Extensor Digitorum Longus Muscle Mass Research Articles

Changes in Skeletal Muscle Protein Metabolism Signaling Induced by Glutamine Supplementation and Exercise.

To evaluate the effects of resistance exercise training (RET) and/or glutamine supplementation (GS) on signaling protein synthesis in adult rat skeletal muscles. The following groups were studied: (1) control, no exercise (C); (2) exercise, hypertrophy resistance exercise training protocol (T); (3) no exercise, supplemented with glutamine (G); and (4) exercise and supplemented with glutamine (GT). The rats performed hypertrophic training, climbing a vertical ladder with a height of 1.1 m at an 80° incline relative to the horizontal with extra weights tied to their tails. The RET was performed three days a week for five weeks. Each training session consisted of six ladder climbs. The extra weight load was progressively increased for each animal during each training session. The G groups received daily L-glutamine by gavage (one g per kilogram of body weight per day) for five weeks. The C group received the same volume of water during the same period. The rats were euthanized, and the extensor digitorum longus (EDL) muscles from both hind limbs were removed and immediately weighed. Glutamine and glutamate concentrations were measured, and histological, signaling protein contents, and mRNA expression analyses were performed. Supplementation with free L-glutamine increased the glutamine concentration in the EDL muscle in the C group. The glutamate concentration was augmented in the EDL muscles from T rats. The EDL muscle mass did not change, but a significant rise was reported in the cross-sectional area (CSA) of the fibers in the three experimental groups. The levels of the phosphorylated proteins (pAkt/Akt, pp70S6K/p70S6K, p4E-BP1/4E-BP1, and pS6/S6 ratios) were significantly increased in EDL muscles of G rats, and the activation of p4E-BP1 was present in T rats. The fiber CSAs of the EDL muscles in T, G, and GT rats were increased compared to the C group. These changes were accompanied by a reduction in the 26 proteasome activity of EDL muscles from T rats. Five weeks of GS and/or RET induced muscle hypertrophy, as indicated by the increased CSAs of the EDL muscle fibers. The increase in CSA was mediated via the upregulated phosphorylation of Akt, 4E-BP1, p70S6k, and S6 in G animals and 4E-BP1 in T animals. In the EDL muscles from T animals, a decrease in proteasome activity, favoring a further increase in the CSA of the muscle fibers, was reported.

Impact of Heat Therapy on Skeletal Muscle Mass and Function in A Mouse Model of Duchenne Muscular Dystrophy

BackgroundDuchenne muscular dystrophy (DMD) is a lethal X‐linked degenerative disease that is characterized by progressive skeletal muscle wasting and weakness. Few non‐invasive and widely available therapies currently exist to combat the skeletal muscle abnormalities in patients with DMD. Repeated exposure to heat therapy (HT) has been shown to attenuate skeletal muscle atrophy during immobilization and unloading, rescue denervation‐induced atrophy, and increase relative muscle mass and strength in models of ischemia‐induced muscle damage. The goal of the present study was to test the hypothesis that repeated exposure to HT would enhance skeletal muscle mass and contractile function in D2‐mdx mice, a model that recapitulates several of the human characteristics of DMD.MethodsMale D2‐mdx and wild‐type DBA/2J mice (8 weeks of age) were obtained from the Jackson Laboratory. In study 1, we sought to define the optimal temperature for HT application using a heat incubator. Male DBA/2J mice (n=24) were randomly allocated to a control group or one of three different HT regimens (37, 39, 41℃). The treatment regimen consisted of exposure to HT for 30 min/day, 5 days/week during 3 consecutive weeks. Mice assigned to the control group underwent a similar intervention, but the incubator was maintained at room temperature. Based upon the findings of the first experiment, the goal of study 2 was to examine the effects of daily HT at 39℃ for 3 weeks on skeletal muscle mass and function in D2.mdx mice. Twenty‐four D2.mdx mice were randomized to either a HT group (n=12) or a control group (n=12) and were treated as described in study 1. Approximately 48 hrs after the last treatment session, the soleus and extensor digitorum longus (EDL) muscles were isolated and harvested for the measurement of wet mass and the assessment of contractile function.ResultsIn study 1, DBA/2J mice exposed to HT at 39ºC, but not 37º or 41ºC, displayed an increase in relative muscle mass (the ratio of muscle mass to body weight) of both EDL (p=0.03) and soleus (p=0.02) muscles when compared to the control group. Moreover, the EDL maximal specific force was greater in animals treated with HT at 39ºC relative to the 41℃ group (p=0.01). There were no significant differences between groups in soleus muscle force development. In study 2, D2.mdx mice exposed HT at 39ºC tended to display greater relative EDL muscle mass when compared to the control group (p=0.06). However, the relative mass of the soleus muscle was similar between the group treated with HT and the control regimen (p=0.84). The maximal force of both EDL and soleus muscles was also comparable between treated animals and the control group.ConclusionsOur findings indicate that exposure to HT at 39℃ for 3 weeks has limited effects on skeletal muscle mass and strength in a mouse model of DMD. Additional studies are warranted to determine whether different treatment regimens (e.g. higher temperature or longer HT sessions) are necessary to elicit beneficial changes in skeletal muscle function in DMD.

CARM1 Regulates AMPK Signaling in Skeletal Muscle.

SummaryCoactivator-associated arginine methyltransferase 1 (CARM1) is an emerging mediator of skeletal muscle plasticity. We employed genetic, physiologic, and pharmacologic approaches to determine whether CARM1 regulates the master neuromuscular phenotypic modifier AMP-activated protein kinase (AMPK). CARM1 skeletal muscle-specific knockout (mKO) mice displayed reduced muscle mass and dysregulated autophagic and atrophic processes downstream of AMPK. We observed altered interactions between CARM1 and AMPK and its network, including forkhead box protein O1, during muscle disuse. CARM1 methylated AMPK during the early stages of muscle inactivity, whereas CARM1 mKO mitigated progression of denervation-induced atrophy and was accompanied by attenuated phosphorylation of AMPK targets such as unc-51 like autophagy-activating kinase 1Ser555. Lower acetyl-coenzyme A corboxylaseSer79 phosphorylation, as well as reduced peroxisome proliferator-activated receptor-γ coactivator-1α, was also observed in mKO animals following acute administration of the direct AMPK activator MK-8722. Our study suggests that targeting CARM1-AMPK interplay may have broad impacts on neuromuscular health and disease.

Age-related changes in isolated mouse skeletal muscle function are dependent on sex, muscle, and contractility mode.

The present study aimed to simultaneously examine the age-related, muscle-specific, sex-specific, and contractile mode-specific changes in isolated mouse skeletal muscle function and morphology across multiple ages. Measurements of mammalian muscle morphology, isometric force and stress (force/cross-sectional area), absolute and normalized (power/muscle mass) work-loop power across a range of contractile velocities, fatigue resistance, and myosin heavy chain (MHC) isoform concentration were measured in 232 isolated mouse (CD-1) soleus, extensor digitorum longus (EDL), and diaphragm from male and female animals aged 3, 10, 30, 52, and 78 wk. Aging resulted in increased body mass and increased soleus and EDL muscle mass, with atrophy only present for female EDL by 78 wk despite no change in MHC isoform concentration. Absolute force and power output increased up to 52 wk and to a higher level for males. A 23-36% loss of isometric stress exceeded the 14-27% loss of power normalized to muscle mass between 10 wk and 52 wk, although the loss of normalized power between 52 and 78 wk continued without further changes in stress (P > 0.23). Males had lower power normalized to muscle mass than females by 78 wk, with the greatest decline observed for male soleus. Aging did not cause a shift toward slower contractile characteristics, with reduced fatigue resistance observed in male EDL and female diaphragm. Our findings show that the loss of muscle quality precedes the loss of absolute performance as CD-1 mice age, with the greatest effect seen in male soleus, and in most instances without muscle atrophy or an alteration in MHC isoforms.

Muscle‐Specific Calpastatin Overexpression Prevents Sarcopenia of Aging

Sarcopenia is a major cause of disability. The precise mechanisms responsible for aging‐related muscle weakness and atrophy are not known. However, recent studies suggest that pathologic activation of calpain modulates skeletal muscle dysfunction in aging. We tested the hypothesis that skeletal muscle‐specific overexpression of calpastatin, the endogenous inhibitor of calpain, would ameliorate aging‐associated decrements in muscle function. Five month old and 25 month old wild type (WT) and muscle‐specific calpastatin overexpressing (CalpOX) male mice (n=23) were studied. Grip strength was used as an index of total forelimb strength. To assess hindlimb strength, we measured extensor digitorum longus (EDL) mass and muscle specific force generation. Aging significantly reduced fore‐limb grip strength (0.94 ± 0.06 vs 1.47 ± 0.06N for old WT vs young WT, respectively, p<0.001). Importantly, calpastatin overexpression mitigated the aging‐induced reductions in fore‐limb grip strength (0.94 ± 0.06 vs 1.22 ± 0.05 for old WT vs old CalpOX, respectively, p=0.007). Aging also induced loss of EDL mass; calpastatin overexpression significantly ameliorated these reductions (7.63 ± 0.33 vs. 9.17 ± 0.45 mg for old WT vs old CalpOX, respectively, p=0.02). While calpastatin overexpression increased EDL muscle mass, EDL force per unit mass was similar between old WT and old CalpOX animals. To our knowledge, this is the first report of the effects of muscle‐specific calpastatin overexpression on skeletal muscle function in aging rodents. These findings support the concept that calpain activation plays a role in aging‐induced sarcopenia. We speculate that therapeutic agents which attenuate calpain activation may preserve muscle function in the elderly.Support or Funding InformationR01HL112085, 5I01BX002132, R01HL113494

The Effect of Short Nerve Grafts in Series on Axonal Regeneration Across Isografts or Acellular Nerve Allografts

To evaluate the regenerative effect of the additional suture line when using either isografts (ISOs) or acellular nerve allografts (ANAs) placed end-to-end to span a short gap in a rat model. Rat sciatic nerves were transected and repaired with 2-cm nerve grafts (ISO or ANA). The grafts were 2 cm in length or a 1-cm segment was connected end-to-end to a 1-cm segment to yield a 2-cm length. At 8 weeks, extensor digitorum longus (EDL) muscle force and mass were measured. Nerves were harvested for histomorphometry. In a separate parallel study, the nerves were harvested 2 weeks following graft implantation to assess gene expression changes. All grafts demonstrated regeneration across the 2-cm segment(s). The additional suture line did not result in statistical differences in the number of myelinated nerve fibers that reached the distal nerve. However, when the graft types were compared, there was a significant decrease in nerve fibers in the ANA groups. The EDL muscle mass was significantly greater by using nerve ISOs compared with ANAs, regardless of an additional suture line, but there were no statistical differences noted in EDL muscle force. Gene expression analysis did not differ owing to an additional suture line. Minimal axonal loss and no functional deficits were identified with an additional suture line in this rodent short nerve gap model. Placing nerve grafts in series is a viable option for treating short nerve gaps; however, the use of autografts remains preferable over the use of ANAs.

Altered contractile properties of fast and slow skeletal muscles from db/db mice (1102.16)

In the db/db mouse, leptin receptor mutation results in a model of metabolic syndrome. Initial hyperinsulinemia brought on by hyperphagia is followed by peripheral insulin resistance, elevated blood glucose and increased body weight. The purpose of this study was to determine the skeletal muscle contractile properties in the BKS.Cg‐Dock7m +/+Leprdb/J mouse model of metabolic syndrome (i.e. db/db mouse). Muscle contractile properties were determined ex vivo in the fast extensor digitorum longus (EDL) muscle and the slow soleus muscle in 17 week old mice. Body weight was 43% greater in db/db mice compared to wild type control mice. Soleus muscle mass was 49% lower and EDL muscle mass was 55% lower in the db/db mouse. EDL twitch force was 58% lower than control with an associated 20% longer contraction time and 25% longer 1/2 relaxation time. There were no differences in soleus twitch force. However, contraction time was 1.7‐fold longer and 1/2 relaxation time was 1.6‐fold longer than control. The force‐frequency relationship in the soleus muscle was significantly shifted to the left toward lower stimulation frequencies, with no differences in the EDL. The rate of force loss with repeated contractions was also consistent with a less oxidative profile in both muscle types. Collectively, these data demonstrate the effects of risk factors for metabolic syndrome on muscle contractile properties in the db/db mouse.

Selective modulation through the glucocorticoid receptor ameliorates muscle pathology inmdxmice

The over-expression of NF-κB signalling in both muscle and immune cells contribute to the pathology in dystrophic muscle. The anti-inflammatory properties of glucocorticoids, mediated predominantly through monomeric glucocorticoid receptor inhibition of transcription factors such as NF-κB (transrepression), are postulated to be an important mechanism for their beneficial effects in Duchenne muscular dystrophy. Chronic glucocorticoid therapy is associated with adverse effects on metabolism, growth, bone mineral density and the maintenance of muscle mass. These detrimental effects result from direct glucocorticoid receptor homodimer interactions with glucocorticoid response elements of the relevant genes. Compound A, a non-steroidal selective glucocorticoid receptor modulator, is capable of transrepression without transactivation. We confirm the in vitro NF-κB inhibitory activity of compound A in H-2K(b) -tsA58 mdx myoblasts and myotubes, and demonstrate improvements in disease phenotype of dystrophin deficient mdx mice. Compound A treatment in mdx mice from 18 days of post-natal age to 8 weeks of age increased the absolute and normalized forelimb and hindlimb grip strength, attenuated cathepsin-B enzyme activity (a surrogate marker for inflammation) in forelimb and hindlimb muscles, decreased serum creatine kinase levels and reduced IL-6, CCL2, IFNγ, TNF and IL-12p70 cytokine levels in gastrocnemius (GA) muscles. Compared with compound A, treatment with prednisolone, a classical glucocorticoid, in both wild-type and mdx mice was associated with reduced body weight, reduced GA, tibialis anterior and extensor digitorum longus muscle mass and shorter tibial lengths. Prednisolone increased osteopontin (Spp1) gene expression and osteopontin protein levels in the GA muscles of mdx mice and had less favourable effects on the expression of Foxo1, Foxo3, Fbxo32, Trim63, Mstn and Igf1 in GA muscles, as well as hepatic Igf1 in wild-type mice. In conclusion, selective glucocorticoid receptor modulation by compound A represents a potential therapeutic strategy to improve dystrophic pathology.

Effects of creatine supplementation on muscle wasting and glucose homeostasis in rats treated with dexamethasone

We aimed to investigate the possible role of creatine (CR) supplementation in counteracting dexamethasone-induced muscle wasting and insulin resistance in rats. Also, we examined whether CR intake would modulate molecular pathways involved in muscle remodeling and insulin signaling. Animals were randomly divided into four groups: (1) dexamethasone (DEX); (2) control pair-fed (CON-PF); (3) dexamethasone plus CR (DEX-CR); and (4) CR pair-fed (CR-PF). Dexamethasone (5 mg/kg/day) and CR (5 g/kg/day) were given via drinking water for 7 days. Plantaris and extensor digitorum longus (EDL) muscles were removed for analysis. Plantaris and EDL muscle mass were significantly reduced in the DEX-CR and DEX groups when compared with the CON-PF and CR-PF groups (P<0.05). Dexamethasone significantly decreased phospho-Ser473-Akt protein levels compared to the CON-PF group (P<0.05) and CR supplementation aggravated this response (P<0.001). Serum glucose was significantly increased in the DEX group when compared with the CON-PF group (DEX 7.8±0.6 vs. CON-PF 5.2±0.5 mmol/l; P<0.05). CR supplementation significantly exacerbated hyperglycemia in the dexamethasone-treated animals (DEX-CR 15.1±2.4 mmol/l; P<0.05 vs. others). Dexamethasone reduced GLUT-4 translocation when compared with the CON-PF and CR-PF (P<0.05) groups and this response was aggravated by CR supplementation (P<0.05 vs. others). In conclusion, supplementation with CR resulted in increased insulin resistance and did not attenuate muscle wasting in rats treated with dexamethasone. Given the contrast with the results of human studies that have shown benefits of CR supplementation on muscle atrophy and insulin sensitivity, we suggest caution when extrapolating this animal data to human subjects.

Attenuation of Age-Related Muscle Wasting and Weakness in Rats After Formoterol Treatment: Therapeutic Implications for Sarcopenia

We investigated the potential of the beta(2)-adrenoceptor agonist formoterol to increase mass and force-producing capacity of extensor digitorum longus (EDL) and soleus muscles from young, adult, and old rats. In addition, we examined the result of formoterol withdrawal. Young (3 month), adult (16 month), and old (27 month) F344 rats were treated with either formoterol (25 microg/kg/day, i.p.) or saline vehicle for 4 weeks. Another group of rats (for each age) was similarly treated with formoterol, followed by a withdrawal period of 4 weeks. Formoterol treatment increased EDL muscle mass and the force-producing capacity of both EDL and soleus muscles, without a concomitant increase in heart mass in adult and old rats. The hypertrophy and increased force-producing capacity of EDL muscles persisted 4 weeks after withdrawal of treatment. The findings have major implications for potential clinical trials utilizing beta(2)-agonists for sarcopenia.

Relative effects of exercise training and alendronate treatment on skeletal muscle function of ovariectomized rats

Increased physical activity and bisphosphonate treatment are both effective in reducing the risk of fracture in postmenopausal women. Physical activity reduces the risk of fracture by improving both bone and skeletal muscle strength. The effects of bisphosphonates have primarily been evaluated on bone. In this study the authors investigated the combined effects of increased physical activity and bisphosphonate treatment on skeletal muscle function using an animal model of postmenopausal osteoporosis. Seven-month-old ovariectomized rats were assigned to one of four combinations of physical activity treatment (sedentary or endurance running) and alendronate treatment (vehicle or 0.015 mg/kg alendronate, two treatments per week). After 14 weeks slow (soleus) and fast (extensor digitorum longus) muscles were isolated from each animal and electrically stimulated for evaluation of twitch, low-frequency, and tetanic force and resistance to fatiguing stimulation. Exercise training reduced body mass, increased soleus and extensor digitorum longus muscle mass, increased soleus tetanic force, reduced the twitch contraction time of the soleus and extensor digitorum longus, and facilitated relaxation of the soleus during fatiguing stimulation. Bisphosphonate treatment had no independent effect on muscle function and interacted with training for only a single variable (twitch half-relaxation time). Endurance training increased muscle mass in ovariectomized rats and had subtle effects on slow and fast muscle function. Alendronate treatment, at a dosage previously shown to improve bone strength in ovariectomized rats, neither blunted nor enhanced these training adaptations and had no independent effect on muscle contractility.

Leucine supplementation mitigates atrophy of non‐weight‐bearing skeletal muscle in rats

Hindlimb unloading (HU) is commonly used to simulate weightless condition which induces severe muscle atrophy. Dietary leucine enhances muscle growth by stimulating mammalian target of rapamycin ( mTOR) signaling and protein synthesis. We hypothesize that dietary supplementation of leucine enhances mTOR signaling and mitigates the atrophy of non-weight-bearing muscle. Thirty Spraque-Dawley rats were separated into 5 treatments: control rats without treatment; rats with HU by tail suspension; rats with HU plus 5% leucine supplement; rat with HU plus 5% casein supplement; rats with HU plus 5% starch supplement based on dry feed weight. Trial was lasted for 2 weeks. HU induced severe loss of soleus muscle mass (P<0.01) but only had mild effect on extensor digitorum longus muscle mass, showing that unloading mainly induced atrophy of red muscle fibers. Rats treated with 5% of leucine mitigated muscle atrophy in soleus muscle (P<0.05). Leucine treatment reduced (P<0.05) the phosphorylation of AMP- activated protein kinase (AMPK). No difference in total AMPK, Akt and acetyl CoA carboxylase (ACC) was observed among treatment groups. Currently, we are measuring the phosphorylation of mTOR, ACC and ribosomal protein S6 kinase to further examine signaling pathways leading to the mitigation of muscle atrophy due to leucine supplementation.

Mechanical function of muscle reinnervated by end-to-side neurorrhaphy.

End-to-side neurorrhaphy is a surgical technique for peripheral nerve reconstruction when end-to-end neurorrhaphy is not an option. To define the effectiveness of end-to-side neurorrhaphy as a method of nerve repair, the authors tested the null hypothesis: there is no difference in the mechanical function of skeletal muscle denervated and reinnervated by end-to-side versus end-to-end neurorrhaphy. Adult Lewis rats underwent either transection and end-to-end epineurial repair of the left peroneal nerve (n = 9) or end-to-side repair of the distal stump of the peroneal nerve to the side of the tibial nerve (n = 8). After a 6-month recovery period, isometric force (Fo) was measured, and specific force (sFo) was calculated for the extensor digitorum longus muscle of each animal. Immunohistochemical staining for neural cell adhesion molecule (NCAM) was performed to identify populations of denervated muscle fibers. The mean extensor digitorum longus muscle mass in the end-to-end group (195 +/- 32 g) was significantly greater than that of the end-to-side group (146 +/- 55 g) (p < 0.05). A significantly greater percentage of denervated fibers was identified in the extensor digitorum longus muscles of animals in the end-to-side group (9.4 +/- 3.2 percent) than in those in the end-to-end group (3.8 +/- 1.0 percent) (p < 0.05). Despite a lower muscle mass and a higher percentage of denervated fibers, neither Fo nor sFo was significantly different in the two groups. These data support the null hypothesis that, under appropriate circumstances, there is no difference in the recovery of whole muscle force and specific force production in muscles reinnervated by end-to-side versus end-to-end neurorrhaphy.

Effects of growth hormone on rat skeletal muscle after hindlimb suspension.

To examine the effects of growth hormone (GH) on the preferential atrophy of the soleus muscle (SOL) occurring after hindlimb suspension (HS), two groups of male rats received daily injections of 2 IU.kg-1 body mass of recombinant human growth hormone (rhGH). Rats were either suspended by the tail for 21 days (HS-GH, n = 5) or nonsuspended (C-GH, n = 5). The effects of rhGH treatment on SOL and extensor digitorum longus muscles (EDL) were compared in two groups of animals receiving daily injections of saline, either suspended by the tail (HS-SA, n = 5) or nonsuspended (C-SA, n = 5). The results showed that the SOL hypertrophy in response to rhGH administration was mostly observed in C rats (+33%, P < 0.01). This increase in muscle mass was correlated with a concomitant increase in the size of type I fibres (+21%, P < 0.05). Although SOL mass decreased during HS in rhGH treated animals (-44%, P < 0.001), the mean normalized mass of this muscle did not significantly differ between C-SA and HS-GH groups. A statistically significant increase in the absolute mass of EDL occurred with rhGH treatment in C-GH (+12%, P < 0.05). The HS-induced decrease in the percentage distribution of type I fibres in SOL was unaffected by the rhGH treatment. In addition, a decrease in the citrate synthase activity in the whole SOL was observed in the two groups of tail-suspended rats (-31%, P < 0.05; -21%, P < 0.05 in SA and GH animals, respectively).2+ f1p4

Muscle atrophy and hypoplasia with aging: impact of training and food restriction.

The purpose of this work is to study the influence of aging, training, and food restriction on skeletal muscle mass and fiber number. Male Fischer 344 rats (n = 49) at 3 mo postpartum were assigned to three groups: 1) sedentary control (confined to cage), 2) exercise trained (18 m/min, 8 degrees grade, 20 min/day, 5 days/wk), or 3) food restricted (alternate days of free access and no access to food). At 12 and 27 mo postpartum the soleus and extensor digitorum longus (EDL) muscles were excised, weighed, and fiber number was quantified after HNO3 digestion. At 27 mo the masses of soleus and EDL muscles of sedentary control rats were 83 and 70%, respectively, of 12-mo values (138 +/- 5 and 151 +/- 4 mg). At 27 mo, soleus muscle mass of trained rats was 113% of sedentary control values, whereas EDL muscle mass was unaffected by training. At 27 mo, food restriction had no effect on the mass of both muscles compared with 27-mo sedentary control values. Fiber number was not affected by training or food restriction in both muscles. Fiber number for soleus and EDL muscles of combined groups declined with age by 5.6 and 4.2%, respectively. With aging, the small loss of muscle fibers can account at most for approximately 25% of the observed skeletal muscle atrophy.