Adult Rat Mesenchymal Stem Cells Delay Denervated Muscle Atrophy

To evaluate the function of rat mesenchymal stem cells (rMSCs) on denervated gastrocnemius muscles and to address the role of ciliary neurotrophic factor (CNTF) in rMSCs, denervated Wistar rats were separately injected with culture media (sham control), CNTF protein, 2.5?×?10⁵ siCNTF-treated rMSCs, 2.5?×?10⁵ GFP-transfected rMSCs, or 2.5?×?10⁵ untreated rMSCs. Muscle function was assessed at different time points post-surgery. Tibial nerve and gastrocnemius muscle samples were taken at 4, 8, and 12?weeks for histochemistry, and neuromuscular junction repair was also examined by electron microscopy. Fluorescence immunocytochemistry on tissue sections confirmed neurotrophin expression in rMSCs but with little evidence of neuronal differentiation. The engraftment of rMSCs significantly preserved the function of denervated gastrocnemius muscle based both on evaluation of muscle function and direct examination of muscle tissue. Further, the density and depth of the junctional folds were visibly reduced 12?weeks after surgery and transplantation, especially in control group. Knockdown of CNTF expression in rMSCs failed to block muscle preservation, although administration of CNTF protein alone inhibited muscle atrophy, which indicating that delivery of rMSCs could preserve gastrocnemius muscle function following denervation and post-junctional mechanisms involved in the repairing capability of rMSCs.     

Impaired Bone Homeostasis in Amyotrophic Lateral Sclerosis Mice with Muscle Atrophy

There is an intimate relationship between muscle and bone throughout life. However, how alterations in muscle functions in disease impact bone homeostasis is poorly understood. Amyotrophic lateral sclerosis (ALS) is a neuromuscular disease characterized by progressive muscle atrophy. In this study we analyzed the effects of ALS on bone using the well established G93A transgenic mouse model, which harbors an ALS-causing mutation in the gene encoding superoxide dismutase 1. We found that 4-month-old G93A mice with severe muscle atrophy had dramatically reduced trabecular and cortical bone mass compared with their sex-matched wild type (WT) control littermates. Mechanically, we found that multiple osteoblast properties, such as the formation of osteoprogenitors, activation of Akt and Erk1/2 pathways, and osteoblast differentiation capacity, were severely impaired in primary cultures and bones from G93A relative to WT mice; this could contribute to reduced bone formation in the mutant mice. Conversely, osteoclast formation and bone resorption were strikingly enhanced in primary bone marrow cultures and bones of G93A mice compared with WT mice. Furthermore, sclerostin and RANKL expression in osteocytes embedded in the bone matrix were greatly up-regulated, and β-catenin was down-regulated in osteoblasts from G93A mice when compared with those of WT mice. Interestingly, calvarial bone that does not load and long bones from 2-month-old G93A mice without muscle atrophy displayed no detectable changes in parameters for osteoblast and osteoclast functions. Thus, for the first time to our knowledge, we have demonstrated that ALS causes abnormal bone remodeling and defined the underlying molecular and cellular mechanisms.     

Effects of Nandrolone in the Counteraction of Skeletal Muscle Atrophy in a Mouse Model of Muscle Disuse: Molecular Biology and Functional Evaluation

Muscle disuse produces severe atrophy and a slow-to-fast phenotype transition in the postural Soleus (Sol) muscle of rodents. Antioxidants, amino-acids and growth factors were ineffective to ameliorate muscle atrophy. Here we evaluate the effects of nandrolone (ND), an anabolic steroid, on mouse skeletal muscle atrophy induced by hindlimb unloading (HU). Mice were pre-treated for 2-weeks before HU and during the 2-weeks of HU. Muscle weight and total protein content were reduced in HU mice and a restoration of these parameters was found in ND-treated HU mice. The analysis of gene expression by real-time PCR demonstrates an increase of MuRF-1 during HU but minor involvement of other catabolic pathways. However, ND did not affect MuRF-1 expression. The evaluation of anabolic pathways showed no change in mTOR and eIF2-kinase mRNA expression, but the protein expression of the eukaryotic initiation factor eIF2 was reduced during HU and restored by ND. Moreover we found an involvement of regenerative pathways, since the increase of MyoD observed after HU suggests the promotion of myogenic stem cell differentiation in response to atrophy. At the same time, Notch-1 expression was down-regulated. Interestingly, the ND treatment prevented changes in MyoD and Notch-1 expression. On the contrary, there was no evidence for an effect of ND on the change of muscle phenotype induced by HU, since no effect of treatment was observed on the resting gCl, restCa and contractile properties in Sol muscle. Accordingly, PGC1α and myosin heavy chain expression, indexes of the phenotype transition, were not restored in ND-treated HU mice. We hypothesize that ND is unable to directly affect the phenotype transition when the specialized motor unit firing pattern of stimulation is lacking. Nevertheless, through stimulation of protein synthesis, ND preserves protein content and muscle weight, which may result advantageous to the affected skeletal muscle for functional recovery.     

Variations of Glucocorticoid Receptors in Intact or Denervated Muscles: Lack of Cause-Effect Relationship with Muscle Atrophy in the Rat

Abstract

The process of muscular atrophy following denervation has been tentatively ascribed to the influence of glucocorticoids (G) because of the rapid increase of cytosolic G receptors (R^G) after sciatic nerve section. It appears however that the level of muscular atrophy is similar: 1. in slow or fast-twitch muscles in spite of huge variations in R^G; 2. in intact or adrenalectomized (ADR-X) rats. Moreover, the protein muscle profile of intact or ADR-X rats after gel electrophoresis is similar but drastically decreased after 3 weeks of denervation. We conclude that there is no causeeffect relationship between muscle atrophy and R^G elevation after nerve section.     

Effect of Estrogen on Denervated Muscle

Abstract: The rate of increase of glucose 6-phosphate dehydrogenase activity in denervated rat extensor digitorum longus muscle shows sexual dimorphism. This phenomenon is further investigated in this report by assessing the effects of ovariectomy, hypophysectomy, hormone replacement therapy, and treatment with an estrogen antagonist, MER-25. The data demonstrate that physiologic doses of estrogens enhance the rate and extent of the increase in glucose 6-phosphate dehydrogenase activity after denervation. The data further indicate that aromatization of androgens may be a significant source of estrogen involved in hormonal modulation of the neural control of glucose 6-phosphate dehydrogenase and other processes in muscle. Furthermore, choline acetyltransferase activity, a marker for the neuromuscular synapse, decreased in rat extensor digitorum longus muscles after denervation, but was unaffected by ovariectomy.     

The Posterior Cricoarytenoid Muscle Is Spared from MuRF1-Mediated Muscle Atrophy in Mice with Acute Lung Injury

Background

Skeletal muscle wasting in acute lung injury (ALI) patients increases the morbidity and mortality associated with this critical illness. The contribution of laryngeal muscle wasting to these outcomes is unknown, though voice impairments and aspiration are common in intensive care unit (ICU) survivors. We evaluated the intrinsic laryngeal abductor (PCA, posterior cricoarytenoid), adductor (CT, cricothyroid) and limb (EDL, extensor digitorum longus) muscles in a mouse model of ALI.

Methods

Escherichia coli lipopolysaccharides were instilled into the lungs of adult male C57Bl6J mice (ALI mice). Limb and intrinsic laryngeal muscles were analyzed for fiber size, type, protein expression and myosin heavy chain (MyHC) composition by SDS-PAGE and mass spectroscopy.

Results

Marked muscle atrophy occurred in the CT and EDL muscles, while the PCA was spared. The E3 ubiquitin ligase muscle ring finger-1 protein (MuRF1), a known mediator of limb muscle atrophy in this model, was upregulated in the CT and EDL, but not in the PCA. Genetic inhibition of MuRF1 protected the CT and EDL from ALI-induced muscle atrophy. MyHC-Extraocular (MyHC-EO) comprised 27% of the total MyHC in the PCA, distributed as hybrid fibers throughout 72% of PCA muscle fibers.

Conclusion

The vocal cord abductor (PCA) contains a large proportion of fibers expressing MyHC-EO and is spared from muscle atrophy in ALI mice. The lack of MuRF1 expression in the PCA suggests a previously unrecognized mechanism whereby this muscle is spared from atrophy. Atrophy of the vocal cord adductor (CT) may contribute to the impaired voice and increased aspiration observed in ICU survivors. Further evaluation of the sparing of muscles involved in systemic wasting diseases may lead to potential therapeutic targets for these illnesses.     

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.     

The Ubiquitin Ligase Nedd4-1 Participates in Denervation-Induced Skeletal Muscle Atrophy in Mice

Skeletal muscle atrophy is a consequence of muscle inactivity resulting from denervation, unloading and immobility. It accompanies many chronic disease states and also occurs as a pathophysiologic consequence of normal aging. In all these conditions, ubiquitin-dependent proteolysis is a key regulator of the loss of muscle mass, and ubiquitin ligases confer specificity to this process by interacting with, and linking ubiquitin moieties to target substrates through protein∶protein interaction domains. Our previous work suggested that the ubiquitin-protein ligase Nedd4-1 is a potential mediator of skeletal muscle atrophy associated with inactivity (denervation, unloading and immobility). Here we generated a novel tool, the Nedd4-1 skeletal muscle-specific knockout mouse (myo^Cre;Nedd4-1^flox/flox) and subjected it to a well validated model of denervation induced skeletal muscle atrophy. The absence of Nedd4-1 resulted in increased weights and cross-sectional area of type II fast twitch fibres of denervated gastrocnemius muscle compared with wild type littermates controls, at seven and fourteen days following tibial nerve transection. These effects are not mediated by the Nedd4-1 substrates MTMR4, FGFR1 and Notch-1. These results demonstrate that Nedd4-1 plays an important role in mediating denervation-induced skeletal muscle atrophy in vivo.     

Neurite Outgrowth-Promoting Factors in Extracts of Denervated Chick Skeletal Muscle

1. An extract of denervated skeletal muscle contained activity for promotion of neurite outgrowth from telencephalic neurons, as well as that from neurons in the spinal cord. A factor responsible for the activity was characterized in cultures of dissociated neurons.2. The factor acted on neurons only when they were attached to the surface of culture dishes. Since treatments with proteases and lectins reduced the outgrowth-promoting activity, the factor was thought to be a glycoprotein.3. Among the monoclonal antibodies raised against the partially purified extract, five antibodies were found to inhibit the activity for spinal and telencephalic neurons. The most potent antibody, 4D2a, recognized mainly a 63-kD protein and other minor proteins in the extract. Although the 63-kD protein was confirmed to be chick serum albumin by analysis of amino acid sequence, the purified albumin exhibited no activity.4. From these observations, the factor was found to be a glycoprotein recognized by the neutralizing antibody as one of the minor components of the extract. This factor exhibits its activity in a substrate-bound form but not in a diffusible one.     

肌萎缩时的蛋白质降解通路

肌萎缩主要是因为蛋白质降解增强,主要涉及到泛素-蛋白酶体系统、依赖于钙离子的蛋白酶系统、溶酶体系统。在肌萎缩时这些蛋白质降解系统可能是平行作用,也可能按多步骤形式进行。弄清其机制无疑对开发抗肌萎缩药物是有帮助的。     

Nerve Stump Length and Membrane Changes in Denervated Skeletal Muscle

THE effect of nerve stump length on the rate of failure of neuromuscular transmission and on the degeneration of the neuromuscular apparatus has been studied in some detail^1–4 and there is general agreement that neuromuscular transmission fails and the nerve terminals degenerate more rapidly if the nerve is cut close to the muscle than if it is cut far away.     

Dietary Enrichment with Fish Oil Prevents High Fat-Induced Metabolic Dysfunction in Skeletal Muscle in Mice

High saturated fat (HF-S) diets increase intramyocellular lipid, an effect ameliorated by omega-3 fatty acids in vitro and in vivo, though little is known about sex- and muscle fiber type-specific effects. We compared effects of standard chow, HF-S, and 7.5% HF-S replaced with fish oil (HF-FO) diets on the metabolic profile and lipid metabolism gene and protein content in red (soleus) and white (extensor digitorum longus) muscles of male and female C57BL/6 mice (n = 9-12/group). Weight gain was similar in HF-S- and HF-FO-fed groups. HF-S feeding increased mesenteric fat mass and lipid marker, Oil Red O, in red and mixed muscle; HF-FO increased interscapular brown fat mass. Compared to chow, HF-S and HF-FO increased expression of genes regulating triacylglycerol synthesis and fatty acid transport, HF-S suppressed genes and proteins regulating fatty acid oxidation, whereas HF-FO increased oxidative genes, proteins and enzymes and lipolytic gene content, whilst suppressing lipogenic genes. In comparison to HF-S, HF-FO further increased fat transporters, markers of fatty acid oxidation and mitochondrial content, and reduced lipogenic genes. No diet-by-sex interactions were observed. Neither diet influenced fiber type composition. However, some interactions between muscle type and diet were observed. HF-S induced changes in triacylglycerol synthesis and lipogenic genes in red, but not white, muscle, and mitochondrial biogenesis and oxidative genes were suppressed by HF-S and increased by HF-FO in red muscle only. In conclusion, HF-S feeding promotes lipid storage in red muscle, an effect abrogated by the fish oil, which increases mediators of lipolysis, oxidation and thermogenesis while inhibiting lipogenic genes. Greater storage and synthesis, and lower oxidative genes in red, but not white, muscle likely contribute to lipid accretion encountered in red muscle. Despite several gender-dimorphic genes, both sexes exhibited a similar HF-S-induced metabolic and gene expression profile; likewise fish oil was similarly protective in both sexes.     

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.     

Preservation of Metabolic Flexibility in Skeletal Muscle by a Combined Use of n-3 PUFA and Rosiglitazone in Dietary Obese Mice

Insulin resistance, the key defect in type 2 diabetes (T2D), is associated with a low capacity to adapt fuel oxidation to fuel availability, i.e., metabolic inflexibility. This, in turn, contributes to a further damage of insulin signaling. Effectiveness of T2D treatment depends in large part on the improvement of insulin sensitivity and metabolic adaptability of the muscle, the main site of whole-body glucose utilization. We have shown previously in mice fed an obesogenic high-fat diet that a combined use of n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) and thiazolidinediones (TZDs), anti-diabetic drugs, preserved metabolic health and synergistically improved muscle insulin sensitivity. We investigated here whether n-3 LC-PUFA could elicit additive beneficial effects on metabolic flexibility when combined with a TZD drug rosiglitazone. Adult male C57BL/6N mice were fed an obesogenic corn oil-based high-fat diet (cHF) for 8 weeks, or randomly assigned to various interventions: cHF with n-3 LC-PUFA concentrate replacing 15% of dietary lipids (cHF+F), cHF with 10 mg rosiglitazone/kg diet (cHF+ROSI), cHF+F+ROSI, or chow-fed. Indirect calorimetry demonstrated superior preservation of metabolic flexibility to carbohydrates in response to the combined intervention. Metabolomic and gene expression analyses in the muscle suggested distinct and complementary effects of the interventions, with n-3 LC-PUFA supporting complete oxidation of fatty acids in mitochondria and the combination with n-3 LC-PUFA and rosiglitazone augmenting insulin sensitivity by the modulation of branched-chain amino acid metabolism. These beneficial metabolic effects were associated with the activation of the switch between glycolytic and oxidative muscle fibers, especially in the cHF+F+ROSI mice. Our results further support the idea that the combined use of n-3 LC-PUFA and TZDs could improve the efficacy of the therapy of obese and diabetic patients.     

Prevention of Milk Fever by Dietary Means

A concentrate was fortified with mineral salts by adding CaCl2, Al2(SO4)3 and MgSO4 to standard concentrate A and pelleting the product When used as feed supplement from 3 weeks pre partum to 1 week post partum this concentrate mixture produced the intended negative alkali alkalinity in the total feed ration in 20 out of 25 cows. Of these 17 remained healthy in spite of a history of milk fever for the majority of them. Two cows got milk fever and 1 showed weak symptoms.     

Lecithin Prevents Cortical Cytoskeleton Reorganization in Rat Soleus Muscle Fibers under Short-Term Gravitational Disuse

The aim of this study was to prevent the cortical cytoskeleton reorganization of rat soleus muscle fibers under short-term gravitational disuse. Once a day, we injected the right soleus muscle with 0.5 ml lecithin at a concentration of 200 mg/ml and the left soleus muscle with a diluted solution in an equal volume for 3 days prior to the experiment. To simulate microgravity conditions in rats, an anti-orthostatic suspension was used according to the Ilyin-Novikov method modified by Morey-Holton et al. for 6 hours. The following groups of soleus muscle tissues were examined: «C», «C+L», «HS», and «HS+L». The transversal stiffness of rat soleus muscle fibers after 6 hours of suspension did not differ from that of the control group for the corresponding legs; there were no differences between the groups without lecithin «C» and «HS» or between the groups with lecithin «C+L» and «HS+L». However, lecithin treatment for three days resulted in an increase in cell stiffness; in the «C+L» group, cell stiffness was significantly higher by 22.7% (p < 0.05) compared with that of group «C». The mRNA content of genes encoding beta- and gamma-actin and beta-tubulin did not significantly differ before and after suspension in the corresponding groups. However, there was a significant increase in the mRNA content of these genes after lecithin treatment: the beta-actin and gamma-actin mRNA content in group «C+L» increased by 200% compared with that of group «C», and beta-tubulin increased by 100% (as well as the mRNA content of tubulin-binding proteins Ckap5, Tcp1, Cct5 and Cct7). In addition, desmin mRNA content remained unchanged in all of the experimental groups. As a result of the lecithin injections, there was a redistribution of the mRNA content of genes encoding actin monomer- and filament-binding proteins in the direction of increasing actin polymerization and filament stability; the mRNA content of Arpc3 and Lcp1 increased by 3- and 5-fold, respectively, but the levels of Tmod1 and Svil decreased by 2- and 5-fold, respectively. However, gravitational disuse did not result in changes in the mRNA content of Arpc3, Tmod1, Svil or Lcp1. Anti-orthostatic suspension for 6 hours resulted in a decrease in the mRNA content of alpha-actinin-4 (Actn4) and alpha-actinin-1 (Actn1) in group «HS» compared with that of group «C» by 25% and 30%, respectively, as well as a decrease and increase in the ACTN4 protein content in the membrane and cytoplasmic fractions, respectively. Lecithin injection resulted in an increase in the Actn1 and Actn4 mRNA content in group «C+L» by 1.5-fold and more than 2-fold, respectively, compared with the levels in group «C». Moreover, in group «HS+L», the mRNA content did not change in these genes compared with the levels in group «C+L», and the ACTN4 protein content in the membrane and cytoplasmic fractions also remained unchanged. Thus, lecithin prevented the reduction of Actn1 and Actn4 mRNA and the migration of ACTN4 from the cortical cytoskeleton to the cytoplasm.     

Aging Is Accompanied by a Blunted Muscle Protein Synthetic Response to Protein Ingestion

Purpose

Progressive loss of skeletal muscle mass with aging (sarcopenia) forms a global health concern. It has been suggested that an impaired capacity to increase muscle protein synthesis rates in response to protein intake is a key contributor to sarcopenia. We assessed whether differences in post-absorptive and/or post-prandial muscle protein synthesis rates exist between large cohorts of healthy young and older men.

Procedures

We performed a cross-sectional, retrospective study comparing in vivo post-absorptive muscle protein synthesis rates determined with stable isotope methodologies between 34 healthy young (22±1 y) and 72 older (75±1 y) men, and post-prandial muscle protein synthesis rates between 35 healthy young (22±1 y) and 40 older (74±1 y) men.

Findings

Post-absorptive muscle protein synthesis rates did not differ significantly between the young and older group. Post-prandial muscle protein synthesis rates were 16% lower in the older subjects when compared with the young. Muscle protein synthesis rates were >3 fold more responsive to dietary protein ingestion in the young. Irrespective of age, there was a strong negative correlation between post-absorptive muscle protein synthesis rates and the increase in muscle protein synthesis rate following protein ingestion.

Conclusions

Aging is associated with the development of muscle anabolic inflexibility which represents a key physiological mechanism underpinning sarcopenia.