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.     

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.     

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.     

A physiological level of clenbuterol does not prevent atrophy or loss of force in skeletal muscle of old rats.

Supraphysiological levels of clenbuterol (CL) reduce muscle degradation in both young and old animals; however, these pharmacological levels induce side effects that are unacceptable in the elderly. In this study, we tested the hypothesis that a "physiological" dose of CL (10 microg. kg(-1). day(-1)) would attenuate the loss of in situ isometric force and mass in muscles of senescent rats during hindlimb suspension (HS). Adult (3 mo) and senescent (38 mo) Fischer 344 x Brown Norway rats received CL or a placebo during 21 days of normal-weight-bearing or HS conditions (8 rats/age group). HS reduced soleus muscle weight-to-body weight ratio by 31%, muscle cross-sectional area by 37%, and maximal isometric tetanic force (P(o)) by 76% in senescent rats. CL attenuated the loss of P(o) and muscle weight by 17 and 8%, respectively, in the soleus of senescent rats relative to HS+placebo conditions, but it did not improve muscle weight normalized for body weight. CL did not reduce the decrease in soleus P(o) or mass after HS in adult rats. CL failed to reduce the loss of plantaris weight (-20%) and P(o) (-46%) in senescent rats after HS. Our data support the conclusion that physiological levels of CL do not improve fast muscle atrophy and only modestly reduce slow muscle atrophy, and, therefore, it is largely an ineffective countermeasure for preventing muscle wasting from HS in senescent rats.     

Skeletal muscle IGF-binding protein-3 and -5 expressions are age,muscle, and load dependent

The purpose of the current study was to examine IGFBP-3, -4, and -5 mRNA and protein expression levels as a function of muscle type, age, and regrowth from an immobilization-induced atrophy in Fischer 344 x Brown Norway rats. IGFBP-3 mRNA expression in the 4-mo-old animals was significantly higher in the red and white portions of the gastrocnemius muscle compared with the soleus muscle. However, there were no significant differences in IGFBP-3 mRNA expression among any of the muscle groups in the 30-mo-old animals. There were no significant differences in IGFBP-5 mRNA expression in any of the muscle groups, whereas in the 30-mo-old animals there was significantly less IGFBP-5 mRNA expression in the white gastrocnemius compared with the red gastrocnemius muscles. Although IGFBP-3 and -5 proteins were detected in the type I soleus muscle with Western blot analyses, no detection was observed in the type II red and white portions of the gastrocnemius muscle. Aging from adult (18 mo) to old animals (30 mo) was associated with decreases in IGFBP-3 mRNA and protein and IGFBP-5 protein only in the soleus muscle. After 10 days of recovery from 10 days of hindlimb immobilization, IGFBP-3 mRNA and protein increased in soleus muscles from young (4-mo) rats; however, only IGFBP-3 protein increased in the old (30-mo) rats. Whereas there were no changes in IGFBP-5 mRNA expression during recovery, IGFBP-5 protein in the 10-day-recovery soleus muscle did increase in the young, but not in the old, rats. Because one of the functions of IGFBPs is to modulate IGF-I action on muscle size and phenotype, it is hypothesized that IGFBP-3 and -5 proteins may have potential modulatory roles in type I fiber-dominated muscles, aging, and regrowth from 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.     

Interleukin-15 responses to aging and unloading-induced skeletal muscle atrophy

Interleukin-15 (IL-15) mRNA is constitutively expressed in skeletal muscle. Although IL-15 has proposed hypertrophic and anti-apoptotic roles in vitro, its role in skeletal muscle cells in vivo is less clear. The purpose of this study was to determine if skeletal muscle aging and unloading, two conditions known to promote muscle atrophy, would alter basal IL-15 expression in skeletal muscle. We hypothesized that IL-15 mRNA expression would increase as a result of both aging and muscle unloading and that muscle would express the mRNA for a functional trimeric IL-15 receptor (IL-15R). Two models of unloading were used in this study: hindlimb suspension (HS) in rats and wing unloading in quail. The absolute muscle wet weight of plantaris and soleus muscles from aged rats was significantly less when compared with muscles from young adult rats. Although 14 days of HS resulted in reduced muscle mass of plantaris and soleus muscles from young adult animals, this effect was not observed in muscles from aged animals. A significant aging times unloading interaction was observed for IL-15 mRNA in both rat soleus and plantaris muscles. Patagialis (PAT) muscles from aged quail retained a significant 12 and 6% of stretch-induced hypertrophy after 7 and 14 days of unloading, respectively. PAT muscles from young quail retained 15% hypertrophy at 7 days of unloading but regressed to control levels following 14 days of unloading. A main effect of age was observed on IL-15 mRNA expression in PAT muscles at 14 days of overload, 7 days of unloading, and 14 days of unloading. Skeletal muscle also expressed the mRNAs for a functional IL-15R composed of IL-15R, IL-2/15R-, and -c. Based on these data, we speculate that increases in IL-15 mRNA in response to atrophic stimuli may be an attempt to counteract muscle mass loss in skeletal muscles of old animals. Additional research is warranted to determine the importance of the IL-15/IL-15R system to counter muscle wasting. atrophy; interleukins; sarcopenia; gene signaling     

Effect of cold environment on skeletal muscle mitochondria in growing rats

Summary Growing rats (4 weeks old) were kept for 3 weeks at 11° C and 24° C respectively. The cold-adapted animals showed a significantly higher oxygen consumption (64%). Volume density of subsarcolemmal and interfibrillar mitochondria as well as volume density of fat droplets were estimated in M. soleus and the diaphragm of both groups. In cold-adapted animals, the total volume of mitochondria was significantly increased by 24% in diaphragm and 37% in M. soleus. The volume of subsarcolemmal mitochondria was almost doubled in each muscle, but the volume of interfibrillar mitochondria did not change significantly. The surface of the inner mitochondrial membranes per unit volume of mitochondrion in M. soleus was significantly increased both in interfibrillar and subsarcolemmal mitochondria, whereas the surface of the outer mitochondrial membranes per unit volume of mitochondrion was increased only in the subsarcolemmal mitochondria. The volume of fat droplets in the diaphragm and M. soleus of cold adapted animals increased significantly by 62% and 150% respectively.     

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

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

Nuclear DNA fragmentation and morphological alterations in adult rabbit skeletal muscle after short-term immobilization

Nuclear DNA fragmentation and ultrastructural changes, indicative of myonuclear apoptosis, were examined in adult skeletal muscle in response to short-term immobilization. Adult rabbits were allocated to 2 days (n=5) or 6 days (n=5) of unilateral casting of the ankle in full plantar flexion or were used as untreated controls (n=2). Atrophy of the soleus muscle was apparent by significant reductions in wet mass of 15% and 26% after 2 days and 6 days of casting (P< or =0.05), respectively. Mean fibre cross-sectional area and myonuclear number per section were also lower (17% and 9.1%, respectively) after 6 days of casting, in comparison with contralateral control muscles (P< or =0.05). Electron-microscopic examination showed condensed chromatin and irregularly shaped myonuclei in muscles immobilized for either 2 days or 6 days. Myofibrillar disruption and abnormalities of the subsarcolemmal mitochondria were also apparent in the absence of inflammation or plasma membrane alterations in cast muscles. Longitudinal and transverse sections showed abundant in situ end-labelling of DNA strand breaks (TUNEL) after 2 days, with less after 6 days, of immobilization. Positive labelling corresponded to myonuclear locations within fibres, yet the number of TUNEL-positive nuclei indicated DNA fragmentation in additional cell types such as capillary endothelial cells or fibroblasts. The data indicate that the immobilization of slow-twitch skeletal muscle in a shortened position rapidly induces morphological alterations consistent with mitochondrial injury and apoptotic myonuclear elimination.     

Effects of aging and denervation on the expression of uncoupling proteins in slow- and fast-twitch muscles of rats

We investigated the effects of aging and denervation on the gene expression of uncoupling proteins (UCPs) in slow-twitch soleus and fast-twitch gastrocnemius muscles. In a comparison between the control limbs of 6- and 24-month-old rats, the mRNA levels of UCP3, heart-type fatty acid binding protein (HFABP), and glucose transporter-4 (GLUT4) were considerably lower in the gastrocnemius muscles of the older rats, whereas no significant differences in the mRNA levels of those genes as well as UCP2 and cytochrome oxidase subunit IV (COX-IV) were observed in the soleus muscles of young and old rats. The UCP3 and COX-IV protein levels were also reduced considerably in the aged gastrocnemius muscles with atrophy. Denervation of the sciatic nerve caused an increase in UCP3 mRNA levels in both muscles, but the regulation of other genes contrasted between the two types of skeletal muscles. In spite of the increased mRNA level, a remarkable reduction in UCP3 protein was found in the denervated gastrocnemius muscles. These results indicate that the effects of aging and denervation on the gene expression of UCPs, HFABP, GLUT4, and COX-IV are different between the muscle types. The reduction in the mitochondrial UCP3 and COX proteins in aged fast-twitch muscles may have a negative effect on energy metabolism and thermogenesis in old animals.     

Muscle regeneration during hindlimb unloading results in a reduction in muscle size after reloading.

The hindlimb-unloading model was used to study the ability of muscle injured in a weightless environment to recover after reloading. Satellite cell mitotic activity and DNA unit size were determined in injured and intact soleus muscles from hindlimb-unloaded and age-matched weight-bearing rats at the conclusion of 28 days of hindlimb unloading, 2 wk after reloading, and 9 wk after reloading. The body weights of hindlimb-unloaded rats were significantly (P < 0.05) less than those of weight-bearing rats at the conclusion of hindlimb unloading, but they were the same (P > 0.05) as those of weight-bearing rats 2 and 9 wk after reloading. The soleus muscle weight, soleus muscle weight-to-body weight ratio, myofiber diameter, number of nuclei per millimeter, and DNA unit size were significantly (P < 0.05) smaller for the injured soleus muscles from hindlimb-unloaded rats than for the soleus muscles from weight-bearing rats at each recovery time. Satellite cell mitotic activity was significantly (P < 0.05) higher in the injured soleus muscles from hindlimb-unloaded rats than from weight-bearing rats 2 wk after reloading, but it was the same (P > 0.05) as in the injured soleus muscles from weight-bearing rats 9 wk after reloading. The injured soleus muscles from hindlimb-unloaded rats failed to achieve weight-bearing muscle size 9 wk after reloading, because incomplete compensation for the decrease in myonuclear accretion and DNA unit size expansion occurred during the unloading period.     

Anatomic capillarization is maintained in relative excess of fiber oxidative capacity in some skeletal muscles of late middle-aged rats.

The anatomic size of the capillary-to-fiber (C/F) interface plays an important role in O(2) flux from blood to tissue by determining the surface area available for diffusion and is maintained in relative proportion to fiber mitochondrial volume across a wide range of muscle aerobic capacity. In the present study, we examined an estimate of the anatomic size of the C/F interface [the quotient of the individual C/F ratio and fiber perimeter, C/F perimeter exchange (CFPE) index] and fiber oxidative capacity in different skeletal muscles, or muscle regions, to test the hypothesis that capillarization would be maintained in relative excess of reduced fiber oxidative capacity in aged muscles. The right gastrocnemius, plantaris, and soleus muscles from young adult (8 mo old) and late middle-aged (28-30 mo old) Fischer 344 x Brown Norway F1 hybrid rats were excised for evaluation of flux through electron transport chain complexes I-III and/or morphometric estimation of capillarization. Muscle mass was lower in the gastrocnemius muscles of the older animals (2,076 +/- 32 vs. 1,825 +/- 47 mg in young adult vs. late middle-aged, respectively; mean +/- SE) but not the plantaris or soleus muscles. Fibers were smaller in the white region of gastrocnemius muscles but larger in the red region of gastrocnemius muscles of the older animals. There was no difference in the number of capillaries around a fiber, the individual C/F ratio, or the CFPE index between groups for any muscle/region, whereas flux through complexes I-III was reduced by 29-43% in late middle-aged animals. Thus the greater quotient of indexes of anatomic capillarity (individual C/F ratio or CFPE index) and fiber oxidative capacity in soleus and the white region of gastrocnemius muscles, but not in the red region of gastrocnemius muscles of the older animals, shows that anatomic capillarity is maintained in relative excess of oxidative capacity in some muscle regions in late middle-aged rats.     

Distribution of myonuclei and microtubules in live muscle fibers of young, middle-aged, and old mice.

We have recently published a new technique for visualizing nuclei in living muscle fibers of intact animals, based on microinjection of labeled DNA into single myofibers, excluding satellite cells (Bruusgaard JC, Liestol K, Ekmark M, Kollstad K, and Gundersen K. J Physiol 551: 467-478, 2003). In the present study, we use this technique to study fiber segments of soleus and extensor digitorum longus (EDL) muscles from mice aged 2, 14, and 23 mo. As the animals maturing from 2 to 14 mo, they displayed an increase in size and number of nuclei. Soleus showed little change in nuclear domain size, whereas this increased by 88% in the EDL. For 14-mo-old animals, no significant correlation between fiber size and nuclear number was observed (R2=0.18, P=0.51) despite a fourfold variation in cytoplasmic volume. This suggests that size and nuclear number is uncoupled in middle-aged mice. When animals aged from 14 to 23 mo, EDL IIb, but not soleus, fibers atrophied by 41%. Both EDL and soleus displayed a reduction in number of nuclei: 20 and 16%, respectively. A positive correlation between number of nuclei and size was observed at 2 mo, and this reappeared in old mice. The atrophy in IIb fibers at old age was accompanied by a disturbance in the orderly positioning of nuclei that is so prominent in glycolytic fibers at younger age. In old animals, changes in nuclear shape and in the peri- and internuclear microtubule network were also observed. Thus changes in myonuclear number and distribution, perhaps related to alterations in the microtubular network, may underlie some of the adverse consequences of aging on skeletal muscle size and function.     

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.     

Angiotensin-converting enzyme inhibition attenuates myonuclear addition in overloaded slow-twitch skeletal muscle

Because optimal overload-induced skeletal muscle hypertrophy requires ANG II, we aimed to determine the effects of blocking ANG II production [via angiotensin-converting enzyme (ACE) inhibition] on potential mediators of hypertrophy in overloaded skeletal muscle, namely, myonuclear addition and fibroblast content. In a 2 x 2 design, adult (200-225 g) female Sprague-Dawley rats were placed into one of four groups (n = 8/group): 7-day skeletal muscle overload, sham operation, 7-day skeletal muscle overload with ACE inhibition, or sham operation with ACE inhibition. Functional overloads of the plantaris and soleus muscles were produced via bilateral surgical ablation of the synergistic gastrocnemius muscle, and ACE inhibition was accomplished by the addition of the ACE inhibitor enalapril maleate to the animals' daily drinking water (0.3 mg/ml). Myonuclear addition and extrasarcolemmal nuclear proliferation, as measured by in vivo 5-bromo-2'-deoxyuridine labeling, were significantly (P < or = 0.05) increased by overload in both the slow-twitch soleus and fast-twitch plantaris muscles. Furthermore, ACE inhibition attenuated these overload-induced increases in the soleus muscle but not in the plantaris muscle. However, the effect of ACE inhibition on soleus extrasarcolemmal nuclei was not likely due to differences in fibroblast content because overload elicited significant increases in vimentin-positive areas in soleus and plantaris muscles, and these areas were unaffected by ACE inhibition in either muscle. There was no effect of ACE inhibition on any measure in sham-operated muscles. Collectively, these data indicate that ANG II may mediate the satellite cell response to overload in slow-twitch soleus but not in fast-twitch plantaris muscles and that this effect may occur independently of changes in fibroblast content.     

Effect of flywheel-based resistance exercise on processes contributing to muscle atrophy during unloading in adult rats.

Flywheel-based resistance exercise (RE) attenuates muscle atrophy during hindlimb suspension. We have previously shown that protein synthesis is elevated in response to RE, but the effect on protein degradation, cell proliferation, or apoptosis was not investigated. We hypothesized that, in addition to affecting protein synthesis, RE inhibits processes that actively contribute to muscle atrophy during hindlimb suspension. Male rats were housed in regular cages (control), tail suspended for 2 wk (HS), or HS with RE every other day for 2 wk (HSRE). Although RE attenuated soleus muscle atrophy during HS, the observed fivefold elevation in apoptosis and the 53% decrease in cell proliferation observed with HS were unaffected by RE. Expression of genes encoding components of the ubiquitin-proteasome pathway of protein degradation were elevated with HS, including ubiquitin, MAFbx, Murf-1, Nedd4, and XIAP, and proteasome subunits C2 and C9. Total ubiquitinated protein was increased with HS, but proteasome activity was not different from control. RE selectively altered the expression of different components of this pathway: MAFbx, Murf-1, and ubiquitin mRNA abundance were downregulated, whereas C2 and C9 subunits remained elevated. Similarly, Nedd4 and XIAP continued to be upregulated, potentially accounting for the observed augmentation in total ubiquitinated protein with RE. Thus a different constellation of proteins is likely ubiquitinated with RE due to altered ubiquitin ligase composition. In summary, the flywheel-based resistance exercise paradigm used in this study is associated with the inhibition of some mechanisms associated with muscle atrophy, such as the increase in MAFbx and Murf-1, but not with others, such as proteasome subunit remodeling, apoptosis, and decreased proliferation, potentially accounting for the inability to completely restore muscle mass. Identifying specific exercise parameters that affect these latter processes may be useful in designing effective exercise strategies in the elderly or during spaceflight.     

Subsarcolemmal mitochondria and capillarization of soleus muscle fibers in young rats subjected to an endurance training

Summary Rats, 6 weeks old, were subjected to a program of endurance running for 3, 6 and 12 weeks. 0.5 to 0.8 m thick sections of Epon embedded soleus muscles were studied with morphometric methods.In cross-sections the area occupied by subsarcolemmal mitochondria was independent of the age, but was 53% higher after 12 weeks of training. The mean depth of the zones with subsarcolemmal mitochondria increased only 15% to about 0.9 m. Thus, the subsarcolemmal mitochondria showed a pronounced spreading at the muscle fiber surface in trained muscles. — The number of capillaries per fiber decreased slightly in controls and increased not significantly in trained muscles.It is concluded that the subsarcolemmal mitochondria supply the energy for the active transport of metabolites through the sarcolemma in oxidative muscle fibers, and that they are the limiting factor for endurance performance of the soleus muscle fibers because the changes in the capillarization were only small. It is suggested that the subsarcolemmal and the interfibrillar mitochondria have different functions and may therefore represent different types of mitochondria which can be distinguished by their morphology as well as by their biochemical properties.