IGF-I treatment improves the functional properties of fast- and slow-twitch skeletal muscles from dystrophic mice

Although insulin-like growth factor-I (IGF-I) has been proposed for use by patients suffering from muscle wasting conditions, few studies have investigated the functional properties of dystrophic skeletal muscle following IGF-I treatment. 129P1 ReJ-Lama2(dy) (129 ReJ dy/dy) dystrophic mice suffer from a deficiency in the structural protein, laminin, and exhibit severe muscle wasting and weakness. We tested the hypothesis that 4 weeks of IGF-I treatment ( approximately 2 mg/kg body mass, 50 g/h via mini-osmotic pump, subcutaneously) would increase the mass and force producing capacity of skeletal muscles from dystrophic mice. IGF-I treatment increased the mass of the extensor digitorum longus (EDL) and soleus muscles of dystrophic mice by 20 and 29%, respectively, compared with untreated dystrophic mice (administered saline-vehicle only). Absolute maximum force (P(o)) of the EDL and soleus muscle was increased by 40 and 32%, respectively, following IGF-I treatment. Specific P(o) (sP(o)) was increased by 23% in the EDL muscles of treated compared with untreated mice, but in the soleus muscle sP(o) was unchanged. IGF-I treatment increased the proportion of type IIB and type IIA fibres and decreased the proportion of type I fibres in the EDL muscles of dystrophic mice. In the soleus muscles of dystrophic mice, IGF-I treatment increased the proportion of type IIA fibres and decreased the proportion of type I fibres. Average fibre cross-sectional area was increased in the EDL and soleus muscles of treated compared with untreated mice. We conclude that IGF-I treatment ameliorates muscle wasting and improves the functional properties of skeletal muscles of dystrophic mice. The findings have important implications for the role of IGF-I in ameliorating muscle wasting associated with the muscular dystrophies.     

Clenbuterol reduces soleus muscle fatigue during disuse in aged rats

High levels of clenbuterol have been shown to preserve muscle mass and function during disuse. In this study we report that a low dose of clenbuterol (10 microg/kg per day) lessened the loss of in situ soleus muscle isometric force normalized to wet muscle weight (P(o)/g wet weight) by 8% and reduced isometric fatigue by approximately 30% in senescent rats after 21 days of hindlimb suspension (HS). Clenbuterol did not reduce the loss of relative force in the soleus of adult rats or the plantaris of old or adult rats. Furthermore, clenbuterol failed to improve muscle force or isometric fatigue in the soleus of adult rats or in the plantaris of either age group after HS. We conclude that low levels of clenbuterol reduce muscle fatigue in slow muscles during disuse and this beta-agonist may also have therapeutic value for reducing fatigue in slow muscles (e.g., postural muscles) in the elderly during disuse.     

Low dose formoterol administration improves muscle function in dystrophic mdx mice without increasing fatigue

The beta(2)-adrenoceptor agonist (beta(2)-agonist), formoterol, has been shown to cause muscle hypertrophy in rats even when administered at the micromolar dose of 25 micro g/kg/day. We investigated whether a similar low dose of formoterol could improve muscle function in the dystrophic mdx mouse. Ten-week-old male mdx and wild-type (C57BL/10) mice were administered formoterol (25 micro g/kg/day, i.p.) for 4 weeks. Formoterol treatment increased extensor digitorum longus (EDL) and soleus muscle mass, increased median muscle fibre size in diaphragm, EDL, and soleus muscles, and increased maximum force producing capacity in skeletal muscles of both wild-type and mdx mice. In contrast to other studies where beta(2)-agonists have been administered to mice and rats, generally at higher doses, low dose formoterol treatment did not increase the fatiguability of EDL, soleus or diaphragm muscles. Although others have found formoterol can decrease ubiquitin mRNA and proteasome activity when administered to tumour bearing rats at high doses (2mg/kg/day), in the present study low dose formoterol treatment did not alter ubiquitin or the E1 and E3 ubiquitin ligases in diaphragm muscles of wild-type or mdx mice, but it did reduce the level of ubiquitinated proteins in diaphragm of wild-type mice. The findings indicate that formoterol has considerably more powerful anabolic effects on skeletal muscle than older generation beta(2)-agonists (like clenbuterol and albuterol), and has considerable therapeutic potential for muscular dystrophies and other neuromuscular disorders where muscle wasting is indicated.     

Muscle fiber type differentiation and satellite cell populations in normally grown and neonatally denervated muscles in the rat

Summary To examine the neural influence upon fiber type differentiation in developing muscles, newborn rats were subjected to sciatic nerve dissection, and the denervated extensor digitorum longus (EDL) (white) and soleus (red) muscles were examined in chronologic sequence by means of histochemistry and electron microscopy. The skeletal muscles in the newborn rats were undifferentiated (type 2C fibers seen on ATPase staining) and contained numerous myotubes. In the controls, the type 2C fibers started to differentiate at around 5 days and had almost completed type differentiation by 30 days in EDL and by 90 days in soleus muscles. On the other hand, none of the fibers in the neonatally denervated muscles developed into well differentiated type 1 and 2A fibers, but both the EDL and soleus showed longlasting type 2C and 2B populations. The satellite cells in the denervated EDL and soleus muscles decreased in number at the same rate as in the control muscles with maturation. The absence of a neural supply in the developing muscles induced a delay in muscle fiber type differentiation but did not influence the satellite cell populations in either EDL or soleus muscles.     

Choline acetyltransferase activity in muscles of old rats

The total activity of choline acetyltransferase (ChAc) in the rat extensor digitorum longus (EDL) and soleus muscles increased by 50 and 55%, respectively, between 3 and 9 months of age. In rats 28 to 29 months old, the activity of ChAc in EDL and soleus diminished to 41 and 40%, respectively, of the activity observed in 9-month-old animals. Age changes of ChAc activity in the diaphragm were not significant. The number of muscle fibers in EDL and soleus muscles of rats 28 to 29 months old decreased by 44 and 38% respectively, in comparison with younger animals. Mean muscle fiber diameters did not change between 3 and 9 months of age and decreased by 24, 35 and 9% in the EDL, soleus and diaphragm, respectively, in the 28- to 29-month-old rats. The activity of ChAc expressed in relation to one muscle fiber was about the same in the EDL and soleus muscles. It increased between 3 and 9 months and decreased between 9 and 28 to 29 months of age. The observation that ChAc activity per muscle fiber was identical in the fast EDL and slow soleus muscle suggests that the physiological differences between the two muscles are not caused by a difference in the capacity of their motor nerves to synthesize ACh. In the diaphragm the activity of ChAc per muscle fiber apparently did not diminish in old age. The decrease in the total ChAc activity in the limb muscles of old animals seems due both to a decrease in the number of nerve terminals in the muscles and to a decrease in the amount of enzyme present in individual terminals. We suggest that the maintenance of ChAc activity in the motor nerve terminals in the diaphragm of old rats is due to the continuous activity of this muscle and its motor nerves.     

The muscle spindle in cortisone-induced muscular atrophy

The muscle spindles in serially sectioned rat extensor digitorum longus (EDL) and soleus (SOL) muscles were studied histologically and histochemically after chronic cortisone administration. Nuclear chain fibers were found to be significantly atrophied in the EDL but not in the SOL. Nuclear bag fibers were not significantly affected in either muscle. Nuclear chain fibers all stained darkly with the pH 9.4 myofibrillar adenosine triphosphatase (ATPase) reaction (type II) and also stained darkly with the succine dehydrogenase (SDH) reaction. Nuclear bag fibers showed mostly light ATPase staining (type I) and dark, light, or intermediate SDH staining. Extrafusal fibers showed preferential type II fiber atrophy in EDL with relative sparing of type II fibers in SOL. The results suggest that histochemical characteristics that we have evaluated are alone insufficient to explain the differential susceptibility of skeletal muscle fibers to corticosteroids and, therefore, do not in themselves define either a myopathic or neurogenic etiology for the observed muscle wasting.     

Recovery of rat skeletal muscles after partial denervation is enhanced by treatment with nifedipine

Following partial denervation of adult rat skeletal muscle intact axons sprout to reinnervate denervated muscle fibres and increase their territory. The extent of this increase is limited and may depend on the ability of axon terminals to form and maintain synaptic contacts with the denervated muscle fibres. Here we tested the possibility whether reducing Ca²⁺ entry into presynaptic nerve terminals through dihydropyridine sensitive channels may allow more nerve–muscle contacts to be formed and maintained. Hindlimb muscles of adult Wistar rats were partially denervated by removing a small segment of the L4 or L5 spinal nerve on one side. A nifedipine-containing silastic rubber strip was subsequently implanted close to the partially denervated soleus or extensor digitorum longus (EDL) muscles in some animals. In control experiments silastic strips which did not contain nifedipine were used. Several weeks later isometric contractions were recorded, to determine the effect of (a) partial denervation and (b) nifedipine treatment on force output and motor unit numbers. The tension produced by nifedipine treated partially denervated muscles was 82% and 79% of the unoperated contralateral value for soleus and EDL, respectively. This was significantly greater than in untreated muscles, which only produced 61% and 48%, respectively. Mean motor unit force was also significantly larger with nifedipine treatment. Histological analysis revealed that a significantly larger proportion of the total number of muscle fibres remained in nifedipine-treated partially denervated muscles (soleus, 90% and EDL, 101%) compared with untreated muscles (soleus, 51% and EDL, 66%). Thus the number of neuromuscular contacts was increased with nifedipine treatment.     

Chemical sympathectomy further increases muscle protein degradation of acutely diabetic rats

The present work investigated the role of the sympathetic nervous system (SNS) in the control of protein degradation in skeletal muscles from rats with streptozotocin (STZ)-induced diabetes. Diabetes (1, 3, and 5 days after STZ) induced a significant increase in the norepinephrine content of soleus and EDL muscles, but it did not affect plasma catecholamine levels. Chemical sympathectomy induced by guanethidine (100 mg/kg body weight, for 1 or 2 days) reduced muscle norepinephrine content to negligible levels (less than 5%), decreased plasma epinephrine concentration, and further increased the high rate of protein degradation in muscles from acutely diabetic rats. The rise in the rate of proteolysis (nmol.mg wet wt(-1).2h(-1)) in soleus from 1-day diabetic sympathectomized rats was associated with increased activities of lysosomal (0.127 +/- 0.008 vs. 0.086 +/- 0.013 in diabetic control) and ubiquitin (Ub)-proteasome-dependent proteolytic pathways (0.154 +/- 0.007 vs. 0.121 +/- 0.006 in diabetic control). Increases in Ca2+-dependent (0.180 +/- 0.007 vs. 0.121 +/- 0.011 in diabetic control) and Ub-proteasome-dependent proteolytic systems (0.092 +/- 0.003 vs. 0.060 +/- 0.002 in diabetic control) were observed in EDL from 1-day diabetic sympathectomized rats. The lower phosphorylation levels of AKT and Foxo3a in EDL muscles from 3-day diabetic rats were further decreased by sympathectomy. The data suggest that the SNS exerts acute inhibitory control of skeletal muscle proteolysis during the early stages of diabetes in rats, probably involving the AKT/Foxo signaling pathway.     

Contractile properties of slow and fast skeletal muscles from protease activated receptor‐1 null mice

Introduction: Protease‐activated receptors (PARs) may play a role in skeletal muscle development. We compared the contractile properties of slow‐twitch soleus muscles and fast‐twitch extensor digitorum longus (EDL) muscles from PAR‐1 null and littermate control mice. Methods: Contractile function was measured using a force transducer system. Fiber type proportions were determined using immunohistochemistry. Results: Soleus muscles from PAR‐1 null mice exhibited longer contraction times, a leftward shift in the force–stimulation frequency relationship, and decreased fatiguability compared with controls. PAR‐1 null soleus muscles also had increased type 1 and decreased type IIb/x fiber numbers compared with controls. In PAR‐1 null EDL muscles, no differences were found, except for a slower rate of fatigue compared with controls. Conclusions: The absence of PAR‐1 results in a slower skeletal muscle contractile phenotype, likely due to an increase in type I and a decrease in type IIb/x fiber numbers. Muscle Nerve 50: 991–998, 2014     

Cycling exercise and fetal spinal cord transplantation act synergistically on atrophied muscle following chronic spinal cord injury in rats

The potential of two interventions, alone or in combination, to restore chronic spinal cord transection-induced changes in skeletal muscles of adult Sprague-Dawley rats was studied. Hind limb skeletal muscles were examined in the following groups of animals: rats with a complete spinal cord transection (Tx) for 8 weeks; Tx with a 4-week delay before initiation of a 4-week motor-assisted cycling exercise (Ex) program; Tx with a 4-week delay before transplantation (Tp) of fetal spinal cord tissue into the lesion cavity; Tx with a 4-week delay before Tp and Ex; and uninjured control animals. Muscle mass, muscle to body mass ratios, and mean myofiber cross-sectional areas were significantly reduced 8 weeks after transection. Whereas transplantation of fetal spinal cord tissue did not reverse this atrophy and exercise alone had only a modest effect in restoring lost muscle mass, the combination of exercise and transplantation significantly increased muscle mass, muscle to body mass ratios, and mean myofiber cross-sectional areas in both soleus and plantaris muscles. Spinal cord injury (SCI) also caused changes in myosin heavy chain (MyHC) expression toward faster isoforms in both soleus and plantaris and increased soleus myofiber succinate dehydrogenase (SDH) activity. Combined exercise and transplantation led to a change in the expression of the fastest MyHC isoform in soleus but had no effect in the plantaris. Exercise alone and in combination with transplantation reduced SDH activity to control levels in the soleus. These results suggest a synergistic action of exercise and transplantation of fetal spinal cord tissue on skeletal muscle properties following SCI, even after an extended post-injury period before intervention.     

A specific force deficit exists in skeletal muscle after partial denervation.

Skeletal muscle demonstrates a specific force deficit after repair of injured peripheral nerves, microneurovascular muscle transfer, and normal aging. Because atrophy cannot account for deficits in specific force, other, unknown, mechanisms are responsible for the resulting muscle contractile dysfunction under these circumstances. We tested the hypothesis that a subpopulation of denervated fibers is partially or completely responsible for the specific force deficit after partial denervation of the rat extensor digitorum longus muscle (EDL). Adult Fisher rats underwent either sham exposure or partial transection of 80% of the cross-sectional area of the left deep peroneal nerve. After a 2-week recovery period, maximum isometric force (F(0)) was measured in situ and maximum specific force (sF(0)) was calculated for EDL from both control (n = 8) and partial denervation (n = 7) groups. Innervated fiber cross-sectional area (CSA(inn)) was measured directly from whole EDL cross sections after immunohistochemical labeling for neural cell adhesion molecule (NCAM), a marker of muscle fiber denervation. A corrected specific force value (sF(0-inn)) was calculated by normalizing F(0) to CSA(inn). Partial skeletal muscle denervation resulted in significant reductions in muscle mass, F(0), and sF(0). The percentage of muscle fibers expressing NCAM in the extrajunctional sarcolemma increased from 1.0 +/- 0.8% in control to 49 +/- 15% in partially denervated EDL muscles. A 62.7% deficit in EDL specific force was observed after partial denervation. Denervated muscle fibers accounted for 59.3% of this deficit, but sF(0-inn) still differed significantly between control and partially denervated muscles, with a 25.5% difference between groups. In partially denervated muscles, the specific force deficit is partially but not fully explained by a subpopulation of noncontractile, denervated fibers.     

Oxidative Enzyme Activity and Soma Size in Motoneurons Innervating the Rat Slow-Twitch and Fast-Twitch Muscles After Chronic Activity

The effects of chronic activity induced by running training on the activity of the mitochondrial enzyme succinate dehydrogenase (SDH) and soma size in motoneurons innervating the slow-twitch soleus (SOL) and fast-twitch extensor digitorum longus (EDL) muscles were studied in rats using the retrograde neuronal tracer Nuclear Yellow. Rats were assigned to control and trained groups that were subjected to treadmill running for 10 weeks (2 h/day, 30 m/min, 5 days/week). After training, both SOL and EDL muscles showed clear adaptations (citrate synthase activity in the SOL muscle, and the fast-twitch oxidative-glycolytic fiber area of the EDL muscle increased significantly after training). The SDH activity of the motoneurons innervating both SOL and EDL muscles was unchanged by training. However, SOL motoneurons of trained rats had a significantly larger soma size and a significantly higher total SDH activity (SDH activity × soma size) than those of control. Total SDH activity was calculated to examine the absolute SDH protein content of the motoneurons. On the other hand, there was no difference in both soma size and total SDH activity of EDL motoneurons between the two groups. These data demonstrate that chronic activity has a considerably stronger impact on soma size and total oxidative enzyme activity of motoneurons innervating slow-twitch rather than fast-twitch muscles.     

Morphology of stable muscle grafts of rats: effects of gender and muscle type

Our purpose was to quantify morphological characteristics of extensor digitorum longus (EDL) and soleus (SOL) muscle grafts in female (N = 8) and male (N = 8) rats. Muscles were grafted orthotopically, with the nerve remaining intact, and were studied 56 days later. The mass of EDL and SOL grafts and control muscles of females was 60% to 65% of male values; this difference was directly related to gender differences in body mass. The fiber composition of EDL and SOL grafts did not differ from control, and no gender effects were noted. The mean fiber area (MFA) of control EDL and SOL muscles of females averaged 65% of male values. The MFA of grafts did not differ due to gender, and averaged 60% of control value for SOL and 70% for EDL grafts. We conclude there are no substantial differences in the regenerative capacity of EDL and SOL muscles grafted with the nerve intact.     

Brain-Derived Neurotrophic Factor mRNA Levels Are Up-Regulated in Hindlimb Skeletal Muscle of Diabetic Rats: Effect of Denervation

In a previous study the levels of brain-derived neurotrophic factor (BDNF) mRNA were shown to be elevated in skeletal muscle of the diabetic rat compared with age-matched control animals. It was proposed that diabetes-induced changes in nerve function may initiate changes in nerve/muscle contact akin to those following denervation of target skeletal muscle. In this study hindlimb skeletal muscles were denervated by sciatic nerve crush or transection and the effect on BDNF mRNA levels in control and diabetic rats was observed using Northern blotting. Contralateral to the side of nerve injury, the levels of BDNF mRNA in soleus muscle of diabetic rats were higher than in controls (three- to sevenfold), as has been seen previously in diabetic rats without any axotomy. Sciatic nerve crush or transection, of 1 week or of 3 weeks duration, lowered the levels of BDNF mRNA by 50% in ipsilateral soleus muscle of diabetic rats. BDNF mRNA levels in contralateral gastrocnemius muscle were not similarly raised in diabetic rats compared with controls and nerve injury had no effect. In control animals, ipsilaterally, the BDNF mRNA levels of soleus muscle were raised approximately twofold at 1 week and were lowered by approximately 50% at 3 weeks following nerve injury. Neurotrophin-3 mRNA levels were reduced approximately 50% in soleus muscle of diabetic rats compared with control rats, and nerve injury had no significant effect. The specific up-regulation of BDNF mRNA in soleus muscle of diabetic rats is discussed in terms of a proposed diabetes-induced ischemia within hindlimb skeletal muscle, with a protective role for BDNF in muscle and/or nerve being introduced.     

Factors affecting muscle fiber transformation in cross. Reinnervated muscle

Nerves of two fast muscles [peroneus longus (PL) and extensor digitorum longus (EDL)], having different type 2 muscle fiber compositions, were used to cross-reinnervate the slow soleus muscle in the rat. Contraction characteristics, histochemical muscle fiber type compsotions and myosin heavy chain (MHC) isoform compositions were determined for the reinnervated muscles. Shortening velocity increased in soleus muscles crossreinnervated with EDL nerve [X-SOL(EDL)] but not in muscles cross-reinnervated with PL nerve [X-SOL(PL)]. Type 2A MHC isoform content was increased in X-SOL(EDL) but not in X-SOL(PL), where MHC isoform composition remained similar to normal soleus. The complement of type 1 (slow) muscle fibers was reduced and that of type 2 (fast) fibers increased in both types of X-SOL muscle, but this change was significantly greater in X-SOL(EDL); the majority of the type 2 fibers in X-SOL muscles were of type 2A. Results show that “the type 2 composition” of the reinnervating motoneuron pool is an important factor in determining the transformation of a target slow muscle after cross-reinnervation. © 1993 John Wiley & Sons, Inc.     

Long-term effects of estrogen on rat skeletal muscle

The long-term effects of estrogen on the development of rat extensor digitorum longus (EDL) and soleus (SOL) muscles were examined using physiological and histochemical methods. The rats were in three groups: group 1, ovariectomized; group 2, sham-operated; and group 3, ovariectomized followed by estradiol administration. Isometric twitch and tetanic tensions of both the EDL and SOL obtained from 10-week-old rats were evoked by electrical stimulation. The isometric twitch tensions of the EDL in groups 2 and 3 were significantly lower than in group 1, however, there was no significant differences in isometric twitch tensions of the SOL among the three groups. The isometric tetanic tensions of the EDL in groups 2 and 3 were also significantly lower than in group 1, and the isometric tetanic tension of the SOL in group 3 was significantly lower than in group 1. There were no changes in the total number of muscle fibers or in the ratios of fiber types. But the mean fiber diameter of all fiber types (particularly in types IIA and IIB) was significantly lower in group 3 than in groups 1 and 2. Therefore, the possibility that estrogen may play an inhibitory role in the development of skeletal muscle fibers has to be considered.     

Histochemical and morphometrical analysis of skeletal muscle in spontaneous diabetic WBN/Kob rat

Spontaneous diabetic WBN/Kob rats develop diabetic peripheral neuropathy characterized by primary segmental demyelination and secondary axonal degeneration. The objective of this study was to evaluate the histochemical and morphometric characteristics of the lesions of skeletal muscles innervated by the affected nerves in diabetic rats. The following groups of rats were investigated: 24-month-old males that had been diabetic for less than 12 months, 10-month-old pre-diabetic males, 24-month-old non-diabetic females, and 10-month-old nondiabetic females. The soleus (SOL), extensor digitorum longus (EDL) and biceps femoris (BF) muscles were studied by light and electron microscopy, including histochemical and morphometric analyses. Muscle weight was reduced with age to a remarkable degree in diabetic BF and EDL. Dispersed atrophy of muscle fiber was observed in type 2a fibers of BF and EDL, and type 2c fibers of SOL, and the incidence was higher in diabetic rats. Multi-core, myofibrillar disorientation and an increased number of central nucleus of SOL, along with connective tissue proliferation of BF perimysium were noted in diabetic rats. The fiber population and type of composition varied with age, but no remarkable changes attributable to diabetic conditions were observed. Electron microscopically, an abnormal arrangement of myofibrils, a number of myelin figures, mitochondrial swelling and lysis of mitochondrial cristae were seen in diabetic rats. However, the neuromuscular junction and capillaries were intact. These findings indicate that the diabetic skeletal muscle lesion in WBN/Kob rats was mainly myogenic in nature, and was aggravated by the age-related change.     

Upregulation of MHC class I in transgenic mice results in reduced force‐generating capacity in slow‐twitch muscle

Expression of major histocompatibility complex (MHC) class I in skeletal muscle fibers is an early and consistent finding in inflammatory myopathies. To test if MHC class I has a primary role in muscle impairment, we used transgenic mice with inducible overexpression of MHC class I in their skeletal muscle cells. Contractile function was studied in isolated extensor digitorum longus (EDL, fast‐twitch) and soleus (slow‐twitch) muscles. We found that EDL was smaller, whereas soleus muscle was slightly larger. Both muscles generated less absolute force in myopathic compared with control mice; however, when force was expressed per cross‐sectional area, only soleus muscle generated less force. Inflammation was markedly increased, but no changes were found in the activities of key mitochondrial and glycogenolytic enzymes in myopathic mice. The induction of MHC class I results in muscle atrophy and an intrinsic decrease in force‐generation capacity. These observations may have important implications for our understanding of the pathophysiological processes of muscle weakness seen in inflammatory myopathies. Muscle Nerve, 2008     

Hypogravity-induced atrophy of rat soleus and extensor digitorum longus muscles

Prolonged exposure of humans to hypogravity causes weakening of their skeletal muscles. This problem was studied in rats exposed to hypogravity for 7 days aboard Spacelab 3. Hindlimb muscles were harvested 12-16 hours postflight for histochemical, biochemical, and ultrastructural analyses. The majority of the soleus and extensor digitorum longus fibers exhibited simple cell shrinkage. However, approximately 1% of the fibers in flight soleus muscles appeared necrotic. Flight muscle fibers showed increased glycogen, lower subsarcolemmal staining for mitochondrial enzymes, and fewer subsarcolemmal mitochondria. During atrophy, myofibrils were eroded by multiple focal losses of myofilaments; lysosomal autophagy was not evident. Tripeptidylaminopeptidase and calcium-activated protease activities of flight soleus fibers were significantly increased, implying a role in myofibril breakdown. Simple fiber atrophy appears to account for muscle weakening during spaceflight, but fiber necrosis is also a contributing factor.