Absence of staircase following disuse in rat gastrocnemius muscle

Repetitive stimulation of mammalian fast-twitch skeletal muscles will normally result in a positive staircase response. This phenomenon was investigated in the rat gastrocnemius muscle following a 2-week period of tetrodotoxin-induced disuse. Muscle inactivity was imposed by superfusing tetrodotoxin in saline over the left sciatic nerve via an implanted osmotic pump. In situ isometric contractile responses to double pulse stimulation and repetitive stimulation at 10 Hz were determined the day after removal of the pump. Two weeks of disuse resulted in 40% muscle weight loss. A twitch contraction gave the same force when expressed per gram of wet muscle weight in control muscles, 317 +/- 24.6 (means +/- SE) g/g, as compared with tetrodotoxin-treated muscles, 328 +/- 24.2 g/g. Both contraction time and half-relaxation time were prolonged following treatment with tetrodotoxin. Repetitive stimulation at 10 Hz resulted in a positive staircase response in the control muscles, but not in muscles of the tetrodotoxin-treated rats. The observed changes in the time course of the twitch contraction with repetitive stimulation following tetrodotoxin-induced disuse are consistent with alterations in sarcoplasmic reticulum handling of calcium. It is not certain if there is a change following disuse in the mechanism normally associated with staircase or if this mechanism is merely opposed by an early fatigue.     

Acute antibody-directed myostatin inhibition attenuates disuse muscle atrophy and weakness in mice

Counteracting the atrophy of skeletal muscle associated with disuse has significant implications for minimizing the wasting and weakness in plaster casting, joint immobilization, and other forms of limb unloading, with relevance to orthopedics, sports medicine, and plastic and reconstructive surgery. We tested the hypothesis that antibody-directed myostatin inhibition would attenuate the loss of muscle mass and functional capacity in mice during 14 or 21 days of unilateral hindlimb casting. Twelve-week-old C57BL/10 mice were subjected to unilateral hindlimb plaster casting or served as controls. Mice received subcutaneous injections of saline or a mouse chimera of anti-human myostatin antibody (PF-354, 10 mg/kg; n = 6-9) on days 0 and 7 and were tested for muscle function on day 14, or were treated on days 0, 7, and 14 and tested for muscle function on day 21. Hindlimb casting reduced muscle mass, fiber size, and function of isolated soleus and extensor digitorum longus (EDL) muscles (P < 0.05). PF-354 attenuated the loss of muscle mass, fiber size, and function with greater effects after 14 days than after 21 days of casting, when wasting and weakness had plateaued (P < 0.05). Antibody-directed myostatin inhibition therefore attenuated the atrophy and loss of functional capacity in muscles from mice subjected to unilateral hindlimb casting with reductions in muscle size and strength being most apparent during the first 14 days of disuse. These findings highlight the therapeutic potential of antibody-directed myostatin inhibition for disuse atrophy especially within the first 2 wk of disuse.     

Disuse atrophy increases the muscle mechanoreflex in rats.

We investigated the effect of disuse atrophy on the magnitude of the muscle mechanoreflex. The left leg of eight rats (6-7 wk, male) was put in a plaster cast for 1 wk. The rats were decerebrated at the midcollicular level. We recorded the pressor and cardioaccelerator responses to 30-s stretch of the calcaneal tendon, which selectively stimulated the muscle mechanosensitive receptors in the left atrophied and right control triceps surae muscles. Atrophied muscles showed significantly lower mass control muscles (1.0 +/- 0.1 vs. 1.4 +/- 0.1 g; P < 0.05). At the same stretch tension (229 +/- 20 g), the pressor response to stretch was significantly greater in the atrophied muscles than in the control muscles (13 +/- 3 vs. 4 +/- 2 mmHg, P < 0.05). The cardioaccelerator response was not significantly different (8 +/- 4 vs. 4 +/- 2 beats/min). Comparing responses at the same relative tension (57 +/- 6 vs. 51 +/- 8% of maximal tension), the pressor response was still significantly greater in the atrophied triceps surae than in the control (14 +/- 4 vs. 4 +/- 2 mmHg; P < 0.05). These results suggest that disuse atrophy increases the magnitude of muscle mechanoreflex.     

Effect of hindlimb unweighting on anaerobic metabolism in rat skeletal muscle.

Female Sprague-Dawley rats (250 g) were hindlimb suspended for 14 days, and the effects of hindlimb unweighting (HU) on skeletal muscle anaerobic metabolism were investigated and compared with nonsuspended controls (C). Soleus (SOL), plantaris (PL), and red and white portions of the gastrocnemius (RG, WG) were sampled from resting and stimulated limbs. Muscle atrophy after HU was 46% in SOL, 22% in PL, and 24% in the gastrocnemius compared with nonsuspended C animals. The muscles innervated by the sciatic nerve were stimulated to contract with an occluded circulation for 60 s with trains of supramaximal impulses (100 ms, 80 Hz) at a train rate of 1.0 Hz. Peak tension development by the gastrocnemius-PL-SOL muscle group was similar in HU and C animals (13.0 +/- 1.2, 12.2 +/- 0.8 N/g wet muscle). Occlusion of the circulation before stimulation created a predominantly anaerobic environment, and in situ glycogenolysis and glycolysis were estimated from accumulations of glycolytic intermediates. Total glycogenolysis and glycolysis were higher in the RG muscle of HU animals (74.6 +/- 3.3, 58.1 +/- 1.1) relative to C (57.1 +/- 4.6, 46.1 +/- 2.9 mumol glucosyl units/g dry muscle). Consequently, total anaerobic ATP production was also increased (HU, 251.3 +/- 1.1; C, 204.6 +/- 8.9 mumol ATP/g dry muscle). Total ATP production, glycogenolysis, and glycolysis were unaffected by HU in SOL, PL, and WG muscles. The enhanced glycolytic activity in RG after HU may be attributed to a shift in the metabolic profile from oxidative to glycolytic in the fast oxidative-glycolytic fiber population.     

Satellite cell regulation of muscle mass is altered at old age.

Muscle mass is decreased with advancing age, likely due to altered regulation of muscle fiber size. This study was designed to investigate cellular mechanisms contributing to this process. Analysis of male Fischer 344 X Brown Norway rats at 6, 20, and 32 mo of age demonstrated that, even though significant atrophy had occurred in soleus muscle by old age, myofiber nuclear number did not change, resulting in a decreased myonuclear domain. Also, the number of centrally located nuclei was significantly elevated in soleus muscle of 32-mo-old rats, correlating with an increase in gene expression of MyoD and myogenin. Whereas total 5'-bromo-2'deoxyuridine (BrdU)-positive nuclei were decreased at older ages, BrdU-positive myofiber nuclei were increased. These results suggest that, with age, loss of muscle mass is accompanied by increased myofiber nuclear density that involves fusion of proliferative satellite cells, resembling ongoing regeneration. Interestingly, centrally located myofiber nuclei were not BrdU labeled. Rats were subjected to hindlimb suspension (HS) for 7 or 14 days and intermittent reloading during HS for 1 h each day (IR) to investigate how aging affects the response of soleus muscle to disuse and an atrophy-reducing intervention. After 14 days of HS, soleus muscle size was decreased to a similar extent at all three ages. However, myofiber nuclear number and the total number of BrdU-positive nuclei decreased with HS only in the young rats. IR was associated with an attenuation of atrophy in soleus muscles of 6- and 20- but not 32-mo-old rats. Furthermore, IR was associated with an increase in BrdU-positive myofiber nuclei only in young rats. These data indicate that altered satellite cell function with age contributes to the impaired response of soleus muscle to an intervention that attenuates muscle atrophy in young animals during imposed disuse.     

Preventive effect of dietary quercetin on disuse muscle atrophy by targeting mitochondria in denervated mice

Quercetin is a major dietary flavonoid in fruits and vegetables. We aimed to clarify the preventive effect of dietary quercetin on disuse muscle atrophy and the underlying mechanisms. We established a mouse denervation model by cutting the sciatic nerve in the right leg (SNX surgery) to lack of mobilization in hind-limb. Preintake of a quercetin-mixed diet for 14 days before SNX surgery prevented loss of muscle mass and atrophy of muscle fibers in the gastrocnemius muscle (GM). Phosphorylation of Akt, a key phosphorylation pathway of suppression of protein degradation, was activated in the quercetin-mixed diet group with and without SNX surgery. Intake of a quercetin-mixed diet suppressed the generation of hydrogen peroxide originating from mitochondria and elevated mitochondrial peroxisome proliferator-activated receptor-γ coactivator 1α mRNA expression as well as NADH dehydrogenase 4 expression in the GM with SNX surgery. Quercetin and its conjugated metabolites reduced hydrogen peroxide production in the mitochondrial fraction obtained from atrophied muscle. In C2C12 myotubes, quercetin reached the mitochondrial fraction. These findings suggest that dietary quercetin can prevent disuse muscle atrophy by targeting mitochondria in skeletal muscle tissue through protecting mitochondria from decreased biogenesis and reducing mitochondrial hydrogen peroxide release, which can be related to decreased hydrogen peroxide production and/or improvements on antioxidant capacity of mitochondria.     

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.     

Effects of Resveratrol on the Recovery of Muscle Mass Following Disuse in the Plantaris Muscle of Aged Rats

Aging is associated with poor skeletal muscle regenerative ability following extended periods of hospitalization and other forms of muscular disuse. Resveratrol (3,5,4’-trihydroxystilbene) is a natural phytoalexin which has been shown in skeletal muscle to improve oxidative stress levels in muscles of aged rats. As muscle disuse and reloading after disuse increases oxidative stress, we hypothesized that resveratrol supplementation would improve muscle regeneration after disuse. A total of thirty-six male Fisher 344 × Brown Norway rats (32 mo.) were treated with either a water vehicle or resveratrol via oral gavage. The animals received hindlimb suspension for 14 days. Thereafter, they were either sacrificed or allowed an additional 14 day period of cage ambulation during reloading. A total of six rats from the vehicle and the resveratrol treated groups were used for the hindlimb suspension and recovery protocols. Furthermore, two groups of 6 vehicle treated animals maintained normal ambulation throughout the experiment, and were used as control animals for the hindlimb suspension and reloading groups. The data show that resveratrol supplementation was unable to attenuate the decreases in plantaris muscle wet weight during hindlimb suspension but it improved muscle mass during reloading after hindlimb suspension. Although resveratrol did not prevent fiber atrophy during the period of disuse, it increased the fiber cross sectional area of type IIA and IIB fibers in response to reloading after hindlimb suspension. There was a modest enhancement of myogenic precursor cell proliferation in resveratrol-treated muscles after reloading, but this failed to reach statistical significance. The resveratrol-associated improvement in type II fiber size and muscle mass recovery after disuse may have been due to decreases in the abundance of pro-apoptotic proteins Bax, cleaved caspase 3 and cleaved caspase 9 in reloaded muscles. Resveratrol appears to have modest therapeutic benefits for improving muscle mass after disuse in aging.     

Age-related differences in apoptosis with disuse atrophy in soleus muscle

Muscle atrophy is associated with a loss of muscle fiber nuclei, most likely through apoptosis. We investigated age-related differences in the extent of apoptosis in soleus muscle of young (6 mo) and old (32 mo) male Fischer 344 x Brown Norway rats subjected to acute disuse atrophy induced by 14 days of hindlimb suspension (HS). HS-induced atrophy (reduction in muscle weight and cross-sectional area) was associated with loss of myofiber nuclei in soleus muscle of young, but not old, rats. This resulted in a significant decrease in the myonuclear domain (cross-sectional area per nucleus) in young and old rats, with changes being more pronounced in old animals. Levels of apoptosis (TdT-mediated dUTP nick end labeling and DNA fragmentation) were higher in soleus muscles of old control rats than young animals. Levels were significantly increased with HS in young and old rats, with the greatest changes in old animals. Caspase-3 activity in soleus muscle tended to be increased with age, but changes were not statistically significant (P=0.052). However, with HS, caspase-3 activity significantly increased in young, but not old, rats. Immunohistochemistry showed that the proapoptotic endonuclease G (EndoG, a mitochondrion-specific nuclease) was localized in the subsarcolemmal mitochondria in control muscles, and translocation to the nucleus occurred in old, but not young, control animals. There was no difference between EndoG total protein content in young and old control rats, but EndoG increased almost fivefold in soleus muscle of old, but not young, rats after HS. These results show that deregulation of myonuclear number occurs in old skeletal muscle and that the pathways involved in apoptosis are distinct in young and old muscles. Apoptosis in skeletal muscle is partly mediated by the subsarcolemmal mitochondria through EndoG translocation to the nucleus in response to HS.     

Hindlimb unloading-induced muscle atrophy and loss of function: protective effect of isometric exercise.

The primary objective of this study was to determine the effectiveness of isometric exercise (IE) as a countermeasure to hindlimb unloading (HU)-induced atrophy of the slow (soleus) and fast (plantaris and gastrocnemius) muscles. Rats were assigned to either weight-bearing control, 7-day HU (H7), H7 plus IE (I7), 14-day HU (H14), or H14 plus IE (I14) groups. IE consisted of ten 5-s maximal isometric contractions separated by 90 s, administered three times daily. Contractile properties of the soleus and plantaris muscles were measured in situ. The IE attenuated the HU-induced decline in the mass and fiber diameter of the slow-twitch soleus muscle, whereas the gastrocnemius and plantaris mass were not protected. These results are consistent with the mean electromyograph recordings during IE that indicated preferential recruitment of the soleus over the gastrocnemius and plantaris muscles. Functionally, the IE significantly protected the soleus from the HU-induced decline in peak isometric force (I14, 1.49 +/- 0.12 vs. H14, 1.15 +/- 0.07 N) and peak power (I14, 163 +/- 17 vs. H14, 75 +/- 11 mN.fiber length.s-1). The exercise protocol showed protection of the plantaris peak isometric force at H7 but not H14. The IE also prevented the HU-induced decline in the soleus isometric contraction time, which allowed the muscle to produce greater tension at physiological motoneuron firing frequencies. In summary, IE resulted in greater protection from HU-induced atrophy in the slow soleus than in the fast gastrocnemius or plantaris.     

Hindlimb unloading decreases thioredoxin-related antioxidant proteins and increases thioredoxin-binding protein-2 in rat skeletal muscle

To investigate role(s) of thioredoxin-related antioxidant proteins in disuse muscle atrophy, we examined the levels of thioredoxin-1 (Trx-1), peroxiredoxin-3/SP-22 (Prx-3) and thioredoxin-binding protein-2 (TBP-2) in rat soleus muscle subjected to hindlimb unloading (HU) for 2, 4, 7 or 14 days. The muscle weight loss was initially observed on day 4. The increases in aclorein- and malondialdehyde-modified proteins, and the decreases in the levels of Trx-1, Prx-3 and Mn-SOD were observed in the late phase of muscle atrophy, whereas, the increase in mRNA expression of TBP-2, a negative regulator of thioredoxin, preceded muscle atrophy. These findings suggest that the decrease of those antioxidant proteins, particularly a marked decrease of Trx-1, may be responsible for the enhanced oxidative damage during the late phase of disuse muscle atrophy. Furthermore, the increase in TBP-2 preceding the muscle atrophy may suppress the thioredoxin-mediated redox signaling, which can be an initial trigger leading to disuse muscle atrophy.     

Regulation of skeletal muscle dihydropyridine receptor gene expression by biomechanical unloading.

Biomechanical unloading of the rat soleus by hindlimb unweighting is known to induce atrophy and a slow- to fast-twitch transition of skeletal muscle contractile properties, particularly in slow-twitch muscles such as the soleus. The purpose of this study was to determine whether the expression of the dihydropyridine (DHP) receptor gene is upregulated in unloaded slow-twitch soleus muscles. A rat DHP receptor cDNA was isolated by screening a random-primed cDNA lambda gt10 library from denervated rat skeletal muscle with oligonucleotide probes complementary to the coding region of the rabbit DHP receptor cDNA. Muscle mass and DHP receptor mRNA expression were assessed 1, 4, 7, 14, and 28 days after hindlimb unweighting in rats by tail suspension. Isometric twitch contraction times of soleus muscles were measured at 28 days of unweighting. Northern blot analysis showed that tissue distribution of DHP receptor mRNA was specific for skeletal muscle and expression was 200% greater in control fast-twitch extensor digitorum longus (EDL) than in control soleus muscles. A significant stimulation (80%) in receptor message of the soleus was induced as early as 24 h of unloading without changes in muscle mass. Unloading for 28 days induced marked atrophy (control = 133 +/- 3 vs. unweighted = 62.4 +/- 1.8 mg), and expression of the DHP receptor mRNA in the soleus was indistinguishable from levels normally expressed in EDL muscles. These changes in mRNA expression are in the same direction as the 37% reduction in time to peak tension and 28% decrease in half-relaxation time 28 days after unweighting. Our results suggest that muscle loading necessary for weight support modulates the expression of the DHP receptor gene in the soleus muscle.     

Impact of reduced cytochrome oxidase activity on peak oxygen consumption of muscle

The impact of reduced muscle oxidative capacity on peak oxygen consumption and isometric performance was evaluated using an isolated rat hindlimb preparation perfused with a high oxygen delivery. Capacity for electron transport was reduced with chloramphenicol (CAP), an inhibitor of mitochondrial gene-coded protein synthesis. The activity of cytochrome oxidase, a mitochondrial cristae component, was reduced approximately 45% (P less than 0.005) in the mixed-fiber-type plantaris muscle. Several facets of muscle remodeling were also evident with the 10- to 14-day CAP treatment, including decreased citrate synthase activity, increased capillarity, and increased numbers of type IIc fibers. Perfusion of CAP (n = 6) and control (n = 7) rat hindlimbs of similar size with similar total flows (10-11 ml/min) and oxygen contents (20-21 vol%) resulted in similarly high oxygen deliveries to contracting muscles of the hindlimbs (CAP, 9.66 +/- 0.83 mumols.min-1.g-1; control, 8.74 +/- 0.75). Performance of the gastrocnemius-plantaris-soleus group declined in a similar fashion for both groups during increasingly intense near-steady-state tetanic contraction (100 ms at 100 Hz) conditions of 4, 8, 15, 30, 45, and 60 per minute. Oxygen consumption was similar for both groups at rest and increased similarly at each contraction condition. Peak oxygen consumption was not different between CAP (5.34 +/- 0.55 mumols.min-1.g-1) and control (5.74 +/- 0.43) groups and required only 56-68% of the oxygen delivered. This implies that rat skeletal muscle can suffer a significant reduction in its electron transport capacity without impairing peak oxygen consumption and muscle performance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrical stimulation influences satellite cell proliferation and apoptosis in unloading-induced muscle atrophy in mice

Muscle atrophy caused by disuse is accompanied by adverse physiological and functional consequences. Satellite cells are the primary source of skeletal muscle regeneration. Satellite cell dysfunction, as a result of impaired proliferative potential and/or increased apoptosis, is thought to be one of the causes contributing to the decreased muscle regeneration capacity in atrophy. We have previously shown that electrical stimulation improved satellite cell dysfunction. Here we test whether electrical stimulation can also enhance satellite cell proliferative potential as well as suppress apoptotic cell death in disuse-induced muscle atrophy. Eight-week-old male BALB/c mice were subjected to a 14-day hindlimb unloading procedure. During that period, one limb (HU-ES) received electrical stimulation (frequency: 20 Hz; duration: 3 h, twice daily) while the contralateral limb served as control (HU). Immunohistochemistry and western blotting techniques were used to characterize specific proteins in cell proliferation and apoptosis. The HU-ES soleus muscles showed significant improvement in muscle mass, cross-sectional area, and peak tetanic force relative to the HU limb (p<0.05). The satellite cell proliferative activity as detected within the BrdU+/Pax7+ population was significantly higher (p<0.05). The apoptotic myonuclei (detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) and the apoptotic satellite cells (detected by cleaved Poly [ADP-ribose] polymerase co-labeled with Pax7) were reduced (p<0.05) in the HU-ES limb. Furthermore the apoptosis-inducing factor and cleaved caspase-3 were down-regulated while the anti-apoptotic Bcl-2 protein was up-regulated (p<0.05), in the HU-ES limb. These findings suggest that the electrical stimulation paradigm provides an effective stimulus to rescue the loss of myonuclei and satellite cells in disuse muscle atrophy, thus maintaining a viable satellite cell pool for subsequent muscle regeneration. Optimization of stimulation parameters may enhance the outcome of the intervention.     

Hot and steady: Elevated temperatures do not enhance muscle disuse atrophy during prolonged aestivation in the ectotherm Cyclorana alboguttata

Animals that undergo prolonged dormancy experience minimal muscle disuse atrophy (MDA) compared to animals subjected to artificial immobilisation over shorter timeframes. An association between oxidative stress and MDA suggests that metabolic depression presumably affords dormant animals some protection against muscle disuse. Because aerobic metabolism is temperature sensitive, we proposed that MDA in dormant (aestivating) ectotherms would be enhanced at elevated temperatures. In the green‐striped burrowing frog, Cyclorana alboguttata, the thermal sensitivity of skeletal muscle metabolic rate is muscle‐specific. We proposed that the degree of atrophy experienced during aestivation would correlate with the thermal sensitivity of muscle metabolic rate such that muscles with a relatively high metabolic rate at high temperatures would experience more disuse atrophy. To test this hypothesis, we examined the effect of temperature and aestivation on the extent of MDA in two functionally different muscles: the M. gastrocnemius (jumping muscle) and M. iliofibularis (non‐jumping muscle), in C. alboguttata aestivating at 24 or 30°C for 6 months. We compared a range of morphological parameters from muscle cross‐sections stained with succinic dehydrogenase to show that muscle‐specific patterns of disuse atrophy were consistent with the relative rates of oxygen consumption of those muscle types. However, despite muscle‐specific differences in thermal sensitivity of metabolic rate, aestivation temperature did not influence the extent of atrophy in either muscle. Our results suggest that the muscles of frogs aestivating at high temperatures are defended against additional atrophy ensuring protection of muscle function during long periods of immobilisation. J. Morphol., 2013. © 2012 Wiley Periodicals, Inc.     

Free radicals may contribute to oxidative skeletal muscle fatigue

We used mouse soleus in vitro (n = 30) and canine gastrocnemius-plantaris preparations (n = 20) pump-perfused at the animal's blood pressure to establish if free radicals contribute to fatigue in oxidative skeletal muscle. The soleus from each leg contracted for 200 ms (70 Hz) once every minute for 60 min in Hepes buffer gassed with 100% oxygen at 27 degrees C. When contracting in Hepes alone, both muscles fatigued at 0.9 mN/mm2.min over the 60 min. The addition of purines to the bath increased the rate to 1.4 mN/mm2.min and the addition of xanthine oxidase to generate free radicals increased the rate again to 1.9 mN/mm2.min. Thus free radicals appeared to attenuate oxidative skeletal muscle function. Each canine muscle contracted isometrically at 4 Hz for 30 min and then rested for 45 min before contracting for a second 30 min at 4 Hz. In each experiment, we infused saline at 0.76 mL/min into resting muscle and at 1.91 mL/min during the first contraction period. During the remainder of the experiment, we infused, at the same rates, saline (n = 4), 10 microM dimethyl sulfoxide (DMSO) (n = 4) to identify the effect of scavenging hydroxyl radicals, 1 mM allopurinol to establish the effect of blocking xanthine oxidase (n = 4), or 200 microM desferoxamine to determine the effect of chelating iron (n = 4). With saline, the fatigue rate over the 30 min of contractions increased from 5.0 +/- 0.2 to 6.3 +/- 0.5 N/kg.min from the first to the second stimulation period. The fatigue rate was slower in the second period with each of the three experimental substances (DMSO, 5.9 +/- 0.8 to 3.2 +/- 0.3; allopurinol, 7.3 +/- 1.1 to 4.6 +/- 0.6; desferoxamine, 6.8 +/- 0.8 to 4.4 +/- 0.8 N/kg.min). The fatigue rate was the same as control when DMSO was infused only during the second contraction period. Therefore, free radicals appeared to contribute to fatigue in oxidative skeletal muscle.     

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.     

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.     

Effect of low extracellular calcium and ryanodine on muscle contraction of the mouse during postnatal development.

We have examined the effects of low Ca2+ solutions, Co2+, and ryanodine on the isometric tension and contraction speed of isolated, developing mouse EDL muscles. Twitch responses of young muscles (7-14 days postnatal) were more sensitive to lowered [Ca2+]o than those of more fully developed muscles (22-35 days postnatal). Responses of EDL muscles from a middle-aged group (15-21 days postnatal) were intermediate between the two other groups. Overall, the time course of contraction in a single twitch was accelerated by low [Ca2+]o. Ca(2+)-free solution induced a 7.95 and 9.25 mV depolarization in young and "old" muscle fibres, respectively. The presence of cobalt ions (5 mM) in the Krebs solution had a similar effect as Ca(2+)-free Krebs in terms of reduction of the isometric twitch and tetanic tensions of EDL muscles from the various age groups. In contrast, the shortening of the contraction time seen with Ca(2+)-free solution did not take place following exposure to Co(2+)-containing solutions. Finally, young (7-14 days postnatal) muscles were less sensitive to the inhibitory action of ryanodine on the twitch compared with more fully developed muscles (22-35 days postnatal). Taken together, our results indicate that from birth to maturity, there is a gradual change in the spectrum of calcium utilization for the contractile process.     

Limited resistance of hypertrophied skeletal muscle to glucocorticoids

Male hypophysectomized rats were initially assigned to a control or an overloaded group that underwent compensatory hypertrophy of plantaris muscles to steady-state levels following removal of synergistic musculature. Plantaris muscle mass of overloaded animals was higher than that of controls by 38% (391 +/- 8 vs 284 +/- 7 mg) and glucocorticoid cytosol specific binding concentrations, using [3H]triamcinolone acetonide (TA) as the labeled steroid, was also significantly higher in hypertrophied muscles (83.3 +/- 3.9 fmol . mg protein-1) than in control muscles 56.3 +/- 3.9 fmol . mg protein-1). Cortisone acetate (CA) was then administered daily subcutaneously in high, 100 mg; intermediate, 10 mg; or low, 1.0 mg . kg-1 body wt doses. Groups of rats were killed after 1/4, 2 days and 7 days. Absolute muscle mass losses after 7 days of CA treatment were approx 80 mg with high doses and 60 mg with intermediate doses in both hypertrophied and control muscles. The low CA dose did not produce atrophy. The absolute depletion of [3H]TA binding activity with CA treatment was always greater in hypertrophied muscles of high and intermediate dose treated than those of their respective controls, but TA binding capacities remained higher in hypertrophied muscles than in controls at almost all time points in all treatment groups. Unlike previous findings in which the simultaneous initiation of overload prevented glucocorticoid induced muscle wasting, no resistance to the effect of CA treatment was observed when treatment was begun after hypertrophy had occurred.