Egg components reverse the atrophy induced by injury in skeletal muscles.

Skeletal muscle atrophy is the loss of muscle tissue caused by factors such as inactivity, malnutrition, aging, and injury. In this study, we aimed to investigate whether egg components exert inhibitory effects on muscle atrophy. An egg mix solution was orally administered for 10 consecutive days to male C57BL/6J mice injected with cardiotoxin in the tibialis anterior (TA) muscle. The administration of egg mixture significantly decreased the atrogin-1 and MuRF-1 protein levels, key factors in muscle atrophy, as observed by western blotting. Furthermore, we investigated the effects of egg components such as avidin, lecithin, biotin, 3-sn-phosphatidylcholine, and L-α-phosphatidylcholine on dexamethasone (DEX)-treated C2C12 myotubes. Lecithin, biotin, 3-sn-phosphatidylcholine, and L-α-phosphatidylcholine in egg yolk significantly recovered the diameters of C2C12 myotubes decreased upon DEX application. Avidin did not show such reversal. Biotin, 3-sn-phosphatidylcholine, and L-α-phosphatidylcholine also attenuated atrogin-1 protein expression enhanced by DEX. Our findings reveal that egg yolk components could contribute to the reversal of skeletal muscle atrophy induced by muscle injury.

Loss of MuRF1 in Duroc pigs promotes skeletal muscle hypertrophy.

Muscle mass development depends on increased protein synthesis and reduced muscle protein degradation. Muscle ring-finger protein-1 (MuRF1) plays a key role in controlling muscle atrophy. Its E3 ubiquitin ligase activity recognizes and degrades skeletal muscle proteins through the ubiquitin-proteasome system. The loss of Murf1, which encodes MuRF1, in mice leads to the accumulation of skeletal muscle proteins and alleviation of muscle atrophy. However, the function of Murf1 in agricultural animals remains unclear. Herein, we bred F1 generation Murf1+/- and F2 generation Murf1-/- Duroc pigs from F0 Murf1-/- pigs to investigate the effect of Murf1 knockout on skeletal muscle development. We found that the Murf1+/- pigs retained normal levels of muscle growth and reproduction, and their percentage of lean meat increased by 6% compared to that of the wild type (WT) pigs. Furthermore, the meat color, pH, water-holding capacity, and tenderness of the Murf1+/- pigs were similar to those of the WT pigs. The drip loss rate and intramuscular fat decreased slightly in the Murf1+/- pigs. However, the cross-sectional area of the myofibers in the longissimus dorsi increased in the adult Murf1+/- pigs. The skeletal muscle proteins MYBPC3 and actin, which are targeted by MuRF1, accumulated in the Murf1+/- and Murf1-/- pigs. Our findings show that inhibiting muscle protein degradation in MuRF1-deficient Duroc pigs increases the size of their myofibers and their percentage of lean meat without influencing their growth or pork quality. Our study demonstrates that Murf1 is a target gene for promoting skeletal muscle hypertrophy in pig breeding.

Mur ligase F as a new target for the flavonoids quercitrin, myricetin, and (-)-epicatechin.

MurC, D, E, and F are ATP-dependent ligases involved in the stepwise assembly of the tetrapeptide stem of forming peptidoglycan. As highly conserved targets found exclusively in bacterial cells, they are of significant interest for antibacterial drug discovery. In this study, we employed a computer-aided molecular design approach to identify potential inhibitors of MurF. A biochemical inhibition assay was conducted, screening twenty-four flavonoids and related compounds against MurC-F, resulting in the identification of quercitrin, myricetin, and (-)-epicatechin as MurF inhibitors with IC50 values of 143 µM, 139 µM, and 92 µM, respectively. Notably, (-)-epicatechin demonstrated mixed type inhibition with ATP and uncompetitive inhibition with D-Ala-D-Ala dipeptide and UM3DAP substrates. Furthermore, in silico analysis using Sitemap and subsequent docking analysis using Glide revealed two plausible binding sites for (-)-epicatechin. The study also investigated the crucial structural features required for activity, with a particular focus on the substitution pattern and hydroxyl group positions, which were found to be important for the activity. The study highlights the significance of computational approaches in targeting essential enzymes involved in bacterial peptidoglycan synthesis.

iPSCs ameliorate hypoxia-induced autophagy and atrophy in C2C12 myotubes via the AMPK/ULK1 pathway.

BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked lethal genetic disorder for which there is no effective treatment. Previous studies have shown that stem cell transplantation into mdx mice can promote muscle regeneration and improve muscle function, however, the specific molecular mechanisms remain unclear. DMD suffers varying degrees of hypoxic damage during disease progression. This study aimed to investigate whether induced pluripotent stem cells (iPSCs) have protective effects against hypoxia-induced skeletal muscle injury. RESULTS: In this study, we co-cultured iPSCs with C2C12 myoblasts using a Transwell nested system and placed them in a DG250 anaerobic workstation for oxygen deprivation for 24 h. We found that iPSCs reduced the levels of lactate dehydrogenase and reactive oxygen species and downregulated the mRNA and protein levels of BAX/BCL2 and LC3II/LC3I in hypoxia-induced C2C12 myoblasts. Meanwhile, iPSCs decreased the mRNA and protein levels of atrogin-1 and MuRF-1 and increased myotube width. Furthermore, iPSCs downregulated the phosphorylation of AMPKα and ULK1 in C2C12 myotubes exposed to hypoxic damage. CONCLUSIONS: Our study showed that iPSCs enhanced the resistance of C2C12 myoblasts to hypoxia and inhibited apoptosis and autophagy in the presence of oxidative stress. Further, iPSCs improved hypoxia-induced autophagy and atrophy of C2C12 myotubes through the AMPK/ULK1 pathway. This study may provide a new theoretical basis for the treatment of muscular dystrophy in stem cells.

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.

Effect of enhanced muscle tone on the expression of atrogenes and cytoskeletal proteins during postural muscle unloading.

Skeletal muscle unloading leads to the decreased electrical activity and decline of muscle tone. AIMS: Current study evaluated the effect of muscle tone preservation achieved by tetanus toxin (TeNT) treatment on signaling pathways regulating atrophic processes during unloading. MAIN METHODS: Four groups of rats were used: non-treated control (C), control rats with TeNT administration (CT), 7 days of unloading/hindlimb suspension with placebo (HS), and 7 days of unloading with TeNT administration (HST). KEY FINDINGS: Absolute and relative force of tetanic contractions was decreased by 65% in soleus muscle of HS rats when compared with C. Treatment with TeNT significantly lessened force decline in soleus muscle of HST rats when compared with HS. TeNT administration increased myosin heavy chain I beta (MyHC Iß) expression in CT rats and prevented MyHC Iß loss in HST group when compared with C rats. Desmin content was lower by 31.4% (p < 0.05) in HS group when compared with HST. Calpain-1 expression was increased in HS group when compared with C, CT and HST. There was a decrease in p-p70S6K content (41%, p < 0,05) and an increase in p-eEF2 content (77%, p < 0,05) in HS group when compared with C, while there were no significant differences in the content of these proteins between HST, CT and C groups. SIGNIFICANCE: Treatment with TeNT significantly diminished unloading-induced decline of soleus muscle mass and mechanical properties and affected the regulation of MyHC Iß expression. These effects are mediated by signaling pathways regulating protein synthesis and degradation.

Changes in AMPKα and Ubiquitin Ligases in Myocyte Reverse Remodeling after Surgical Ventricular Reconstruction in rats with ischemic cardiomyopathy.

BACKGROUND: The change in myocardial protein degradation systems after ventricular unloading has been unknown. We aimed to evaluate the anti-hypertrophic protein adenosine monophosphate-activated protein kinase (AMPK) and two major protein degradation systems (ubiquitin proteasome system and autophagy) in a model of surgical ventricular reconstruction (SVR) in rats with ischemic cardiomyopathy. METHODS AND RESULTS: Rats were randomized into the following groups: sham/sham (control group), myocardial infarction (MI)/sham (sham group) and MI/SVR (SVR group), with an interval of 4 weeks. Two (early, n = 5 for each) and 28 days (late, n = 5 for each) after SVR, ventricular size, and wall stress were assessed. Myocyte area, protein expression of AMPKα and autophagy markers, and gene expression of ubiquitin ligases (Atrogin-1 and Murf-1) were evaluated in the late phase. In the early phase, left ventricular dimensions and wall stress were smaller in the SVR group than in the sham group, whereas they were comparable in the late period. Myocyte area in the SVR group was reduced to the value in the control group, while it was larger in the sham group than in the control group. Total-AMPKα, p-AMPKα, and AMPKα phosphorylation rates were higher, and Atrogin-1 and Murf-1 were lower in the SVR group than in the sham group, while the autophagy markers were not different between the groups. p-AMPKα had strong negative correlations with myocyte area, Atrogin-1, and Murf-1. CONCLUSIONS: In myocyte reverse remodeling after SVR, AMPKα phosphorylation increased in association with reduced gene expression of ubiquitin ligases.

Taurine Supplementation Inhibits the Expression of Atrogin-1 and MURF-1, Protein Degradation Marker Genes, in Skeletal Muscle of C26-Induced Cachexia Mouse Model.

This study was designed to investigate the therapeutic effects of taurine in attenuating muscle atrophy. C26 carcinoma cells were cultured and injected into the scapulae of Balb/c mice with 1 × 106 cells. Taurine (200 µl suspension) was orally administered at the concentration of 200 mg/kg of body weight for 2 weeks. Femur muscle tissue, spleen, and gonadal fat tissue were collected and weighed. Muscle tissue was stained by H&E for histopathological analysis. The transcriptional expression of atrogin-1 and MuRF-1 gene was checked by real-time PCR. C26 cells, which induced tumor growth, caused a loss in muscle mass and gonadal fat tissue mass. Simultaneously, there was an increase in spleen and tumor tissue mass. In contrast, taurine supplementation showed a downregulatory effect on the transcriptional expression profile of muscle degradative factors atrogin-1 and MuRF-1. Our findings suggest that taurine has the potential to inhibit muscle atrophy and can be developed as a safe treatment option against muscle loss in sarcopenia patients.

Empagliflozin Preserves Skeletal Muscle Function in a HFpEF Rat Model.

Besides structural alterations in the myocardium, heart failure with preserved ejection fraction (HFpEF) is also associated with molecular and physiological alterations of the peripheral skeletal muscles (SKM) contributing to exercise intolerance often seen in HFpEF patients. Recently, the use of Sodium-Glucose-Transporter 2 inhibitors (SGLT2i) in clinical studies provided evidence for a significant reduction in the combined risk of cardiovascular death or hospitalization for HFpEF. The present study aimed to further elucidate the impact of Empagliflozin (Empa) on: (1) SKM function and metabolism and (2) mitochondrial function in an established HFpEF rat model. At the age of 24 weeks, obese ZSF1 rats were randomized either receiving standard care or Empa in the drinking water. ZSF1 lean animals served as healthy controls. After 8 weeks of treatment, echocardiography and SKM contractility were performed. Mitochondrial function was assessed in saponin skinned fibers and SKM tissue was snap frozen for molecular analyses. HFpEF was evident in the obese animals when compared to lean-increased E/é and preserved left ventricular ejection fraction. Empa treatment significantly improved E/é and resulted in improved SKM contractility with reduced intramuscular lipid content. Better mitochondrial function (mainly in complex IV) with only minor modulation of atrophy-related proteins was seen after Empa treatment. The results clearly documented a beneficial effect of Empa on SKM function in the present HFpEF model. These effects were accompanied by positive effects on mitochondrial function possibly modulating SKM function.

Removal of MuRF1 Increases Muscle Mass in Nemaline Myopathy Models, but Does Not Provide Functional Benefits.

Nemaline myopathy (NM) is characterized by skeletal muscle weakness and atrophy. No curative treatments exist for this debilitating disease. NM is caused by mutations in proteins involved in thin-filament function, turnover, and maintenance. Mutations in nebulin, encoded by NEB, are the most common cause. Skeletal muscle atrophy is tightly linked to upregulation of MuRF1, an E3 ligase, that targets proteins for proteasome degradation. Here, we report a large increase in MuRF1 protein levels in both patients with nebulin-based NM, also named NEM2, and in mouse models of the disease. We hypothesized that knocking out MuRF1 in animal models of NM with muscle atrophy would ameliorate the muscle deficits. To test this, we crossed MuRF1 KO mice with two NEM2 mouse models, one with the typical form and the other with the severe form. The crosses were viable, and muscles were studied in mice at 3 months of life. Ultrastructural examination of gastrocnemius muscle lacking MuRF1 and with severe NM revealed a small increase in vacuoles, but no significant change in the myofibrillar fractional area. MuRF1 deficiency led to increased weights of various muscle types in the NM models. However, this increase in muscle size was not associated with increased in vivo or in vitro force production. We conclude that knocking out MuRF1 in NEM2 mice increases muscle size, but does not improve muscle function.

Celastrol and Rhynchophylline in the mitigation of simulated muscle atrophy under in vitro.

Muscular atrophy (MA) is a disease of various origins, i.e., genetic or the most common, caused by mechanical injury. So far, there is no universal therapeutic model because this disease is often progressive with numerous manifested symptoms. Moreover, there is no safe and low-risk therapy dedicated to muscle atrophy. For this reason, our research focuses on finding an alternative method using natural compounds to treat MA. This study proposes implementing natural substances such as celastrol and Rhynchophylline on the cellular level, using a simulated and controlled atrophy process. Methods: Celastrol and Rhynchophylline were used as natural compounds against simulated atrophy in C2C12 cells. Skeletal muscle C2C12 cells were stimulated for the differentiation process. Atrophic conditions were obtained by the exposure to the low concertation of doxorubicin and validated by FoxO3 and MAFbx. The protective and regenerative effect of drugs on cell proliferation was determined by the MTT assay and MT-CO1, VDAC1, and prohibitin expression. Results: The obtained results revealed that both natural substances reduced atrophic symptoms. Rhynchophylline and celastrol attenuated atrophic cells in the viability studies, morphology analysis by diameter measurements, modulated prohibitin VDAC, and MT-CO1 expression. Conclusions: The obtained results revealed that celastrol and Rhynchophylline could be effectively used as a supportive treatment in atrophy-related disorders. Thus, natural drugs seem promising for muscle regeneration.

DCAF8, a novel MuRF1 interaction partner, promotes muscle atrophy.

The muscle-specific RING-finger protein MuRF1 (also known as TRIM63) constitutes a bona fide ubiquitin ligase that routes proteins like several different myosin heavy chain proteins (MyHC) to proteasomal degradation during muscle atrophy. In two unbiased screens, we identified DCAF8 as a new MuRF1-binding partner. MuRF1 physically interacts with DCAF8 and both proteins localize to overlapping structures in muscle cells. Importantly, similar to what is seen for MuRF1, DCAF8 levels increase during atrophy, and the downregulation of either protein substantially impedes muscle wasting and MyHC degradation in C2C12 myotubes, a model system for muscle differentiation and atrophy. DCAF proteins typically serve as substrate receptors for cullin 4-type (Cul4) ubiquitin ligases (CRL), and we demonstrate that DCAF8 and MuRF1 associate with the subunits of such a protein complex. Because genetic downregulation of DCAF8 and inhibition of cullin activity also impair myotube atrophy in C2C12 cells, our data imply that the DCAF8 promotes muscle wasting by targeting proteins like MyHC as an integral substrate receptor of a Cul4A-containing ring ubiquitin ligase complex (CRL4A).This article has an associated First Person interview with the first author of the paper.

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.

Role of Pannexin 1 ATP-Permeable Channels in the Regulation of Signaling Pathways during Skeletal Muscle Unloading.

Skeletal muscle unloading results in atrophy. We hypothesized that pannexin 1 ATP-permeable channel (PANX1) is involved in the response of muscle to unloading. We tested this hypothesis by blocking PANX1, which regulates efflux of ATP from the cytoplasm. Rats were divided into six groups (eight rats each): non-treated control for 1 and 3 days of the experiments (1C and 3C, respectively), 1 and 3 days of hindlimb suspension (HS) with placebo (1H and 3H, respectively), and 1 and 3 days of HS with PANX1 inhibitor probenecid (PRB; 1HP and 3HP, respectively). When compared with 3C group there was a significant increase in ATP in soleus muscle of 3H and 3HP groups (32 and 51%, respectively, p < 0.05). When compared with 3H group, 3HP group had: (1) lower mRNA expression of E3 ligases MuRF1 and MAFbx (by 50 and 38% respectively, p < 0.05) and MYOG (by 34%, p < 0.05); (2) higher phosphorylation of p70S6k and p90RSK (by 51 and 35% respectively, p < 0.05); (3) lower levels of phosphorylated eEF2 (by 157%, p < 0.05); (4) higher level of phosphorylated GSK3ß (by 189%, p < 0.05). In conclusion, PANX1 ATP-permeable channels are involved in the regulation of muscle atrophic processes by modulating expression of E3 ligases, and protein translation and elongation processes during unloading.

Ubiquitin Ligases at the Heart of Skeletal Muscle Atrophy Control.

Skeletal muscle loss is a detrimental side-effect of numerous chronic diseases that dramatically increases mortality and morbidity. The alteration of protein homeostasis is generally due to increased protein breakdown while, protein synthesis may also be down-regulated. The ubiquitin proteasome system (UPS) is a master regulator of skeletal muscle that impacts muscle contractile properties and metabolism through multiple levers like signaling pathways, contractile apparatus degradation, etc. Among the different actors of the UPS, the E3 ubiquitin ligases specifically target key proteins for either degradation or activity modulation, thus controlling both pro-anabolic or pro-catabolic factors. The atrogenes MuRF1/TRIM63 and MAFbx/Atrogin-1 encode for key E3 ligases that target contractile proteins and key actors of protein synthesis respectively. However, several other E3 ligases are involved upstream in the atrophy program, from signal transduction control to modulation of energy balance. Controlling E3 ligases activity is thus a tempting approach for preserving muscle mass. While indirect modulation of E3 ligases may prove beneficial in some situations of muscle atrophy, some drugs directly inhibiting their activity have started to appear. This review summarizes the main signaling pathways involved in muscle atrophy and the E3 ligases implicated, but also the molecules potentially usable for future therapies.

Identification and characterization of Fbxl22, a novel skeletal muscle atrophy-promoting E3 ubiquitin ligase.

Muscle-specific E3 ubiquitin ligases have been identified in muscle atrophy-inducing conditions. The purpose of the current study was to explore the functional role of F-box and leucine-rich protein 22 (Fbxl22), and a newly identified splice variant (Fbxl22-193), in skeletal muscle homeostasis and neurogenic muscle atrophy. In mouse C2C12 muscle cells, promoter fragments of the Fbxl22 gene were cloned and fused with the secreted alkaline phosphatase reporter gene to assess the transcriptional regulation of Fbxl22. The tibialis anterior muscles of male C57/BL6 mice (12-16 wk old) were electroporated with expression plasmids containing the cDNA of two Fbxl22 splice variants and tissues collected after 7, 14, and 28 days. Gastrocnemius muscles of wild-type and muscle-specific RING finger 1 knockout (MuRF1 KO) mice were electroporated with an Fbxl22 RNAi or empty plasmid and denervated 3 days posttransfection, and tissues were collected 7 days postdenervation. The full-length gene and novel splice variant are transcriptionally induced early (after 3 days) during neurogenic muscle atrophy. In vivo overexpression of Fbxl22 isoforms in mouse skeletal muscle leads to evidence of myopathy/atrophy, suggesting that both are involved in the process of neurogenic muscle atrophy. Knockdown of Fbxl22 in the muscles of MuRF1 KO mice resulted in significant additive muscle sparing 7 days after denervation. Targeting two E3 ubiquitin ligases appears to have a strong additive effect on protecting muscle mass loss with denervation, and these findings have important implications in the development of therapeutic strategies to treat muscle atrophy.

Irisin prevents dexamethasone-induced atrophy in C2C12 myotubes.

Irisin is a myokine secreted mainly from skeletal muscle that is known for having beneficial metabolic effects via enhancement of energy expenditure and insulin sensitivity. Studies show that irisin also acts as an autocrine/paracrine to promote myogenesis and muscle growth. However, the protective role of irisin against muscular wasting remains unclear. We confirmed that irisin secretion was upregulated by electrical pulse stimulation an in vitro exercise mimetic model. Next, we tested if irisin exerted an anti-atrophic effect on cultured C2C12 myotubes treated with dexamethasone (DEX), a representative inducer of muscular atrophy. Treatment of cultured myotubes with DEX reduced myotube size and increased proteasome activity, which were attenuated by irisin. Also, irisin effectively prevented dephosphorylation of forkhead box O (FoxO) 3α and upregulation of muscle-specific ubiquitin ligases in DEX-treated myotubes. The protective effect of irisin on DEX-mediated myotube atrophy was partially regulated by insulin-like growth factor-1-dependent signaling. These results suggested that irisin may prevent glucocorticoid-induced muscle atrophy by inhibiting FoxO-mediated ubiquitin-proteasome overactivity.

(-)-Epicatechin reduces muscle waste after complete spinal cord transection in a murine model: role of ubiquitin-proteasome system.

The skeletal muscle mass reduces 30-60% after spinal cord injury, this is mostly due to protein degradation through ubiquitin-proteasome system. In this work, we propose that the flavanol (-)-epicatechin, due its widespread biological effects on muscle health, can prevent muscle mass decrease after spinal cord injury. Thirty-six female Long Evans rats were randomized into 5 groups: (1) Spinal cord injury 7 days, (2) Spinal cord injury + (-)-epicatechin 7 days, (3) Spinal cord injury 30 days, (4) Spinal cord injury + (-)-epicatechin 30 days and (5) Sham (Only laminectomy). Hind limb perimeter, muscle cross section area, fiber cross section area and ubiquitin-proteasome system protein expression together with total protein ubiquitination were assessed. At 30 days Spinal cord injury group lost 49.52 ± 2.023% of muscle cross section area (-)-epicatechin treated group lost only 24.28 ± 15.45% being a significant difference. Ubiquitin-proteasome markers showed significant changes. FOXO1a increased in spinal cord injury group vs Sham (-)-epicatechin reduced this increase. In spinal cord injury group MAFbx increased significantly vs Sham but decrease in (-)-epicatechin treatment group at 30 days. At 7 and 30 days MuRF1 increased in the spinal cord injury and decreased in the (-)-epicatechin group. The global protein ubiquitination increases after spinal cord injury, epicatechin treatment induce a significant decrease in protein ubiquitination. These results suggest that (-)-epicatechin reduces the muscle waste after spinal cord injury through down regulation of the ubiquitin-proteasome system.

Exosome-Mediated miR-29 Transfer Reduces Muscle Atrophy and Kidney Fibrosis in Mice.

Our previous study showed that miR-29 attenuates muscle wasting in chronic kidney disease. Other studies found that miR-29 has anti-fibrosis activity. We hypothesized that intramuscular injection of exosome-encapsulated miR-29 would counteract unilateral ureteral obstruction (UUO)-induced muscle wasting and renal fibrosis. We used an engineered exosome vector, which contains an exosomal membrane protein gene Lamp2b that was fused with the targeting peptide RVG (rabies viral glycoprotein peptide). RVG directs exosomes to organs that express the acetylcholine receptor, such as kidney. The intervention of Exo/miR29 increased muscle cross-sectional area and decreased UUO-induced upregulation of TRIM63/MuRF1 and FBXO32/atrogin-1. Interestingly, renal fibrosis was partially depressed in the UUO mice with intramuscular injection of Exo/miR29. This was confirmed by decreased TGF-ß, alpha-smooth muscle actin, fibronectin, and collagen 1A1 in the kidney of UUO mice. When we used fluorescently labeled Exo/miR29 to trace the Exo/miR route in vivo and found that fluorescence was visible in un-injected muscle and in kidneys. We found that miR-29 directly inhibits YY1 and TGF-ß3, which provided a possible mechanism for inhibition of muscle atrophy and renal fibrosis by Exo/miR29. We conclude that Exo/miR29 ameliorates skeletal muscle atrophy and attenuates kidney fibrosis by downregulating YY1 and TGF-ß pathway proteins.

Emerging Strategies Targeting Catabolic Muscle Stress Relief.

Skeletal muscle wasting represents a common trait in many conditions, including aging, cancer, heart failure, immobilization, and critical illness. Loss of muscle mass leads to impaired functional mobility and severely impedes the quality of life. At present, exercise training remains the only proven treatment for muscle atrophy, yet many patients are too ill, frail, bedridden, or neurologically impaired to perform physical exertion. The development of novel therapeutic strategies that can be applied to an in vivo context and attenuate secondary myopathies represents an unmet medical need. This review discusses recent progress in understanding the molecular pathways involved in regulating skeletal muscle wasting with a focus on pro-catabolic factors, in particular, the ubiquitin-proteasome system and its activating muscle-specific E3 ligase RING-finger protein 1 (MuRF1). Mechanistic progress has provided the opportunity to design experimental therapeutic concepts that may affect the ubiquitin-proteasome system and prevent subsequent muscle wasting, with novel advances made in regards to nutritional supplements, nuclear factor kappa-light-chain-enhancer of activated B cells (NFB) inhibitors, myostatin antibodies, ß2 adrenergic agonists, and small-molecules interfering with MuRF1, which all emerge as a novel in vivo treatment strategies for muscle wasting.