Adult Mouse Muscle Research Articles

Impact of Extremely Low-Frequency Electromagnetic Fields on Skeletal Muscle of Sedentary Adult Mice: A Pilot Study.

Extremely low-frequency electromagnetic fields (ELF-EMFs) are ubiquitous in industrialized environments due to the continuous use of electrical devices. Our previous studies demonstrated that ELF-EMFs affect muscle cells by modulating oxidative stress and enhancing myogenesis. This pilot study investigated these effects on the skeletal muscles of sedentary adult mice, assessing physiological responses to ELF-EMF exposure and potential modulation by antioxidant supplementation. Male C57BL/6 mice were exposed to ELF-EMFs (0.1 or 1.0 mT) for 1 h/day for up to 5 weeks and fed a standard diet without or with N-acetyl-cysteine (NAC). The results showed transient increases in muscle strength (after 2 weeks of exposure at 1.0 mT), potentially linked to muscle fiber recruitment and activation, revealed by higher PAX7 and myosin heavy chain (MyH) expression levels. After ELF-EMF exposure, oxidative status assessment revealed transient increases in the expression levels of SOD1 and catalase enzymes, in total antioxidant capacity, and in protein carbonyl levels, markers of oxidative damage. These effects were partially reduced by NAC. In conclusion, ELF-EMF exposure affects skeletal muscle physiology and NAC supplementation partially mitigates these effects, highlighting the complex interactions between ELF-EMFs and antioxidant pathways in vivo. Further investigations on ELF-EMFs as a therapeutic modality for muscle health are necessary.

Better maintenance of enzymatic capacity and higher levels of substrate transporter proteins in skeletal muscle of aging female mice.

This study investigated sex-specific differences in high-energy phosphate, glycolytic, and mitochondrial enzyme activities and also metabolite transporter protein levels in the skeletal muscles of adult (5 months old), middle-aged (12 months old), and advanced-aged (24 months old) mice. While gastrocnemius glycogen content increased with age regardless of sex, gastrocnemius triglyceride levels increased only in advanced-aged female mice. Aging decreased creatine kinase and adenylate kinase activities in the plantaris muscle of both sexes and in the soleus muscle of male mice but not in female mice. Irrespective of sex, phosphofructokinase and lactate dehydrogenase (LDH) activities decreased in the plantaris and soleus muscles. Additionally, hexokinase activity in the plantaris muscle and LDH activity in the soleus muscle decreased to a greater extent in aged male mice compared with those in aged female mice. Mitochondrial enzyme activities increased in the plantaris muscle of aged female mice but did not change in male mice. The protein content of the glucose transporter 4 in the aged plantaris muscle and fatty acid translocase/cluster of differentiation 36 increased in the aged plantaris and soleus muscles of both sexes, with a significantly higher content in female mice. These findings suggest that females possess a better ability to maintain metabolic enzyme activity and higher levels of metabolite transport proteins in skeletal muscle during aging, despite alterations in lipid metabolism. Our data provide a basis for studying muscle metabolism in the context of age-dependent metabolic perturbations and diseases that affect females and males differently.

A mechano- and heat-gated two-pore domain K+ channel controls excitability in adult zebrafish skeletal muscle.

TRAAK channels are mechano-gated two-pore-domain K+ channels. Up to now, activity of these channels has been reported in neurons but not in skeletal muscle, yet an archetype of tissue challenged by mechanical stress. Using patch clamp methods on isolated skeletal muscle fibers from adult zebrafish, we show here that single channels sharing properties of TRAAK channels, i.e., selective to K+ ions, of 56 pS unitary conductance in the presence of 5 mM external K+, activated by membrane stretch, heat, arachidonic acid, and internal alkaline pH, are present in enzymatically isolated fast skeletal muscle fibers from adult zebrafish. The kcnk4b transcript encoding for TRAAK channels was cloned and found, concomitantly with activity of mechano-gated K+ channels, to be absent in zebrafish fast skeletal muscles at the larval stage but arising around 1 mo of age. The transfer of the kcnk4b gene in HEK cells and in the adult mouse muscle, that do not express functional TRAAK channels, led to expression and activity of mechano-gated K+ channels displaying properties comparable to native zebrafish TRAAK channels. In whole-cell voltage-clamp and current-clamp conditions, membrane stretch and heat led to activation of macroscopic K+ currents and to acceleration of the repolarization phase of action potentials respectively, suggesting that heat production and membrane deformation associated with skeletal muscle activity can control muscle excitability through TRAAK channel activation. TRAAK channels may represent a teleost-specific evolutionary product contributing to improve swimming performance for escaping predators and capturing prey at a critical stage of development.

Unilateral Hypofunction of the Masseter Leads to Molecular and 3D Morphometric Signs of Atrophy in Ipsilateral Agonist Masticatory Muscles in Adult Mice.

Mice are commonly used to study mandibular dynamics due to their similarity in chewing cycle patterns with humans. Adult mice treated unilaterally with botulinum toxin type A (BoNTA) in the masseter exhibit atrophy of this muscle characterized by an increase in the gene expression of atrophy-related molecular markers, and a reduction in both muscle fiber diameter and muscle mass at 14d. However, the impact of this muscle imbalance on the non-treated masticatory muscles remains unexplored. Here, we hypothesize that the unilateral masseter hypofunction leads to molecular and 3D morphometric signs of atrophy of the masseter and its agonist masticatory muscles in adult mice. Twenty-three 8-week-old male BALB/c mice received a single injection of BoNTA in the right masseter, whereas the left masseter received the same volume of saline solution (control side). Animals were euthanized at 2d, 7d, and 14d, and the masticatory muscles were analyzed for mRNA expression. Five heads were harvested at 14d, fixed, stained with a contrast-enhanced agent, and scanned using X-ray microtomography. The three-dimensional morphometric parameters (the volume and thickness) from muscles in situ were obtained. Atrogin-1/MAFbx, MuRF-1, and Myogenin mRNA gene expression were significantly increased at 2 and 7d for both the masseter and temporalis from the BoNTA side. For medial pterygoid, increased mRNA gene expression was found at 7d for Atrogin-1/MAFbx and at 2d-7d for Myogenin. Both the volume and thickness of the masseter, temporalis, and medial pterygoid muscles from the BoNTA side were significantly reduced at 14d. In contrast, the lateral pterygoid from the BoNTA side showed a significant increase in volume at 14d. Therefore, the unilateral hypofunction of the masseter leads to molecular and morphological signs of atrophy in both the BoNTA-injected muscle and its agonistic non-injected masticatory muscles. The generalized effect on the mouse masticatory apparatus when one of its components is intervened suggests the need for more clinical studies to determine the safety of BoNTA usage in clinical dentistry.

Multiple Factors Influence the Incubation Period of ALS Prion-like Transmission in SOD1 Transgenic Mice.

Mutations in superoxide dismutase 1 (SOD1) that are associated with amyotrophic lateral sclerosis (ALS) cause its misfolding and aggregation. Prior studies have demonstrated that the misfolded conformation of ALS-SOD1 can template with naïve SOD1 "host proteins" to propagate, spread, and induce paralysis in SOD1 transgenic mice. These observations have advanced the argument that SOD1 is a host protein for an ALS conformer that is prion-like and experimentally transmissible. Here, we investigated the propagation of different isolates of G93A-SOD1 ALS conformers using a paradigm involving transmission to mice expressing human G85R-SOD1 fused to yellow fluorescent protein (G85R-SOD1:YFP). In these studies, we also utilized a newly developed line of mice in which the G85R-SOD1:YFP construct was flanked by loxp sites, allowing its temporal and spatial regulation. We used methods in which the G93A ALS conformers were injected into the sciatic nerve or hindlimb muscle of adult transgenic mice. We observed that the incubation period to paralysis varied significantly depending upon the source of inoculum containing misfolded G93A SOD1. Serial passage and selection produced stable isolates of G93A ALS conformers that exhibited a defined minimum incubation period of ~2.5 months when injected into the sciatic nerve of young adult mice. As expected, neuronal excision of the transgene in loxpG85R-SOD1:YFP mice blocked induction of paralysis by transmission of G93A ALS conformers. Our findings indicate that G93A ALS conformers capable of inducing disease require neuronal expression of a receptive host SOD1 protein for propagation, with a defined incubation period to paralysis.

A High-Phosphorus Diet Moderately Alters the Lipidome and Transcriptome in the Skeletal Muscle of Adult Mice.

A high phosphorus intake has been associated with various metabolic disorders, including chronic kidney disease, cardiovascular disease, and osteoporosis. Recent studies have demonstrated the effects of dietary phosphorus on lipid and glucose metabolism. This study investigated the impact of a high-phosphorus diet on mouse skeletal muscle lipid composition and gene transcription. Adult male mice (n = 12/group) received either a diet with an adequate (0.3%) or a high (1.2%) phosphorus concentration for 6 weeks. The lipidome analysis showed that among the 17 analyzed lipid classes, the concentrations of three classes were reduced in the high phosphorus group compared to the adequate phosphorus group. These classes were phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and lysophosphatidylcholine (LPC) (p < 0.05). Out of the three hundred and twenty-three individual lipid species analyzed, forty-nine showed reduced concentrations, while three showed increased concentrations in the high phosphorus group compared to the adequate phosphorus group. The muscle transcriptome analysis identified 142 up- and 222 down-regulated transcripts in the high phosphorus group compared to the adequate phosphorus group. Gene set enrichment analysis identified that genes that were up-regulated in the high phosphorus group were linked to the gene ontology terms "mitochondria" and "Notch signaling pathway", whereas genes that were down-regulated were linked to the "PI3K-AKT pathway". Overall, the effects of the high-phosphorus diet on the muscle lipidome and transcriptome were relatively modest, but consistently indicated an impact on lipid metabolism.

Dexmedetomidine alleviates anxiety-like behaviors in female mice with musculoskeletal pain through SIRT1/p53 axis

BackgroundMusculoskeletal pain is the most common form of chronic pain. Anxiety increases pain intensity and appears to have a major impact on the prevalence and also disability of musculoskeletal pain in women. We examined the effect of dexmedetomidine (DEX) on anxiety-like behaviors associated with musculoskeletal pain and the underlying molecular mechanism in female mice. MethodsMusculoskeletal pain was induced by injection of acidified saline into the gastrocnemius muscle in adult female mice, and the von Frey filament test is used to measure mechanical sensitivity. DEX and EX527 (SIRT1 inhibitor) were administered after modelling. Behavioral tests were used for anxiety and motor activity tests. SIRT1, p53 and acetyl-p53 were quantified by Western blot. ResultsAdult female mice with musculoskeletal pain exhibit increased fear-like behavior by reducing SIRT1 expression in the medial prefrontal cortex (mPFC). While administration of DEX was able to alleviate mechanical hypersensitivity and anxiety-like behaviors by blocking SIRT1 decline and acetyl-p53 upregulation in mPFC, EX527 inhibited acetyl-p53 rise and reversed the antinociceptive and anxiolytic effects of DEX. ConclusionDEX may alleviate anxiety-like behaviors in mice with musculoskeletal pain via the SIRT1/p53 axis. These results suggest that DEX may have a potential therapeutic role in musculoskeletal pain-induced anxiety.

A novel mitochondrial complex I ROS inhibitor partially improves muscle regeneration in adult but not old mice

It is unclear whether mitochondrial dysfunction and redox stress contribute to impaired age-related muscle regenerative capacity. Here we characterized a novel compound, BI4500, that inhibits the release of reactive oxygen species (ROS) from the quinone site in mitochondrial complex I (site IQ). We tested the hypothesis that ROS release from site IQ contributes to impaired regenerative capacity in aging muscle. Electron transfer system site-specific ROS production was measured in adult and aged mouse isolated muscle mitochondria and permeabilized gastrocnemius fibers. BI4500 inhibited ROS production from site IQ in a concentration-dependent manner (IC50 = ∼985 nM) by inhibiting ROS release without impairing complex I-linked respiration. In vivo BI4500 treatment decreased ROS production from site IQ. Muscle injury and sham injury were induced using barium chloride or vehicle injection to the tibialis anterior (TA) muscle in adult and aged male mice. On the same day as injury, mice began a daily gavage of 30 mg/kg BI4500 (BI) or placebo (PLA). Muscle regeneration (H&E, Sirius Red, Pax7) was measured at 5 and 35 days after injury. Muscle injury increased centrally nucleated fibers (CNFs) and fibrosis with no treatment or age effect. There was a significant age by treatment interaction for CNFs at 5- and 35-days post injury with significantly more CNFs in BI adults compared to PLA adults. Muscle fiber cross-sectional area (CSA) recovered significantly more in adult BI mice (−89 ± 365 μm2) compared to old PLA (−599 ± 153 μm2) and old BI (−535 ± 222 μm2, mean ± SD). In situ TA force recovery was measured 35 days after injury and was not significantly different by age or treatment. Inhibition of site IQ ROS partially improves muscle regeneration in adult but not old muscle demonstrating a role for CI ROS in the response to muscle injury. Site IQ ROS does not contribute to impaired regenerative capacity in aging.

Targeted regulation of TAK1 counteracts dystrophinopathy in a DMD mouse model.

Muscular dystrophies make up a group of genetic neuromuscular disorders that involve severe muscle wasting. TGF-β-activated kinase 1 (TAK1) is an important signaling protein that regulates cell survival, growth, and inflammation. TAK1 has been recently found to promote myofiber growth in the skeletal muscle of adult mice. However, the role of TAK1 in muscle diseases remains poorly understood. In the present study, we have investigated how TAK1 affects the progression of dystrophic phenotype in the mdx mouse model of Duchenne muscular dystrophy (DMD). TAK1 is highly activated in the dystrophic muscle of mdx mice during the peak necrotic phase. While targeted inducible inactivation of TAK1 inhibits myofiber injury in young mdx mice, it results in reduced muscle mass and contractile function. TAK1 inactivation also causes loss of muscle mass in adult mdx mice. By contrast, forced activation of TAK1 through overexpression of TAK1 and TAB1 induces myofiber growth without having any deleterious effect on muscle histopathology. Collectively, our results suggest that TAK1 is a positive regulator of skeletal muscle mass and that targeted regulation of TAK1 can suppress myonecrosis and ameliorate disease progression in DMD.

Overexpression of GRK2 in vascular smooth muscle leads to inappropriate hypertension and acute heart failure as in clinical scenario 1

Clinical scenario 1 (CS1) is acute heart failure (HF) characterized by transient systolic blood pressure (SBP) elevation and pulmonary congestion. Although it is managed by vasodilators, the molecular mechanism remains unclear. The sympathetic nervous system plays a key role in HF, and desensitization of cardiac β-adrenergic receptor (AR) signaling due to G protein-coupled receptor kinase 2 (GRK2) upregulation is known. However, vascular β-AR signaling that regulates cardiac afterload remains unelucidated in HF. We hypothesized that upregulation of vascular GRK2 leads to pathological conditions similar to CS1. GRK2 was overexpressed in vascular smooth muscle (VSM) of normal adult male mice by peritoneally injected adeno-associated viral vectors driven by the myosin heavy chain 11 promoter. Upregulation of GRK2 in VSM of GRK2 overexpressing mice augmented the absolute increase in SBP (+ 22.5 ± 4.3 mmHg vs. + 36.0 ± 4.0 mmHg, P < 0.01) and lung wet weight (4.28 ± 0.05 mg/g vs. 4.76 ± 0.15 mg/g, P < 0.01) by epinephrine as compared to those in control mice. Additionally, the expression of brain natriuretic peptide mRNA was doubled in GRK2 overexpressing mice as compared to that in control mice (P < 0.05). These findings were similar to CS1. GRK2 overexpression in VSM may cause inappropriate hypertension and HF, as in CS1.

Skeletal muscle transcriptome is affected by age in severely burned mice

Severe burn results in muscle wasting affecting quality of life in both children and adults. Biologic metabolic profiles are noticeably distinctive in childhood. We posit that muscle gene expression profiles are differentially regulated in response to severe burns in young animals. Twelve C57BL6 male mice, including young (5 weeks-old) and adults (11 weeks-old), received either scald burn, or sham procedure. Mouse muscle tissue was harvested 24 h later for Next Generation Sequence analysis. Our results showed 662 downregulated and 450 upregulated genes in gastrocnemius of young mice compared to adults without injury. After injury, we found 74/75 downregulated genes and 107/128 upregulated genes in both burned groups compared to respective uninjured age groups. VEGFA-VEGFR2, focal adhesion, and nuclear receptor meta-pathways were the top 3 gene pathways undergoing a differential change in response to age. Of note, the proteasome degradation pathway showed the most similar changes in both adult and young burned animals. This study demonstrates the characteristic profile of gene expression in skeletal muscle in young and adult burned mice. Prominent age effects were revealed in transcriptional levels with increased alterations of genes, miRNAs, pathways, and interactions.

Prenatal EGCG consumption causes obesity and perturbs glucose homeostasis in adult mice

Epigallocatechin gallate (EGCG) has a wide consumption for its health advantages. The current study investigates the effects of prenatal EGCG administration on glucose metabolism and obesity in adulthood. Pregnant C57BL/6J mice were supplemented with EGCG in drinking water (3 µg/mL) for 16 d. Abdominal obesity was observed in both male and female adult mice, which was associated with the upregulation of adipose-specific genes, including C/ebpα and Srebf1 (Srebf1 only in males), and the downregulation of genes related to lipolysis, such as Acox1, Atgl and Pdk4 (only in males) in visceral adipose tissue. Elevated fasting glucose levels and hyperinsulinemia were observed in adult males, while females exhibit lower glucose level in glucose tolerance test, which might be due to reduced glucagon levels. Though hepatic expression of the insulin receptor signaling pathway was upregulated in males and was not altered in females, prenatal treatment with EGCG downregulated the expression of this signaling pathway in the skeletal muscle of adult mice, which was further demonstrated in primary human skeletal muscle cells treated with EGCG. The methylation levels in promotor of genes related to the insulin receptor signaling were matched with their transcription in mice, while the expression of acetylated histones was downregulated in human skeletal muscle cells. These results suggest that EGCG consumption during pregnancy should be a risk factor for the disruption of glucose homeostasis in adulthood.

Effect of Estrogen on Bronchiolar Smooth Muscle and Peri-Bronchial Lymphocytic Infiltration in Adult Male Mice

Objective: To study the effect of estrogen on lungs of adult male mice by assessing and comparing the following histological parameters including bronchiolar smooth muscle size and peri-bronchial lymphocytic Infiltration. Study Design: Randomized control trial Place and Duration of Study: National institute of Health sciences, Islamabad from 1st October 2018 to 31st July 2019. Methodology: Ninety BALB/c mice divided into 2 groups (n=30 per group) were enrolled. The control (male) group received only distilled water, while the interventional groups received pills (estradiol valerate) mixed in distal water according to body weight of the mice for (60) day. Results: 10% of the total mice had nil, 60% had mild, 30% of the total showed moderate while no severe peri-bronchiolar lymphocytic infiltration in response to estrogen. The results also showed that estrogen produced marked hyperplasia of bronchial smooth muscle cells. Conclusion: Estrogen is the sexual hormones which modulate inflammatory processes in the lungs producing pulmonary inflammatory responses leading to asthma and causes hyperplasia of the bronchiolar smooth muscles. Keywords: Estrogen, Asthma, Bronchiolar smooth muscle, Peri-bronchial lymphocytic infiltration, BALB/c mice

FACS-isolation and Culture of Fibro-Adipogenic Progenitors and Muscle Stem Cells from Unperturbed and Injured Mouse Skeletal Muscle.

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along fibrogenic, adipogenic, osteogenic, or chondrogenic lineage. Together with muscle stem cells (MuSCs), FAPs play a critical role in muscle homeostasis, repair, and regeneration, while actively maintaining and remodeling the extracellular matrix (ECM). In pathological conditions, such as chronic damage and muscular dystrophies, FAPs undergo aberrant activation and differentiate into collagen-producing fibroblasts and adipocytes, leading to fibrosis and intramuscular fatty infiltration. Thus, FAPs play a dual role in muscle regeneration, either by sustaining MuSC turnover and promoting tissue repair or contributing to fibrotic scar formation and ectopic fat infiltrates, which compromise the integrity and function of the skeletal muscle tissue. A proper purification of FAPs and MuSCs is a prerequisite for understanding the biological role of these cells in physiological as well as in pathological conditions. Here, we describe a standardized method for the simultaneous isolation of FAPs and MuSCs from limb muscles of adult mice using fluorescence-activated cell sorting (FACS). The protocol describes in detail the mechanical and enzymatic dissociation of mononucleated cells from whole limb muscles and injured tibialis anterior (TA) muscles. FAPs and MuSCs are subsequently isolated using a semi-automated cell sorter to obtain pure cell populations. We additionally describe an optimized method for culturing quiescent and activated FAPs and MuSCs, either alone or in coculture conditions.

Characterizing Subsynaptic Myonuclei in Aged Muscle

IntroductionDuring aging, the neuromuscular junction (NMJ) undergoes structural fragmentation and functional deficits that alter neuromuscular communication, impair muscle contraction, and contribute to muscle atrophy. At the NMJ, myonuclei accumulate and localize in a fashion that juxtaposes three‐to‐six myonuclei in the postsynaptic region. Although the entire role of these so‐called subsynaptic myonuclei remains enigmatic, previous literature suggests they contribute to gene regulation of synapse‐specific proteins required for maintaining the structural integrity of the synaptic architecture. Interestingly, a recent report find that NMJs in muscles of aged mice contain fewer subsynaptic myonuclei compared with muscles of adult mice in parallel with degenerating or degenerated endplates. Furthermore, disturbance of the nuclear envelope by in‐vivo knock‐out of Lmna appears to accelerate NMJ degeneration and reduces the number of myonuclei confined to the endplate. Therefore, with the observed reductions in subsynaptic myonuclei in degenerated NMJs, our study aims to further examine and characterize subsynaptic myonuclei and their role in NMJ degeneration.MethodsGastrocnemius muscles were harvested from Young (6 months) and Aged (28 months) wild type mice and 6‐ and 12‐month‐old Sod1KO mice (N=3/group) and briefly fixed in 4% PFA for 10 minutes. Myofiber bundles were mechanically teased under a dissecting microscope and subjected to immunohistochemistry for visualization of motor neurons (neurofilament‐NF and synaptic vesicle glycoprotein 2‐SV2), motor end plates (alpha‐bungarotoxin), and myonuclei (nesprin 1 and 4’,6‐diamidino‐2‐phenylindole‐DAPI). Myofiber bundles were then mounted onto a slide and imaged via High‐sensitivity confocal microscopy. Captured images were then 3D projected in FIJI for assessment and quantification of subsynaptic myonuclei, innervation status, and post synaptic area assessed via aNMJ morph.ResultsIn young mice with primarily fully innervated endplates, the number of myonuclei associated with the endplate was lower than the number of synaptic myonuclei in muscles of aged and Sod1KO mice, both of which contain greater numbers of partially and non‐innervated endplates.ConclusionThere may be a greater number of subsynaptic myonuclei localized to degenerating or degenerated endplates, although whether the change in nuclear number is a consequence of NMJ degeneration or contributes to the denervation cannot be established from this study.

Older Mice Show Decreased Regeneration of Neuromuscular Junctions Following Lengthening‐Contraction Injury

Progressive muscle atrophy and loss of muscle strength associated with old age have been well documented. Although impairments in the ability of old mice to regenerate skeletal muscle following injury have been demonstrated, less is known about the way age modulates the regenerative response of motor neurons and the neuromuscular junctions (NMJ) of mice following contraction‐induced injury. The inability for NMJs to effectively regenerate following an injury could lead to the death of the denervated muscle fibers and therefore play a contributing role to age‐related sarcopenia. To investigate the relationship between age and NMJ regeneration following injury, extensor digitorum longus (EDL) muscles of adult (8‐9 months), middle‐aged (18‐19 months), and old mice (27‐28 months) were subjected to a protocol of repeated lengthening‐contractions that resulted in an acute force deficit of ~70%. After 28 days, muscles were evaluated for maximum force generating capacity, muscle mass, NMJ innervation, and endplate fragmentation. Cross sections of muscles taken at three days post injury showed extensive infiltration of mononuclear cells and widespread tissue disruption consistent with the induction of severe injury to the muscles. At 28 days, maximum isometric force recovered completely in adult mice, but was significantly reduced in middle aged and old mice when compared to muscles of adult mice. In each case, approximately 29% of fibers in the cross sections displayed containing central nuclei, whereas controls showed only 2.9%, indicating that the injured muscle fibers had undergone degeneration and regeneration. Analysis of the innervation of the NMJs in the regenerated muscles showed a moderate decrease in the number of fully innervated endplates in old mice as compared to adult and middle‐aged mice. When the architecture of the endplates in regenerated muscles was examined, the relative number of NMJs with intact postsynaptic structure varied little between the age groups, although control muscles of old mice showed an increase in the number of fragmented endplates compared with the adult and middle aged mice. Thus, the diminished ability of the skeletal muscle of old mice to recover following injury may be, in part, due to an age‐related decrease in the ability to adequately regenerate motor neurons and perhaps form NMJs in the injured muscle.

Combined transcriptomic, proteomic and synthesis profiling reveal distinct proteostatic signatures for skeletal muscle of adult and old mice

Muscle mass is reduced with age and numerous studies have examined the potential role of global changes in proteostasis, through protein synthesis and/or degradation in the overall loss of muscle mass with age, although no consensus has been reached.We hypothesise that changes in expression, synthesis and degradation may be very different for individual proteins and such findings may be overshadowed by examining global changes in protein turnover. In addition, we hypothesize that alterations in the synthesis rates of specific individual proteins in the muscles of old mice when compared with that of adult are likely to impact on the age‐related loss of muscle mass. We used deuterated water labelling to determine the synthesis rates of individual muscle proteins and have integrated these data with RNA Sequencing data and protein abundances to provide a powerful analysis of individual proteins in muscles of adult (6‐8months) and old (26months) C57BL/6J mice by which time ~15% of gastrocnemius muscle had been lost.For protein synthesis analysis, all animals received an initial loading dose of 2H2O via two IP injections of 99% 2H2O in PBS, 4hrs apart. Mice were then given 8% 2H2O in normal drinking water to maintain plasma enrichment of 2H2O at around 4.5% and gastrocnemius muscles were harvested for up to 60 days when mice were culled. Muscle RNA was isolated, RNA libraries generated using poly‐A selection, sequenced using Illumina HiSeq 4000 platform and analysed with DESEQ2 and gene ontology and pathway tools. To determine the effect of age on differentially expressed genes, protein abundance and turnover, we analysed the common list of 185 proteins and cognate transcripts for which we had data on transcript and turnover parameters.Functional annotation of proteins in the unlabelled dataset demonstrated an upregulation of protein degradation pathways with aging, including ubiquitin protein ligase binding and the unfolded protein response suggesting a clear role of protein degradation as a major contributor to the loss of muscle mass with age in mice. The top four KEGG disease pathways enriched when comparing protein abundance in the muscles of adult and old mice were all degenerative diseases caused or exacerbated by protein misfolding and accumulation. For most proteins, synthesis rates were unchanged, and thus, any change in abundance has to be reconciled with a commensurate change in degradation rate. These global data demonstrate that the net negative protein balance within the muscles of old mice is driven by elevated protein degradation rather than reduced translation. However, there were some notable outliers from the analysis of protein synthesis data showing a decrease in the synthesis of several structural proteins and an increase in the synthesis of metabolic or cytoprotective proteins in muscles of old compared with adult mice. The rate of synthesis of a protein was not always associated with transcript level suggesting caution in extrapolating the suggestion that static mRNA levels reflect the rate of protein synthesis or the protein levels in skeletal muscle.

Deletion of the Vitamin D Receptor in Skeletal Muscle is Associated with Improved Glucose Tolerance and Reduced Muscle Function

Skeletal muscle insulin resistance contributes to the pathogenesis of type 2 diabetes mellitus. Epidemiological studies have demonstrated that vitamin D deficiency is associated with the presence of type 2 diabetes in humans. To probe the role of the vitamin D receptor (VDR), an intracellular and nuclear protein that mediates the bioactivity of the active vitamin D ligand, 1,25‐dihydroxyvitamin D, in skeletal muscle mediated glucose disposal, we deleted the skeletal muscle VDR in adult mice. We assessed glucose disposal, insulin concentrations and sensitivity, and skeletal muscle gene expression in Vdr knockout and wild‐type Vdr mice. Following the induction of a skeletal muscle‐specific Crerecombinase with tamoxifen in adult male Vdrfl/flmice, efficient deletion of the Vdrwas demonstrated. In skeletal muscle‐specific Vdrknockout mice, basal glucose concentrations were similar to those in wild‐type Vdrmice. In Vdrknockout mice, blood glucose concentrations decreased more rapidly following the administration of intraperitoneal glucose than in control mice expressing wild‐type Vdr (Figure 1A and Figure 1B). The AUC for glucose concentrations in Vdrfl/fl.actin iCre mice treated with tamoxifen was reduced by 20% (p=0.015) compared to the AUC of blood glucose concentrations prior to tamoxifen treatment. Pre‐and post‐treatment AUC’s for blood glucose in Vdrfl/fl.actin iCre mice treated with vehicle and in Vdrfl/flmice treated with tamoxifen were unchanged (p=0.97,0.23). Plasma insulin concentrations were identical in knockout and wild‐type Vdr mice following a bolus of glucose, demonstrating that deletion of the Vdr in skeletal muscle did not influence the release of insulin in response to glucose (one‐way ANOVA, p=0.45). Insulin sensitivity was similar in Vdrknockout and wild‐type Vdrmice. Gene expression analysis of skeletal muscle mRNA derived from Vdrknockout mice and wild‐type mice demonstrated an increase in the expression of genes associated with oxidative phosphorylation, fatty acid oxidation and adipogenesis pathways that could potentially alter glucose utilization. We conclude that selective deletion of the Vdr in the skeletal muscle of adult mice results in more efficient glucose disposal than in wild‐type Vdrmice; basal and glucose‐stimulated insulin serum concentrations are identical in knock‐out and wild type VDR mice with no changes in insulin sensitivity. The data suggest that modulation of the vitamin‐D endocrine system is unlikely to improve glucose utilization.

Botulinum toxin injection in masseter muscle evokes musculoskeletal impairment of the masticatory system

The masticatory system is a finely coordinated machine, where minimal deregulation induces alterations of the whole system. Botulinum toxin type A (BoNTA) injection in masseter muscle is widely used as a clinical/aesthetic procedure. However, the non‐desirable effects of masseter muscle paralysis in neighbor masticatory muscles or mandibular bone are unknown. In the current work, we aimed to evaluate musculoskeletal loss at the masticatory system after BoNTA‐injection in masseter muscle of adult mice.Unilateral paralysis of masseter muscles was induced in male BalbC mice (8 weeks‐old) by single‐injection of BoNTA (0.2U/10 µL) in the right muscle and saline solution in the left muscle. Masticatory muscles (masseter, temporalis, lateral‐ and medial‐pterygoid) and mandibles were dissected after 2‐7‐14d for molecular measurements (qPCR), histology, or morphology (microcomputed tomography). All procedures were approved by the IACUC‐Universidad de Chile (# 20381‐ODO‐UCH). T‐test or Wilcoxon‐test were used for statistical comparisons between BoNTA‐ and Control‐side (n=3‐10, p&lt;0,05).The expression of molecular markers of atrophy (Atrogin1, Murf1) and regeneration (myogenin) significantly increased in the BoNTA‐side, as early as 2‐7d, in the intervened masseter but also in the ipsilateral temporalis and medial pterygoid muscles. There was a time‐dependent reduction in masseter fibers diameter from 2d to 14d. Masseter, temporalis, and medial pterygoid’s volumes significantly reduced in the BoNTA‐injected side at 14d. Interestingly, the volume of lateral pterygoid increased in the BoNTA side, with no change in atrophy/regeneration markers. Microstructural analysis of mandibular condyles demonstrated a progressive bone loss in the BoNTA‐injected side, starting as early as 7d and worsening 14d after the intervention.In conclusion, BoNTA injection in the masseter muscle impairs the whole masticatory system, inducing atrophy of masseter but also the ipsilateral temporalis and medial pterygoid, and even early‐bone loss of the mandibular condyle at the injected side. Then, clinical uses of BoNTA in masticatory muscles should be avoided or carried out under strict morpho‐functional monitoring.

Supraphysiological activation of TAK1 promotes skeletal muscle growth and mitigates neurogenic atrophy

Skeletal muscle mass is regulated through coordinated activation of multiple signaling pathways. TAK1 signalosome has been found to be activated in various conditions of muscle atrophy and hypertrophy. However, the role and mechanisms by which TAK1 regulates skeletal muscle mass remain less understood. Here, we demonstrate that supraphysiological activation of TAK1 in skeletal muscle of adult mice stimulates translational machinery, protein synthesis, and myofiber growth. TAK1 causes phosphorylation of elongation initiation factor 4E (eIF4E) independent of mTOR. Inactivation of TAK1 disrupts neuromuscular junction morphology and causes deregulation of Smad signaling. Using genetic approaches, we demonstrate that TAK1 prevents excessive loss of muscle mass during denervation. TAK1 favors the nuclear translocation of Smad4 and cytoplasmic retention of Smad6. TAK1 is also required for the phosphorylation of eIF4E in denervated skeletal muscle. Collectively, our results demonstrate that TAK1 supports skeletal muscle growth and prevents neurogenic muscle atrophy in adult mice.