Reduced Muscle Weight Research Articles

CLIC5 promotes myoblast differentiation and skeletal muscle regeneration via the BGN-mediated canonical Wnt/β-catenin signaling pathway

Myoblast differentiation plays a vital role in skeletal muscle regeneration. However, the protein-coding genes controlling this process remain incompletely understood. Here, we showed that chloride intracellular channel 5 (CLIC5) exerts a critical role in mediating myogenesis and skeletal muscle regeneration. Deletion of CLIC5 in skeletal muscle leads to reduced muscle weight and decreases the number and differentiation potential of satellite cells. In vitro, CLIC5 consistently inhibits myoblast proliferation while promoting myotube formation. CLIC5 promotes myogenic differentiation by activating the canonical Wnt/β-catenin signaling pathway in a biglycan (BGN)–dependent manner. CLIC5 deletion impairs muscle regeneration. Paired box gene 7 (Pax7) expression and the activity of BGN-mediated canonical Wnt/β-catenin signaling are reduced in CLIC5-deficient mice. Conversely, increasing CLIC5 levels in skeletal muscles enhances muscle regeneration capacity. In conclusion, our findings underscore CLIC5 as a pivotal regulator of myogenesis and skeletal muscle regeneration, functioning through interaction with BGN to activate the canonical Wnt/β-catenin signaling pathway.

Muscle Atrophy and mRNA-miRNA Network Analysis of Vascular Endothelial Growth Factor (VEGF) in a Mouse Model of Denervation-Induced Disuse.

Skeletal muscle atrophy is frequently caused by the disuse of muscles.It impacts quality of life, especially in aging populations and those with chronic diseases. Understanding the molecular mechanisms underlying muscle atrophy is crucial for developing effective therapies. To investigate the roles of vascular endothelial growth factor (VEGF) and various microRNAs (miRNAs) in muscle atrophy using a mouse model of denervation (DEN)-induced disuse, and to elucidate their interactions and regulatory functions through comprehensive network analysis. The right sciatic nerve of C57BL/6J mice (n=6) was excised to simulate DEN, with the left serving as a sham surgery control (Sham). Following a two-week period, wet muscle weight was measured. Total RNA was extracted from the tibialis anterior muscle for microarray analysis. Significant expression changes were analyzed via Kyoto Encyclopedia of Genes and Genomes (KEGG)pathway analysis and miRNet for miRNAs. Denervated limbs showed a significant reduction in muscle weight. Over 1,000 genes displayed increased expression, while 527 showed reductions to less than half of control levels. VEGF, along with specific miRNAs such as miR-106a-5p, miR-mir20a-5p, mir93-5p and mir17-5p, occupied central regulatory nodes within the gene network. Functional analysis revealed that these molecules are involved in key biological processes including regulation of cell migration, vasculature development, and regulation of endothelial cell proliferation. The increased miRNAswere subjected to further network analysis that revealed significant regulatory interactions with target mRNAs. VEGF and miRNAs play crucial roles in the progression of skeletal muscle atrophy, offering potential targets for therapeutic interventions aimed at reducing atrophy and enhancing muscle regeneration.

Abstract 6816: Prevention of cancer cachexia by inhibition of soluble epoxide hydrolase

Abstract Background: Cancer cachexia is a devastating syndrome characterized by progressive muscle wasting and hyperinflammation. The underlying mechanisms remain poorly understood with no treatment options available. Cachexia is driven by systemic inflammation, pro-inflammatory cytokines, and apoptotic cell death (debris). The resolution of inflammation as an active biochemical process is regulated by novel pro-resolving lipid mediators. Arachidonic acid-derived epoxyeicosatrienoic acids (EETs) are lipid mediators stimulating inflammation resolution. EETs are rapidly metabolized by the enzyme soluble epoxide hydrolase (sEH). sEH is a critical cause of inflammation and a biomarker in several chronic inflammatory diseases. sEH inhibitors promote tissue regeneration and counter-regulate pro-inflammatory cytokines. We hypothesized that pharmacological inhibition of the sEH may prevent cachexia. Methods: We investigated murine cancer cachexia models using genetically engineered pancreatic (KPC) and prostate (TRAMP C1) tumor cell lines, and “TRAMP” mice (transgenic adenocarcinoma of the mouse prostate). We performed LC-MS/MS-based profiling of bioactive lipids in plasma from KPC and colon cancer (CT26)-induced cachectic mice. Results: KPC cells injected intraperitoneally induced 19.7% and 54.1% reduction in muscle weight in the tibialis anterior and soleus, respectively, in immunocompetent C57BL/6 mice compared to non-tumor bearing (NTB) mice. LC-MS/MS revealed KPC and CT26 cachexia induced a pro-inflammatory eicosanoid-driven cytokine storm. The human sEH inhibitor EC5026 delayed the onset of cachexia, leading to sustained survival over 250 days post-injection in the KPC model (n=15 mice/group). sEH expression was increased in the gastrocnemius of KPC mice by day 22 post-tumor cell injection vs. NTB. EC5026 counter-regulated sEH expression compared to vehicle-treated. In the TRAMP model, 5/5 of mice treated with EC5026 survived 230 days post-treatment compared to no survival of vehicle-treated mice (n=5). The transplanted KPC and TRAMP showed reduced CD4+ and CD8+ T lymphocytes and B lymphocytes in the tibialis anterior and gastrocnemius vs. NTB after flow cytometry analysis. EC5026 stimulated a general increase in CD3+ T lymphocytes and counter-regulated an eicosanoid-driven cytokine storm as shown by cytokine assays. EC5026 treatment increased tibialis anterior and gastrocnemius muscle weights and body weights compared to vehicle-treated KPC and TRAMP mice. Conclusions: sEH inhibition may be a novel therapeutic approach to cachexia by targeting the immune response via stimulation of resolution of inflammation without toxicity or immunosuppression. Since sEH inhibitors have proven safe and effective in clinical trials for controlling multiple inflammatory diseases, this study provides a basis for the rapid clinical translation of sEH inhibitors to prevent/reverse cachexia in cancer patients. Citation Format: Rachel L. Bayer, Sarina Virani, Michael Gillespie, Jacqueline Capuano, Keira Smith, Katherine Quinlivan, Kimberly Vasquez, Jun Yang, Haixia Yang, Nicholas Mitsiades, Bruce D. Hammock, Dipak Panigrahy. Prevention of cancer cachexia by inhibition of soluble epoxide hydrolase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6816.

The effectiveness of acellular nerve allografts compared to autografts in animal models: A systematic review and meta-analysis.

Treatment of nerve injuries proves to be a worldwide clinical challenge. Acellular nerve allografts are suggested to be a promising alternative for bridging a nerve gap to the current gold standard, an autologous nerve graft. To systematically review the efficacy of the acellular nerve allograft, its difference from the gold standard (the nerve autograft) and to discuss its possible indications. PubMed, Embase and Web of Science were systematically searched until the 4th of January 2022. Original peer reviewed paper that presented 1) distinctive data; 2) a clear comparison between not immunologically processed acellular allografts and autologous nerve transfers; 3) was performed in laboratory animals of all species and sex. Meta analyses and subgroup analyses (for graft length and species) were conducted for muscle weight, sciatic function index, ankle angle, nerve conduction velocity, axon count diameter, tetanic contraction and amplitude using a Random effects model. Subgroup analyses were conducted on graft length and species. Fifty articles were included in this review and all were included in the meta-analyses. An acellular allograft resulted in a significantly lower muscle weight, sciatic function index, ankle angle, nerve conduction velocity, axon count and smaller diameter, tetanic contraction compared to an autologous nerve graft. No difference was found in amplitude between acellular allografts and autologous nerve transfers. Post hoc subgroup analyses of graft length showed a significant reduced muscle weight in long grafts versus small and medium length grafts. All included studies showed a large variance in methodological design. Our review shows that the included studies, investigating the use of acellular allografts, showed a large variance in methodological design and are as a consequence difficult to compare. Nevertheless, our results indicate that treating a nerve gap with an allograft results in an inferior nerve recovery compared to an autograft in seven out of eight outcomes assessed in experimental animals. In addition, based on our preliminary post hoc subgroup analyses we suggest that when an allograft is being used an allograft in short and medium (0-1cm, > 1-2cm) nerve gaps is preferred over an allograft in long (> 2cm) nerve gaps.

Bezafibrate attenuates immobilization-induced muscle atrophy in mice

Muscle atrophy due to fragility fractures or frailty worsens not only activity of daily living and healthy life expectancy, but decreases life expectancy. Although several therapeutic agents for muscle atrophy have been investigated, none is yet in clinical use. Here we report that bezafibrate, a drug used to treat hyperlipidemia, can reduce immobilization-induced muscle atrophy in mice. Specifically, we used a drug repositioning approach to screen 144 drugs already utilized clinically for their ability to inhibit serum starvation-induced elevation of Atrogin-1, a factor related to muscle atrophy, in myotubes in vitro. Two candidates were selected, and here we demonstrate that one of them, bezafibrate, significantly reduced muscle atrophy in an in vivo model of muscle atrophy induced by leg immobilization. In gastrocnemius muscle, immobilization reduced muscle weight by an average of ~ 17.2%, and bezafibrate treatment prevented ~ 40.5% of that atrophy. In vitro, bezafibrate significantly inhibited expression of the inflammatory cytokine Tnfa in lipopolysaccharide-stimulated RAW264.7 cells, a murine macrophage line. Finally, we show that expression of Tnfa and IL-1b is induced in gastrocnemius muscle in the leg immobilization model, an activity significantly antagonized by bezafibrate administration in vivo. We conclude that bezafibrate could serve as a therapeutic agent for immobilization-induced muscle atrophy.

Oral glutamine supplementation relieves muscle loss in immobilized rats, altering p38MAPK and FOXO3a signaling pathways

BackgroundSkeletal muscle synthesizes, stores, and releases body L-glutamine (GLN). Muscle atrophy due to disabling diseases triggers the activation of proteolytic and pro-apoptotic cell signaling, thus impairing the body's capacity to manage GLN content. This situation has a poor therapeutic prognosis. ObjectiveEvaluating if oral GLN supplementation can attenuate muscle wasting mediated by elevated plasma cortisol and activation of caspase-3, p38MAPK, and FOXO3a signaling pathways in soleus and gastrocnemius muscles of rats submitted to 14-day bilateral hindlimbs immobilization. MethodsAnimals were randomly distributed into six groups: non-immobilized rats (Control), control orally supplemented with GLN (1 g kg−1) in solution with L-alanine (ALA: 0.61 g kg−1; GLN+ALA), control orally supplemented with dipeptide L-alanyl-L-glutamine (DIP; 1.49 g kg−1), hindlimbs immobilized rats (IMOB), IMOB orally GLN+ALA supplemented (GLN+ALA-IMOB), and IMOB orally DIP supplemented (DIP-IMOB). Plasma and muscle GLN concentration, plasma cortisol level, muscle caspase-3 activity, muscle p38MAPK and FOXO3a protein content (total and phosphorylated forms), and muscle cross-sectional area (CSA) were measured. ResultsCompared to controls, IMOB rats presented: a) increased plasma cortisol levels; b) decreased plasma and muscle GLN concentration; c) increased muscle caspase-3 activity; d) increased total and phosphorylated p38MAPK protein content; e) increased FOXO3a and decreased phosphorylated FOXO3a protein content; f) reduced muscle weight and CSA befitting to atrophy. Oral supplementation with GLN+ALA and DIP was able to significantly attenuate these effects. ConclusionsThese findings attest that oral GLN supplementation in GLN+ALA solution or DIP forms attenuates rats' skeletal muscle mass wasting caused by disuse-mediated muscle atrophy.

Effects of C-Peptide on Dexamethasone-Induced In Vitro and In Vivo Models as a Potential Therapeutic Agent for Muscle Atrophy.

This study aimed to investigate the effects of C-peptide on C2C12 myotubes and a mouse model. Both in vitro and in vivo experiments were conducted to elucidate the role of C-peptide in muscle atrophy. Various concentrations (0, 0.01, 0.1, 1, 10, and 100 nM) of C-peptide were used on the differentiated C2C12 myotubes with or without dexamethasone (DEX). C57BL/6J mice were administered with C-peptide and DEX for 8 days, followed by C-peptide treatment for 12 days. Compared to the DEX group, C-peptide increased the fusion and differentiation indices and suppressed atrophic factor expression in C2C12 myotubes. However, 100 nM C-peptide decreased the fusion and differentiation indices and increased atrophic factor expression regardless of DEX treatment. In C57BL/6J mice, DEX + C-peptide co-treatment significantly attenuated the body and muscle weight loss and improved the grip strength and cross-sectional area of the gastrocnemius (Gas) and quadriceps (Quad) muscles. C-peptide downregulated the mRNA and protein levels of muscle degradation-related markers, particularly Atrogin-1, in Gas and Quad muscles. This study underscores the potential of C-peptides in mitigating muscle weight reduction and preserving muscle function during muscle atrophy via molecular regulation. In addition, the work presents basic data for future studies on the effect of C-peptide on diabetic muscular dystrophy.

Lack of adipocyte IP3R1 reduces diet-induced obesity and greatly improves whole-body glucose homeostasis

The normal function of skeletal muscle and adipose tissue ensures whole-body glucose homeostasis. Ca2+ release channel inositol 1,4,5-trisphosphate receptor 1 (IP3R1) plays a vital role in regulating diet-induced obesity and disorders, but its functions in peripheral tissue regulating glucose homeostasis remain unexplored. In this study, mice with Ip3r1 specific knockout in skeletal muscle or adipocytes were used for investigating the mediatory role of IP3R1 on whole-body glucose homeostasis under normal or high-fat diet. We reported that IP3R1 expression levels were increased in the white adipose tissue and skeletal muscle of diet-induced obese mice. Ip3r1 knockout in skeletal muscle improved glucose tolerance and insulin sensitivity of mice on a normal chow diet, but worsened insulin resistance in diet-induced obese mice. These changes were associated with the reduced muscle weight and compromised Akt signaling activation. Importantly, Ip3r1 deletion in adipocytes protected mice from diet-induced obesity and glucose intolerance, mainly due to the enhanced lipolysis and AMPK signaling pathway in the visceral fat. In conclusion, our study demonstrates that IP3R1 in skeletal muscle and adipocytes exerts divergent effects on systemic glucose homeostasis, and characterizes adipocyte IP3R1 as a promising target for treating obesity and type 2 diabetes.

Effects Of Hindlimb Unloading And Long Acting Reversible Contraception On Markers Of Mitophagy In Muscle

Spaceflight has been shown to cause severe muscle atrophy. While loss of contractile proteins has been well documented, there is little research on the effects on the mitochondria and mitophagy. Long-acting reversible contraceptives (LARC; progestin-only) will be likely be recommended for female astronauts on deep space mission, however, the impact on muscle is unknown. PURPOSE: The purpose of this study was to examine the effects of hindlimb unloading (HU) and LARC on muscle mitophagy. METHODS: Female Sprague-Dawley rats (n = 52; 4-mo-old) were singly housed and randomly assigned to placebo (PL) and LARC groups, via an implanted slow-release etonogestrel pellet (0.00ug/d vs. 0.30ug/d). A week later, animals were further randomized to weight bearing (WB) and HU groups for 6 weeks. At the end of the 6-week HU period animals were euthanized. Tissues collected and snap frozen for later analysis. Western blot analysis was run to quantify markers of mitophagy (Parkin, NIX) as well as glycolytic enzymes (GAPDH). Signal values were normalized against the loading control for each gel. Statistics were conducted using RStudio. RESULTS: Despite increasing their food intake during HU (p < 0.01), HU animals lost weight and weighed less than WB animals starting on HU week 2 (p < 0.01). Weight wet of the gastrocnemius was significantly different after HU (WB: 2.07 g +/- 0.08 vs HU: 1.58 g +/- 0.11). A significant difference was observed between the WB and HU groups for both Parkin (1.01 +/- 0.33 vs 0.74 +/- .25, p = .006) and NIX (1.10 +/- 0.38 vs 0.82 +/- 0.43, p = .040), respectively. No significant difference was found in GAPDH. LARC had no effect on mitophagy or glycolytic enzyme content. There was no significant interaction between HU and LARC on mitophagy or glycolytic markers. CONCLUSION: After 6-weeks of unloading, markers of mitophagy were significantly decreased. This could be due to either impairments in the mitophagy process or reduced enzyme content due to reduced muscle fiber size as indicated by reduced muscle weight after HU. LARC implantation did not alter the impact of HU on mitophagy markers. Further analysis into mitochondrial protein content as well as respiratory capacity will provide insight into mitochondrial health and function.

Physicochemical characteristics of lamb meat fed with cottonseed associated with calcium lignosulphonate

The physicochemical characteristics of the meat from lambs fed diets containing whole or disintegrated cottonseed, associated or not with calcium lignosulfonate (LignoCaSO3), were evaluated. Thirty non-castrated Dorper x Santa Inês lambs, with an average of 24.9 ± 3.6 kg and four months of age were confined for 60 days in collective stalls and distributed in a completely randomized design with six replications. After slaughter, by means of contrasts, the averages of the parameters of the semimembranous and semitendinosus muscles were analyzed. The cottonseed increased cooking loss and ash, and reduced muscle weight, water holding capacity and red intensity. The disintegration of the cottonseed reduced the shear force in diets without LignoCaSO3, increased the protein and the loss by cooking and reduced the pH in the diets with the additive. The luminosity values increased with the disintegration of the cottonseed in diets with and without LignoCaSO3. The addition of LignoCaSO3 increased the weight of the muscle, protein, ash, pH, shear strength and the intensity of red. Moisture, lipids and yellow intensity were not influenced by the diets. Even changing the physical-chemical characteristics, the cottonseed with or without LignoCaSO3 does not change the quality of the meat

Extensor carpi ulnaris muscle shows unexpected slow-to-fast fiber-type switch in Duchenne muscular dystrophy dogs.

ABSTRACTAged dystrophin-null canines are excellent models for studying experimental therapies for Duchenne muscular dystrophy, a lethal muscle disease caused by dystrophin deficiency. To establish the baseline, we studied the extensor carpi ulnaris (ECU) muscle in 15 terminal age (3-year-old) male affected dogs and 15 age/sex-matched normal dogs. Affected dogs showed histological and anatomical hallmarks of dystrophy, including muscle inflammation and fibrosis, myofiber size variation and centralized myonuclei, as well as a significant reduction of muscle weight, muscle-to-body weight ratio and muscle cross-sectional area. To rigorously characterize the contractile properties of the ECU muscle, we developed a novel in situ assay. Twitch and tetanic force, contraction and relaxation rate, and resistance to eccentric contraction-induced force loss were significantly decreased in affected dogs. Intriguingly, the time-to-peak tension and half-relaxation time were significantly shortened in affected dogs. Contractile kinetics predicted an unforeseen slow-to-fast myofiber-type switch, which we confirmed at the protein and transcript level. Our study establishes a foundation for studying long-term and late-stage therapeutic interventions in dystrophic canines. The unexpected myofiber-type switch highlights the complexity of muscle remodeling in dystrophic large mammals. This article has an associated First Person interview with the first author of the paper.

Mountain ginseng inhibits skeletal muscle atrophy by decreasing muscle RING finger protein-1 and atrogin1 through forkhead box O3 in L6 myotubes

Ethnopharmacological relevanceMountain ginseng (Panax ginseng C.A. Meyer) is a medicinal herb with immune effects, muscle damage protection and energy metabolism effects. However, the pharmacological role of mountain ginseng in dexamethasone (DEXA)-induced muscle atrophy through the forkhead box O (FOXO) family is not understood. Therefore, we hypothesized that mountain ginseng inhibits skeletal muscle atrophy by decreasing muscle RING finger protein-1 (MuRF1) and atrogin1 through FOXO3 in L6 myotubes. MethodsRat myoblast (L6) cells or Sprague-Dawley (SD) rats were exposed to DEXA and mountain ginseng. The expressions of muscle atrophy targets such as MuRF1, atrogin1, MyHC (myosin heavy chain), HSP90, p-Akt, Akt, p-ERK1/2, ERK, FOXO3a, FOXO1, myostatin, and follistatin were analyzed by using Western blot analysis or real-time PCR. The diameter of myotubes was measured. Recruitment of glucocorticoid receptor (GR) or FOXO3a was analyzed by performing a chromatin immunoprecipitation (ChIP) assay. ResultsMountain ginseng treatment reduced muscle weight loss and collagen deposition in DEXA-induced rats. Mountain ginseng treatment led to decreases in MuRF1, atrogin1, p-ERK1/2, FOXO3a, FOXO1, and myostatin. Also, mountain ginseng treatment led to increases in the diameter of myotubes, MyHC, HSP90, p-Akt, and follistatin. Treatment with mountain ginseng reduced enrichment of GR, FOXO3a, and RNA polymerase II on the promoters. ConclusionsThese results suggest that mountain ginseng inhibits skeletal muscle atrophy by decreasing MuRF1 and atrogin1 through FOXO3a in L6 myotubes.

Early life fluoxetine treatment causes long-term lean phenotype in skeletal muscle of rats exposed to maternal lard-based high-fat diet

There is a concern about early life exposure to Selective Serotonin Reuptake Inhibitors (SSRI) in child development and motor system maturation. Little is known, however, about the interaction of environmental factors, such as maternal nutrition, associated with early exposure to SSRI. The increased maternal consumption of high-fat diets is worrisome and affects serotonin system development with repercussions in body phenotype. This study aimed to assess the short- and long-term effects of neonatal fluoxetine treatment on the body and skeletal muscle phenotype of rats exposed to a maternal lard-based high-fat (H) diet during the perinatal period. A maternal lard-based high-fat diet causes reduced birth weight, a short-term reduction in type IIA fibers in the soleus muscle, and in type IIB fibers in the Extensor Digitorum Longus (EDL) muscle, reducing Lactate Dehydrogenase (LDH) activity in both muscles. In the long-term, the soleus showed reduced muscle weight, smaller area and perimeter of muscle fibers, while the EDL muscle showed reduced Citrate Synthase (CS) activity in offspring from the rats on the maternal lard-based high-fat diet. Early-life exposure to fluoxetine reduced body weight and growth and reduced soleus weight and enzymatic activity in young rats. Exposure to neonatal fluoxetine in adult rats caused a decreased body mass index, less food intake, and reduced muscle weight with reduced CS and LDH activity. Neonatal fluoxetine in young rats exposed to a maternal lard-based high-fat diet caused reduced body weight and growth, reduced soleus weight as well as area and perimeter of type I muscle fibers. In adulthood, there was a reduction in food intake, increased proportion of IIA type fibers, reduced area and perimeter of type IIB, and reduction in levels of CS activity in EDL muscle. Neonatal fluoxetine treatment in rats exposed to a maternal lard-based, high-fat diet induces a reduction in muscle weight, an increase in the proportion of oxidative fibers and greater oxidative enzymatic activity in adulthood.

Syndecan-4-/- Mice Have Smaller Muscle Fibers, Increased Akt/mTOR/S6K1 and Notch/HES-1 Pathways, and Alterations in Extracellular Matrix Components.

BackgroundExtracellular matrix (ECM) remodeling is essential for skeletal muscle development and adaption in response to environmental cues such as exercise and injury. The cell surface proteoglycan syndecan-4 has been reported to be essential for muscle differentiation, but few molecular mechanisms are known. Syndecan-4–/– mice are unable to regenerate damaged muscle, and display deficient satellite cell activation, proliferation, and differentiation. A reduced myofiber basal lamina has also been reported in syndecan-4–/– muscle, indicating possible defects in ECM production. To get a better understanding of the underlying molecular mechanisms, we have here investigated the effects of syndecan-4 genetic ablation on molecules involved in ECM remodeling and muscle growth, both under steady state conditions and in response to exercise.MethodsTibialis anterior (TA) muscles from sedentary and exercised syndecan-4–/– and WT mice were analyzed by immunohistochemistry, real-time PCR and western blotting.ResultsCompared to WT, we found that syndecan-4–/– mice had reduced body weight, reduced muscle weight, muscle fibers with a smaller cross-sectional area, and reduced expression of myogenic regulatory transcription factors. Sedentary syndecan-4–/– had also increased mRNA levels of syndecan-2, decorin, collagens, fibromodulin, biglycan, and LOX. Some of these latter ECM components were reduced at protein level, suggesting them to be more susceptible to degradation or less efficiently translated when syndecan-4 is absent. At the protein level, TRPC7 was reduced, whereas activation of the Akt/mTOR/S6K1 and Notch/HES-1 pathways were increased. Finally, although exercise induced upregulation of several of these components in WT, a further upregulation of these molecules was not observed in exercised syndecan-4–/– mice.ConclusionAltogether our data suggest an important role of syndecan-4 in muscle development.

Volumetric muscle loss disrupts length-dependent architectural and functional characteristics of skeletal muscle

ABSTRACT Purpose/Aim: Skeletal muscle architecture is a primary determinant of function. Volumetric muscle loss (VML) injury is destructive; however, the impact on muscle architecture is uncharacterized. Methods: Architectural and functional effects of VML were assessed in rat tibialis anterior (TA) muscle model 4 weeks post-injury. Results: VML caused a 31% and 33% reduction in muscle weight (p < 0.001) and fiber length (p = 0.002), respectively, culminating a 34% reduction of fiber to muscle length ratio (FL:ML; p < 0.001). Fiber pennation angle (+14%; p = 0.150) and physiological cross-sectional area (PCSA; −12%; p = 0.220) were unchanged. VML injury reduced peak isometric force (Po) by 36% (p < 0.001), specific force (sPo = Po/PCSA) by 41% (vs. Po, p > 0.999), and force per gram muscle weight (Po/mw) by 18% (vs. Po, p < 0.001). VML injury increased the length at which Po was produced (Lo) by 8% (p = 0.009), and reduced functional excursion by 35% (p = 0.035). Conclusion: The architectural changes after VML injury preserved PCSA, and therefore preserved “potential” maximal force-producing capacity. At most, only half the Po deficit was due directly to the cumulative effect of horizontal and longitudinal tissue loss. Highlighting the impact of longitudinal muscle loss, VML injury reduced fiber length, and FL:ML and grossly disrupted length-dependent functional properties. These findings raise the importance of augmenting length-dependent muscle properties to optimize functional recovery after VML injury.

M1 macrophage infiltration exacerbate muscle/bone atrophy after peripheral nerve injury

BackgroundPeripheral nerve injury causes limb muscle/bone atrophy, leading to chronic pain. However, the mechanisms underlying muscle/bone atrophy after peripheral nerve injury remain unknown. It was recently reported that M1 macrophages are the main factors responsible for neurogenic inflammation after peripheral nerve injury. We hypothesized that M1 macrophages are important in muscle/bone atrophy after nerve injury. Therefore, we investigated the influence of M1 macrophages on muscle/bone atrophy after nerve injury in mice to prevent muscle/bone atrophy by suppressing M1 macrophages.MethodsHindlimb muscle weight and total bone density were measured in a chronic constriction injury (CCI) mouse model. Immunohistochemical analysis and intravital microscopy were performed to visualize hindlimb muscles/bones, and cells were quantified using flow cytometry. We compared M1 macrophage infiltration into muscles/bones and muscle/bone atrophy between macrophage depletion and untreated groups. We also investigated muscle/bone atrophy using administration models for anti-inflammatory and neuropathic pain drugs.ResultsPeripheral nerve injury caused significant reduction in muscle weight and total bone density at 1 and 3 weeks after CCI, respectively, compared with that in controls. Osteoclast numbers were significantly higher at 1 week after CCI in the CCI group than in the control group. M1 macrophage infiltration into muscles was observed from 2 h after CCI via intravital microscopy and 1 week after CCI, and it was significantly higher 1 week after CCI than in the control group. In the macrophage depletion group, dexamethasone, pregabalin, and loxoprofen groups, M1 macrophage infiltration into muscles/bones was significantly lower and muscle weight and total bone density were significantly higher than in the untreated group.ConclusionsM1 macrophage infiltration exacerbates muscle/bone atrophy after peripheral nerve injury. By suppressing M1 macrophages at the neural injury local site, muscle/bone atrophy could be avoided.

Therapeutic glucocorticoids prevent bone loss but drive muscle wasting when administered in chronic polyarthritis

BackgroundPatients with rheumatoid arthritis (RA) experience extra-articular manifestations including osteoporosis and muscle wasting, which closely associate with severity of disease. Whilst therapeutic glucocorticoids (GCs) reduce inflammation in RA, their actions on muscle and bone metabolism in the context of chronic inflammation remain unclear. We utilised the TNF-tg model of chronic polyarthritis to ascertain the impact of therapeutic GCs on bone and muscle homeostasis in the context of systemic inflammation.MethodsTNF-tg and wild-type (WT) animals received either vehicle or the GC corticosterone (100 μg/ml) in drinking water at onset of arthritis. Arthritis severity and clinical parameters were measured, serum collected for ELISA and muscle and bone biopsies collected for μCT, histology and mRNA analysis. In vivo findings were examined in primary cultures of osteoblasts, osteoclasts and myotubes.ResultsTNF-tg mice receiving GCs showed protection from inflammatory bone loss, characterised by a reduction in serum markers of bone resorption, osteoclast numbers and osteoclast activity. In contrast, muscle wasting was markedly increased in WT and TNF-tg animals receiving GCs, independently of inflammation. This was characterised by a reduction in muscle weight and fibre size, and an induction in anti-anabolic and catabolic signalling.ConclusionsThis study demonstrates that when given in early onset chronic polyarthritis, oral GCs partially protect against inflammatory bone loss, but induce marked muscle wasting. These results suggest that in patients with inflammatory arthritis receiving GCs, the development of interventions to manage deleterious side effects in muscle should be prioritised.

Acteoside Improves Muscle Atrophy and Motor Function by Inducing New Myokine Secretion in Chronic Spinal Cord Injury.

Chronic spinal cord injury (SCI) is difficult to cure, even by several approaches effective at the acute or subacute phase. We focused on skeletal muscle atrophy as a detrimental factor in chronic SCI and explored drugs that protect against muscle atrophy and activate secretion of axonal growth factors from skeletal muscle. We found that acteoside induced the secretion of axonal growth factors from skeletal muscle cells and proliferation of these cells. Intramuscular injection of acteoside in mice with chronic SCI recovered skeletal muscle weight reduction and motor function impairment. We also identified pyruvate kinase isoform M2 (PKM2) as a secreted factor from skeletal muscle cells, stimulated by acteoside. Extracellular PKM2 enhanced proliferation of skeletal muscle cells and axonal growth in cultured neurons. Further, we showed that PKM2 might cross the blood–brain barrier. These results indicate that effects of acteoside on chronic SCI might be mediated by PKM2 secretion from skeletal muscles. This study proposes that the candidate drug acteoside and a new myokine, PKM2, could be used for the treatment of chronic SCI.

OR09-3 11β-HSD1 Mediates a Partial Suppression of Muscle Atrophy in Chronic Inflammatory Disease in Response to Therapeutic Glucocorticoids

Objective: Therapeutic glucocorticoids (GCs) are routinely used in the treatment of chronic inflammatory disease due to their potent anti-inflammatory effects. Despite their efficacy, chronic exposure to GCs elicits undesirable side effects, including muscle atrophy (1). 11 beta-hydroxysteroid dehydrogenase 1 (11β-HSD1) activates GCs in muscle, is induced by inflammation, and has previously shown to induce muscle wasting (2). We examined the role of 11β-HSD1 in mediating muscle wasting in a mouse model of inflammatory myopathy receiving therapeutic GCs. Methods: Wild type (WT) and global knock out (KO) of 11β-HSD1 animals were crossed onto the TNF-tg murine model of polyarthritis and inflammatory myopathy. Animals received vehicle or the GC corticosterone (100µg/ml) over three weeks in drinking water at therapeutic doses. Quadriceps and tibialis anterior (TA) were examined at 7 weeks and histology assessed. Anabolic, catabolic and inflammatory gene and protein expression were examined by RT-qPCR and western blot. Results: GC activation by 11β-HSD1 was completely attenuated in muscles from TNF-tg11β-HSD1KO animals. Both TNF-tg and TNF-tg11β-HSD1KO animals developed inflammatory myopathy characterised by reduced muscle weights and fibre size. In the TNF-tg mouse, therapeutic GC treatment further exacerbated muscle wasting with reduced muscle weight and fibre size relative to vehicle treated controls. This was characterised by elevated gene expression of atrophy markers (FoxO1, Trim63) and anti-anabolic signalling (REDD1), whilst pro-inflammatory gene expression of IL-6 was markedly suppressed. In contrast TNF-tg11β-HSD1KO animals receiving therapeutic GCs were protected from further muscle wasting, with muscle weights and fibre sizes significantly greater than TNF-tg counterparts. These animals showed a partial protection from the elevated expression of the atrophy markers FoxO1, Trim63 and anti-anabolic marker REDD1. They were also resistant to the suppression of the pro-inflammatory gene of IL-6 and induction of GC response gene GILZ, a mediator of anti-inflammatory effects. These data suggest that whilst therapeutic GCs supress inflammation within muscles of TNF-tg mice, they further exacerbate muscle wasting during chronic inflammation. This was dependant on 11β-HSD1 expression, with TNF-tg/11β-HSD1 KO animals being protected from further muscle wasting in response to therapeutic GCs. Reference: (1) Cushing, H. Obes Res. 1932;2(5):486-508. (2) Morgan et al., Proc Natl Acad Sci U S A. 2014;111(24):E2482-91.

Mitochondrial DNA Mutations Induce Neuromuscular Junction Degeneration

IntroductionMitochondrial dysfunction is frequently posited as a cause of aging muscle atrophy. One of the proposed mechanisms involves an age‐related accumulation of mitochondrial DNA (mtDNA) mutations and subsequent mitochondrial dysfunction. Consistent with this notion, previous studies have reported co‐localization of fiber segments with high mtDNA mutation load, severe oxidative impairment, up‐regulation of necrotic and apoptotic pathways, fiber atrophy and breakage in aged muscle. However, the significance of this mechanism is dubious as the majority of fibers harbouring high mtDNA mutation loads and oxidative damage do not exhibit atrophy. In contrast, we have reported previously that persistent denervation of muscle fibers correlates with fiber atrophy in advanced age, where over 90% of severely atrophied fibers show evidence of persistent denervation. These latter findings raise the question of what causes denervation in aging muscle. As virtually nothing is known about the potential for accumulation of mtDNA mutations to cause denervation in aging muscle, we studied neuromuscular junction (NMJ) morphology in a mouse model, exhibiting a rapid accumulation of mtDNA mutations and markedly accelerated muscle atrophy, the polymerase gamma mutator (PolGmut).FindingsOur analysis revealed significantly lower (p&lt;0.05) muscle mass in 16‐mo old PolGmut (n=4) versus 5‐mo old PolGmut mice (n=8) in tibialis anterior (TA; mean ±SD: 25.5±7.4 mg versus 44.3 ±4.7 mg), soleus (Sol: 4.5 ±0.6 mg versus 6.5 mg ±1.1 mg) and gastrocnemius (90.9 ±8.5 mg versus 122.9 ±19.7 mg). In contrast, there was not a significant reduction in muscle weight in 15 to 17‐mo old (n=11) versus 5‐mo old (n=8) WT mice in the TA (42.5 ±7.9 mg versus 43.3 ±9.1 mg), soleus (7.3 ±1.8 mg versus 5.9 ±1.9 mg) and gastrocnemius (mean weight=123.1 ±29.6 mg versus 136.4 ±26.5 mg). Furthermore, we tested the impact of aging on NMJ morphology by immunofluorescent labelling and quantitative assessment of NMJ morphology from the TA of these mice. Significant alterations (p&lt;0.05) were found in WT mice between 5‐mo and 16‐mo including reductions in pre‐synaptic variables: nerve terminal area, nerve terminal perimeter; and in post‐synaptic variables: AChR area, AChR perimeter, AChR compactness and nerve terminal area outside the endplate. Increased nerve terminal area outside AChRs and axonal area within the endplate were also detected. Some of these changes were potentiated in 16‐mo old PolGmut, characterized by increased pre‐synaptic axon diameter and post‐synaptic fragmentation, and reductions in post‐synaptic: unoccupied AChR area, endplate area and endplate perimeter. Most of the NMJ morphology changes were associated with post‐synaptic variables in both WT and PolGmut.ConclusionOur findings show that PolGmut mice exhibit an accelerated appearance of NMJ degeneration, consistent with the possibility that mtDNA mutations may be a cause of denervation in aging muscle. Further work is underway to determine whether NMJ degeneration is related to mtDNA alterations and mitochondrial dysfunction in motor neurons versus muscle fibers.Support or Funding InformationSupported by UF start‐up funds to RTH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.