Length-tension relationships are altered in regenerating muscles of the rat after bupivacaine injection.

Intramuscular injection of bupivacaine causes complete degeneration of fibers in extensor digitorum longus (EDL) muscles of rats, followed by complete regeneration within 60 days. Previous studies have shown that regenerated EDL muscles are protected from contraction-induced injury 60 days after bupivacaine injection. It is possible that these regenerated muscles have altered length-tension relations because of fiber remodeling. We tested the hypothesis that length-tension relations are different in bupivacaine-injected and noninjected control muscles. EDL and soleus muscles of the right hindlimb of deeply anesthetized rats were injected with bupivacaine and then allowed to recover for 7, 14, 21, or 60 days (7D, 14D, 21D, 60D), and isometric contractile properties were assessed. Muscles of the contralateral limb were not injected and served as control. EDL muscles recovered from bupivacaine injection more rapidly than soleus muscles, with mass restored to control levels at 21D, and isometric tetanic force (P(o)) restored to control at 60D. In contrast, mass and P(o) of injected soleus muscles was not restored to control even at 60D. In 7D EDL muscles, length-tension curves were shifted leftward compared with control, but in 21D and 60D EDL muscles length-tension curves were right shifted significantly (treatment x muscle length: P < 0.001). Although no clear shift in the position of the length-tension curve was observed in regenerating soleus muscles, force production was enhanced on the descending limb of the curve in 60D soleus muscles (treatment x relative muscle length: P < 0.01). The rightward shift in the length-tension curve of EDL muscles 60 days after bupivacaine injection is likely to contribute to the mechanism for their previously observed protection from contraction-induced injury.     

Isometric training of young rats — Effects upon hind limb muscles

The soleus, rectus femoris, and gastrocnemius muscles of young rats trained isometrically for 4 weeks were studied by light and electron microscopy.--The percentage of fast-twitch oxidative muscle fibers decreased at the cost of the fast-twitch glycolytic fibers in the rectus femoris muscle. The percentages of the slow-twitch oxidative fibers did not change significantly in any of the muscles studied. The changes in the areas of the muscle fibers were specific for the muscle and the fiber type and indicate geometrical rearrangements of the fibers in the trained muscles. The Z and M lines were broader in the soleus (containing about 85% slow-twitch oxidative fibers) than in the rectus femoris muscle (containing about 90% fast-twitch glycolytic fibers), while the sarcomere length and the pseudo-H zone were similar. The length of the myosin filaments appeared to be slightly shorter in the fast rectus femoris than in the slow soleus muscle.--The hypothesis on the temporal progress of muscle adaptation to training (Müller, 1974) was substantiated. Correlations between biochemical (Exner et al., 1973a) and histochemical parameters measuring the oxidative capacity were preserved during adaptation to training. The comparison of the histochemical results with the physiological data on similar animals (Exner et al., 1973a) suggests a complex relationship between the contraction time and the percentage of fast-twitch muscle fibers.     

Proteomic analysis of slow- and fast-twitch skeletal muscles

Skeletal muscles are composed of slow- and fast-twitch muscle fibers, which have high potential in aerobic and anaerobic ATP production, respectively. To investigate the molecular basis of the difference in their functions, we examined protein profiles of skeletal muscles using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis with pH 4-7 and 6-11 isoelectric focusing gels. A comparison between rat soleus and extensol digitorum longus (EDL) muscles that are predominantly slow- and fast-twitch fibers, respectively, showed that the EDL muscle had higher levels of glycogen phosphorylase, most glycolytic enzymes, glycerol 3-phosphate dehydrogenase, and creatine kinase; while the soleus muscle had higher levels of myoglobin, TCA cycle enzymes, electron transfer flavoprotein, and carbonic anhydrase III. The two muscles also expressed different isoforms of contractile proteins including myosin heavy and light chains. These protein patterns were further compared with those of red and white gastrochnemius as well as red and white quadriceps muscles. It was found that metabolic enzymes showed a concerted regulation dependent on muscle fiber types. On the other hand, expression of contractile proteins seemed to be independent of the metabolic characteristics of muscle fibers. These results suggest that metabolic enzymes and contractile proteins show different expression patterns in skeletal muscles.     

Skeletal muscle response to spaceflight, whole body suspension, and recovery in rats

Comparisons of soleus and extensor digitorum longus (EDL) muscles from male Sprague-Dawley rats (350-400 g) after 7 days of weightlessness, 7 and 14 days of whole body suspension (WBS), and 7 days of recovery from WBS and from vivarium controls were made. Muscle mass loss of approximately 30% was observed in soleus after 7 and 14 days of WBS. Measurement of slow- and fast-twitch fibers showed significant alterations. Reductions in cross-sectional areas and increases in fiber densities in soleus after spaceflight and WBS were related to previous findings of muscle atrophy during unloading. Capillary density also showed a marked increase with unloading. Seven days of weightlessness were sufficient to effect a 20 and 15% loss in absolute muscle mass in soleus and EDL, respectively. However, the antigravity soleus was more responsive in terms of cross-sectional area reductions. After 7 days of recovery from WBS, with normal ambulatory loading, the parameters studied showed a reversal to control levels. Muscle plasticity, in terms of fiber and capillary responses, indicated differences in responses in the two types of muscles and further amplified that antigravity posture muscles are highly susceptible to unloading. Studies of recovery from spaceflight for both muscle metabolism and microvascular modifications are further justified.     

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.     

Expression of nestin,desmin and vimentin in intact and regenerating muscle spindles of rat hind limb skeletal muscles

We describe the expression and distribution patterns of nestin, desmin and vimentin in intact and regenerating muscle spindles of the rat hind limb skeletal muscles. Regeneration was induced by intramuscular isotransplantation of extensor digitorum longus (EDL) or soleus muscles from 15-day-old rats into the EDL muscle of adult female inbred Lewis rats. The host muscles with grafts were excised after 7-, 16-, 21- and 29-day survival and immunohistochemically stained. Nestin expression in intact spindles in host muscles was restricted to Schwann cells of sensory and motor nerves. In transplanted muscles, however, nestin expression was also found in regenerating “spindle fibers”, 7 and 16 days after grafting. From the 21st day onwards, the regenerated spindle fibers were devoid of nestin immunoreactivity. Desmin was detected in spindle fibers at all developmental stages in regenerating as well as in intact spindles. Vimentin was expressed in cells of the outer and inner capsules of all muscle spindles and in newly formed myoblasts and myotubes of regenerating spindles 7 days after grafting. Our results show that the expression pattern of these intermediate filaments in regenerating spindle fibers corresponds to that found in regenerating extrafusal fibers, which supports our earlier suggestion that they resemble small-diameter extrafusal fibers.     

Seven skeletal muscles rich in slow muscle fibers may function to sustain neutral position in the rodent hindlimb

Skeletal muscles consist of slow-twitch and fast-twitch muscle fibers, which have distinct physiological and biochemical properties. The muscle fiber composition determines the contractile velocity and fatigability of a particular skeletal muscle. We analyzed the systemic distribution of slow muscle fibers in all rodent skeletal muscles by myosin ATPase staining and found that only seven hindlimb skeletal muscles were extremely rich in slow muscle fibers. These included the mouse piriformis (56.5%), gluteus minimus (35.7%), vastus intermedius (24.7%), quadratus femoris (69.9%), adductor brevis (44.3%), gracilis (24.6%), and soleus muscles (35.1%). In mice, the relative proportion of slow muscle fibers did not exceed 15% in skeletal muscles in other regions. The distribution of slow muscle fibers was well conserved in rats and rabbits. The soleus muscle is an important antigravity muscle in both rodents and humans; therefore, these skeletal muscles rich in slow muscle fibers might play an important role in sustaining neutral alignment of the lower extremity.     

Life-long calorie restriction in Fischer 344 rats attenuates age-related loss in skeletal muscle-specific force and reduces extracellular space.

The decline in muscle function is associated with an age-related decrease in muscle mass and an age-related decline in strength. However, decreased strength is not solely due to decreased muscle mass. The age-related decline in muscle-specific force (force/muscle cross-sectional area), a measure of intrinsic muscle function, also contributes to age-related strength decline, and the mechanisms by which this occurs are only partially known. Moreover, changes in the extracellular space could have a profound effect on skeletal muscle function. Life-long calorie restriction in rodents has shown to be a powerful anti-aging intervention. In this study, we examine whether calorie restriction is able to attenuate the loss of muscle function and elevations in extracellular space associated with aging. We hypothesize that calorie restriction attenuates the age-associated decline in specific force and increases in extracellular space. Measurements of in vitro contractile properties of the extensor digitorum longus (type II) and soleus (type I) muscles from 12-mo and 26- to 28-mo-old ad libitum-fed, as well as 27- to 28-mo-old life-long calorie-restricted male Fischer 344 rats, were performed. We found that calorie restriction attenuated the age-associated decline in muscle mass-to-body mass ratio (mg/g) and strength-to-body mass ratio (N/kg) in the extensor digitorum longus muscle (P < 0.05) but not in the soleus muscle (P > 0.05). Importantly, muscle-specific force (N/cm2) in the extensor digitorum longus, but not in the soleus muscle, of the old calorie-restricted rats was equal to that of the young 12-mo-old animals. Moreover, the age-associated increase in extracellular space was reduced in the fast-twitch extensor digitorum longus muscle (P < 0.05) but not in the soleus muscle with calorie restriction. We also found a significant correlation between the extracellular space and the muscle-specific force in the extensor digitorum longus (r = -0.58; P < 0.05) but not in the soleus muscle (r = -0.38; P > 0.05). Hence, this study shows a loss of muscle function with age and suggests that long-term calorie restriction is an effective intervention against the loss of muscle function with age.     

Expression of myosin isoforms during notexin-induced regeneration of rat soleus muscles

Myosin isozymes and their fiber distribution were studied during regeneration of the soleus muscle of young adult (4-6 week old) rats. Muscle degeneration and regeneration were induced by a single subcutaneous injection of a snake toxin, notexin. If reinnervation of the regenerating muscle was allowed to occur (functional innervation nearly complete by 7 days), then fiber diameters continued to increase and by 28 days after toxin treatment they attained the same values as fibers in the contralateral soleus. If the muscles were denervated at the time of toxin injection, the early phases of regeneration still took place but the fibers failed to continue to increase in size. Electrophoresis of native myosin showed multiple bands between 3 and 21 days of regeneration which could be interpreted as indicating the presence of embryonic, neonatal, fast and slow myosins in the innervated muscles. Adult slow myosin became the exclusive from in innervated regenerates. In contrast, adult fast myosin became the predominant form in denervated regenerating muscles. Immunocytochemical localization of myosin isozymes demonstrated that in innervated muscles the slow form began to appear in a heterogeneous fashion at about 7 days, and became the major form in all fibers by 21-28 days. Thus, the regenerated muscle was almost entirely composed of slow fibers, in clear contrast to the contralateral muscle which was still substantially mixed. In denervated regenerating muscles, slow myosin was not detected biochemically or immunocytochemically whereas fast myosin was detected in all denervated fibers by 21-28 days. The regenerating soleus muscle therefore is clearly different from the developing soleus muscle in that the former is composed of a uniform fiber population with respect to myosin transitions. Moreover the satellite cells which account for the regeneration process in the soleus muscle do not appear to be predetermined with respect to myosin heavy chain expression, since the fibers they form can express either slow or fast isoforms. The induction of the slow myosin phenotype is entirely dependent on a positive, extrinsic influence of the nerve.     

Effects of a chronic wasting infection on skeletal muscle size and contractile properties

To evaluate the effects of chronic infection on skeletal muscle dimensions and contractile properties, we used a hamster model of visceral leishmaniasis, a parasitic infection of the reticuloendothelial system produced by Leishmania donovani (LD). To distinguish between effects of reduced caloric intake and infection per se, we also studied healthy control animals and noninfected animals subjected to caloric restriction. Three muscles were tested in vitro: plantaris, soleus, and diaphragm. Both caloric restriction and LD infection caused loss of body weight and reduced muscle cross-sectional areas and wet weights. The interventions had variable effects on in vitro contractile properties, the most pronounced being reduction in peak tension in response to tetanic stimulation. Tension loss was 35-45%, except for a loss of 65% in plantaris of LD-infected animals. We conclude that chronic LD infection affects skeletal muscles in both indirect and direct ways. 1) Reduced caloric intake due to anorexia decreases muscle size and active tension. Disuse probably enhances this effect in limb muscles. 2) Infection produces profound weakness of inactive fast-twitch muscle by unknown mechanisms.     

Effect of hindlimb unweighting on tissue blood flow in the rat.

The purpose of this study was to characterize the distribution of blood flow in the rat during hindlimb unweighting (HU) and post-HU standing and exercise and examine whether the previously reported (Witzmann et al., J. Appl. Physiol. 54: 1242-1248, 1983) elevation in anaerobic metabolism observed with contractile activity in the atrophied soleus muscle was caused by a reduced hindlimb blood flow. After either 15 days of HU or cage control, blood flow was measured with radioactive microspheres during unweighting, normal standing, and running on a treadmill (15 m/min). In another group of control and experimental animals, blood flow was measured during preexercise (PE) treadmill standing and treadmill running (15 m/min). Soleus muscle blood flow was not different between groups during unweighting, PE standing, and running at 15 m/min. Chronic unweighting resulted in the tendency for greater blood flow to muscles composed of predominantly fast-twitch glycolytic fibers. With exercise, blood flow to visceral organs was reduced compared with PE values in the control rats, whereas flow to visceral organs in 15-day HU animals was unaltered by exercise. These higher flows to the viscera and to muscles composed of predominantly fast-twitch glycolytic fibers suggest an apparent reduction in the ability of the sympathetic nervous system to distribute cardiac output after chronic HU. In conclusion, because 15 days of HU did not affect blood flow to the soleus during exercise, the increased dependence of the atrophied soleus on anerobic energy production during contractile activity cannot be explained by a reduced muscle blood flow.     

Expression of slow myosin heavy chain during muscle regeneration is not always dependent on muscle innervation and calcineurin phosphatase activity

In the literature, there is an ambiguity as to the respective roles played by calcineurin phosphatase activity (CPA) and muscle innervation in the reestablishment of the slow-twitch muscle phenotype after muscle regeneration in different species. In this study, we wanted to determine the role of calcineurin and muscle innervation on the appearance and maintenance of the slow phenotype during mouse muscle regeneration. The pattern of myosin expression and CPA was analyzed in adult (n=15), regenerating (n=45) and denervated-regenerating (n=32) slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles. Moreover, in a second group of denervated-regenerating mice (n=9), the animals were treated with a calcineurin inhibitor. Regeneration was induced by injection of cardiotoxin and in the denervated-regenerating group, denervation was carried out by cutting the sciatic nerve before the administration of cardiotoxin. In innervated-regenerating soleus muscle, CPA increased continuously after 10 days postinjury and by 21 days, there was a 3.5-fold increase in CPA compared with adult basal level, whereas in innervated-regenerating EDL muscle, CPA remained unchanged. Moreover, our results show that in denervated-regenerating muscles, the MyHC profile was identical in spite of the functional differences inherent in these muscles. In long-term denervated-regenerating muscles, a slow muscle phenotype was reexpressed both in the presence or absence of calcineurin inhibitor. Our results show that although in innervated-regenerating mouse muscle, the appearance of a slow phenotype is correlated with a peak of CPA, in denervated-regenerating muscles, a slow phenotype is triggered and maintained in a calcineurin- and nerve-independent manner.     

No effects of lifelong creatine supplementation on sarcopenia in senescence-accelerated mice (SAMP8)

Oral creatine supplementation can acutely ameliorate skeletal muscle function in older humans, but its value in the prevention of sarcopenia remains unknown. We evaluated the effects of lifelong creatine supplementation on muscle mass and morphology, contractility, and metabolic properties in a mouse model of muscle senescence. Male senescence-accelerated mice (SAMP8) were fed control or creatine-supplemented (2% of food intake) diet from the age of 10 to 60 wk. Soleus and extensor digitorum longus muscles were tested for in vitro contractile properties, creatine content, and morphology at weeks 25 and 60. Both muscle types showed reduced phosphocreatine content at week 60 that could not be prevented by creatine. Accordingly, age-associated decline in muscle mass and contractility was not influenced by treatment. Aged soleus muscles had fewer and smaller fast-twitch glycolytic fibers irrespective of treatment received. It is concluded that lifelong creatine supplementation is no effective strategy to prevent sarcopenia in senescence-accelerated mice.     

Adaptation of rat skeletal muscle to creatine depletion: AMP deaminase and AMP deamination.

AMP deaminase catalyzes deamination of the AMP formed in contracting muscles to inosine 5'-monophosphate (IMP). Slow-twitch muscle has only approximately 30% as high a level of AMP deaminase activity as fast-twitch muscle in the rat, and rates of IMP formation during intense contractile activity are much lower in slow-twitch muscle. We found that feeding the creatine analogue beta-guanidinopropionic acid (beta-GPA) to rats, which results in creatine depletion, causes a large decrease in muscle AMP deaminase. This adaptation was used to evaluate the role of AMP deaminase activity level in accounting for differences in IMP production in slow-twitch and fast-twitch muscles. beta-GPA feeding for 3 wk lowered AMP deaminase activity in fast-twitch epitrochlearis muscle to a level similar to that found in the normal slow-twitch soleus muscle but had no effect on the magnitude of the increase in IMP in response to intense contractile activity. Despite a similar decrease in ATP in the normal soleus and the epitrochlearis from beta-GPA-fed rats, the increase in IMP was only approximately 30% as great in the soleus in response to intense contractile activity. These results demonstrate that the accumulation of less IMP in slow- compared with fast-twitch skeletal muscle during contractile activity is not due to the lower level of AMP deaminase in slow-twitch muscle.     

Fast-to-Slow Transition of Skeletal Muscle Contractile Function and Corresponding Changes in Myosin Heavy and Light Chain Formation in the R6/2 Mouse Model of Huntington’s Disease

Huntington´s disease (HD) is a hereditary neurodegenerative disease resulting from an expanded polyglutamine sequence (poly-Q) in the protein huntingtin (HTT). Various studies report atrophy and metabolic pathology of skeletal muscle in HD and suggest as part of the process a fast-to-slow fiber type transition that may be caused by the pathological changes in central motor control or/and by mutant HTT in the muscle tissue itself. To investigate muscle pathology in HD, we used R6/2 mice, a common animal model for a rapidly progressing variant of the disease expressing exon 1 of the mutant human gene. We investigated alterations in the extensor digitorum longus (EDL), a typical fast-twitch muscle, and the soleus (SOL), a slow-twitch muscle. We focussed on mechanographic measurements of excised muscles using single and repetitive electrical stimulation and on the expression of the various myosin isoforms (heavy and light chains) using dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole muscle and single fiber preparations. In EDL of R6/2, the functional tests showed a left shift of the force-frequency relation and decrease in specific force. Moreover, the estimated relative contribution of the fastest myosin isoform MyHC IIb decreased, whereas the contribution of the slower MyHC IIx isoform increased. An additional change occurred in the alkali MyLC forms showing a decrease in 3f and an increase in 1f level. In SOL, a shift from fast MyHC IIa to the slow isoform I was detectable in male R6/2 mice only, and there was no evidence of isoform interconversion in the MyLC pattern. These alterations point to a partial remodeling of the contractile apparatus of R6/2 mice towards a slower contractile phenotype, predominantly in fast glycolytic fibers.     

Nitric oxide synthase expression and effects of nitric oxide modulation on contractility of rat extraocular muscle.

Extraocular muscles (EOMs) are specialized skeletal muscles that are constantly active, generate low levels of force for cross sectional area, have rapid contractile speeds, and are highly fatigue resistant. The neuronal isoform of nitric oxide synthase (nNOS) is concentrated at the sarcolemma of fast-twitch muscles fibers, and nitric oxide (NO) modulates contractility. This study evaluated nNOS expression in EOM and the effect of NO modulation on lateral rectus muscle's contractility. nNOS activity was highest in EOM compared with diaphragm, extensor digitorum longus, and soleus. Neuronal NOS was concentrated to the sarcolemma of orbital and global singly innervated fibers, but not evident in the multi-innervated fibers. The NG-nitro-L-arginine methyl ester (L-NAME, a NOS inhibitor), increased submaximal tetanic and peak twitch forces. The NO donors S-nitroso-N-acetylcysteine (SNAC) and spermineNONOate reduced submaximal tetanic and peak twitch forces. The effect of NO on the contractile force of lateral rectus muscle is greater than previously observed on other skeletal muscle. NO appears more important in modulating contraction of EOM compared with other skeletal muscles, which could be important for the EOM's specialized role in generation of eye movements.     

Recovery of skeletal muscle mass after extensive injury: positive effects of increased contractile activity

The present study was designed to test the hypothesis that increasing physical activity by running exercise could favor the recovery of muscle mass after extensive injury and to determine the main molecular mechanisms involved. Left soleus muscles of female Wistar rats were degenerated by notexin injection before animals were assigned to either a sedentary group or an exercised group. Both regenerating and contralateral intact muscles from active and sedentary rats were removed 5, 7, 14, 21, 28 and 42 days after injury (n = 8 rats/group). Increasing contractile activity through running exercise during muscle regeneration ensured the full recovery of muscle mass and muscle cross-sectional area as soon as 21 days after injury, whereas muscle weight remained lower even 42 days postinjury in sedentary rats. Proliferator cell nuclear antigen and MyoD protein expression went on longer in active rats than in sedentary rats. Myogenin protein expression was higher in active animals than in sedentary animals 21 days postinjury. The Akt-mammalian target of rapamycin (mTOR) pathway was activated early during the regeneration process, with further increases of mTOR phosphorylation and its downstream effectors, eukaryotic initiation factor-4E-binding protein-1 and p70(s6k), in active rats compared with sedentary rats (days 7-14). The exercise-induced increase in mTOR phosphorylation, independently of Akt, was associated with decreased levels of phosphorylated AMP-activated protein kinase. Taken together, these results provided evidence that increasing contractile activity during muscle regeneration ensured early and full recovery of muscle mass and suggested that these beneficial effects may be due to a longer proliferative step of myogenic cells and activation of mTOR signaling, independently of Akt, during the maturation step of muscle regeneration.     

Comparison of premodulated interferential and pulsed current electrical stimulation in prevention of deep muscle atrophy in rats

The goal of this study was to compare the effects of electrical stimulation using pulsed current (PC) and premodulated interferential current (IC) on prevention of muscle atrophy in the deep muscle layer of the calf. Rats were randomly divided into 3 treatment groups: control, hindlimb unloading for 2 weeks (HU), and HU plus electrical stimulation for 2 weeks. The animals in the electrical stimulation group received therapeutic stimulation of the left (PC) or right (IC) calf muscles twice a day during the unloading period. Animals undergoing HU for 2 weeks exhibited significant loss of muscle mass, decreased cross-sectional area (CSA) of muscle fibers, and increased expression of ubiquitinated proteins in the gastrocnemius and soleus muscles compared with control animals. Stimulation with PC attenuated the effects on the muscle mass, fiber CSA, and ubiquitinated proteins in the gastrocnemius muscle. However, PC stimulation failed to prevent atrophy of the deep layer of the gastrocnemius muscle and the soleus muscle. In contrast, stimulation with IC inhibited atrophy of both the gastrocnemius and soleus muscles. In addition, the IC protocol inhibited the HU-induced increase in ubiquitinated protein expression in both gastrocnemius and soleus muscles. These results suggest that electrical stimulation with IC is more effective than PC in preventing muscle atrophy in the deep layer of limb muscles.