damage, with the actual damage elicited during postflight reloading. Evidence in rats indicates that SF increases fatigability and reduces the capacity for fat oxidation in skeletal muscles. Future studies will be required to establish the cellular and molecular mechanisms of the SF-induced muscle atrophy and functional loss and to develop effective exercise countermeasures.     

Effect of heart failure on the regulation of skeletal muscle protein synthesis,breakdown, and apoptosis

Heart failure is often characterized by skeletal muscle atrophy. The mechanisms underlying muscle wasting, however, are not fully understood. We studied 30 Dahl salt-sensitive rats (10 male, 20 female) fed either a high-salt (HS; n = 15) or a low-salt (LS; n = 15) diet. This strain develops cardiac hypertrophy and failure when fed a HS diet. LS controls were matched to HS rats for gender and duration of diet. Body mass, food intake, and muscle mass and composition were measured. Skeletal muscle protein synthesis was measured by isotope dilution. An additional group of 27 rats (HS, n = 16; LS; n = 11) were assessed for expression of genes regulating protein breakdown and apoptosis. Gastrocnemius and plantaris muscles weighed less (16 and 22%, respectively) in HS than in LS rats (P < 0.01). No differences in soleus or tibialis anterior weights were found. Differences in muscle mass were abolished after data were expressed relative to body size, because HS rats tended (P = 0.094) to weigh less. Lower body mass in HS rats was related to a 16% reduction (P < 0.01) in food intake. No differences in muscle protein or DNA content, the protein-to-DNA ratio, or muscle protein synthesis were found. Finally, no differences in skeletal muscle gene expression were found to suggest increased protein breakdown or apoptosis in HS rats. Our results suggest that muscle wasting in this model of heart failure is not associated with alterations in skeletal muscle metabolism. Instead, muscle atrophy was related to reduced body weight secondary to decreased food intake. These findings argue against the notion that heart failure is characterized by a skeletal muscle myopathy that predisposes to atrophy.     

Space travel directly induces skeletal muscle atrophy.

Space travel causes rapid and pronounced skeletal muscle wasting in humans that reduces their long-term flight capabilities. To develop effective countermeasures, the basis of this atrophy needs to be better understood. Space travel may cause muscle atrophy indirectly by altering circulating levels of factors such as growth hormone, glucocorticoids, and anabolic steroids and/or by a direct effect on the muscle fibers themselves. To determine whether skeletal muscle cells are directly affected by space travel, tissue-cultured avian skeletal muscle cells were tissue engineered into bioartificial muscles and flown in perfusion bioreactors for 9 to 10 days aboard the Space Transportation System (STS, i.e., Space Shuttle). Significant muscle fiber atrophy occurred due to a decrease in protein synthesis rates without alterations in protein degradation. Return of the muscle cells to Earth stimulated protein synthesis rates of both muscle-specific and extracellular matrix proteins relative to ground controls. These results show for the first time that skeletal muscle fibers are directly responsive to space travel and should be a target for countermeasure development.     

Effect of resistance exercise training on expression of Hsp70 and inflammatory cytokines in skeletal muscle and adipose tissue of STZ-induced diabetic rats

Impairment of adipose tissue and skeletal muscles accrued following type 1 diabetes is associated with protein misfolding and loss of adipose mass and skeletal muscle atrophy. Resistance training can maintain muscle mass by changing both inflammatory cytokines and stress factors in adipose tissue and skeletal muscle. The purpose of this study was to determine the effects of a 5-week ladder climbing resistance training program on the expression of Hsp70 and inflammatory cytokines in adipose tissue and fast-twitch flexor hallucis longus (FHL) and slow-twitch soleus muscles in healthy and streptozotocin-induced diabetic rats. Induction of diabetes reduced body mass, while resistance training preserved FHL muscle weight in diabetic rats without any changes in body mass. Diabetes increased Hsp70 protein content in skeletal muscles, adipose tissue, and serum. Hsp70 protein levels were decreased in normal and diabetic rats by resistance training in the FHL, but not soleus muscle. Furthermore, resistance training decreased inflammatory cytokines in FHL skeletal muscle. On the other hand, Hsp70 and inflammatory cytokine protein levels were increased by training in adipose tissue. Also, significant positive correlations between inflammatory cytokines in adipose tissue and skeletal muscles with Hsp70 protein levels were observed. In conclusion, we found that in diabetic rats, resistance training decreased inflammatory cytokines and Hsp70 protein levels in fast skeletal muscle, increased adipose tissue inflammatory cytokines and Hsp70, and preserved FHL muscle mass. These results suggest that resistance training can maintain skeletal muscle mass in diabetes by changing inflammatory cytokines and stress factors such as Hsp70 in skeletal muscle and adipose tissue.     

Expression of uncoupling protein 3 is upregulated in skeletal muscle during sepsis

Uncoupling protein 3 (UCP3) is a member of the mitochondrial transporter superfamily that is expressed primarily in skeletal muscle. UCP3 is upregulated in various conditions characterized by skeletal muscle atrophy, including hyperthyroidism, fasting, denervation, diabetes, cancer, lipopolysaccharide (LPS), and treatment with glucocorticoids (GCs). The influence of sepsis, another condition characterized by muscle cachexia, on UCP3 expression and activity is not known. We examined UCP3 gene and protein expression in skeletal muscles from rats after cecal ligation and puncture and from sham-operated control rats. Sepsis resulted in a two- to threefold increase in both mRNA and protein levels of UCP3 in skeletal muscle. Treatment of rats with the glucocorticoid receptor antagonist RU-38486 prevented the sepsis-induced increase in gene and protein expression of UCP3. The UCP3 mRNA and protein levels were increased 2.4- to 3.6-fold when incubated muscles from normal rats were treated with dexamethasone (DEX) and/or free fatty acids (FFA) ex vivo. In addition, UCP3 mRNA and protein levels were significantly increased in normal rat muscles in vivo with treatment of either DEX or FFA. The results suggest that sepsis upregulates the gene and protein expression of UCP3 in skeletal muscle, which may at least in part be mediated by GCs and FFA.     

Translational suppression of atrophic regulators by microRNA-23a integrates resistance to skeletal muscle atrophy

Muscle atrophy is caused by accelerated protein degradation and occurs in many pathological states. Two muscle-specific ubiquitin ligases, MAFbx/atrogin-1 and muscle RING-finger 1 (MuRF1), are prominently induced during muscle atrophy and mediate atrophy-associated protein degradation. Blocking the expression of these two ubiquitin ligases provides protection against muscle atrophy. Here we report that miR-23a suppresses the translation of both MAFbx/atrogin-1 and MuRF1 in a 3'-UTR-dependent manner. Ectopic expression of miR-23a is sufficient to protect muscles from atrophy in vitro and in vivo. Furthermore, miR-23a transgenic mice showed resistance against glucocorticoid-induced skeletal muscle atrophy. These data suggest that suppression of multiple regulators by a single miRNA can have significant consequences in adult tissues.     

S100a0 (αα) Protein, a Calcium-Binding Protein, Is Localized in the Slow-Twitch Muscle Fiber

We previously showed that, in contrast to the distribution of S100b (beta beta), S100a0 (alpha alpha) is mainly present in human skeletal and heart muscles at the level of 1-2 micrograms/mg of soluble protein and is universally distributed at high levels in skeletal and heart muscles of various mammals. To elucidate cellular and ultrastructural localizations of the alpha subunit of S100 protein (S100-alpha) in skeletal muscle, we used immunohistochemical and enzyme immunoassay methods. The immunohistochemical study revealed that S100-alpha is mainly localized in slow-twitch muscle fibers, whereas the beta subunit of S100 protein (S100-beta) was not detected in both types of muscle fibers, an observation indicating that the predominant form of S100 protein in the slow-twitch muscle fiber is not S100a or S100b, but S100a0. The quantitative analysis using enzyme immunoassay corroborates the immunohistochemical finding: The S100-alpha concentration of mouse soleus muscle (mainly composed of slow-twitch muscle fibers) is about threefold higher than that of mouse rectus femoris muscle (mainly composed of fast-twitch muscle fibers). At the ultrastructural level, S100-alpha is associated with polysomes, sarcoplasmic reticulum, the plasma membrane, the pellicle around lipid droplets, the outer membrane of mitochondria, and thin and thick filaments, by immunoelectron microscopy.     

An X-linked myopathy with postural muscle atrophy and generalized hypertrophy, termed XMPMA, is caused by mutations in FHL1

We have identified a large multigenerational Austrian family displaying a novel form of X-linked recessive myopathy. Affected individuals develop an adult-onset scapulo-axio-peroneal myopathy with bent-spine syndrome characterized by specific atrophy of postural muscles along with pseudoathleticism or hypertrophy and cardiac involvement. Known X-linked myopathies were excluded by simple-tandem-repeat polymorphism (STRP) and single-nucleotide polymorphism (SNP) analysis, direct gene sequencing, and immunohistochemical analysis. STRP analysis revealed significant linkage at Xq25-q27.1. Haplotype analysis based on SNP microarray data from selected family members confirmed this linkage region on the distal arm of the X chromosome, thereby narrowing down the critical interval to 12 Mb. Sequencing of functional candidate genes led to the identification of a missense mutation within the four and a half LIM domain 1 gene (FHL1), which putatively disrupts the fourth LIM domain of the protein. Mutation screening of FHL1 in a myopathy family from the UK exhibiting an almost identical phenotype revealed a 3 bp insertion mutation within the second LIM domain. FHL1 on Xq26.3 is highly expressed in skeletal and cardiac muscles. Western-blot analysis of muscle biopsies showed a marked decrease in protein expression of FHL1 in patients, in concordance with the genetic data. In summary, we have to our knowledge characterized a new disorder, X-linked myopathy with postural muscle atrophy (XMPMA), and identified FHL1 as the causative gene. This is the first FHL protein to be identified in conjunction with a human genetic disorder and further supports the role of FHL proteins in the development and maintenance of muscle tissue. Mutation screening of FHL1 should be considered for patients with uncharacterized myopathies and cardiomyopathies.     

Clenbuterol induces muscle-specific attenuation of atrophy through effects on the ubiquitin-proteasome pathway.

The ubiquitin-proteasome pathway is primarily responsible for myofibrillar protein degradation during hindlimb unweighting (HU). Beta-adrenergic agonists such as clenbuterol (CB) induce muscle hypertrophy and attenuate muscle atrophy due to disuse or inactivity. However, the molecular mechanism by which CB exerts these effects remains poorly understood. The aims of this study were to investigate whether CB attenuates HU-induced muscle atrophy through an inhibition of the ubiquitin-proteasome pathway and whether insulin-like growth factor I (IGF-I) mediates this inhibition. Rats were randomized to the following groups: weight-bearing control, 14-day CB-treated, 14-day HU, and CB + HU. HU-induced atrophy was associated with increased proteolysis and upregulation of components of the ubiquitin-proteasome pathway (ubiquitin conjugates, ubiquitin conjugating enzyme E2-14 kDa, and 20S proteasome activity). Upregulation of the ubiquitin proteasome occurred in all muscles tested but was more pronounced in muscles composed primarily of slow-twitch fibers (soleus) than in fast-twitch muscles (plantaris and tibialis anterior). Although CB induced hypertrophy in all muscles, CB attenuated the HU-induced atrophy and reduced ubiquitin conjugates only in the fast plantaris and tibialis anterior and not in the slow soleus muscle. CB did not elevate IGF-I protein content in either of the muscles examined. These results suggest that CB induces hypertrophy and alleviates HU-induced atrophy, particularly in the fast muscles, at least in part through a muscle-specific inhibition of the ubiquitin-proteasome pathway and that these effects are not mediated by the local production of IGF-I in skeletal muscle.