Prevent Muscle Atrophy Research Articles

Resistance Training's Ability to Prevent Cancer-induced Muscle Atrophy Extends Anabolic Stimulus.

This study aimed to determine the role of mammalian target of rapamycin (mTORC1) activation and catabolic markers in resistance training's (RT) antiatrophy effect during cachexia-induced muscle loss. Myofiber atrophy was induced by injecting Walker 256 tumor cells into rats exposed or not exposed to the RT protocol of ladder climbing. The role of RT-induced anabolic stimulation was investigated in tumor-bearing rats with the mTORC1 inhibitor rapamycin, and cross-sectional areas of skeletal muscle were evaluated to identify atrophy or hypertrophy. Components of the mTORC1 and ubiquitin-proteasome pathways were assessed by real-time polymerase chain reaction or immunoblotting. Although RT prevented myofiber atrophy and impaired the strength of tumor-bearing rats, in healthy rats, it promoted activated mTORC1, as demonstrated by p70S6K's increased phosphorylation and myofiber's enlarged cross-sectional area. However, RT promoted no changes in the ratio of p70S6K to phospho-p70S6K protein expression while prevented myofiber atrophy in tumor-bearing rats. Beyond that, treatment with rapamycin did not preclude RT's preventive effect on myofiber atrophy in tumor-bearing rats. Thus, RT's ability to prevent cancer-induced myofiber atrophy seems to be independent of mTORC1's and p70S6K's activation. Indeed, RT's preventive effect on cancer-induced myofiber atrophy was associated with its capacity to attenuate elevated tumor necrosis factor α and interleukin 6 as well as to prevent oxidative damage in muscles and an elevated abundance of atrogin-1. By inducing attenuated myofiber atrophy independent of mTORC1's signaling activation, RT prevents muscle atrophy during cancer by reducing inflammation, oxidative damage, and atrogin-1 expression.

Adipose Stem Cell–Derived Exosomes Recover Impaired Matrix Metabolism of Torn Human Rotator Cuff Tendons by Maintaining Tissue Homeostasis

Background: Adipose stem cell–derived exosomes (ASC-Exos) are reported to effectively prevent muscle atrophy and degeneration of torn rat rotator cuff, but their influence on human samples and their potential mechanism are still unclear. Purpose: We aimed to investigate the effects of ASC-Exos on the metabolic activities of torn human rotator cuff tendons and explore the potential mechanism behind it. Study Design: Controlled laboratory study. Methods: Diseased supraspinatus tendons were harvested from 15 patients with a mean ± SD age of 65.8 ± 3.2 years who underwent reverse shoulder arthroplasty for chronic rotator cuff tears associated with glenohumeral pathological changes. Each tendon was dissected into 3 × 4 × 4–mm explants: the ones derived from the same tendon were placed into 12-well plates and cultured in complete culture media (control) or in complete culture media supplemented with ASC-Exos for 72 hours. Afterward, the concentrations of cytokines secreted into the culture media—including interleukin 1β (IL-1β), IL-6, IL-8, and matrix metalloproteinase 9 (MMP-9)—were measured using enzyme-linked immunosorbent assay (ELISA). Tendons were stained with hematoxylin and eosin and immunohistochemistry (type I and III collagens) for histological analyses. Moreover, the expression of anabolic genes (TIMP-1 and TIMP-3; type I and III collagen encoding) and catabolic genes (MMP-9 and MMP-13) in tendons were measured using real-time quantitative polymerase chain reaction. Phosphorylated AMPKα and Wnt/β-catenin pathways were assayed by western blotting to explore the potential mechanism of action of ASC-Exos. Results: Secretion of proinflammatory cytokines, including IL-1β, IL-6, and MMP-9, was significantly reduced in the ASC-Exos group as compared with the control group. Supraspinatus tendons in the ASC-Exos group exhibited superior histological properties, as demonstrated by higher tendon maturing scores and more type I collagen content, but there was no significant difference in type III collagen content between groups. Expression of MMP-9 and MMP-13 genes was decreased in the ASC-Exos group versus the control group. Increased expression of type I and III collagens and an elevated type I/III ratio were found in the ASC-Exos group when compared with the control group. There was no significant difference in the secretion of IL-8 and expression of TIMP-1 and TIMP-3 genes between the ASC-Exos and control groups. Western blotting revealed that ASC-Exos enhanced phosphorylated AMPKα and decreased β-catenin levels to prevent tendon degeneration. Conclusion: ASC-Exos maintained metabolic homeostasis of torn human rotator cuff tendons to improve their histological properties, which might be achieved by enhancing AMPK signaling to suppress Wnt/β-catenin activity. Clinical Relevance: ASC-Exos could be used as an effective biological tool to promote healing in torn human rotator cuff tendons.

Transgenic expression of SOD1 specifically in neurons of Sod1 deficient mice prevents defects in muscle mitochondrial function and calcium handling

Aging is accompanied by loss of muscle mass and force, known as sarcopenia. Muscle atrophy, weakness, and neuromuscular junction (NMJ) degeneration reminiscent of normal muscle aging are observed early in adulthood for mice deficient in Cu, Zn-superoxide dismutase (SOD, Sod1−/−). Muscles of Sod1−/− mice also display impaired mitochondrial ATP production and increased mitochondrial reactive oxygen species (ROS) generation implicating oxidative stress in sarcopenia. Restoration of CuZnSOD specifically in neurons of Sod1−/− mice (SynTgSod1−/−) prevents muscle atrophy and loss of force, but whether muscle mitochondrial function is preserved is not known. To establish links among CuZnSOD expression, mitochondrial function, and sarcopenia, we examined contractile properties, mitochondrial function and ROS production, intracellular calcium transients (ICT), and NMJ morphology in lumbrical muscles of 7–9 month wild type (WT), Sod1−/−, and SynTgSod1−/− mice. Compared with WT values, mitochondrial ROS production was increased 2.9-fold under basal conditions and 2.2-fold with addition of glutamate and malate in Sod1−/− muscle fibers while oxygen consumption was not significantly altered. In addition, NADH recovery was blunted following contraction and the peak of the ICT was decreased by 25%. Mitochondrial function, ROS generation and calcium handling were restored to WT values in SynTgSod1−/− mice, despite continued lack of CuZnSOD in muscle. NMJ denervation and fragmentation were also fully rescued in SynTgSod1−/− mice suggesting that muscle mitochondrial and calcium handling defects in Sod1−/− mice are secondary to neuronal oxidative stress and its effects on the NMJ rather than the lack of muscle CuZnSOD. We conclude that intact neuronal function and innervation are key to maintaining excitation-contraction coupling and muscle mitochondrial function.

Effect of Protein Supplement on Paraspinal Muscles in Spine Fusion Surgery: A Randomized, Double-Blind, Placebo-Controlled Trial.

Dysfunction and weakness due to atrophy of the paraspinal muscles is a major issue after posterior spinal fusion (PSF) surgery, resulting in pain and disability. Considering the role of protein in muscle regeneration, it seems that protein supplements after surgery may prevent muscle atrophy. To date, to our knowledge, no intervention study has investigated the effect of protein supplementation on the volume of paraspinal muscles, pain, or disability after PSF. In this randomized, double-blind, placebo-controlled clinical trial, patients were randomly assigned to a control (placebo + diet with 1.2 g/kg body weight of protein, n = 40) or a protein supplementation (36 g/day + a diet with 1.2 g/kg body weight of protein, n = 40) group, which received intervention from 48 hours before to 1 month after surgery. The cross-sectional area (CSA) of the paraspinal muscles was measured by thin-slice computed tomography, and pain and disability were assessed using the visual analog scale and Oswestry Disability Index. After 4 weeks of protein supplementation, the CSAs of multifidus and psoas muscles on both sides were significantly higher in the supplementation group than the placebo group (P <.001). Less atrophy was seen in the right erector spinae and quadratus lumborum muscles in the group receiving protein supplements than the placebo group (P < .001). In addition, protein supplementation was significantly negatively correlated with both pain (P < .001) and disability (P < .001). In conclusion, we demonstrated that 36 g/day protein supplementation significantly increased the CSA of muscles and reduced the atrophy, pain, and disability after PSF surgery. 2.

Astaxanthin Prevents Atrophy in Slow Muscle Fibers by Inhibiting Mitochondrial Reactive Oxygen Species via a Mitochondria-Mediated Apoptosis Pathway.

Astaxanthin (AX) is a carotenoid that exerts potent antioxidant activity and acts in the lipid bilayer. This study aimed to investigate the effects of AX on muscle-atrophy-mediated disturbance of mitochondria, which have a lipid bilayer. Tail suspension was used to establish a muscle-atrophied mouse model. AX diet fed to tail-suspension mice prevented loss of muscle weight, inhibited the decrease of myofiber size, and restrained the increase of hydrogen peroxide (H2O2) production in the soleus muscle. Additionally, AX improved downregulation of mitochondrial respiratory chain complexes I and III in the soleus muscle after tail suspension. Meanwhile, AX promoted mitochondrial biogenesis by upregulating the expressions of adenosine 5′-monophosphate–activated protein kinase (AMPK) α-1, peroxisome proliferator–activated receptor (PPAR)-γ, and creatine kinase in mitochondrial (Ckmt) 2 in the soleus muscle of tail-suspension mice. To confirm the AX phenotype in the soleus muscle, we examined its effects on mitochondria using Sol8 myotubes derived from the soleus muscle. We found that AX was preferentially detected in the mitochondrial fraction; it significantly suppressed mitochondrial reactive oxygen species (ROS) production in Sol8 myotubes. Moreover, AX inhibited the activation of caspase 3 via inhibiting the release of cytochrome c into the cytosol in antimycin A–treated Sol8 myotubes. These results suggested that AX protected the functional stability of mitochondria, alleviated mitochondrial oxidative stress and mitochondria-mediated apoptosis, and thus, prevented muscle atrophy.

Genistein–a supplement improving efficiency of the human body: A review

In our review, we have compiled and discussed studies relating to the effects of genistein on the efficiency of the human body. Genistein is a compound classified to isoflavones, phytoestrogens and polyphenols. It is found in many products, such as soy, potatoes or beans. (So far, many studies confirming the healthy effects of this compound to the body have been conducted. One of the aspects of genistein is its effect on exercise capacity. It reduces adipose tissue, inhibits cytodifferentiation of adipocytes and also prevents lipogenetic processes. It also lowers cholesterol and triglycerides levels. It shows myoprotective effect, preventing muscle atrophy and damage, and also affects the growth of muscle mass. It reduces the concentration of cortisol, commonly called stress hormone, which has an negative effect on fat metabolism and skeletal muscle. It also supports glucose transport and regulates carbohydrate metabolism, reducing insulin resistance. However, due to estrogen-like action, it lowers testosterone levels. Genistein is a potential nutrient for physically active people. Dans notre revue, nous avons compilé et discuté des études relatives aux effets de la génistéine sur l’efficacité du corps humain. La génistéine est un composé classé parmi les isoflavones, les phytoestrogènes et les polyphénols. On le trouve dans de nombreux produits, tels que le soja, les pommes de terre ou les haricots. Jusqu’à présent, de nombreuses études confirmant les effets sains de ce composé sur le corps ont été menées. L’un des aspects de la génistéine est son effet sur la capacité d’exercice. Il réduit le tissu adipeux, inhibe la cytodifférenciation des adipocytes et empêche également les processus lipogénétiques. Il abaisse également les niveaux de cholestérol et de triglycérides. Il montre un effet myoprotecteur, prévenant l’atrophie musculaire et les dommages, et affecte également la croissance de la masse musculaire. Il réduit la concentration de cortisol, communément appelée hormone du stress, qui a un effet négatif sur le métabolisme des graisses et les muscles squelettiques. Il prend également en charge le transport du glucose et régule le métabolisme des glucides, réduisant la résistance à l’insuline. Cependant, en raison de son action semblable à celle des œstrogènes, elle abaisse les niveaux de testostérone. La génistéine est un nutriment potentiel pour les personnes physiquement actives.

Exercise training in dialysis patients: impact on cardiovascular and skeletal muscle health.

Dialysis patients show a high rate of reduced functional capacity, morbidity and mortality. Cardiovascular disorders, muscle atrophy and malnutrition play an essential role among the aetiological factors. Sedentary lifestyle characterizes them and contributes to the aggravation of the disorders. On the contrary, exercise training is an important preventive and therapeutic tool both for cardiovascular problems and for the appearance of muscle atrophy in dialysis patients. Regular exercise causes both central (cardiac) and peripheral (muscular) adaptations, improving functional capacity. In particular, circulatory system clinical trials in haemodialysis (HD) patients documented that exercise has favourable effects on heart function, promotes balance on the cardiac autonomic nervous system and contributes to the management of arterial hypertension. In the muscular system, it prevents muscle atrophy or contributes significantly to its treatment. The main preventive mechanisms of the beneficial effect of exercise on the muscles constitute the inhibition of the apoptotic processes and protein degradation. Exercise training in HD patients leads to an increase of muscle fibers, mitochondria and capillaries, and the combination of regular exercise and dietary strategies is even more effective in preventing or treating muscle atrophy. Finally, an improvement in functional capacity and quality of life was found also in peritoneal dialysis patients following exercise training.

Effect of neuromuscular electrical stimulation on the recovery of people with COVID-19 admitted to the intensive care unit: A narrative review.

The rehabilitation of patients with COVID-19 after prolonged treatment in the intensive care unit is often complex and challenging. Patients may develop a myriad of long-term multi-organ impairments, affecting the respiratory, cardiac, neurological, digestive and musculoskeletal systems. Skeletal muscle dysfunction of respiratory and limb muscles, commonly referred to as intensive care unit acquired weakness, occurs in approximately 40% of all patients admitted to intensive care. The impact on mobility and return to activities of daily living is severe. Furthermore, many patients experience ongoing symptoms of fatigue, weakness and shortness of breath, in what is being described as “long COVID”. Neuromuscular electrical stimulation is a technique in which small electrical impulses are applied to skeletal muscle to cause contractions when voluntary muscle contraction is difficult or impossible. Neuromuscular electrical stimulation can prevent muscle atrophy, improve muscle strength and function, maintain blood flow and reduce oedema. This review examines the evidence, current guidelines, and proposed benefits of using neuromuscular electrical stimulation with patients admitted to the intensive care unit. Practical recommendations for using electrical muscle stimulation in patients with COVID-19 are provided, and suggestions for further research are proposed.Evidence suggests NMES may play a role in the weaning of patients from ventilators and can be continued in the post-acute and longer-term phases of recovery. As such, NMES may be a suitable treatment modality to implement within rehabilitation pathways for COVID-19, with consideration of the practical and safety issues highlighted within this review. LAY ABSTRACTMany patients with COVID-19 are admitted to the intensive care unit with ongoing symptoms of fatigue, weakness and shortness of breath. Neuromuscular electrical stimulation is a technique in which small electrical impulses are applied to skeletal muscle to cause contractions when voluntary muscle contraction is difficult or impossible. It can prevent muscle atrophy, improve muscle strength and function, maintain blood flow and reduce oedema. This review examines the evidence, current guidelines, and proposed benefits of using neuromuscular electrical stimulation with patients admitted to the intensive care unit. Practical recommendations for using electrical muscle stimulation with COVID-19 patients are provided and suggestions for further research are proposed. Evidence suggests NMES may play a role in the weaning of patients from ventilators and can be continued in the post-acute and longer-term phases of recovery. As such, NMES may be a suitable treatment modality to implement within rehabilitation pathways for COVID-19, with consideration of the practical and safety issues highlighted within this review.

High-dose leucine supplementation does not prevent muscle atrophy or strength loss over 7 days of immobilization in healthy young males

Unavoidable periods of disuse lead to muscle atrophy and functional decline. Preventing such declines can reduce the risk of re-injury and improve recovery of normal physiological functioning. We aimed to determine the effectiveness of high-dose leucine supplementation on muscle morphology and strength during 7 d of unilateral lower-limb immobilization, and the role of myofibrillar (MyoPS) and mitochondrial (MitoPS) protein synthesis in disuse atrophy. Sixteen healthy males (mean±SEM age: 23±1 y) underwent 7 d of unilateral lower-limb immobilization, with thrice-daily leucine (LEU; n=8) or placebo (PLA; n=8) supplementation (15 g/d). Before and after immobilization, muscle strength and compartmental tissue composition were assessed. A primed continuous infusion of l-[ring-13C6]-phenylalanine with serial muscle biopsies was used to determine postabsorptive and postprandial (20 g milk protein) MyoPS and MitoPS, fiber morphology, markers of protein turnover, and mitochondrial function between the control leg (CTL) and the immobilized leg (IMB). Leg fat-free mass was reduced in IMB (mean±SEM: -3.6%±0.5%; P=0.030) but not CTL with no difference between supplementation groups. Isometric knee extensor strength declined to a greater extent in IMB (-27.9%±4.4%) than in CTL (-14.3%±4.4%; P=0.043) with no difference between groups. In response to 20 g milk protein, postprandial MyoPS rates were significantly lower in IMB than in CTL (-22%±4%; P<0.01) in both LEU and PLA. Postabsorptive MyoPS rates did not differ between legs or groups. Postabsorptive MitoPS rates were significantly lower in IMB than in CTL (-14%±5%; P<0.01) and postprandial MitoPS rates significantly declined in response to 20 g milk protein ingestion (CTL: -10%±8%; IMB: -15%±10%; P=0.039), with no differences between legs or groups. There were no significant differences in measures of mitochondrial respiration between legs, but peroxisome proliferator-activated receptor γ coactivator 1-α and oxidative phosphorylation complex II and III were significantly lower in IMB than in CTL (P<0.05), with no differences between groups. High-dose leucine supplementation (15 g/d) does not appear to attenuate any functional declines associated with 7 d of limb immobilization in young, healthy males.This trial was registered at clinicaltrials.gov as NCT03762278.

A Diet Including Red Bell Pepper Juice and Soy Protein Suppress Physiological Markers of Muscle Atrophy in Mice.

Although muscle atrophy can be caused by disuse and lifestyle-related syndromes, it may be possible to prevent this condition through dietary intervention. We hypothesized that a diet including red bell pepper juice (RBPJ) and soy protein isolate (SPI) would prevent muscle atrophy. Accordingly, an experimental diet containing RBPJ and/or SPI was administered for 18 d to normal C57BL/6J mice. The control group was administered a casein diet. Four days before the end of the test period, denervation-induced muscle atrophy and/or sham operation were performed. Anterior tibialis muscle samples were then obtained to assess muscle degradation and perform metabolome analysis. Under the denervation condition, the 20% SPI diet did not alter the mRNA expression levels of muscle atrophy marker genes compared with the 20% casein group. Although the diet comprising RBPJ and 20% casein did not prevent muscle atrophy compared with the control group, the diet containing RBPJ and 20% SPI did. Metabolome analysis revealed that a diet including RBPJ and SPI induced a greater than 1.5-fold change in the levels of 20 muscle atrophy-related metabolites. In particular, the level of S-adenosylmethionine, which concerned with energy metabolism and lifespan, showed a strong positive correlation with the muscle atrophy marker. These findings suggest that a diet including RBPJ and soy protein suppress gene expressions related with muscle atrophy. Further research in humans is needed to confirm whether a combination of RBPJ and SPI can indeed prevent muscle atrophy.

Overexpression of Dnmt3a ameliorates diabetic muscle atrophy by modulating the Pten/Akt pathway.

What is the central question of this study? Does Dnmt3a play a crucial role in regulating diabetic muscle atrophy? What is the main finding and its importance? Muscle atrophy is one of the major long-term complications of diabetes mellitus. However, little is known about the molecular mechanism involved. In this paper, we demonstrated that Dnmt3a overexpression effectively improves the diabetic muscle health in mice and documented the underlying mechanisms. DNMT3A might become a promising target to prevent muscle atrophy in patients with diabetes. Muscle atrophy is one of the major long-term complications of diabetes mellitus, which greatly affects the mobility of patients. Epigenetic processes mediated by DNA methyltransferases (DNMTs) play crucial roles in the locomotor system, but little is known about the functions of DNMTs in diabetic muscle atrophy. Here, we investigated the function of Dnmt3a in diabetic muscle atrophy and explored the mechanisms involved. Adeno-associated virus AAV2 overexpressing Dnmt3a or its vector control was injected into the tibialis anterior muscle of streptozotocin-induced diabetic mice. Muscle mass and muscle cross-sectional area were used to evaluate muscle atrophy. In vitro, adeno-associated virus AAV2 overexpressing Dnmt3a or its vector control was transfected into C2C12 myoblasts. Horse serum was used to induce differentiation and palmitate to stimulate the C2C12 myoblasts. The expressions of myogenic regulatory factors were examined by real-time PCR and western blot analysis. Overexpression of Dnmt3a attenuated muscle atrophy in diabetic mice and promoted myotube formation of C2C12 myoblasts. Overexpression of Dnmt3a restored the expressions of myogenic regulatory factors atrogin-1, MuRF1, Pax7, Myod1 and myogenin, both in vivo and in vitro. Moreover, overexpression of Dnmt3a activated the phosphorylation of Akt by inhibiting the activation of Pten. This study demonstrates that overexpression of Dnmt3a prevents diabetic muscle atrophy by modulating the Pten/Akt pathway.

Satellite cell proliferation and myofiber cross-section area increase after electrical stimulation following sciatic nerve crush injury in rats.

BackgroundElectrical stimulation has been recommended as an effective therapy to prevent muscle atrophy after nerve injury. However, the effect of electrical stimulation on the proliferation of satellite cells in denervated muscles has not yet been fully elucidated. This study was aimed to evaluate the changes in satellite cell proliferation after electrical stimulation in nerve injury and to determine whether these changes are related to the restoration of myofiber cross-section area (CSA).MethodsSciatic nerve crush injury was performed in 48 male Sprague-Dawley rats. In half (24/48) of the rats, the gastrocnemius was electrically stimulated transcutaneously on a daily basis after injury, while the other half were not stimulated. Another group of 24 male Sprague-Dawley rats were used as sham operation controls without injury or stimulation. The rats were euthanized 2, 4, and 6 weeks later. After 5-bromo-2’-deoxyuridine (BrdU) labeling, the gastrocnemia were harvested for the detection of paired box protein 7 (Pax7), BrdU, myofiber CSA, and myonuclei number per fiber. All data were analyzed using two-way analysis of variance and Bonferroni post-hoc test.ResultsThe percentages of Pax7-positive nuclei (10.81 ± 0.56%) and BrdU-positive nuclei (34.29 ± 3.87%) in stimulated muscles were significantly higher compared to those in non-stimulated muscles (2.58 ± 0.33% and 1.30 ± 0.09%, respectively, Bonferroni t = 15.91 and 18.14, P < 0.05). The numbers of myonuclei per fiber (2.19 ± 0.24) and myofiber CSA (1906.86 ± 116.51 μm2) were also increased in the stimulated muscles (Bonferroni t = 3.57 and 2.73, P < 0.05), and both were positively correlated with the Pax7-positive satellite cell content (R2 = 0.52 and 0.60, P < 0.01). There was no significant difference in the ratio of myofiber CSA/myonuclei number per fiber among the three groups.ConclusionsOur results indicate that satellite cell proliferation is promoted by electrical stimulation after nerve injury, which may be correlated with an increase in myonuclei number and myofiber CSA.

Effects of astaxanthin on the protection of muscle health (Review).

Sarcopenia refers to the involuntary and generalized deterioration of skeletal muscle mass and strength, which may lead to falls, frailty, physical disability, loss of independence, morbidity and mortality. The majority of molecular and cellular changes involved in the degeneration of muscle tissues are mediated by oxidative stress. Therefore, astaxanthin may act as a potential adjunct therapy for sarcopenia owing to its antioxidant activity. The present review examines the effects of astaxanthin on the promotion of skeletal muscle performance and prevention of muscle atrophy and the potential mechanisms underlying these effects. The available evidence till date was retrieved from PubMed and Medline electronic databases. The present review reported the beneficial effects of astaxanthin in preventing muscle degeneration in various animal models of sarcopenia. In humans, the effects of astaxanthin in combination with other antioxidants on muscle health are mixed, wherein positive and negligible effects were reported. Mechanistic studies revealed that astaxanthin promotes muscle health by reducing oxidative stress, myoblast apoptosis and proteolytic pathways while promoting mitochondria regeneration and formation of blood vessels. Thus, astaxanthin is a potential therapeutic agent for sarcopenia but its effects in humans require further validation.

20(S)-ginsenoside Rg3 promotes myoblast differentiation and protects against myotube atrophy via regulation of the Akt/mTOR/FoxO3 pathway

We previously found that 20(S)-ginsenoside Rg3 (S-Rg3) promotes myoblast differentiation via an unknown mechanism. Here we measured levels of myosin heavy chain (MHC) and myogenin, markers of myoblast differentiation, using Western blot analysis and immunofluorescence staining. Notably, S-Rg3 treatment of C2C12 myoblasts led to increased muscle differentiation and protection from muscle atrophy in a dexamethasone (DEX)-treated C2C12 myotube-based muscle atrophy model. This effect was likely caused by S-Rg3 treatment-induced promotion of Akt/mTOR phosphorylation and inhibition of FoxO3 nuclear transcription. Additionally, S-Rg3 treatment also led to increased fruit fly climbing distances (Drosophila melanogaster) and prevented muscle atrophy in aged fruit flies. Our study provides a mechanistic framework for understanding how S-Rg3 enhances myoblast differentiation and inhibits myotube atrophy through activation of the Akt/mTOR/FoxO3 signaling pathway, as demonstrated in vitro in C2C12 cells and in vivo in fruit flies.

Functional effect of nobiletin as a food-derived allosteric modulator of mouse CRFR2β in skeletal muscle

Activation of corticotropin-releasing factor receptor 2β (CRFR2β) results in increased skeletal muscle mass and the prevention of muscle atrophy. Using a luciferase reporter assay, we screened 357 functional food factors that activate CRFR2β and, subsequently, confirmed that nobiletin (NBT) increases CRFR2β activity. Additionally, we found that NBT augments the activity of the endogenous peptide ligand urocortin 2 (Ucn2) in a concentration-dependent manner. Computational simulation of CRFR2β confirmed that transmembrane domains (TMs) 1 and 2 are important for the synergistic activity of NBT and also identified important amino acids in these domains. Finally, we demonstrated that a co-administration of Ucn2 and NBT increases the hypertrophic signal in mouse skeletal muscle. These observations demonstrate that NBT can activate CRFR2β and amplify the agonistic activity of Ucn2 and that such food-derived molecules have the potential to enhance endogenous G protein-coupled receptor ligand activities and contribute to the maintenance of skeletal muscle mass and function.

Neuromuscular Electrical Stimulation At Long Pulse Duration Is Associated With Higher Muscle Oxygen Utilization

Conventionally Neuromuscular Electrical Stimulation (NMES), delivered on the muscle belly at low frequency and short pulse duration, is used in rehabilitation protocols to prevent muscle atrophy. However, conventional NMES causes rapid on-set of fatigue and possibly activates muscle fibers in a synchronous, spatially fixed order (i.e. reverse of the size principle). To overcome these limitations, in the past years, a novel approach that utilizes both high frequency (100Hz) and long pulse duration (1000μs) (Wide Pulse High Frequency Stimulation, WPHF) was proposed. During WPHF NMES muscle contraction is achieved via afferent nerve fibers, allowing the reflexive discharge of motor units, with the contribution of spinal circuitries, and mimic the recruitment order of a voluntary effort (i.e. first type I fibers). PURPOSE: Evaluating muscle oxygen consumption via Near-Infrared Spectroscopy (NIRS) technique during two NMES bouts at fixed frequency (100Hz), applying Short (200μs) and Long (1000μs) Pulse duration, while concurrently matching the external force output. METHODS: Two 5 minutes intermittent (1 sec on - 3 sec off) NMES bouts at Short and Long pulse duration were delivered on the right quadricep muscle of 14 healthy subjects while sitting on an isometric chair. NIRS recorded oxygen extraction (i.e. Deoxygenated Hemoglobin - Myoglobin) from the vastus lateralis of the stimulated limb. Equal muscle force between protocols was achieved by carefully selecting the individual stimulation intensity. RESULTS: We selected 10 consecutive contractions for Short and Long Pulse NMES in order to have equal muscle output and evaluate the corresponding oxygen consumption. In the selected contractions, Peak Force was 15.3 ± 5.7 and 15.4 ± 5.6 % (p: ns) of the Maximal Voluntary Contraction while Force Time Integral was 1522.6 ± 593.4 and 1539.4 ± 597.3 Nm∗s (p: ns) for Short and Long Pulse respectively. At the same force output, Long Pulse NMES exhibited higher oxygen consumption, in percentage of the physiological calibration, than Short Pulse NMES (36.71 ± 11.82 % vs 28.79 ± 17.07 %, p:0.041). CONCLUSION: Applying Long Pulse NMES can elicit higher muscle oxygen utilization compared to Short Pulse duration, suggesting preferential recruitment of oxidative, type I muscle fibers, likewise to a voluntary contraction.

299-OR: Loss of Insulin and IGF1 Receptors in Muscle Impairs Complex-I Dependent Mitochondrial Bioenergetics and Supercomplex Formation via Foxo Transcription Factors

Diabetes is associated with decreased muscle strength and mitochondrial metabolism which can lead to disability. Recently, we reported that insulin receptor (IR) and IGF-1 receptor (IGF1R) prevent muscle atrophy via suppression of FoxOs, but how FoxOs interact with mitochondrial function remains unclear. To test whether IR/IGF1R suppression of FoxOs coordinates muscle strength with mitochondrial function, we measured mitochondrial bioenergetics and RNA-Seq in mice lacking IR, IGF1R, and/or FoxO1/3/4 in muscle. Perinatal muscle-specific deletion of IR (MIRKO) showed a mild 20% decrease in pyruvate-driven respiratory capacity in soleus permeabilized fibers, whereas succinate/Rotenone respiration was not different. Deletion of both IR/IGF1R (MIGIRKO) showed a marked 60% decrease in pyruvate respiration and ATP production in soleus fibers. These mitochondrial abnormalities were reversed in muscle-specific IR/IGF1R and FoxO1/3/4 quintuple knockout (QKO), suggesting FoxO activation mediates the mitochondrial decline. FoxOs are known inducers of autophagy, but mitophagy quantification using MitoTIMER or mitoKeima biosensors was not altered in Day 21 Tamoxifen-inducible IR/IGF1R KO in adult muscle (IND-IGIRKO) or by acute in-vitro deletion of IR/IGF1R primary myotubes. Complex-I activity was decreased 50-70% in MIGIRKO and Day 21 IND-IGIRKO muscle, but other complex activities were unchanged. Furthermore, 14 complex-I subunits were decreased in MIGIRKO by RNA-Seq, and 13 of these were reversed in QKO. IND-IGIRKO also showed a decrease in pyruvate respiration, ATP production, complex-I activity, and complex-I/supercomplex content by blue-native gels 21 days after tamoxifen. Thus, loss of IR or both IR/IGF1R in muscle decreases complex-I driven mitochondrial respiration and supercomplex assemblies, at least in part by FoxO-mediated downregulation of mitochondrial genes. Disclosure G. Bhardwaj: None. C.M. Penniman: None. P.A. Suarez Beltran: None. J. Jena: None. C.M. Foster: None. K. Poro: None. T. Junck: None. A. Hinton: None. R. Souvenir: None. J. Fuqua: None. P.E. Morales: None. R. Bravo-Sagua: None. V.A. Lira: None. E.D. Abel: None. B.T. ONeill: None.

1722-P: Loss of Foxos in Muscle Maintains Strength and Mitochondrial Function during Aging, but Does Not Alter Glucose or Insulin Tolerance

Muscle mitochondrial decline plays an important role in aging-related muscle weakness and is associated with insulin resistance. FoxO transcription factors are targets in insulin action and are determinants of longevity across species. However, the role of FoxOs in age-related strength and muscle mitochondrial decline is unknown. We measured strength, glucose/insulin tolerance, and muscle mitochondrial bioenergetics in 4.5- month (Young) and 24-month-old (Aged) muscle specific FoxO1/3/4 triple KO (TKO) and littermate control mice. Lean mass and muscle weights were increased 22-48% in young and aged TKO mice compared to age-matched controls. Ad libitum glucose levels were ∼12 mg/dL higher in aged groups compared to young, without differences between TKO and controls. Similarly, glucose and insulin tolerance were unchanged between TKO and control mice in both young and aged groups. While treadmill tests were not different between TKO and controls at either age, the aged control mice show a 20% decrease in max force of ankle dorsi-flexor muscles compared to young controls which was prevented in aged TKOs. Reductions in muscle mitochondrial respiration using glutamate/malate/succinate substrates were also prevented in aged TKO mice compared to aged controls. Mitochondrial Complex I activity was higher in both young and aged TKO compared to their respective controls. We did not see any differences in activity of citrate synthase, and protein expression of subunits from complex I and III, but not other complexes, showed an age-related decline in both TKO and control muscle. These data indicate that loss of FoxOs in muscle prevents age-related mitochondrial dysfunction and muscle weakness, without altering glucose or insulin tolerance, and may be a therapeutic target to prevent muscle atrophy. Disclosure C.M. Penniman: None. G. Bhardwaj: None. T. Junck: None. C. Boyer: None. J. Jena: None. J. Fuqua: None. V.A. Lira: None. B.T. ONeill: None.

EGCG down-regulates MuRF1 expression through 67-kDa laminin receptor and the receptor signaling is amplified by eriodictyol.

(-)-epigallocatechin-3-O-gallate (EGCG) is a bioactive polyphenol in green tea. Previous studies have demonstrated the beneficial effects of EGCG on muscle mass and muscle atrophy. In the current study, we investigated the mechanisms underlying effect of EGCG on muscle atrophy. It was demonstrated that EGCG suppressed muscle-specific ubiquitin ligase, muscle RING Finger 1 (MuRF1) expression through 67-kDa laminin receptor (67LR). Previous studies have shown that eriodictyol potentiates the anti-tumor activities of EGCG by amplifying 67LR signaling. Therefore, we investigated the effects of EGCG and eriodictyol on the MuRF1 expression in C2C12 myotubes. The combined treatment of EGCG and eriodictyol significantly suppressed MuRF1 expression in dexamethasone-treated C2C12 myotubes. Tail suspension was maintained for 10 consecutive days using C57BL6/J mice, and during this time EGCG and eriodictyol were orally administered. In the gastrocnemius muscle, the muscle mass loss was inhibited by the combination of EGCG and eriodictyol. Therefore, EGCG may prevent muscle atrophy by inducing 67LR signaling and eriodictyol amplifies this pathway.

The Use of Recombinant Human Growth Hormone to Protect Against Muscle Weakness in Patients Undergoing Anterior Cruciate Ligament Reconstruction: A Pilot, Randomized Placebo-Controlled Trial

Background: Anterior cruciate ligament (ACL) tears are common knee injuries. Despite undergoing extensive rehabilitation after ACL reconstruction (ACLR), many patients have persistent quadriceps muscle weakness that limits their successful return to play and are also at an increased risk of developing knee osteoarthritis (OA). Human growth hormone (HGH) has been shown to prevent muscle atrophy and weakness in various models of disuse and disease but has not been evaluated in patients undergoing ACLR. Hypothesis: Compared with placebo treatment, a 6-week perioperative treatment course of HGH would protect against muscle atrophy and weakness in patients undergoing ACLR. Study Design: Randomized controlled trial; Level of evidence, 2. Methods: A total of 19 male patients (aged 18-35 years) scheduled to undergo ACLR were randomly assigned to the placebo (n = 9) or HGH (n = 10) group. Patients began placebo or HGH treatment twice daily 1 week before surgery and continued through 5 weeks after surgery. Knee muscle strength and volume, patient-reported outcome scores, and circulating biomarkers were measured at several time points through 6 months after surgery. Mixed-effects models were used to evaluate differences between treatment groups and time points, and as this was a pilot study, significance was set at P < .10. The Cohen d was calculated to determine the effect size. Results: HGH was well-tolerated, and no differences in adverse events between the groups were observed. The HGH group had a 2.1-fold increase in circulating insulin-like growth factor 1 over the course of the treatment period (P < .05; d = 2.93). The primary outcome measure was knee extension strength, and HGH treatment increased normalized peak isokinetic knee extension torque by 29% compared with the placebo group (P = .05; d = 0.80). Matrix metalloproteinase–3 (MMP3), which was used as an indirect biomarker of cartilage degradation, was 36% lower in the HGH group (P = .05; d = –1.34). HGH did not appear to be associated with changes in muscle volume or patient-reported outcome scores. Conclusion: HGH improved quadriceps strength and reduced MMP3 levels in patients undergoing ACLR. On the basis of this pilot study, further trials to more comprehensively evaluate the ability of HGH to improve muscle function and potentially protect against OA in patients undergoing ACLR are warranted. Registration: NCT02420353 (ClinicalTrials.gov identifier)