Leucine Supplementation Prevents the Development of Skeletal Muscle Dysfunction in a Rat Model of HFpEF.

Heart failure with preserved ejection fraction (HFpEF) is associated with exercise intolerance due to alterations in the skeletal muscle (SKM). Leucine supplementation is known to alter the anabolic/catabolic balance and to improve mitochondrial function. Thus, we investigated the effect of leucine supplementation in both a primary and a secondary prevention approach on SKM function and factors modulating muscle function in an established HFpEF rat model. Female ZSF1 obese rats were randomized to an untreated, a primary prevention, and a secondary prevention group. For primary prevention, leucine supplementation was started before the onset of HFpEF (8 weeks of age) and for secondary prevention, leucine supplementation was started after the onset of HFpEF (20 weeks of age). SKM function was assessed at an age of 32 weeks, and SKM tissue was collected for the assessment of mitochondrial function and histological and molecular analyses. Leucine supplementation prevented the development of SKM dysfunction whereas it could not reverse it. In the primary prevention group, mitochondrial function improved and higher expressions of mitofilin, Mfn-2, Fis1, and miCK were evident in SKM. The expression of UCP3 was reduced whereas the mitochondrial content and markers for catabolism (MuRF1, MAFBx), muscle cross-sectional area, and SKM mass did not change. Our data show that leucine supplementation prevented the development of skeletal muscle dysfunction in a rat model of HFpEF, which may be mediated by improving mitochondrial function through modulating energy transfer.

Leucine Supplementation Improves Diastolic Function in HFpEF by HDAC4 Inhibition.

Heart failure with preserved ejection fraction (HFpEF) is a complex syndrome associated with a high morbidity and mortality rate. Leucine supplementation has been demonstrated to attenuate cardiac dysfunction in animal models of cachexia and heart failure with reduced ejection fraction (HFrEF). So far, no data exist on leucine supplementation on cardiac function in HFpEF. Thus, the current study aimed to investigate the effect of leucine supplementation on myocardial function and key signaling pathways in an established HFpEF rat model. Female ZSF1 rats were randomized into three groups: Control (untreated lean rats), HFpEF (untreated obese rats), and HFpEF_Leu (obese rats receiving standard chow enriched with 3% leucine). Leucine supplementation started at 20 weeks of age after an established HFpEF was confirmed in obese rats. In all animals, cardiac function was assessed by echocardiography at baseline and throughout the experiment. At the age of 32 weeks, hemodynamics were measured invasively, and myocardial tissue was collected for assessment of mitochondrial function and for histological and molecular analyses. Leucine had already improved diastolic function after 4 weeks of treatment. This was accompanied by improved hemodynamics and reduced stiffness, as well as by reduced left ventricular fibrosis and hypertrophy. Cardiac mitochondrial respiratory function was improved by leucine without alteration of the cardiac mitochondrial content. Lastly, leucine supplementation suppressed the expression and nuclear localization of HDAC4 and was associated with Protein kinase A activation. Our data show that leucine supplementation improves diastolic function and decreases remodeling processes in a rat model of HFpEF. Beneficial effects were associated with HDAC4/TGF-ß1/Collagenase downregulation and indicate a potential use in the treatment of HFpEF.

Small-Molecule Inhibition of MuRF1 Prevents Early Disuse-Induced Diaphragmatic Dysfunction and Atrophy.

In clinical conditions such as diaphragm paralysis or mechanical ventilation, disuse-induced diaphragmatic dysfunction (DIDD) is a condition that poses a threat to life. MuRF1 is a key E3-ligase involved in regulating skeletal muscle mass, function, and metabolism, which contributes to the onset of DIDD. We investigated if the small-molecule mediated inhibition of MuRF1 activity (MyoMed-205) protects against early DIDD after 12 h of unilateral diaphragm denervation. Wistar rats were used in this study to determine the compound's acute toxicity and optimal dosage. For potential DIDD treatment efficacy, diaphragm contractile function and fiber cross-sectional area (CSA) were evaluated. Western blotting investigated potential mechanisms underlying MyoMed-205's effects in early DIDD. Our results indicate 50 mg/kg bw MyoMed-205 as a suitable dosage to prevent early diaphragmatic contractile dysfunction and atrophy following 12 h of denervation without detectable signs of acute toxicity. Mechanistically, treatment did not affect disuse-induced oxidative stress (4-HNE) increase, whereas phosphorylation of (ser632) HDAC4 was normalized. MyoMed-205 also mitigated FoxO1 activation, inhibited MuRF2, and increased phospho (ser473) Akt protein levels. These findings may suggest that MuRF1 activity significantly contributes to early DIDD pathophysiology. Novel strategies targeting MuRF1 (e.g., MyoMed-205) have potential therapeutic applications for treating early DIDD.

miR-29c Increases Protein Synthesis in Skeletal Muscle Independently of AKT/mTOR.

microRNAs negatively regulate gene expression by blocking translation or increasing mRNA degradation. In skeletal muscle, these molecules play important roles in adaptive responses, and ongoing investigations are necessary to understand the fine-tune regulation of skeletal muscle mass. Herein we showed that skeletal muscle overexpression of miR-29c increased fiber size and force at 7 and 30 days after electrotransfer. At both time points, AKT/mTOR pathway components were downregulated, and, surprisingly, overall protein synthesis was strongly elevated at day 7, which normalized by day 30 after pCMVmiR-29c electrotransfer. These results indicate that miR-29c expression induces skeletal muscle hypertrophy and gain of function, which involves increased overall protein synthesis in spite of the deactivation of the AKT/mTOR pathway.

Firearms-related skeletal muscle trauma: pathophysiology and novel approaches for regeneration.

One major cause of traumatic injury is firearm-related wounds (i.e., ballistic trauma), common in both civilian and military populations, which is increasing in prevalence and has serious long-term health and socioeconomic consequences worldwide. Common primary injuries of ballistic trauma include soft-tissue damage and loss, haemorrhage, bone fracture, and pain. The majority of injuries are of musculoskeletal origin and located in the extremities, such that skeletal muscle offers a major therapeutic target to aid recovery and return to normal daily activities. However, the underlying pathophysiology of skeletal muscle ballistic trauma remains poorly understood, with limited evidence-based treatment options. As such, this review will address the topic of firearm-related skeletal muscle injury and regeneration. We first introduce trauma ballistics and the immediate injury of skeletal muscle, followed by detailed coverage of the underlying biological mechanisms involved in regulating skeletal muscle dysfunction following injury, with a specific focus on the processes of muscle regeneration, muscle wasting and vascular impairments. Finally, we evaluate novel approaches for minimising muscle damage and enhancing muscle regeneration after ballistic trauma, which may have important relevance for primary care in victims of violence.

Regulation of Glucose Metabolism by MuRF1 and Treatment of Myopathy in Diabetic Mice with Small Molecules Targeting MuRF1.

The muscle-specific ubiquitin ligase MuRF1 regulates muscle catabolism during chronic wasting states, although its roles in general metabolism are less-studied. Here, we metabolically profiled MuRF1-deficient knockout mice. We also included knockout mice for MuRF2 as its closely related gene homolog. MuRF1 and MuRF2-KO (knockout) mice have elevated serum glucose, elevated triglycerides, and reduced glucose tolerance. In addition, MuRF2-KO mice have a reduced tolerance to a fat-rich diet. Western blot and enzymatic studies on MuRF1-KO skeletal muscle showed perturbed FoxO-Akt signaling, elevated Akt-Ser-473 activation, and downregulated oxidative mitochondrial metabolism, indicating potential mechanisms for MuRF1,2-dependent glucose and fat metabolism regulation. Consistent with this, the adenoviral re-expression of MuRF1 in KO mice normalized Akt-Ser-473, serum glucose, and triglycerides. Finally, we tested the MuRF1/2 inhibitors MyoMed-205 and MyoMed-946 in a mouse model for type 2 diabetes mellitus (T2DM). After 28 days of treatment, T2DM mice developed progressive muscle weakness detected by wire hang tests, but this was attenuated by the MyoMed-205 treatment. While MyoMed-205 and MyoMed-946 had no significant effects on serum glucose, they did normalize the lymphocyte-granulocyte counts in diabetic sera as indicators of the immune response. Thus, small molecules directed to MuRF1 may be useful in attenuating skeletal muscle strength loss in T2DM conditions.

Leucine Supplementation Decreases HDAC4 Expression and Nuclear Localization in Skeletal Muscle Fiber of Rats Submitted to Hindlimb Immobilization.

In this study we surveyed a rat skeletal muscle RNA-Seq for genes that are induced by hindlimb immobilization and, in turn, become attenuated by leucine supplementation. This approach, in search of leucine-atrophy protection mediating genes, identified histone deacetylase 4 (HDAC4) as highly responsive to both hindlimb immobilization and leucine supplementation. We then examined the impact of leucine on HDAC4 expression, tissue localization, and target genes. A total of 76 male Wistar rats (~280 g) were submitted to hindlimb immobilization and/or leucine supplementation for 3, 7 and 12 days. These animals were euthanized, and soleus muscle was removed for further analysis. RNA-Seq analysis of hindlimb immobilized rats indicated a sharp induction (log2 = 3.4) of HDAC4 expression which was attenuated by leucine supplementation (~50%). Real-time PCR and protein expression analysis by Western blot confirmed increased HDAC4 mRNA after 7 days of hindlimb immobilization and mitigation of induction by leucine supplementation. Regarding the HDAC4 localization, the proportion of positive nuclei was higher in the immobilized group and decreased after leucine supplementation. Also, we found a marked decrease of myogenin and MAFbx-atrogin-1 mRNA levels upon leucine supplementation, while CAMKII and DACH2 mRNA levels were increased by leucine supplementation. Our data suggest that HDAC4 inhibition might be involved in the anti-atrophic effects of leucine.

Skeletal Muscle Anti-Atrophic Effects of Leucine Involve Myostatin Inhibition.

Lack of mechanical load leads to skeletal muscle atrophy, and one major underlying mechanism involves the myostatin pathway that negatively regulates protein synthesis and also activates Atrogin-1/MAFbx and MuRF1 genes. In hindlimb immobilization, leucine was observed to attenuate the upregulation of the referred atrogenes, thereby shortening the impact on fiber cross-sectional area, nonetheless, the possible connection with myostatin is still elusive. This study sought to verify the impact of leucine supplementation on myostatin expression. Male Wistar rats were supplemented with leucine and hindlimb immobilized for 3 and 7 days, after which soleus muscles were removed for morphometric measurements and analyzed for gene and protein expression by real-time PCR and Western blotting, respectively. Muscle wasting was prominent 7 days after immobilization, as expected, leucine feeding mitigated this effect. Atrogin-1/MAFbx gene expression was upregulated only after 3 days of immobilization, and this effect was attenuated by leucine supplementation. Atrogin-1/MAFbx protein levels were elevated after 7 days of immobilization, which leucine supplementation was not able to lessen. On the other hand, myostatin gene expression was upregulated in immobilization for 3 and 7 days, which returned to normal levels after leucine supplementation. Myostatin protein levels followed gene expression at a 3-day time point only. Follistatin gene expression was upregulated during immobilization and accentuated by leucine after 3 days of supplementation. Concerning protein expression, follistatin was not altered neither by immobilization nor in immobilized animals treated with leucine. In conclusion, leucine protects against skeletal muscle mass loss during disuse, and the underlying molecular mechanisms appear to involve myostatin inhibition and Atrogin-1 normalization independently of follistatin signaling.

Loss of Parkin Results in Altered Muscle Stem Cell Differentiation during Regeneration.

The high capacity of the skeletal muscle to regenerate is due to the presence of muscle stem cells (MuSCs, or satellite cells). The E3 ubiquitin ligase Parkin is a key regulator of mitophagy and is recruited to mitochondria during differentiation of mouse myoblast cell line. However, the function of mitophagy during regeneration has not been investigated in vivo. Here, we have utilized Parkin deficient (Parkin-/-) mice to investigate the role of Parkin in skeletal muscle regeneration. We found a persistent deficiency in skeletal muscle regeneration in Parkin-/- mice after cardiotoxin (CTX) injury with increased area of fibrosis and decreased cross-sectional area (CSA) of myofibres post-injury. There was also a significant modulation of MuSCs differentiation and mitophagic markers, with altered mitochondrial proteins during skeletal muscle regeneration in Parkin-/- mice. Our data suggest that Parkin-mediated mitophagy plays a key role in skeletal muscle regeneration and is necessary for MuSCs differentiation.

Small-Molecule Chemical Knockdown of MuRF1 in Melanoma Bearing Mice Attenuates Tumor Cachexia Associated Myopathy.

: Patients with malignant tumors frequently suffer during disease progression from a syndrome referred to as cancer cachexia (CaCax): CaCax includes skeletal muscle atrophy and weakness, loss of bodyweight, and fat tissues. Currently, there are no FDA (Food and Drug Administration) approved treatments available for CaCax. Here, we studied skeletal muscle atrophy and dysfunction in a murine CaCax model by injecting B16F10 melanoma cells into mouse thighs and followed mice during melanoma outgrowth. Skeletal muscles developed progressive weakness as detected by wire hang tests (WHTs) during days 13-23. Individual muscles analyzed at day 24 had atrophy, mitochondrial dysfunction, augmented metabolic reactive oxygen species (ROS) stress, and a catabolically activated ubiquitin proteasome system (UPS), including upregulated MuRF1. Accordingly, we tested as an experimental intervention of recently identified small molecules, Myomed-205 and -946, that inhibit MuRF1 activity and MuRF1/MuRF2 expression. Results indicate that MuRF1 inhibitor fed attenuated induction of MuRF1 in tumor stressed muscles. In addition, the compounds augmented muscle performance in WHTs and attenuated muscle weight loss. Myomed-205 and -946 also rescued citrate synthase and complex-1 activities in tumor-stressed muscles, possibly suggesting that mitochondrial-metabolic and muscle wasting effects in this CaCax model are mechanistically connected. Inhibition of MuRF1 during tumor cachexia may represent a suitable strategy to attenuate skeletal muscle atrophy and dysfunction.

miR-29c improves skeletal muscle mass and function throughout myocyte proliferation and differentiation and by repressing atrophy-related genes.

AIM: To identify microRNAs (miRs) involved in the regulation of skeletal muscle mass. For that purpose, we have initially utilized an in silico analysis, resulting in the identification of miR-29c as a positive regulator of muscle mass. METHODS: miR-29c was electrotransferred to the tibialis anterior to address its morphometric and functional properties and to determine the level of satellite cell proliferation and differentiation. qPCR was used to investigate the effect of miR-29c overexpression on trophicity-related genes. C2C12 cells were used to determine the impact of miR-29c on myogenesis and a luciferase reporter assay was used to evaluate the ability of miR-29c to bind to the MuRF1 3'UTR. RESULTS: The overexpression of miR-29c in the tibialis anterior increased muscle mass by 40%, with a corresponding increase in fibre cross-sectional area and force and a 30% increase in length. In addition, satellite cell proliferation and differentiation were increased. In C2C12 cells, miR-29c oligonucleotides caused increased levels of differentiation, as evidenced by an increase in eMHC immunostaining and the myotube fusion index. Accordingly, the mRNA levels of myogenic markers were also increased. Mechanistically, the overexpression of miR-29c inhibited the expression of the muscle atrophic factors MuRF1, Atrogin-1 and HDAC4. For the key atrogene MuRF1, we found that miR-29c can bind to its 3'UTR to mediate repression. CONCLUSIONS: The results herein suggest that miR-29c can improve skeletal muscle size and function by stimulating satellite cell proliferation and repressing atrophy-related genes. Taken together, our results indicate that miR-29c might be useful as a future therapeutic device in diseases involving decreased skeletal muscle mass.

TRB3 regulates skeletal muscle mass in food deprivation-induced atrophy.

Tribbles 3 (TRB3) is a pseudokinase that has been found in multiple tissues in response to various stress stimuli, such as nutrient deprivation and endoplasmic reticulum (ER) stress. We recently found that TRB3 has the potential to regulate skeletal muscle mass at the basal state. However, it has not yet been explored whether TRB3 regulates skeletal muscle mass under atrophic conditions. Here, we report that food deprivation for 48 h in mice significantly reduces muscle mass by ∼15% and increases TRB3 expression, which is associated with increased ER stress. Interestingly, inhibition of ER stress in C2C12 myotubes reduces food deprivation-induced expression of TRB3 and muscle-specific E3-ubiquitin ligases. In further in vivo experiments, muscle-specific TRB3 transgenic mice increase food deprivation-induced muscle atrophy compared with wild-type (WT) littermates presumably by the increased proteolysis. On the other hand, TRB3 knockout mice ameliorate food deprivation-induced atrophy compared with WT littermates by preserving a higher protein synthesis rate. These results indicate that TRB3 plays a pivotal role in skeletal muscle mass regulation under food deprivation-induced muscle atrophy and TRB3 could be a pharmaceutical target to prevent skeletal muscle atrophy.-Choi, R. H., McConahay, A., Silvestre, J. G., Moriscot, A. S., Carson, J. A., Koh, H.-J. TRB3 regulates skeletal muscle mass in food deprivation-induced atrophy.

Low-intensity resistance training with partial blood flow restriction and high-intensity resistance training induce similar changes in skeletal muscle transcriptome in elderly humans.

We aimed to investigate the mechanisms underlying muscle growth after 12 weeks of resistance training performed with blood flow restriction (RT-BFR) and high-intensity resistance training (HRT) in older individuals. Participants were allocated into the following groups: HRT, RT-BFR, or a control group. High-throughput transcriptome sequencing was performed by the Illumina HiSeq 2500 platform. HRT and RT-BFR presented similar increases in the quadriceps femoris cross-sectional area, and few genes were differently expressed between interventions. The small differences in gene expression between interventions suggest that similar mechanisms may underpin training-induced muscle growth.

Radicicol enhances the regeneration of skeletal muscle injured by crotoxin via decrease of NF-kB activation

This study evaluated cellular and molecular effects of radicicol, a heat shock protein (HSP) inducer, on the regeneration of skeletal muscle injured by crotoxin, the main toxin isolated from Crotalus durissus terrificus venom. Regenerating muscles treated with radicicol had decreased NF-kB activation. Differentiating myoblasts treated with radicicol showed reduced number of NF-kB positive nuclei and increased fusion index. The results suggest that radicicol enhances regeneration of muscle by attenuating NF-kB activation and increasing myogenic differentiation.

FoxO3a suppression and VPS34 activity are essential to anti-atrophic effects of leucine in skeletal muscle.

Our aim is to gain insight into the mechanisms underlying the anti-atrophic effects of leucine, namely, the way that this amino acid can restrain the up-regulation of MuRF1 and Mafbx/Atrogin-1 in muscle atrophy. Male rats received dietary leucine supplementation for 1-3 days, during which time their hind limbs were immobilized. Our results showed that leucine inhibited Forkhead Box O3 (FoxO3a) translocation to cell nuclei. In addition, leucine was able to reverse the expected reduction of FoXO3a ubiquitination caused by immobilization. Unexpectedly, leucine promoted these effects independently of the Class I PI3K/Akt pathway. Vacuolar protein sorting 34 (VPS34; a Class III PI3K) was strongly localized in nuclei after immobilization and leucine supplementation was able to prevent this effect. In experiments on cultured primary myotubes, dexamethasone led to the localization of VPS34 in the nucleus. In addition, the pharmacological inhibition of VPS34 blocked VPS34 nuclear localization and impaired the protective effect of leucine upon myotube trophicity. Finally, the pharmacological inhibition of VPS34 in primary myotubes prevented the protective effects of leucine upon MuRF1 and Mafbx/Atrogin-1 gene expression. Autophagy-related target genes were not responsive to leucine. Thus, we demonstrate that the anti-atrophic effect of leucine is dependent upon FoxO3a suppression and VPS34 activity.

S100A8/MYD88/NF-қB: a novel pathway involved in cardiomyocyte hypertrophy driven by thyroid hormone.

Recent studies have evidenced the involvement of inflammation-related pathways to the development of cardiac hypertrophy and other consequences on the cardiovascular system, including the calcium-binding protein S100A8. However, this has never been investigated in the thyroid hormone (TH)-prompted cardiac hypertrophy. Thus, we aimed to test whether S100A8 and related signaling molecules, myeloid differentiation factor-88 (MyD88) and nuclear factor kappa B (NF-қB), could be associated with the cardiomyocyte hypertrophy induced by TH. Our results demonstrate that the S100A8/MyD88/NF-қB signaling pathway is activated in cardiomyocytes following TH stimulation. The knockdown of S100A8 and MyD88 indicates the contribution of those molecules to cardiomyocyte hypertrophy in response to TH, as evaluated by cell surface area, leucine incorporation assay, and gene expression. Furthermore, S100A8 and MyD88 are crucial mediators of NF-қB activation, which is also involved in the hypertrophic growth of TH-treated cardiomyocytes. Supporting the in vitro data, the contribution of NF-қB for TH-induced cardiac hypertrophy is confirmed in vivo, by using transgenic mice with cardiomyocyte-specific suppression of NF-қB. These data identify a novel pathway regulated by TH that mediates cardiomyocyte hypertrophy. However, the potential role of this new pathway in short and long-term cardiac effects of TH remains to be further investigated. KEY MESSAGES: Inflammation-related signaling is activated by T3 in cardiomyocytes. S100A8 and MyD88 have a crucial role in cardiomyocyte hypertrophy by T3. S100A8 and MyD88 mediate NF-қB activation by T3. NF-қB contributes to T3-induced cardiac hypertrophy in vitro and in vivo.

Leucine supplementation improves regeneration of skeletal muscles from old rats.

The decreased regenerative capacity of old skeletal muscles involves disrupted turnover of proteins. This study investigated whether leucine supplementation in old rats could improve muscle regenerative capacity. Young and old male Wistar rats were supplemented with leucine; then, the muscles were cryolesioned and examined after 3 and 10 days. Leucine supplementation attenuated the decrease in the expression of eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) and eukaryotic translation initiation factor 4E (eIF4E) in young and old muscles on day 3 post-injury and promoted an increase in the cross-sectional area of regenerating myofibers from both young and old soleus muscles on day 10 post-injury. This supplementation decreased the levels of ubiquitinated proteins and increased the proteasome activity in young regenerating muscles, but the opposite effect was observed in old regenerating muscles. Moreover, leucine decreased the inflammation area and induced an increase in the number of proliferating satellite cells in both young and old muscles. Our results suggest that leucine supplementation improves the regeneration of skeletal muscles from old rats, through the preservation of certain biological responses upon leucine supplementation. Such responses comprise the decrease in the inflammation area, increase in the number of proliferating satellite cells and size of regenerating myofibers, combined with the modulation of components of the phosphoinositide 3-kinase/Akt-protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway and ubiquitin-proteasome system.

Muscle IGF-1-induced skeletal muscle hypertrophy evokes higher insulin sensitivity and carbohydrate use as preferential energy substrate.

We characterized the metabolic profile of transgenic mice exhibiting enhanced muscle mass driven by increased mIGF-1 expression (MLC/mIGF-1). As expected, 6-month-old MLC/mIGF-1 mice were heavier than age-matched wild type (WT) mice (37.4 ± 0.3 versus 31.8 ± 0.6 g, resp.). MLC/mIGF-1 mice had higher respiratory quotient when compared to WT (0.9 ± 0.03 versus 0.74 ± 0.02, resp.) suggesting a preference for carbohydrate as the major fuel source. MLC/mIGF-1 mice had a higher rate of glucose disposal when compared to WT (3.25 ± 0.14 versus 2.39 ± 0.03%/min, resp.). The higher disposal rate correlated to ∼ 2-fold higher GLUT4 content in the extensor digitorum longus (EDL) muscle. Analysis of mRNA content for the glycolysis-related gene PFK-1 showed ∼ 3-fold upregulation in MLC/mIGF-1 animals. We also found a 50% downregulation of PGC1α mRNA levels in MLC/mIGF-1 mouse EDL muscle, suggesting less abundant mitochondria in this tissue. We found no difference in the expression of PPARα and PPARß/δ, suggesting no modulation of key elements in oxidative metabolism. These data together suggest a shift in metabolism towards higher carbohydrate utilization, and that could explain the increased insulin sensitivity of hypertrophied skeletal muscle in MLC/mIGF-1 mice.

Embryonic stem cells improve skeletal muscle recovery after extreme atrophy in mice.

INTRODUCTION: We injected embryonic stem cells into mouse tibialis anterior muscles subjected to botulinum toxin injections as a model for reversible neurogenic atrophy. METHODS: Muscles were exposed to botulinum toxin for 4 weeks and allowed to recover for up to 6 weeks. At the onset of recovery, a single muscle injection of embryonic stem cells was administered. The myofiber cross-sectional area, single twitch force, peak tetanic force, time-to-peak force, and half-relaxation time were determined. RESULTS: Although the stem cell injection did not affect the myofiber cross-sectional area gain in recovering muscles, most functional parameters improved significantly compared with those of recovering muscles that did not receive the stem cell injection. CONCLUSIONS: Muscle function recovery was accelerated by embryonic stem cell delivery in this durable neurogenic atrophy model. We conclude that stem cells should be considered a potential therapeutic tool for recovery after extreme skeletal muscle atrophy.