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     

PGC-1α and FOXO1 mRNA levels and fiber characteristics of the soleus and plantaris muscles in rats after hindlimb unloading

Fifteen-week-old rats were subjected to unloading induced by hindlimb suspension for 3 weeks. The peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and forkhead box-containing protein O1 (FOXO1) mRNA levels and fiber profiles of the soleus and plantaris muscles in rats subjected to unloading (unloaded group) were determined and compared with those of age-matched control rats (control group). The body weight and both the soleus and plantaris muscle weights were lower in the unloaded group than in the control group. The PGC-1α mRNA was downregulated in the soleus, but not in the plantaris muscle of the unloaded group. The FOXO1 mRNA was upregulated in both the soleus and plantaris muscles of the unloaded group. The oxidative enzyme activity was reduced in the soleus, but not in the plantaris muscle of the unloaded group. The percentage of type I fibers was decreased and the percentages of type IIA and IIC fibers were increased in the soleus muscle of the unloaded group, whereas there was no change in fiber type distribution in the plantaris muscle of the unloaded group. Atrophy of all types of fibers was observed in both the soleus and plantaris muscles of the unloaded group. We conclude that decreased oxidative capacity and fiber atrophy in unloaded skeletal muscles are associated with decreased PGC-1α and increased FOXO1 mRNA levels.     

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.     

Age effects on myosin subunit and biochemical alterations with skeletal muscle hypertrophy.

The purpose of this study was to determine whether skeletal muscle mass, myofibrillar adenosinetriphosphatase activity, and the expression of myosin heavy (MHC) and light chain subunits are differentially affected in juvenile (4 wk) and young adult (12 wk) rats by a hypertrophic growth stimulus. Hypertrophy of the plantaris or soleus was studied 4 wk after ablation of either two [gastrocnemius (GTN) and soleus or plantaris] or one (GTN) synergistic muscle(s). There was no difference in the relative magnitude of hypertrophy because of age. Plantaris myofibrillar adenosinetriphosphatase activity was decreased 21 and 12% in juvenile and adult rats, respectively, as a result of ablation of both the GTN and soleus. Slow myosin light chain isoforms (1s and 2s) were expressed to a greater extent in hypertrophied plantaris muscles of both ages, but the increase in 1s was greater in juvenile rats. The relative expression of slow beta-MHC in hypertrophied plantaris muscles increased by 470 and 350%, whereas MHC IIb decreased by 70 and 33% in juvenile and adult rats, respectively. The relative expression of MHC IIa increased (56%) in the plantaris after ablation in juvenile rats only. These shifts in myosin subunit expression and the increases in mass were generally about one-half the magnitude when only the GTN was removed. There were no detectable myosin shifts in hypertrophied soleus muscles. Although the extent of muscle hypertrophy is similar, the shifts in myosin subunits were greater in juvenile than in young adult rats.     

Effect of burn injury on apoptosis and expression of apoptosis-related genes/proteins in skeletal muscles of rats

The purpose of this study was to investigate the occurrence and possible mechanisms of apoptosis in skeletal muscles after burn injury. After a 40% body surface area burn to rats, TA muscles were examined for apoptosis at varying times by TEM, TUNEL and cell death ELISA assay. Thermal injury was found to induce apoptosis in skeletal muscle on the first day and maximal apoptosis appeared 4 days post-injury. Apoptotic ligands in serum assessed by ELISA revealed rapidly increase of TNF-α and subsequent increase of sFasL to sFas ratio after burn injury. It implied TNF-α induced apoptosis in early stage and FasL induced apoptosis in later stage after burn injury. Apoptosis-related genes/proteins in skeletal muscles examined by real-time PCR array and Western blotting showed pro-apoptotic genes/proteins, including Tnfrsf1a, Tnfrsf1b and Tnfsf6 in TNF ligand and receptor family, Bax and Bid in Bcl-2 family, caspase-3 and caspase-6 in caspase family, Dapk1, FADD and Cidea in death and CIDE domain family, Apaf-1 in CARD family, and Gadd45a were up-regulated, while anti-apoptotic gene Bnip1 was down-regulated compared with that of time-matched controls. In addition, increment of caspase-3, caspase-8 and caspase-9 activity provided further evidence for their role in apoptosis in skeletal muscle. Significant increase in expression in pro-apoptotic genes/proteins and activity of caspases suggested that death receptor-mediated signaling pathways and other apoptotic related pathways participated in apoptosis in skeletal muscle after burn injury. However, it was found that some anti-apoptotic genes such as Bcl2l1, Mcl-1, Nol-3, Il-10 and Prok2 were also up-regulated, which might imply the co-existence of protective response of the body after burns. In conclusion, the data suggest that apoptosis and pro-apoptotic signaling are enhanced in muscles of burned rats. To further elucidate the underlying apoptotic mechanisms mediating the atrophic response is important in establishing potential therapeutic interventions that could prevent and/or reduce skeletal muscle wasting and preserve its physiological function.     

Diminished overload-induced hypertrophy in aged fast-twitch skeletal muscle is associated with AMPK hyperphosphorylation.

Skeletal muscle mass declines with age, as does the potential for overload-induced fast-twitch skeletal muscle hypertrophy. Because 5'-AMP-activated protein kinase (AMPK) activity is thought to inhibit skeletal muscle protein synthesis and may therefore modulate muscle mass and hypertrophy, the purpose of this investigation was to examine AMPK phosphorylation status (a marker of AMPK activity) and its potential association with the attenuated overload-induced hypertrophy observed in aged skeletal muscle. One-week overload of fast-twitch plantaris and slow-twitch soleus muscles was achieved in young adult (8 mo; n = 7) and old (30 mo; n = 7) Fischer344 x Brown Norway male rats via unilateral gastrocnemius ablation. Significant (P < or = 0.05) age-related atrophy (as measured by total protein content) was noted in plantaris and soleus control (sham-operated) muscles. In fast-twitch plantaris muscles, percent hypertrophy with overload was significantly attenuated with age, whereas AMPK phosphorylation status as determined by Western blotting [phospho-AMPK (Thr172)/total AMPK] was significantly elevated with age (regardless of loading status). There was also a main effect of loading on AMPK phosphorylation status in plantaris muscles (overload > control). Moreover, a strong and significant negative correlation (r = -0.82) was observed between AMPK phosphorylation status and percent hypertrophy in the overloaded plantaris muscles of all animals. In contrast to the plantaris, overload-induced hypertrophy of the slow-twitch soleus muscle was similar between ages, and AMPK phosphorylation in this muscle was also unaffected by age or overload. These data support the possibility that an age-related elevation in AMPK phosphorylation may partly contribute to the attenuated hypertrophic response observed with age in overloaded fast-twitch plantaris muscle.     

High-fat feeding does not induce an autophagic or apoptotic phenotype in female rat skeletal muscle

Apoptosis and autophagy are critical in normal skeletal muscle homeostasis; however, dysregulation can lead to muscle atrophy and dysfunction. Lipotoxicity and/or lipid accumulation may promote apoptosis, as well as directly or indirectly influence autophagic signaling. Therefore, the purpose of this study was to examine the effect of a 16-week high-fat diet on morphological, apoptotic, and autophagic indices in oxidative and glycolytic skeletal muscle of female rats. High-fat feeding resulted in increased fat pad mass, altered glucose tolerance, and lower muscle pAKT levels, as well as lipid accumulation and reactive oxygen species generation in soleus muscle; however, muscle weights, fiber type-specific cross-sectional area, and fiber type distribution were not affected. Moreover, DNA fragmentation and LC3 lipidation as well as several apoptotic (ARC, Bax, Bid, tBid, Hsp70, pBcl-2) and autophagic (ATG7, ATG4B, Beclin 1, BNIP3, p70 s6k, cathepsin activity) indices were not altered in soleus or plantaris following high-fat diet. Interestingly, soleus muscle displayed small increases in caspase-3, caspase-8, and caspase-9 activity, as well as higher ATG12-5 and p62 protein, while both soleus and plantaris muscle showed dramatically reduced Bcl-2 and X-linked inhibitor of apoptosis protein (XIAP) levels. In conclusion, this work demonstrates that 16 weeks of high-fat feeding does not affect tissue morphology or induce a global autophagic or apoptotic phenotype in skeletal muscle of female rats. However, high-fat feeding selectively influenced a number of apoptotic and autophagic indices which could have implications during periods of enhanced muscle stress.     

Denervation and reinnervation of fast and slow muscles. A histochemical study in rats.

A histochemical study, using myosin-adenosine triphosphatase activity at pH 9.4, was conducted in soleus and plantaris muscles of adult rats, after bilateral crushing of the sciatic nerve at the sciatic notch. The changes in fiber diameter and per cent composition of type I and type II fibers plus muscle weights were evaluated along the course of denervation-reinnervation curve at 1, 2, 3, 4 and 6 weeks postnerve crush. The study revealed that in the early denervation phase (up to 2 weeks postcrush) both the slow and fast muscles, soleus and plantaris, resepctively, atrophied similarly in muscle mass. Soleus increased in the number of type II fibers, which may be attributed to "disuse" effect. During the same period, the type I fibers of soleus atrophied as much or slightly more than the type II fibers; whereas the type II fibers of plantaris atrophied significantly more than the type I fibers, reflecting that the process of denervation, in its early stages, may affect the two fiber types differentially in the slow and fast muscles. It was deduced that the type I fibers of plantaris may be essentially different in the slow (soleus) and fast (plantaris) muscles under study. The onset of reinnervation, as determined by the increase in muscle weight and fiber diameter of the major fiber type, occurred in soleus and plantaris at 2 and 3 weeks postcrush, respectively, which confirms the earlier hypotheses that the slow muscles are reinnervated sooner than the fast muscles. It is suggested that the reinnervation of muscle after crush injury may be specific to the muscle type or its predominant fiber type.     

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.     

ANG II is required for optimal overload-induced skeletal muscle hypertrophy

ANG II mediates the hypertrophic response of overloaded cardiac muscle, likely via the ANG II type 1 (AT(1)) receptor. To examine the potential role of ANG II in overload-induced skeletal muscle hypertrophy, plantaris and/or soleus muscle overload was produced in female Sprague-Dawley rats (225-250 g) by the bilateral surgical ablation of either the synergistic gastrocnemius muscle (experiment 1) or both the gastrocnemius and plantaris muscles (experiment 2). In experiment 1 (n = 10/group), inhibiting endogenous ANG II production by oral administration of an angiotensin-converting enzyme (ACE) inhibitor during a 28-day overloading protocol attenuated plantaris and soleus muscle hypertrophy by 57 and 96%, respectively (as measured by total muscle protein content). ACE inhibition had no effect on nonoverloaded (sham-operated) muscles. With the use of new animals (experiment 2; n = 8/group), locally perfusing overloaded soleus muscles with exogenous ANG II (via osmotic pump) rescued the lost hypertrophic response in ACE-inhibited animals by 71%. Furthermore, orally administering an AT(1) receptor antagonist instead of an ACE inhibitor produced a 48% attenuation of overload-induced hypertrophy that could not be rescued by ANG II perfusion. Thus ANG II may be necessary for optimal overload-induced skeletal muscle hypertrophy, acting at least in part via an AT(1) receptor-dependent pathway.     

Contractile properties and myosin expression in rats born and reared in hypergravity

The effects of hypergravity (HG) on soleus and plantaris muscles were studied in Long Evans rats aged 100 days, born and reared in 2-g conditions (HG group). The morphological and contractile properties and the myosin heavy chain (MHC) content were examined in whole muscles and compared with terrestrial control (Cont) age-paired rats. The growth of HG rats was slowed compared with Cont rats. A decrease in absolute muscle weight was observed. An increase in fiber cross-sectional area/muscle wet weight was demonstrated, associated with an increase in relative maximal tension. The soleus muscle changed into a slower type both in contractile parameters and in MHC content, since HG soleus contained only the MHC I isoform. The HG plantaris muscle presented a faster contractile behavior. Moreover, the diversity of hybrid fiber types expressing multiple MHC isoforms (including MHC IIB and MHC IIX isoforms) was increased in plantaris muscle after HG. Thus the HG environment appears as an important inductor of muscular plasticity both in slow and fast muscle types.     

Endogenous expression and developmental changes of HSP72 in rat skeletal muscles

The purpose of the present study was to determine whether endogenous factor(s) contributes to the expression of heat shock proteins (HSPs) during the early developmental stages of rat skeletal muscles. HSP72 was expressed in both the soleus and plantaris muscles at embryonic day 22 (E22). On the basis of myosin heavy chain (MHC) immunohistochemistry, HSP72 was specifically expressed in slow type I fibers in both muscles. These slow fibers were observed throughout the entire cross section of the soleus muscle and only in the deep region (close to the bone) of the plantaris muscle. These results indicate that the expression of HSP72 is related to endogenous factors associated with type I fibers, because E22 rats have minimal exogenous influences and the soleus and plantaris muscles of E22 rats have similar metabolic and contractile profiles at this stage of development. We then examined the changes in HSP72 and heat shock cognate (HSC) 73 in the same two muscles from E22 to postnatal day 56 via Western blotting. The level of HSP72 in the soleus muscle gradually increased in parallel with the increment in the type I MHC isoform. Compared with the soleus, only a small amount of HSP72 could be detected in the plantaris muscle throughout the developmental period. For both muscles, HSC73 reached levels observed in adult muscles at postnatal day 3, and these levels were unchanged thereafter. These results indicate that the expression of HSP72, but not HSC73, is influenced by both endogenous and exogenous factors during the embryonic and early developmental periods.     

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.     

Some hormonal factors (hypophysectomy, castration and testosterone administration) modifying the course of "compensatory" muscle hypertrophy in the rat.

1. Maximum compensatory hypertrophy of the soleus and plantaris muscle in male rats is attained seven days after tenotomy of the gastrocnemius muscle (39% and 9% respectively). When tenotomy of the gastrocnemius was performed seven days ater hypophysectomy, hypertrophy in these two muscles was aproximately half that found in control animals. 2. After 81-day castration of young male rats the weight of the saleus and plantaris was reduced and hypertrophy following tenotomy of the gastrocneumius muscle did not develop. 3. Chronically castrated rats received testosterone two weeks prior to tenotomy of the gastrocnemius and a week during the muscle hypertrophy phase. Hypertrophy of the soleus in castrated rats which had received testosterone seven days after tenotomy of the gastrocnemius was 25% as compared with muscles of castrated animals. The corresponding value in the plantaris muscle was 10%. 4. These results indicate that even calf muscles of the rat, namely the soleus and plantaris muscles, are significantly affected by testosterone under these conditions, although it is not, as yet, clear whether its action is direct or indirect.     

Adaptation in synergistic muscles to soleus and plantaris muscle removal in the rat hindlimb.

Although the soleus muscle comprises only 6% of the ankle plantar flexor mass in the rat, a major role in stance and walking has been ascribed to it. The purpose of this study was to determine if removal of the soleus muscle would result in adaptations in the remaining gastrocnemius and plantaris muscles due to the new demands for force production imposed on them during stance or walking. A second purpose was to determine whether the mass or the fiber type of the muscle(s) removed was a more important determinant of compensatory adaptations. Male Sprague-Dawley rats underwent bilateral removal of soleus muscle, plantaris muscle, or both muscles. For comparison, compensatory hypertrophy was induced in soleus and plantaris muscles by gastrocnemius muscle ablation. After forty days, synergist muscles remaining intact were removed. Mass, and oxidative, glycolytic, and contractile enzyme activities were determined. Despite its role in stance and slow walking, removal of the soleus muscle did not elicit a measurable alteration in muscle mass, or in citrate synthase, lactate dehydrogenase, or myofibrillar ATPase activity in gastrocnemius or plantaris muscles. Similarly, removal of the plantaris muscle, or soleus and plantaris muscles, had no effect on the gastrocnemius muscle, suggesting that this muscle was able to easily meet the new demands placed on it. These results suggest that amount of muscle mass removed, rather than fiber type, is the most important stimulus for compensatory hypertrophy. They also suggest that slow-twitch motor units in the gastrocnemius muscle play an important role during stance and locomotion in the intact animal.     

Conversion of rat muscle fiber types

Summary Rats were used in this study to determine the time course of conversion of muscle fiber types. The right or left gastrocnemius muscle was removed thereby causing an overload on the ipsilateral soleus and plantaris muscles. The contralateral limb served as a control. The type II to type I fiber conversion was followed histochemically in the soleus and plantaris muscles for one to six weeks following surgery. Muscle sections were stained for myofibrillar actomyosin ATPase and NADH tetrazolium reductase. The type I population in the soleus muscle was 99.3% six weeks after synergist removal. The plantaris muscle underwent a two fold increase in the percentage of type I fibers after six weeks. Transitional fibers were prominent in the plantaris muscle and reached their peak at 4% (P<0.05) of the total population, four weeks after surgery.This research was funded in part by grants from The Graduate School at Washington State University, and The Society of the Sigma Xi     

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.     

Age‐related differences in synaptic plasticity following muscle unloading

The objective of the present investigation was to determine the effects of muscle unloading—a form of subtotal disuse— on the morphology of the neuromuscular junction (NMJ) in younger and aged animals. Sixteen aged (22 months) and 16 young adult (8 months) male Fischer 344 rats were assigned to control and hindlimb suspension (HS) conditions (n = 8/group). At the conclusion of the 4 week experimental period, soleus muscles were collected, and immunofluorescent procedures were used to visualize acetylcholine (ACh) vesicles and receptors, nerve terminal branching, as well as NCAM and NT‐4 expression. Quantitative analyses revealed that aged controls displayed significant (p < 0.05) reductions in area and perimeter length of ACh vesicle and receptor regions, without affecting nerve terminal branch number or length. In contrast to younger NMJs, which were resilient to the effects of unloading, NMJs of aged HS rats demonstrated significant expansion of ACh vesicle and receptor dimensions compared to aged controls. Qualitative analyses of NCAM staining indicated that aging alone somewhat increased this molecule's expression (aged controls > young controls). Among the four groups, however, the greatest amount of NCAM content was detected among aged HS muscles, matching the degree of synaptic plasticity exhibited in those muscles. Unlike NCAM, the expression of NT‐4 did not appear to differ among the treatment groups. These data suggest that although young adult muscle maintains normal NMJ structure during prolonged exposure to unloading, aged NMJs experience significant adaptation to that stimulus. © 2003 Wiley Periodicals, Inc. J Neurobiol 57: 246–256, 2003     

Calcineurin differentially regulates maintenance and growth of phenotypically distinct muscles

Adequate muscle mass is critical for human health. The molecular pathways regulating maintenance and growth of adult skeletal muscle are little understood. Calcineurin (CN) is implicated as a key signaling molecule in hypertrophy. Whether CN is involved in all forms of muscle growth or in different muscles is unknown. Here, we examine the role of CN in regulating maintenance of muscle size and growth of atrophied muscle in the soleus (slow) and plantaris (fast). The CN inhibitor cyclosporin A (CsA) differentially affects muscle growth and maintenance depending on muscle phenotype. The plantaris is more severely affected by CsA than the soleus in both growth conditions. One-week vs. 2-wk CsA treatment suggests that both CN-dependent and CN-independent growth occur in the atrophied soleus, whereas plantaris growth appears to be totally CN dependent. Our results suggest that CN regulates multiple types of muscle growth, depending both on muscle phenotype and stage of myofiber growth. Differential expression of components of the CN pathway occurs and may contribute to the differences between muscles.