Tibialis Anterior Muscle Weight Research Articles

Tumour-induced alterations in single-nucleus transcriptome of atrophying muscles indicate enhanced protein degradation and reduced oxidative metabolism.

Tumour-induced skeletal muscle wasting in the context of cancer cachexia is a condition with profound implications for patient survival. The loss of muscle mass is a significant clinical obstacle and is linked to reduced tolerance to chemotherapy and increased frailty. Understanding the molecular mechanisms driving muscle atrophy is crucial for the design of new therapeutics. Lewis lung carcinoma tumours were utilized to induce cachexia and muscle atrophy in mice. Single-nucleus libraries of the tibialis anterior (TA) muscle from tumour-bearing mice and their non-tumour-bearing controls were constructed using 10X Genomics applications following the manufacturer's guidelines. RNA sequencing results were analysed with Cell Ranger software and the Seurat R package. Oxygen consumption of mitochondria isolated from TA muscle was measured using an Oroboros O2k-FluoRespirometer. Mouse primary myotubes were treated with a recombinant ectodysplasin A2 (EDA-A2) protein to activate EDA-A2 receptor (EDA2R) signalling and study changes in gene expression and oxygen consumption. Tumour-bearing mice were sacrificed while exhibiting moderate cachexia. Average TA muscle weight was reduced by 11% (P=0.0207) in these mice. A total of 12335 nuclei, comprising 6422 nuclei from the control group and 5892 nuclei from atrophying muscles, were studied. The analysis of single-nucleus transcriptomes identified distinct myonuclear gene signatures and a shift towards type IIb myonuclei. Muscle atrophy-related genes, including Atrogin1, MuRF1 and Eda2r, were upregulated in these myonuclei, emphasizing their crucial roles in muscle wasting. Gene set enrichment analysis demonstrated that EDA2R activation and tumour inoculation led to similar expression patterns in muscle cells, including the stimulation of nuclear factor-kappa B, Janus kinase-signal transducer and activator of transcription and transforming growth factor-beta pathways and the suppression of myogenesis and oxidative phosphorylation. Muscle oxidative metabolism was suppressed by both tumours and EDA2R activation. This study identified tumour-induced transcriptional changes in muscle tissue at single-nucleus resolution and highlighted the negative impact of tumours on oxidative metabolism. These findings contribute to a deeper understanding of the molecular mechanisms underlying muscle wasting.

Astragaloside IV alleviates sepsis-induced muscle atrophy by inhibiting the TGF-β1/Smad signaling pathway.

Muscle atrophy occurs in patients with sepsis and increases mortality and disability. Remission of muscle atrophy may improve the quality of life in patients with sepsis. Astragaloside IV (ASIV) has been shown to have excellent anti-inflammatory and anti-fibrotic effects and to reduce organ damage caused by sepsis. However, the effect of ASIV on sepsis-induced muscle atrophy has not been reported. Therefore, this study explored the pharmacological effects and mechanisms of ASIV in sepsis-induced muscle atrophy. Cecal ligation and puncture (CLP) was used to establish a mouse model of sepsis and lipopolysaccharide (LPS)-stimulated C2C12 myotubes. After administration of ASIV, the body weight, tibialis anterior (TA) and gastrocnemius muscle weight and fiber cross-sectional area of the mice were measured. The diameter of myotubes was observed by immunofluorescence staining. ELISA was used to assess inflammatory factors in plasma and cell culture supernatants. RT-PCR and Western blotting were used to detect the expression of MuRF1, Atrogin-1 and TGF-β1/Smad signaling pathway components in TA and C2C12 myotubes. Our study found that ASIV reduced serum inflammatory factors and improved survival in septic mice. ASIV alleviated muscle mass reduction, myofiber cross-sectional area reduction, and C2C12 myotube atrophy by inhibiting the expression of the E3 ubiquitin ligases MuRF1 and atrogin-1. In addition, we observed that ASIV inhibited TGF-β1/Smad signaling. Inhibition of the TGF-β1/Smad signaling pathway partly blocked the anti-muscle atrophy effect of ASIV. ASIV can alleviate sepsis-induced muscle atrophy, which may be related to the inhibition of the TGF-β1/Smad signaling pathway.

Noninvasive monitoring of muscle atrophy and bone metabolic disorders using dual-energy X-ray absorptiometry in diabetic mice.

Tracking metabolic changes in skeletal muscle and bone using animal models of diabetes mellitus (DM) provides important insights for the management of DM complications. In this study, we aimed to establish a method for monitoring changes in body composition characteristics, such as fat mass, skeletal muscle mass (lean mass), bone mineral density, and bone mineral content, during DM progression using a dual-energy X-ray absorptiometry (DXA) system in a mouse model of streptozotocin (STZ)-induced type 1 DM. In the DM model, STZ administration resulted in increased blood glucose levels, increased water and food intake, and decreased body weight. Serum insulin levels were significantly decreased on day 30 of STZ administration. The DXA analysis revealed significant and persistent decreases in fat mass, lower limb skeletal muscle mass, and bone mineral content in DM mice. We measured tibialis anterior (TA) muscle weight and performed a quantitative analysis of tibial microstructure by micro-computed tomography imaging in DM mice. The TA muscle weight of DM mice was significantly lower than that of control mice. In addition, the trabecular bone volume fraction, trabecular thickness, trabecular number, and cortical thickness were significantly decreased in DM mice. Pearson's product-moment correlation coefficient analysis showed a high correlation between the DXA-measured and actual body composition. In conclusion, longitudinal measurement of body composition changes using a DXA system may be useful for monitoring abnormalities in muscle and bone metabolism in animal models of metabolic diseases such as DM mice.

Conservative analysis of Synaptopodin-2 intron sense-overlapping lncRNA reveals its novel function in promoting muscle atrophy.

BackgroundDissection of the regulatory pathways that control skeletal muscle development and atrophy is important for the treatment of muscle wasting. Long noncoding RNA (lncRNA) play important roles in various stages of muscle development. We previously reported that Synaptopodin‐2 (SYNPO2) intron sense‐overlapping lncRNA (SYISL) regulates myogenesis through an interaction with enhancer of zeste homologue 2 (EZH2). However, it remains unclear whether SYISL homologues exist in humans and pigs, and whether the functions and mechanisms of these homologues are conserved among species.MethodsBioinformatics, cell fractionation, and quantitative real‐time polymerase chain reaction (qRT‐PCR) analyses were used for the identification and molecular characterization of SYISL homologues in humans and pigs. Effects on myogenesis and muscle atrophy were determined via loss‐of‐function or gain‐of‐function experiments using C2C12 myoblasts, myogenic progenitor cells, dexamethasone (DEX), and aging‐induced muscle atrophy models. RNA pulldown, RNA immunoprecipitation, dual luciferase reporting, and co‐transfection experiments were used to explore the mechanisms of SYISL interactions with proteins and miRNAs.ResultsWe identified SYISL homologues in humans (designated hSYISL) and pigs (designated pSYISL). Functional experiments demonstrated that hSYISL and pSYISL regulate myogenesis through interactions with EZH2. Interestingly, we showed that SYISL functions to regulate muscle atrophy and sarcopenia through comparative analysis. SYISL is significantly up‐regulated after muscle atrophy (P < 0.01); it significantly promotes muscle atrophy in DEX‐induced muscle atrophy models (P < 0.01). SYISL knockdown or knockout alleviates muscle atrophy and sarcopenia in DEX‐induced and aged mice. The tibialis anterior (TA) muscle weight of 3‐month‐old wild‐type (WT) mice decreased by 33.24% after DEX treatment (P < 0.001), while the muscle weight loss of 3‐month‐old SYISL knockout mice was only 18.20% after DEX treatment (P < 0.001). SYISL knockout in 18‐month‐old WT mice significantly increased the weights of quadriceps (Qu), gastrocnemius (Gas), and TA muscles by 10.45% (P < 0.05), 13.95% (P < 0.01), and 24.82% (P < 0.05), respectively. Mechanistically, SYISL increases the expression levels of the muscle atrophy genes forkhead box protein O3a (FoxO3a), muscle ring finger 1 (MuRF1), and muscle atrophy‐related F‐box (Atrogin‐1) via sponging of miR‐23a‐3p/miR‐103‐3p/miR‐205‐5p and thus promotes muscle atrophy. Additionally, we verified that human SYISL overexpression in muscles of 18‐month‐old WT mice significantly decreased the weights of Gas, Qu, and TA muscles by 7.76% (P < 0.01), 12.26% (P < 0.05), and 13.44% (P < 0.01), respectively, and accelerates muscle atrophy through conserved mechanisms.ConclusionsOur results identify SYISL as a conserved lncRNA that modulates myogenesis in mice, pigs, and humans. We also demonstrated its previously unknown ability to promote muscle atrophy.

A Collagen Hydrolysate Containing Tripeptides Ameliorates Sarcopenia in Middle-Aged Mice.

Collagen peptide (CP) and collagen tripeptide (CTP) are supplementary health foods that exhibit several biological effects. However, the effects of collagen on age-associated sarcopenia and its underlying mechanisms are unclear. C57BL/6J mice (n = 24, 12 months old) were divided into three dietary groups and administered AIN93G (aging control, AC; JA BIO, Suwon, Korea), AIN93G plus 0.2% CP, and AING93G plus 0.2% CTP supplement for 12 weeks. The results indicated that the CP and CTP supplements significantly increased the weight of the quadriceps tibialis anterior and gastrocnemius muscles and reduced body fat. A morphological analysis revealed that the spaces within the muscle cells were tight with attenuated fibrosis following CP and CTP supplementation. Immunohistochemistry was applied and a Western blot analysis was performed to determine the underlying mechanisms. The CTP supplement increased the expression of IGF-1, PI3K/AKT, and mTOR, whereas the CP supplement increased the expression of IGF-1 and AMPK in the gastrocnemius of aging mice. CP and CTP ameliorate age-associated sarcopenia through different mechanisms.

Sepsis induces interleukin 6, gp130/JAK2/STAT3, and muscle wasting

BackgroundSepsis and inflammation can cause intensive care unit‐acquired weakness (ICUAW). Increased interleukin‐6 (IL‐6) plasma levels are a risk factor for ICUAW. IL‐6 signalling involves the glycoprotein 130 (gp130) receptor and the JAK/STAT‐pathway, but its role in sepsis‐induced muscle wasting is uncertain. In a clinical observational study, we found that the IL‐6 target gene, SOCS3, was increased in skeletal muscle of ICUAW patients indicative for JAK/STAT‐pathway activation. We tested the hypothesis that the IL‐6/gp130‐pathway mediates ICUAW muscle atrophy.MethodsWe sequenced RNA (RNAseq) from tibialis anterior (TA) muscle of cecal ligation and puncture‐operated (CLP) and sham‐operated wildtype (WT) mice. The effects of the IL‐6/gp130/JAK2/STAT3‐pathway were investigated by analysing the atrophy phenotype, gene expression, and protein contents of C2C12 myotubes. Mice lacking Il6st, encoding gp130, in myocytes (cKO) and WT controls, as well as mice treated with the JAK2 inhibitor AG490 or vehicle were exposed to CLP or sham surgery for 24 or 96 h.ResultsAnalyses of differentially expressed genes in RNAseq (≥2‐log2‐fold change, P < 0.01) revealed an activation of IL‐6‐signalling and JAK/STAT‐signalling pathways in muscle of septic mice, which occurred after 24 h and lasted at least for 96 h during sepsis. IL‐6 treatment of C2C12 myotubes induced STAT3 phosphorylation (three‐fold, P < 0.01) and Socs3 mRNA expression (3.1‐fold, P < 0.01) and caused myotube atrophy. Knockdown of Il6st diminished IL‐6‐induced STAT3 phosphorylation (−30.0%; P < 0.01), Socs3 mRNA expression, and myotube atrophy. JAK2 (− 29.0%; P < 0.01) or STAT3 inhibition (−38.7%; P < 0.05) decreased IL‐6‐induced Socs3 mRNA expression. Treatment with either inhibitor attenuated myotube atrophy in response to IL‐6. CLP‐operated septic mice showed an increased STAT3 phosphorylation and Socs3 mRNA expression in TA muscle, which was reduced in septic Il6st‐cKO mice by 67.8% (P < 0.05) and 85.6% (P < 0.001), respectively. CLP caused a loss of TA muscle weight, which was attenuated in Il6st‐cKO mice (WT: −22.3%, P < 0.001, cKO: −13.5%, P < 0.001; WT vs. cKO P < 0.001). While loss of Il6st resulted in a reduction of MuRF1 protein contents, Atrogin‐1 remained unchanged between septic WT and cKO mice. mRNA expression of Trim63/MuRF1 and Fbxo32/Atrogin‐1 were unaltered between CLP‐treated WT and cKO mice. AG490 treatment reduced STAT3 phosphorylation (−22.2%, P < 0.05) and attenuated TA muscle atrophy in septic mice (29.6% relative reduction of muscle weight loss, P < 0.05). The reduction in muscle atrophy was accompanied by a reduction in Fbxo32/Atrogin‐1‐mRNA (−81.3%, P < 0.05) and Trim63/MuRF1‐mRNA expression (−77.6%, P < 0.05) and protein content.ConclusionsIL‐6 via the gp130/JAK2/STAT3‐pathway mediates sepsis‐induced muscle atrophy possibly contributing to ICUAW.

Microenergy acoustic pulses promotes muscle regeneration through in situ activation of muscle stem cells.

Microenergy acoustic pulses (MAP) is a modified low-intensity extracorporeal shock wave therapy that currently used for treating musculoskeletal disorders. However, its function on muscle regeneration after ischemia-reperfusion injury (IRI) remains unknown. This study aimed to explore the effect of MAP on muscle injury after IRI and its underlying mechanisms. Ten-week-old C57BL/6J mice underwent unilateral hindlimb IRI followed with or without MAP treatment. Wet weight of tibialis anterior muscles at both injury and contralateral sides were measured followed with histology analysis at 3 weeks after IRI. In in vitro study, the myoblasts, endothelial cells and fibro-adipogenic progenitors (FAP) were treated with MAP. Cell proliferation and differentiation were assessed, and related gene expressions were measured by real-time PCR. Our results showed that MAP significantly increased the muscle weight and centrally nucleated regenerating muscle fiber size along with a trend in activating satellite cells. In vitro data indicated that MAP promoted myoblast proliferation and differentiation and endothelial cells migration. MAP also induced FAP brown/beige adipogenesis, a promyogenic phenotype of FAPs. Our findings demonstrate the beneficial function of MAP in promoting muscle regeneration after IR injury by inducing muscle stem cells proliferation and differentiation.

Effectiveness and Biocompatibility of Decellularized Nerve Graft Using an In Vivo Rat Sciatic Nerve Model.

Decellularized nerve allografting is one of promising treatment options for nerve defect. As an effort to develop more efficient nerve graft, recently we have developed a new decellularization method for nerve allograft. The aim of this study was to evaluate the effectiveness and biocompatibility of nerve graft decellularized by our newly developed method. Forty-eight inbred male Lewis rats were divided into two groups, Group I (autograft group, n = 25), Group II (decellularized isograft group, n = 23). Decellularized nerve grafts were prepared with our newly developed methods using amphoteric detergent and nuclease treatment. Serum cytokine level measurements at 0, 2, and 4weeks and histologic evaluation for inflammatory cell infiltration at 6 and 16weeks after nerve graft. There was no significant difference in mean maximum isometric tetanic force and weight of tibialis anterior muscle or ankle angle at toe-off phase between two groups at 6 and 16weeks survival time points (p > 0.05). There was no inflammatory cell infiltration in either group and histomorphometric assessments of 6- and 16-week specimens of the isograft group did not differ from those in the autograft group with regard to number of fascicle, cross sectional area, fascicle area ratio, and number of regenerated nerve cells. Based on inflammatory reaction, axonal regeneration, and functional outcomes, our newly developed decellularized nerve grafts were fairly biocompatible and had comparable effectiveness to autografts for nerve regeneration, which suggested it would be suitable for nerve reconstruction as an alternative to autograft.

Polybutylene succinate artificial scaffold for peripheral nerve regeneration.

Regeneration and recovery of nerve tissues are a great challenge for medicine, and positively affect the quality of life of patients. The development of tissue engineering offers a new approach to the problem with the creation of multifunctional artificial scaffolds that act on various levels in the damaged tissue, providing physical and biochemical support for the growth of nerve cells. In this study, the effects of the use of a tubular scaffold made of polybutylene succinate (PBS), surgically positioned at the level of a sciatic nerve injured in rat, between the proximal stump and the distal one, was investigated. Scaffolds characterization was carried out by scanning electron microscopy and X‐ray microcomputed tomography and magnetic resonance imaging, in vivo. The demonstration of the nerve regeneration was based on the evaluation of electroneurography, measuring the weight of gastrocnemius and tibialis anterior muscles, histological examination of regenerated nerves and observing the recovery of the locomotor activity of animals. The PBS tubular scaffold minimized iatrogenic trauma on the nerve, acting as a directional guide for the regenerating fibers by conveying them toward the distal stump. In this context, neurotrophic and neurotropic factors may accumulate and perform their functions, while invasion by macrophages and scar tissue is hampered.

The Role of Coactivator‐associated Arginine Methyltransferase 1 (CARM1) During Sarcopenia

CARM1 is emerging as an important regulator of skeletal muscle plasticity. We recently reported reduced muscle mass, along with aberrant autophagic and atrophic signaling in CARM1 skeletal muscle-specific knockout (mKO) mice. Notably, CARM1 interacts with forkhead box O1 (FOXO1), a master transcriptional regulator of the atrophy program, and knockout of CARM1 in skeletal muscle results in elevated FOXO1 protein content. Although CARM1 appears to be indispensable for preserving muscle mass, the impact of CARM1 deletion during the sarcopenia of aging remains to be elucidated. The purpose of this study was to more comprehensively understand the function of CARM1 during the progressive loss of skeletal muscle mass and strength with age. Survival analysis revealed that mKO mice have a significantly shorter lifespan compared to their wild type (WT) littermates. For instance, the survival rate of mKO and WT animals at 22 months is 17% and 33%, respectively. In order to assess functional and physiological adaptations to aging, grip strength and tibialis anterior (TA) muscle weights were assessed in 3 month (young) and 22 month (old) animals. We detected main effects (p < 0.05) of aging and genotype on forelimb grip strength. In particular, grip strength was significantly lower by 24% in old mKO versus old WT mice. Main effects (p < 0.05) of aging and genotype were also observed for TA muscle weight normalized to body weight. Relative to old WT mice, TA muscle weight was significantly lower by 7% in old mKO animals. We also employed Western blot analyses to assess FOXO1Ser256 activation status (phosphorylated/total), as well as the mammalian target of rapamycin complex 1 (mTORC1)Ser2448, a key regulator of muscle protein synthesis. The phosphorylation status of FOXO1Ser256 was lower (p = 0.07) in old mKO versus old WT mice, suggesting that CARM1 deletion leads to greater FOXO1 nuclear entry and transcription of atrogenes with aging. Compared to old WT animals, phosphorylation levels of mTORC1Ser2448 were also lower (p < 0.05) in old mKO mice, suggesting that CARM1 is important for driving muscle protein synthesis throughout the lifespan. Collectively, these results highlight the importance of CARM1 for combating the progressive and generalized loss of skeletal muscle mass and strength that occurs with age. Manipulating the expression and/or activity of CARM1 in skeletal muscle may offer a viable therapeutic strategy for mitigating sarcopenia.

Cancer-derived exosome miRNAs induce skeletal muscle wasting by Bcl-2-mediated apoptosis in colon cancer cachexia

Cancer cachexia is a kind of whole-body metabolic disorder syndrome accompanied by severe wasting of muscle tissue in which cancer exosomes may be involved. Analysis of clinical samples showed that the serum exosome concentrations were correlated with the development of cancer cachexia. Exosomes secreted by C26 cells could decrease the diameter of C2C12 myotubes in vitro and decrease mouse muscle strength and tibialis anterior (TA) muscle weight in vivo. GW4869, an inhibitor of exosome excretion, ameliorated muscle wasting in C26 tumor-bearing mice. MicroRNA (miRNA) sequencing (miRNA-seq) analysis suggested that miR-195a-5p and miR-125b-1-3p were richer in C26 exosomes than in exosomes secreted from MC38 cells (non-cachexic). Both miR-195a-5p and miR-125b-1-3p mimics could induce atrophy of C2C12 myoblasts. Downregulation of Bcl-2 and activation of the apoptotic signaling pathway were observed in C2C12 myoblasts transfected with miR-195a-5p and miR-125b-1-3p mimics, in the gastrocnemius muscle of C26 tumor-bearing mice and in the TA muscle injected with C26 exosomes. Results of dual-luciferase assay confirmed the targeting of miR-195a-5p/miR-125b-1-3p to Bcl-2. Overexpression of Bcl-2 successfully reversed atrophy of C2C12 myoblasts induced by the two miRNA mimics. These results suggested that cancer exosome enriched miRNAs might induce muscle atrophy by targeting Bcl-2-mediated apoptosis.

ω-3 Polyunsaturated Fatty Acids Facilitate the Repair of Peripheral Nerve Defects with Chemically Extracted Acellular Allograft in Rats.

Acellular allograft (ACA) improves the repair and reconstruction of long peripheral nerve defects. ω-3 Polyunsaturated fatty acids (PUFAs) carry a neuroprotective potential, and their effects on ACA bridging were elucidated. Thirty rats with long gap sciatic nerve defects (15 mm long) were randomly divided into three groups (n = 10): ACA, ACA + PUFAs, and autograft (AU). Limb condition, wet weight of tibialis anterior muscle (TAM), nerve electrophysiology, S-100, horseradish peroxidase (HRP), and percentage of splenic CD4+ and CD8 + T-lymphocytes were evaluated for 12 weeks after the operation. Rats in the AU and ACA + PUFA groups showed superior condition in affected limbs compared to the ACA group. At 12 wk after surgery, the wet weight of TAM in the ACA + PUFA group was higher than that in the ACA group (0.4519 ± 0.1185 vs. 0.3049 ± 0.1272; P < 0.01) but lower than that in the AU group (0.4519 ± 0.1185, 0.5628 ± 0.0092; P < 0.05). In all the three groups, sole irritation elicited withdrawal reflex, and S-100 staining was detected in plantar skin. Moreover, horseradish peroxidase staining was overt in both the ventral horn and dorsal root ganglion of the spinal cord. Nerve conduction velocity (m/s), amplitude of action potential (mV), or somatosensory evoked potentials in ACA + PUFAs (28.81 ± 1.04, 2.20 ± 0.27, 6.98 ± 0.29) were significantly different from that in the AU (35.71 ± 1.28, 1.81 ± 0.19, 8.15 ± 0.52; P < 0.05) and ACA (20.03 ± 1.94, 2.95 ± 0.36, 5.22 ± 0.53; P < 0.01) groups. The percentages of splenic CD4+ and CD8+ cells were similar among the three groups. Omega-3 PUFAs improve the bridging effect of ACA on long gap peripheral nerve defects by promoting neuroprotection without arousing an immune response.

Formononetin ameliorates muscle atrophy by regulating myostatin-mediated PI3K/Akt/FoxO3a pathway and satellite cell function in chronic kidney disease.

Muscle atrophy is a common complication in chronic kidney disease (CKD). Inflammation and myostatin play important roles in CKD muscle atrophy. Formononetin (FMN), which is a major bioactive isoflavone compound in Astragalus membranaceus, exerts anti‐inflammatory effects and the promotion of myogenic differentiation. Our study is based on myostatin to explore the effects and mechanisms of FMN in relation to CKD muscle atrophy. In this study, CKD rats and tumour necrosis factor α (TNF‐α)‐induced C2C12 myotubes were used for in vivo and in vitro models of muscle atrophy. The results showed that FMN significantly improved the renal function, nutritional status and inflammatory markers in CKD rats. Values for bodyweight, weight of tibialis anterior and gastrocnemius muscles, and cross‐sectional area (CSA) of skeletal muscles were significantly larger in the FMN treatment rats. Furthermore, FMN significantly suppressed the expressions of MuRF‐1, MAFbx and myostatin in the muscles of CKD rats and the TNF‐α‐induced C2C12 myotubes. Importantly, FMN significantly increased the phosphorylation of PI3K, Akt, and FoxO3a and the expressions of the myogenic proliferation and differentiation markers, myogenic differentiation factor D (MyoD) and myogenin in muscles of CKD rats and the C2C12 myotubes. Similar results were observed in TNF‐α‐induced C2C12 myotubes transfected with myostatin‐small interfering RNA (si‐myostatin). Notably, myostatin overexpression plasmid (myostatin OE) abolished the effect of FMN on the phosphorylation of the PI3K/Akt/FoxO3a pathway and the expressions of MyoD and myogenin. Our findings suggest that FMN ameliorates muscle atrophy related to myostatin‐mediated PI3K/Akt/FoxO3a pathway and satellite cell function.

Therapeutic Potential Of Different Pericyte Populations In The Recovery Of Skeletal Muscle Mass Following Disuse

Significant loss in skeletal muscle mass and function can occur following periods of extended bed rest or immobilization. Physical therapy is recommended, but recovery may be incomplete in special populations due to injury and functional limitations. Our lab recently demonstrated the capacity for pericytes, or vascular stromal cells, to accelerate recovery of skeletal muscle following disuse in a mouse model. Different pericyte populations exist in skeletal muscle based on localization and unique cell surface markers, yet the most therapeutic population has not been identified. PURPOSE: To identify the pericyte population with the greatest therapeutic benefit when transplanted into muscle following a period of disuse. METHODS: Twenty-four 4-month old C57BL/6 mice were randomly divided into four groups (n=6/group). Mice hindlimbs were immobilized in full dorsiflexion via a surgical staple inserted through the center of the foot and body of the gastrocnemius for 2 weeks. At 2 weeks post immobilization, staples were removed and either pericytes (CD146+Lin-, CD146+NG2-Lin-, NG2+Lin-) or saline (control) were injected into the tibialis anterior (TA) muscle. TA muscles were excised for analysis after 2 weeks of remobilization and the extent of recovery was assessed. One-way ANOVA was used to compare the extent of improvement between treatment groups. RESULTS: There was no significant improvement in TA muscle weight or myofiber CSA with pericyte transplantation in the current study (p>0.05). However, a trend toward significant improvement in myofiber CSA was noted for fibers ranging 2000-3000 μm2 in mice receiving CD146+NG2-Lin-pericytes (p=0.072). Significant improvements in capillarization and collagen remodeling were detected in mice receiving CD146+NG2-Lin- (p<0.05) and CD146+Lin- (p<0.05) pericytes compared to controls. CONCLUSION: CD146+Lin- pericyte transplantation effectively recovered capillary quantity and collagen remodeling following a period disuse compared to controls, whereas NG2+Lin- pericytes did not demonstrate similar capacity for recovery. Supported by NIH Grant NIAMS R01 AR072735 to MDB.

Effect of forced running on rat skeletal muscle with acrylamide neuropathy

This study was performed to evaluate the effect of prolonged forced running on rat lower limb muscles with acrylamide neuropathy. Twenty-four 4-week-old rats were divided into three groups of eight rats. Acrylamide was given to two groups of sixteen rats to induce mild paralysis. Eight rats with acrylamide injections were forced to run 3,200 m/day on a treadmill for five weeks. Running activities slowed the rate of body weight gain and aggravated paralysis. Although the wet weight of tibialis anterior (TA) and extensor digitorum longus (EDL) muscles was reduced by running, that of soleus (SOL) muscles was unchanged. The ratio of their weight to their body weight (BW) remained constant regardless of exercise. Protein content (PC) of muscles was not altered by exercise, either. We postulated that exercise-induced worsening of paralysis in acrylamide neuropathy rats was not caused by muscle pathology. Deterioration of neuropathic condition due to exercise was suggested.

Differentiated adipose-derived stem cells promote peripheral nerve regeneration.

Many reports have indicated that adipose-derived stem cells (ADSCs) are effective for nerve regeneration. We investigated nerve regeneration by combining a polyglycolic acid collagen (PGA-c) tube, which is approved for clinical use, and Schwann cell-like differentiated ADSCs (dADSCs). Fifteen-millimeter-long gaps in the sciatic nerve of rats were bridged in each group using tubes (group I), with tubes injected with dADSCs (group II), or by resected nerve (group III). Axonal outgrowth was greater in group II than in group I. Tibialis anterior muscle weight revealed recovery only in group III. Latency in nerve conduction studies was equivalent in group II and III, but action potential was lower in group II. Transplanted dADSCs maintained Schwann cell marker expression. ATF3 expression level in the dorsal root ganglia was equivalent in groups II and III. dADSCs maintained their differentiated state in the tubes and are believed to have contributed to nerve regeneration.

Long‐Term High‐Fat Feeding Reduces the Numbers of Satellite Cells and Myonuclei in the Adult Murine Skeletal Muscle Fibers

High‐Fat Diet (HFD) affects the skeletal muscle fibers at both morphological and functional levels. At a functional level, it has been reported that HFD reduces the oxidative capacity and leads to insulin resistance in the skeletal muscle tissue. However, there are very limited and conflicting information about the effects of HFD on the morphology of skeletal muscle fibers. This study aimed to clarify the effects of long‐term feeding with HFD on murine skeletal muscle fibers. These effects include the size of skeletal muscle fibers, frequency of satellite cells, and number of myonuclei within the fibers. Adult male mice of the C57BL/6 strain were randomly divided into 2 groups; a control group, and a HFD group. Each group contained four mice. Food and water were provided ad libitum. At the end of a twenty‐week feeding period, the tibialis anterior (TA) muscle was dissected out from each animal and weighed. Immunocytochemical and morphometric image analyses techniques were applied to quantify the fiber diameter, satellite cell frequency, and number of myonuclei per fiber cross‐section. The HFD group showed a significant (P&lt;0.01) decrease in TA muscle weight compared to control group. However, a significant (P&lt;0.05) increase in the muscle fiber diameter was found in the HFD group compared to control one. In addition, significant reductions (P&lt;0.01) in satellite cell frequency and number of myonuclei were detected in the HFD group compared to control group. This study showed that long‐term feeding with HFD increases the size of skeletal muscle fibers, which may be attributed to the accumulation of intramyocellular lipids. In addition, it reveals that HFD decreases the numbers of satellite cells and myonuclei within the murine skeletal muscle fibers.Support or Funding InformationFunds for this study were provided by a seeding grant to MZA from the College of Medicine and Health Sciences at United Arab Emirates University.

840 Myostatin Deficiency Inhibits Muscle Wasting and Improves Bacterial Clearance and Survival in Septic Mice

Abstract Introduction Sepsis is a leading cause of the mortality of burn patients. Muscle wasting is a major complication of sepsis and burn injury and negatively affects clinical outcomes of patients with sepsis and burn injury. Myostatin (MSTN) is a myokine that causes muscle atrophy by activating the activin type 2 receptor (ActRII) pathway and myostatin deficiency increases skeletal muscle mass. However, a role of myostatin in critical illness (e.g., sepsis, burn injury)-induced muscle wasting has not yet been investigated. Moreover, a role of muscle wasting in immune suppression and mortality in sepsis is unknown. Methods Sepsis was induced by cecum ligation and puncture (CLP) in male MSTN-deficient mice at 8 weeks of age and age- and body weight (BW)-matched wild type (WT) mice. Survival was monitored for 14 days. Bacterial clearance was evaluated at 16 h after CLP by measuring bacterial load in the peritoneal cavity and circulation. The weight of gastrocnemius (GC), tibialis anterior (TA) and soleus (SOL) muscle and cross-sectional areas of GC were measured before and at 14 days after CLP. To evaluate neutrophil organ infiltration, myeloperoxidase (MPO) activity was measured in the liver and kidney at 16 h after CLP. To evaluate liver dysfunction, acute kidney injury and inflammatory response, plasma levels of AST, ALT, NGAL and high mobility group box 1 (HMGB1) were measured at 16 h after CLP. Protein expression of Murf-1 and Atrogin-1, key players in muscle wasting, and ActRIIB was evaluated in GC muscle by immunoblotting at 3 days and 16 h after CLP, respectively. Results MSTN deficiency increased skeletal muscle mass and inhibited sepsis-induced muscle wasting compared with BW-matched WT mice. Sepsis-induced increase in Murf-1, but not Atrogin-1, expression in muscle was attenuated by MSTN deficiency. CLP increased protein expression of ActRIIB in muscle. Moreover, MSTN deficiency reduced mortality of septic mice compared with age- and BW-matched WT mice. MSTN deficiency improved bacterial clearance and ameliorated increases in MPO activity in the liver and kidney and plasma concentrations of AST, ALT, NGAL and HMGB1 in septic mice. Conclusions Our data showed that MSTN deficiency inhibited muscle wasting and improved bacterial clearance and survival in septic mice. These data indicate that MSTN plays an important role in sepsis-induced muscle wasting. Moreover, our findings suggest that muscle wasting may not be just a complication of sepsis but a driver of sepsis development contributing to mortality of septic mice. This study also raises the possibility that muscle wasting and/or activation of the MSTN-ActRII pathway may exacerbate sepsis-induced immune dysfunction. Applicability of Research to Practice Our study identifies the MSTN-ActRII pathway as a novel, potential molecular target to ameliorate muscle wasting and improve survival of septic burned patients.

A rat study of the use of end-to-side peripheral nerve repair as a "babysitting" technique to reduce the deleterious effect of chronic denervation.

Functional recovery is disappointing after surgical repair of nerves that are injured far from their target organs and/or after delayed repair. In the former case, a nerve transfer that transects a distal nerve fascicle to innervate denervated targets is one strategy to promote nerve regeneration and functional recovery. An alternate strategy tested in this study is to perform an end-to-side neurorrhaphy to "babysit" (protect) the denervated distal nerve stump at the time of nerve repair and reduce the deleterious effect of chronic denervation on nerve regeneration. In the hindlimbs of Sprague-Dawley rats, the common peroneal (CP) nerve was transected unilaterally and the distal CP nerve stump inserted through a perineurial window into the intact tibial (TIB) nerve, i.e., CP-TIB end-to-side neurorrhaphy. In the first experiment, TIB nerve motoneurons that had regenerated and/or sprouted axons into the CP nerve within 3 months were stimulated to elicit contractions, and thereafter, identified with retrograde dyes for counting. In the second experiment, the intact TIB nerve was transected and cross-sutured to a 3-month chronically denervated distal CP nerve stump that had either been "protected" by ingrown TIB nerves after CP-TIB neurorrhaphy or remained chronically denervated. Thereafter, the number of retrogradely labeled TIB nerve motoneurons that had regenerated their nerves within 3 months were counted and reinnervated tibialis anterior (TA) muscles weighed. A mean (± SE) of 231 ± 83 TIB nerve motoneurons grew into the end-to-side CP distal nerve stump with corresponding ankle flexion; 32% regenerated their axons and 24% sprouted axons from the intact TIB nerve, eliciting ankle flexor-extensor co-contraction. In the second experiment, after a 3-month period of TIB nerve regeneration, significantly more TIB motoneurons regenerated their axons into "protected" than "unprotected" CP distal nerve stumps within 3 months (mean 332 ± 43.6 vs 235 ± 39.3 motoneurons) with corresponding and significantly higher numbers of regenerated nerve fibers, resulting in significantly better recovery of reinnervated TA muscle weight. These experiments in rats demonstrated that delayed nerve repair is more effective when the deleterious effects of chronic denervation of the distal nerve stump are reduced by protecting the nerve stump with ingrowing nerve fibers across an end-to-side insertion of the distal nerve stump into a neighboring intact nerve. Such an end-to-side neurorrhaphy may be invaluable as a means of preventing the atrophy of distal nerve stumps and target organs after chronic denervation, which allows for effective reinnervation of the protected distal nerve stumps and target organs over distance and time.

Comparison of systematically combined detergent and nuclease‐based decellularization methods for acellular nerve graft: An ex vivo characterization and in vivo evaluation

Little consensus exists regarding which decellularization technique best removes the cellular components while maintaining structural integrity. We aimed to identify the most efficient and safest decellularization method by combining previously established chemical (detergent based) and biological (nuclease based) methods in a systematic manner. Sixty sciatic nerves were harvested from Sprague-Dawley rats and prepared in 120 nerve fragments with 1-cm length. Nerve fragments were randomly divided into six groups and decellularized with six different methods: A, nonionic detergent+amphoteric detergent; B, nonionic detergent+anionic detergent; C, anionic detergent+amphoteric detergent; D, nonionic detergent+nuclease; E, amphoteric detergent+nuclease; and F, anionic detergent+nuclease. The remaining cellular components were evaluated with H&E, DAPI, and S-100 immunohistochemical staining, and DNA content was measured in each sample. The remaining extracellular matrix (ECM) integrity was evaluated with H&E, Masson's trichrome, periodic acid-Schiff, Luxol fast blue, and laminin immunohistochemical staining, and collagen content was measured in each sample. The amphoteric detergent+nuclease method was the best protocol for both cell removal and ECM preservation. In the in vivo study, the nerve allograft that was decellularized with amphoteric detergent+nuclease showed an inferior recovery rate based on the tibialis anterior muscle weight to autograft, but considerable recovery was observed. In conclusion, among the possible systematic combinations of detergent- and nuclease-based methods, the combination of amphoteric detergent and nuclease is currently the most suitable for nerve decellularization in terms of adequate cell removal and sufficient preservation of the ECM.