Role of afferent input in muscle atrophy.

It is well known that soleus muscle of rat atrophies following spaceflight or hindlimb suspension (Ohira et al., 1992). It is, further, reported that the electromyogram (EMG) of soleus muscle disappears immediately in response to unloading by exposure to actual micro-g environment (Kawano et al., 2002; Leterme and Falempin, 1998) and by hindlimb suspension of rats (Alford et al., 1987; Ohira et al., 2000). However, the EMG level is increased gradually to the control level following 7-10 days of continuous hindlimb suspension (Alford et al., 1987; Ohira, 2000), while muscle atrophy is progressing (Winiarski et al., 1987). We previously reported that reduction of the EMG level of rat soleus in response to actual micro-g environment, created by a parabolic flight of a jet airplane, was closely associated with a decrease of the afferent input recorded at the L5 segmental level of spinal cord (Kawano et al., 2002). However, it is still unclear how the EMG level of soleus muscle adapts to unloading condition. The current study was performed to investigate the responses of soleus EMG and both afferent and efferent neurogram at the L5 segmental level of spinal cord to acute (20 seconds) and chronic (14 days) unloading.     

Effects of 9 weeks hindlimb suspension on neuromuscular activity patterns in rat

It is well known that gravitational unloading induces muscle atrophy associated with a shift of fiber type in slow-twitch muscle. Ishihara et al. (1996 & 1997) reported that 2 weeks of spaceflight caused a decrease in succinate dehydrogenase activities of ventral horn and dorsal root ganglion neurons in rats. Significant effects on motor performance are also induced in both human (Kozlovskaya et al., 1981) and rats (Canu and Falempin, 1997), but these changes are reversible. However, it is not known how neuromuscular function respond to long-term gravitational unloading. Therefore, the current study was carried out to investigate the effects of 9 weeks of hindlimb suspension on the neuromuscular function and mass in hindlimb muscles in rats.     

The realization of a mechanical signal during gravitational unloading: The response of mTORC1 targets to eccentric contractions

The aim of this study was to assess the response of key mTORC1 substrates to a bout of contractile stimuli under different times of functional unloading. Functional unloading of hind-limb muscles was carried out by the method of antiorthostatic suspension. Twenty-eight Wistar rats were divided into four groups: control, and hindlimb suspension for 1, 3, and 7 days. After hindlimb suspension, isolated soleus muscles of rats were subjected to a bout of ex vivo eccentric contractions. The contents of phosphorylated forms of p70s6k and 4E-BP1 were then determined using western blotting. It was found that an eccentric load resulted in a significant increase in p70s6k phosphorylation and reduced 4E-BP1 phosphorylation both in control and suspended rats, but in the case of suspension the response was dramatically reduced. Thus, it can be concluded that a bout of eccentric contractions of isolated rat soleus muscle during functional unloading causes a weaker activation of the Akt-mTORC1-p70s6k signaling pathway compared with the control. This may indicate that it is important to maintain muscle tone for a more efficient muscle perception of an external mechanical signal and subsequent activation of anabolic signaling pathways.     

Effects of 9 weeks of hindlimb unloading on motor performances in adult rats

It has been reported that abnormal steps associated with an ankle hyper-extension during walking were observed in adult rats after 2 weeks of hindlimb suspension (Canu and Falempin, 1997 & 1998). But such phenomena were normalized after 7 days of reambulation recovery. Canu and Falempin (1996) suggested that the spinal cord has a capacity to generate a well-organized pattern of locomotion even after a period of muscle disuse. There are, however, no reports about the effects of more prolonged suspension on motor performances. In the present study, 7 weeks old male rats were hindlimb-unloaded by tail suspension for 9 consecutive weeks and landing performances in response to drop from head-down, head-up, or supine position were investigated during 8 weeks of recovery. Posture maintenance during right-left translation was also checked.     

Age effects on rat hindlimb muscle atrophy during suspension unloading.

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

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.     

Unloading of juvenile muscle results in a reduced muscle size 9 wk after reloading.

The role of satellite cells and DNA unit size in determining muscle size was examined by inhibiting postnatal skeletal muscle development by using hindlimb suspension. Satellite cell mitotic activity and DNA unit size were determined in the soleus muscles from hindlimb-suspended and age-matched weight-bearing rats before the initiation of hindlimb suspension, at the conclusion of a 28-day hindlimb-suspension period, 2 wk after reloading, and 9 wk after reloading. The body weights of hindlimb-suspended rats were significantly (P < 0.05) less than those of weight-bearing rats at the conclusion of hindlimb suspension, but they were the same (P > 0. 05) as those of weight-bearing rats 9 wk after reloading. The soleus muscle weight, soleus muscle weight-to-body weight ratio, myofiber diameter, nuclei per millimeter, and DNA unit size for the hindlimb-suspended rats were significantly (P < 0.05) smaller than for the weight-bearing rats at all recovery times. Satellite cell mitotic activity was significantly (P < 0.05) higher in the soleus muscles from hindlimb-suspended rats 2 wk after reloading, but it was the same (P > 0.05) as in weight-bearing rats 9 wk after reloading. Juvenile soleus muscles failed to achieve normal muscle size 9 wk after reloading because there was incomplete compensation for the hindlimb-suspension-induced interruptions in myonuclear accretion and DNA unit size expansion.     

Hindlimb suspension reduces muscle regeneration

Exposure of juvenile skeletal muscle to a weightless environment reduces growth and satellite cell mitotic activity. However, the effect of a weightless environment on the satellite cell population during muscle repair remains unknown. Muscle injury was induced in rat soleus muscles using the myotoxic snake venom, notexin. Rats were placed into hindlimb-suspended or weightbearing groups for 10 days following injury. Cellular proliferation during regeneration was evaluated using 5-bromo-2′-deoxyuridine (BrdU) immunohistochemistry and image analysis. Hindlimb suspension reduced (P<0.05) regenerated muscle mass, regenerated myofiber diameter, uninjured muscle mass, and uninjured myofiber diameter compared to weightbearing rats. Hindlimb suspension reduced (P<0.05) BrdU labeling in uninjured soleus muscles compared to weightbearing muscles. However, hindlimb suspension did not abolish muscle regeneration because myofibers formed in the injured soleus muscles of hindlimb-suspended rats, and BrdU labeling was equivalent (P>0.10) on myofiber segments isolated from the soleus muscles of hindlimb-suspended and weightbearing rats following injury. Thus, hindlimb suspension (weightlessness) does not suppress satellite cell mitotic activity in regenerating muscles before myofiber formation, but reduces growth of the newly formed myofibers. Accepted: 11 December 1997     

Metabolic transitions in rat jaw muscles during postnatal development.

The program of acquisition of adult metabolic phenotypes was studied in three jaw muscles in order to determine the link between muscle metabolism and functional development. During early postnatal stages, there were similar transitions in the masseter, anterior digastric, and internal pterygoid muscles with respect to fiber growth, fiber type composition, and whole muscle energy metabolism. Oxidative capacity, as judged by the activities of the enzymes succinate dehydrogenase (SDH), malate dehydrogenase (MDH), and beta-hydroxyacyl CoA dehydrogenase (beta OAC), rose sharply after birth to reach near maximal levels by 3 weeks. The capacities for glycolytic metabolism represented by lactate dehydrogenase (LDH), and for high-energy phosphate metabolism represented by adenylokinase (AK) and creatine kinase (CK) activities, rose gradually, not reaching peak values until 6 weeks or later. Thus, the maturation of oxidative metabolism preceded that of glycolytic metabolism in the developing jaw muscles. This was documented for individual fibers in the masseter muscle. Differential metabolic maturation among the jaw muscles was evident beyond 3 weeks. All three jaw muscles attained their specific adult fiber-type profile by about 6 weeks. This maturation program differed from that of hindlimb muscles [Nemeth et al., J Neurosci 9:2336-2343, 1989] and diaphragm muscle [Kelly et al., J Neurosci 11:1231-1242, 1991], reflecting their differential energy demands for contractile performance.     

Physiological, histological and biochemical properties of rat skeletal muscles in response to hindlimb suspension.

In previous study, we found that the reduced exercise-induced production of reactive oxygen species (ROS) reported in slow-oxidative muscle of hypoxemic rats and also in chronic hypoxemic patients did not simply result from deconditioning. In control rats and after a 3-week period of hindlimb suspension (HS), the slow-oxidative (Soleus, SOL) and fast-glycolytic skeletal muscles (Extensor digitorum longus, EDL) were sampled. We determined the response to direct muscle stimulation (twitch stimulation (TS), Maximal force (Fmax)), twitch amplitude and maximal relaxation rate, tetanic frequency, endurance to fatigue after muscle stimulation (MS), the different fibre types based on their myofibrillar adenosinetriphosphatase (ATPase) activity, and the intra-muscular redox status (Thiobarbituric Acid Reactive Sustances: TBARS, reduced glutathione: GSH, reduced ascorbic acid: RAA). After the 3-w HS period: (1) the contractile properties were modified in SOL only (reduced Fmax and twitch amplitude, increased tetanic frequency); (2) the fibre typology was modified in both muscles (in SOL: increased proportion of IIa and IIc fibres, in EDL: increased proportion of IId/x fibres but decreased proportion of IIb fibres); and (3) only in SOL, the TBARS level increased and the GSH and RAA concentrations decreased at rest and after fatiguing MS. Thus, HS accentuates the exercise-induced ROS production in slow-oxidative muscle in a direction opposite to that measured in chronic hypoxemic rats. This strongly suggests that hypoxemia reduces the ROS production independently from any muscle disuse.     

Mechanical, morphological and biochemical adaptations of bone and muscle to hindlimb suspension and exercise

The influences of weightbearing forces on the structural remodeling, matrix biochemistry, and mechanical characteristics of the rat tibia and femur and surrounding musculature were examined by means of a hindlimb suspension protocol and highly intensive treadmill running. Female, young adult, Sprague-Dawley rats were designated as either normal control, sedentary suspended, or exercise suspended rats. For 4 weeks, sedentary suspended rats were deprived of hindlimb-to-ground contact forces, while the exercise suspended rats experienced hindlimb ground reaction forces only during daily intensive treadmill training sessions. The suspension produced generalized atrophy of hindlimb skeletal muscles, with greater atrophy occurring in predominantly slow-twitch extensors and adductors, as compared with the mixed fiber-type extensors and flexors. Region-specific cortical thinning and endosteal resorption in tibial and femoral diaphyses occurred in conjunction with decrements in bone mechanical properties. Tibial and femoral regional remodeling was related to both the absence of cyclic bending strains due to normal weightbearing forces and the decrease in forces applied to bone by antigravity muscles. To a moderate extent, the superimposed strenuous running counteracted muscular atrophy during the suspension, particularly in the predominantly slow-twitch extensor and adductor muscles. The exercise did not, however, mitigate changes in bone mechanical properties and cross-sectional morphologies, and in some cases exacerbated the changes. Suspension with or without exercise did not alter the normal concentrations of collagen, phosphorus, and calcium in either tibia or femur.     

Contribution of nerve growth factor to upregulation of P2X₃ expression in DRG neurons of rats with femoral artery occlusion

Femoral artery occlusion augments the sympathetic nerve and pressor responses to muscle contraction and muscle metabolites injected into the arterial blood supply of the hindlimb muscles in rats. The underlying mechanism by which these reflex responses are enhanced after muscle vascular insufficiency is unclear. Purinergic P2X(3) receptor has been reported to contribute to the metabolic component of the exercise pressor reflex. Thus the purpose of this study was to examine if chronic femoral occlusion would alter the expression of P2X(3) in dorsal root ganglion (DRG) neurons of rats. Also, P2X(3)-mediated sympathetic responsiveness was examined after femoral occlusion. In addition, the role played by nerve growth factor (NGF) in regulating the expression and response of P2X(3) was examined. Western blot analysis showed that 24 h of femoral ligation increased the levels of P2X(3) (optical density: 0.93 ± 0.07 in control and 1.37 ± 0.10 after occlusion; P < 0.05 vs. control). The fluorescence immunohistochemistry further demonstrated that the occlusion elevated P2X(3) expression in DRG neurons (percentage of P2X(3)-positive cells: 33 ± 3% in control and 51 ± 3% in occlusion; P < 0.05 vs. control). Furthermore, the results showed that responses of renal sympathetic nerve activity and blood pressure to stimulation of P2X were greater in occluded rats than responses in control rats by injection of α,β-methylene ATP into the arterial blood supply of the hindlimb muscle. Finally, infusion of NGF in the hindlimb muscles of healthy rats increased P2X(3) (optical density: 0.98 ± 0.12 in control and 1.37 ± 0.16 with NGF; P < 0.05 vs. control). The pressor response to injection of α,β-methylene ATP was increased in the rats with NGF infusion. Likewise, blocking NGF attenuated exaggeration of the reflex response induced by α,β-methylene ATP in occluded rats. The findings of this study suggest that the levels of P2X(3) in primary afferent neurons are upregulated as the blood supply to the hindlimb is deficient under ischemic conditions, leading to augmentation of the muscle reflex. NGF is closely related to increases in P2X(3) receptor expression and response.     

Profiles of connectin (titin) in atrophied soleus muscle induced by unloading of rats.

Responses of the properties of connectin molecules in the slow-twitch soleus (Sol) and fast-twitch extensor digitorum longus muscles of rats to 3 days of unloading with or without 3-day reloading were investigated. The wet weight (relative to body wt) of Sol, not of extensor digitorum longus, in the unloaded group was significantly less than in the age-matched control (P < 0.05). Immunoelectron microscopic analyses showed that a monoclonal antibody against connectin (SM1) bound to the I-band region close to the edge of the A band at resting length and moved reversibly away from the Z line as the muscle fibers were stretched. In Sol, the displacement of the SM1-bound dense spots in response to stretching decreased after hindlimb suspension. There were no changes in the molecular weights and the percent distributions of alpha- and beta-connectin in both muscles after hindlimb suspension. A significant increment of percent beta-connectin in Sol was observed after 3 days of reloading after hindlimb suspension (P < 0.05). It is suggested that the elasticity of connectin filaments in the I-band region of the atrophied Sol fibers was reduced relative to that of the control fibers. The lack of the elasticity in atrophied muscle fibers may cause a decrease in contractile function.     

Cysteine supplementation prevents unweighting-induced ubiquitination in association with redox regulation in rat skeletal muscle

We have previously reported that spaceflight and tail suspension enhanced degradation of rat myosin heavy chain (MHC) in association with activation of a ubiquitin-dependent proteolytic pathway [Ikemoto et al., FASEB J. 15 (2001), 1279-1281]. To elucidate whether the ubiquitination is accompanied by oxidative stress, we measured markers for oxidative stress, such as thiobarbituric acid-reactive substance (TBARS) and glutathione disulfide (GSSG), in gastrocnemius muscle of tail-suspended rats. Glutathione (GSH) concentration in the muscle significantly decreased from day 5 and reached a minimum value on day 10. Tail suspension reciprocally increased concentrations of TBARS and GSSG in parallel with enhancement of protein ubiquitination, suggesting that oxidative stress may play an important role in protein ubiquitination caused by tail suspension. To prevent ubiquitination associated with oxidative stress, we also administered an antioxidative nutrient, cysteine, to tail-suspended rats. Intragastric supplementation of 140 mg/rat of cysteine for 2 weeks or longer normalized the ratio of GSH to GSSG in the muscle and suppressed protein ubiquitination and MHC fragmentation, compared with supplementation of the equimolar amount of alanine. The cysteine supplementation significantly suppressed the loss of hindlimb muscle mass. Our results suggest that supplementation of antioxidative nutrients, such as cysteine, may be beneficial for preventing ubiquitination of muscle proteins caused by unweighting.     

Diaphragm muscle development in bottlenose dolphins (Tursiops truncatus)

Being born directly into the aquatic environment creates unique challenges for the breathing muscles of neonatal cetaceans. Not only must these muscles be active at the instant of birth to ventilate the lungs, but their activities must also be coordinated with those of the locomotor muscles such that breathing takes place only at the water's surface. At least one major locomotory muscle of bottlenose dolphins (Tursiops truncatus) has been demonstrated to be well developed and, therefore, able to power the neonatal dolphin's early movements (Dearolf et al. [2000] J Morphol 244:203-215). Thus, because of the demands for coordinated behavior with the locomotor muscles, it is hypothesized that the breathing muscles of bottlenose dolphins, represented in this study by the diaphragm, will also demonstrate adult morphology at birth. However, histochemical and biochemical analyses demonstrate that neonatal dolphins have immature diaphragms, with only 52% of the adult slow fiber-type profile (neonates: 34% slow-twitch fibers; adults: 66% slow-twitch fibers). The developmental state of the dolphin diaphragm is compared to those of other neonatal mammals, using a muscle development index (% slow-twitch fibers in neonatal muscle / % slow-twitch fibers in adult muscle). Fiber-type profiles reported in the literature are used to calculate index values for the diaphragms of altricial rats, rabbits, and cats, intermediate baboons and humans, and precocial sheep and horses. The dolphin is not unique in having an immature diaphragm at birth; however, there is a positive relationship between the developmental state of the diaphragm and the overall developmental state of the neonate. The presence of type IIc ("undifferentiated") fibers in the diaphragms of altricial developers (e.g., rats, rabbits, and cats) is correlated with the slow contraction speeds recorded from their diaphragms. The diaphragms of neonatal horses and dolphins express little to no type IIc fibers and, thus, may have the ability to contract at the speeds required for their increased ventilation rates. These results lead to the modification of the criterion for evaluating the developmental state of a muscle at birth. Thus, the developmental state of a neonatal muscle should be based on both its value of Dearolf et al.'s (2000) developmental index, as well as the percentage of type IIc fibers found in that muscle.     

Age effect on expression of myosin heavy and light chain isoforms in suspended rat soleus muscle.

This study was designed to test the hypothesis that myosin heavy (MHC) and light chain (MLC) plasticity resulting from hindlimb suspension (HS) is an age-dependent process. By using an electrophoretic technique, the distribution of MHC and MLC isoforms was quantitatively evaluated in the soleus muscles from 3- or 12-wk-old rats after 1-3 wk of HS treatment was maintained. In normal 12- and 15-wk-old rats, the soleus muscles contained a predominance of MHCI ( approximately 94%) with small amounts of MHCIIa, but not MHCIId or MHCIIb. The suspended muscles of adult rats were characterized by the appearance of MHCIIb and MHCIId, the latter reaching approximately 6% after 3 wk of HS treatment. In contrast to changes in MHC, HS did not induce a transition in the MLC pattern in the soleus muscles from adult rats. Compared with adult rats, in juveniles HS had a much more pronounced effect on the shift toward faster MHC and MLC isoform expression. The soleus muscles of 6-wk-old rats after 3 wk of HS were composed of 37.0% MHCI, 19.1% MHCIIa, 23.7% MHCIId, and 20.2% MHCIIb. Changes in MLC isoforms consisted of an increase in MLC1f and MLC2f concomitant with a decrease in MLC2s. These results indicate the existence of a differential effect of HS on MHC and MLC transitions that appears to be age dependent. They also suggest that the suspended soleus muscles from young rats may acquire the intrinsic contractile properties that are intermediate between those in the normal soleus and typical fast-twitch skeletal muscles.     

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

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

Muscle atrophy by limb immobilization is not caused by insulin resistance

Hindlimbs of adult female rats were immobilized for 1 day. The hindquarter was then perfused either without or with 200 microunits of insulin/ml perfusate. The percentage increase in muscle protein synthesis rates by the inclusion of insulin in the perfusate was similar between control and hindlimb immobilized groups. Thus, the previously reported inability of insulin to stimulate any increase in glucose metabolism in skeletal muscle after 1 day of immobilization ( Seider , Nicholson and Booth 1982) does not also extend to an inability of insulin to stimulate an increase in protein synthesis in muscles of immobilized limbs.     

Muscle adaptations to hindlimb suspension in mature and old Fischer 344rats

Stump, Craig S., Charles M. Tipton, and Erik J. Henriksen.Muscle adaptations to hindlimb suspension in mature and oldFischer 344 rats. J. Appl. Physiol.82(6): 1875-1881, 1997.We examined skeletal and cardiac muscleresponses of mature (8 mo) and old (23 mo) male Fischer 344 rats to 14 days of hindlimb suspension. Hexokinase (HK) and citrate synthase (CS)activities and GLUT-4 glucose transporter protein level, which arecoregulated in many instances of altered neuromuscular activity, wereanalyzed in soleus (Sol), plantaris (Pl), tibialis anterior (TA),extensor digitorum longus (EDL), and left ventricle. Protein contentwas significantly (P < 0.05) lowerin all four hindlimb muscles after suspension compared with controls inboth mature (21-44%) and old (17-43%) rats. Old ratsexhibited significantly lower CS activities than mature rats for theSol, Pl, and TA. HK activities were significantly lower in the old ratsfor the Pl (19%) and TA (33%), and GLUT-4 levels were lower in theold rats for the TA (38%) and EDL (24%) compared with the maturerats. Old age was also associated with a decrease in CS activity (12%)and an increase in HK activity (14%) in cardiac muscle. CS activitieswere lower in the Sol (20%) and EDL (18%) muscles from maturesuspended rats and in the Sol (25%), Pl (27%), and EDL (25%) musclesfrom old suspended rats compared with corresponding controls. However,suspension was associated with significantly higher HK activities forall four hindlimb muscles examined, in both old (16-57%) andmature (10-43%) rats, and higher GLUT-4 concentrations in the TAmuscles of the old rats (68%) but not the mature rats. These resultsindicate that old age is associated with decreased CS and HK activities and GLUT-4 protein concentration for several rat hindlimb muscles, andthese variables are not coregulated during suspension. Finally, old ratskeletal muscle appears to respond to suspension to a similar orgreater degree than mature rat muscle responds.

     

Apoptotic responses to hindlimb suspension in gastrocnemius muscles from young adult and aged rats

Although apoptosis has been demonstrated in soleus during hindlimb suspension (HS), it is not known whether apoptosis is also involved in the loss of muscles dominated by mixed fibers. Therefore, we examined the apoptotic responses in gastrocnemius muscles of young adult and aged Fischer 344 x Brown Norway rats after 14 days of HS. The medial gastrocnemius muscle wet weight significantly decreased by 30 and 32%, and muscle wet weight normalized to the animal body weight decreased by 11 and 15% in young adult and aged animals, respectively, after HS. The extent of apoptotic DNA fragmentation increased by 119 and 61% in suspended muscles from young and aged rats, respectively. Bax mRNA increased by 73% in young muscles after HS. Bax and Bcl-2 protein levels were greater in suspended muscles relative to control muscles in both age groups. The level of cytosolic mitochondria-housed apoptotic factor cytochrome c was significantly increased in the mitochondria-free cytosol of suspended muscles from young and aged rats. In contrast, the release/accumulation of AIF, a caspase-independent apoptogenic factor, was exclusively expressed in the suspended muscles from aged rats. Our data also show that aging favors the proapoptotic signaling in skeletal muscle by altering the contents of Bax, Bcl-2, Apaf-1, AIF, caspases, XIAP, Smac/DIABLO, and cytochrome c. Furthermore, these results indicate that apoptosis occurs not only in slow-twitch soleus muscle but also in the mixed-fiber (predominately fast fibered) gastrocnemius muscle. Our data are consistent with the hypothesis that apoptotic signaling differs in young adult and aged gastrocnemius muscles during HS.