Mouse Gastrocnemius Research Articles

Sema3a-Nrp1 Signaling Mediates Fast-Twitch Myofiber Specificity of Tw2+ Cells.

We previously identified a unique population of interstitial muscle progenitors, marked by expression of the Twist2 transcription factor, which fuses specifically to type IIb/x fast-twitch myofibers. Tw2+ progenitors are distinct from satellite cells, a muscle progenitor that expresses Pax7 and contributes to all myofiber types. Through RNA sequencing and immunofluorescence, we identify the membrane receptor, Nrp1, as a marker of Tw2+ cells but not Pax7+ cells. We also found that Sema3a, a chemorepellent ligand for Nrp1, is expressed by type I and IIa myofibers but not IIb myofibers. Using stripe migration assays, chimeric cell-cell fusion assays, and a Sema3a transgenic mouse model, we identify Sema3a-Nrp1 signaling as a major mechanism for Tw2+ cell fiber-type specificity. Our findings reveal an extracellular signaling mechanism whereby a cell-surface receptor for a chemorepellent confers specificity of intercellular fusion of a specific muscle progenitor with its target tissue.

Mitochondrial transplantation ameliorates acute limb ischemia

ObjectiveAcute limb ischemia (ALI), the most challenging form of ischemia-reperfusion injury (IRI) in skeletal muscle tissue, leads to decreased skeletal muscle viability and limb function. Mitochondrial injury has been shown to play a key role in skeletal muscle IRI. In previous studies, we have demonstrated that mitochondrial transplantation (MT) is an efficacious therapeutic strategy to replace or to augment mitochondria damaged by IRI, allowing enhanced muscle viability and function in cardiac tissue. In this study, we investigated the efficacy of MT in a murine ALI model. MethodsC57BL/6J mice (male, 10-12 weeks) were used in a model of ALI. Ischemia was induced by applying a tourniquet on the left hindlimb. After 2 hours of ischemia, the tourniquet was released, and reperfusion of the hindlimb was re-established; either vehicle alone (n = 15) or vehicle containing mitochondria (n = 33) was injected directly into all the muscles of the hindlimb. Mitochondria were delivered at concentrations of 1 × 106 to 1 × 109 per gram wet weight to each muscle, and the animals were allowed to recover. Sham mice received no ischemia or injections but were anesthetized for 2 hours and allowed to recover. After 24 hours of recovery, limb function was assessed by DigiGait (Mouse Specifics Inc, Boston, Mass), and animals were euthanized; the gastrocnemius, soleus, and vastus medialis muscles were collected for analysis. ResultsAfter 24 hours of hindlimb reperfusion, infarct size (percentage of total mass) and apoptosis were significantly decreased (P < .001, each) in the gastrocnemius, soleus, and vastus medialis muscles in MT mice compared with vehicle mice for all mitochondrial concentrations (1 × 106 to 1 × 109 per gram wet weight). DigiGait analysis at 24 hours of reperfusion showed that percentage shared stance time was significantly increased (P < .001) and stance factor was significantly decreased (P = .001) in vehicle compared with MT and sham mice. No significant differences in percentage shared stance time (P = .160) or stance factor (P = .545) were observed between MT and sham mice. ConclusionsMT ameliorates skeletal muscle injury and enhances hindlimb function in the murine model of ALI.

Resveratrol shifts energy metabolism to increase lipid oxidation in healthy old mice

ObjectivesThe objective of this work was to determine the specific mechanisms by which resveratrol inhibits lipogenesis and stimulates lipolysis. MethodsTwelve male mice were individually introduced into a metabolic cage for 24 h to measure basal metabolic rate, prior to intervention. They were randomly divided into two groups, resveratrol (RSV) and control (C), and administered resveratrol intraperitoneally or vehicle, respectively, for two consecutive days. After 24 h, the metabolic energy expenditure was again determined for 24 h, before mice were sacrificed. Protein and gene expression of different enzymes related to metabolism in the hepatic tissue, adipose tissue and gastrocnemius of mice were analyzed by RT-PCR, western blot or ELISA. ResultsWe report that resveratrol lowers the respiratory quotient in old mice and that this may be due to the activation of fatty acid mobilization from white adipose tissue (because hormone-activated lipase expression is increased) and fatty acid transport into mitochondria and eventual oxidation in muscle and liver (because transport enzymes and beta oxidation enzymes are also increased). Indeed, we have observed that resveratrol in vivo causes an increase in the expression and phosphorylation of AMPKα in liver, muscle and adipose tissue and an increase in the expression of acyl-CoA synthetase, of carnitine palmitoyl transferase 1 and of carnitine acylcarnitine translocase, all enzymes involved in lipid catabolism.On the other hand, the levels of acetyl-CoA carboxylase as well as those its product, i.e. malonyl CoA, are decreased. ConclusionsWe conclude that a controlled dose of resveratrol activates fatty acid mobilization and degradation and inhibits fatty acid synthesis in old mice. This is the first time that these effects of resveratrol in lipid metabolism in healthy old (non-obese) animals are reported.

Adipose Tissue-Derived Stromal Cells in Matrigel Impacts the Regeneration of Severely Damaged Skeletal Muscles.

In case of large injuries of skeletal muscles the pool of endogenous stem cells, i.e., satellite cells, might be not sufficient to secure proper regeneration. Such failure in reconstruction is often associated with loss of muscle mass and excessive formation of connective tissue. Therapies aiming to improve skeletal muscle regeneration and prevent fibrosis may rely on the transplantation of different types of stem cell. Among such cells are adipose tissue-derived stromal cells (ADSCs) which are relatively easy to isolate, culture, and manipulate. Our study aimed to verify applicability of ADSCs in the therapies of severely injured skeletal muscles. We tested whether 3D structures obtained from Matrigel populated with ADSCs and transplanted to regenerating mouse gastrocnemius muscles could improve the regeneration. In addition, ADSCs used in this study were pretreated with myoblasts-conditioned medium or anti-TGFβ antibody, i.e., the factors modifying their ability to proliferate, migrate, or differentiate. Analyses performed one week after injury allowed us to show the impact of 3D cultured control and pretreated ADSCs at muscle mass and structure, as well as fibrosis development immune response of the injured muscle.

Effects of chronic treatment with gold nanoparticles on inflammatory responses and oxidative stress in Mdx mice

Duchenne muscular dystrophy (DMD) is an X-linked recessive hereditary myopathy characterised by progressive muscle degeneration in male children. As a consequence of DMD, increased inflammation and oxidative stress occur in muscle tissue along with morphological changes. Several studies have reported anti-inflammatory and antioxidant effects of gold nanoparticles (GNP) in muscle injury models. The objective of this study was to evaluate these effects along with the impacts of the disease on histopathological changes following chronic administration of GNP to Mdx mice. Two-month-old Mdx mice were separated into five groups of eight individuals each, as follows: wild-type (WT), Mdx-modified without treatment, Mdx + 2.5 mg/kg GNP, Mdx + 7.0 mg/kg GNP and Mdx + 21 mg/kg GNP. GNP with a mean diameter of 20 nm were injected subcutaneously at concentrations of 2.5, 7.0 and 21 mg/kg. Treatments continued for 30 d with injections administered at 48-h intervals. Twenty-four hours after the last injection, the animals were killed and the central region of the gastrocnemius muscle was surgically removed. Chronic administration of GNP reduced inflammation in the gastrocnemius muscle of Mdx mice and reduced morphological alterations due to inflammatory responses to muscular dystrophy. In addition, GNP also demonstrated antioxidant potential by reducing the production of reactive oxygen and nitrogen species, reducing oxidative damage and improving antioxidant activity.

Relationship between Changes of Pericyte Number and Wound Age during Repair of Skeletal Muscle Contusion in Mice.

Objective To observe the change pattern of pericyte number at different time periods after mice skeletal muscle contusion and discuss its role in wound age estimation. Methods A mice gastrocnemius muscle contusion model was established. The form and number changes of pericytes at 1, 3, 5, 7, 9, 14, and 28 d post-injury were detected by multiple immunofluorescence staining. Results Compared with the slender shape of pericytes in normal skeletal muscles, pericytes in the contusion area had increased volume, rounder form and a round nuclei. Part of pericytes were found to express satellite cell markers paired-box transcription factor （Pax7） or myoblast determination 1 （MyoD1）. The changes of pericyte number in skeletal muscles after contusion were time-dependant, and showed unimodal distribution with the extension of wound age. In the central contusion area, the number of pericytes peaked at 5 d post-injury while in the peripheral contusion area, the number of pericytes peaked at 5 d and 7 d post-injury. Conclusion The number of pericytes in contusion area varies time-dependently after skeletal muscle contusion in mice and might be a reference index for muscle wound age estimation, and is involved in the repair and regeneration of skeletal muscle injury.

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia.

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Consumption of Whole Eggs Promotes Insulin Sensitivity in Db/db Mice (OR34-01-19)

ObjectivesChicken eggs have a high nutrient density. Some country guidelines recommend that people with type 2 diabetes (T2D) limit their consumption of eggs due to its high cholesterol content. However, several clinical studies showed that egg intake is associated with a lower risk of type 2 diabetes. In the current study, we sought to explore whether egg consumption improves insulin sensitivity and subsequent blood glucose management in type 2 diabetic db/db mice. MethodsSix-week-old male db/db mice were fed a low-fat diet (LFD, 10 kCal % from fat) or LFD supplemented with 1% whole eggs (Egg) for 8 weeks. At the termination of the study, mice were fasted for 3 hrs prior to euthanization. Blood and other tissues were collected for laboratory assessments. Plasma metabolic parameters and pro-inflammatory cytokines were monitored by a clinical analyzer and ELISA, respectively. Hepatic and skeletal muscle mitochondrial respiratory activity was assessed by a Seahorse XFe Analyzer. Hepatic gene expression was analyzed by transcriptomics and confirmed by real-time PCR and/or Western blot. ResultsEgg consumption significantly increased body weight gain, lowered fasting blood glucose, insulin, and IL-6 levels, and elevated total cholesterol, HDL, LDL, and GLP-1 levels. Only the basal mitochondrial respiratory activity was decreased, and the complex II respiratory activity was increased in gastrocnemius muscles in mice fed Egg. Hepatic mitochondrial activity was not altered by diet. Mechanistically, transcriptomics results revealed that hepatic genes involved in enhanced insulin sensitivity were highly expressed, but genes in endogenous cholesterol synthesis were significantly suppressed after egg consumption. ConclusionsThe results suggest that egg consumption is beneficial to blood glucose control in type 2 diabetic mice. Type 2 diabetic animal could manage the cholesterol level through suppression of de novo cholesterol synthesis when consuming a high cholesterol diet, e.g., egg diet. Funding SourcesNational egg nutrition center grant USDA NIFA grant

Effects of glutamine on skeletal muscle membrane repair in severely burned mice and the functional mechanism

Objective: To observe how glutamine affect the skeletal muscle membrane repair in severely burned mice through promoting the mitsugumin 53 (MG53) dimerization in skeletal muscle and to explore its functional mechanism. Methods: (1) Animal experiments. A total of 179 BALB/c male mice aged 6 to 8 weeks were divided into sham injury group (n=43), burn group (n=73) and burn+ glutamine group (n=63) according to the random number table (the same grouping method below). Mice in sham injury group were sham injured on the back, and mice in burn group and burn+ glutamine group were inflicted with 30% total body surface area full-thickness scald (hereinafter referred to as burn) on the back. Mice in burn+ glutamine group were intragastrically administered with glutamine (1 mg/kg), and the other two groups were given the same amount of amino acid solution once per day for 14 days. On post burn hour 12, 10 mice from burn group were taken for preparation of burn serum, which is used in the following cell experiments. Blood samples were collected from the hearts to prepare serum from 10 mice in sham injury group immediately after burn and from 10 mice in burn group and burn+ glutamine group on post burn day (PBD) 5, 10, and 14, respectively. And then the whole gastrocnemius muscle was harvested after the mice were sacrificed. On PBD 10, the whole flexor brevis digitorum was harvested from 6 mice in the 3 groups respectively after the mice were sacrificed. On PBD 5, 10, and 14, the whole gastrocnemius muscle tissue was harvested from another 9 mice in the 3 groups respectively after the mice were sacrificed. The mass of the whole gastrocnemius muscle of mice was weighed. The total protein content of gastrocnemius muscle of mice was detected by coomassie brilliant blue method. The repair function of myolemma of flexor brevis digitorum of mice was detected by two-photon laser fiber membrane perforating. The serum content of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) of mice was determined with radioimmunoassay. The expressions of MG53 dimer and monomer in gastrocnemius of mice were determined with non-reductive electrophoresis-Western blotting. The protein expressions of endoplasmic reticulum stress sign proteins CCAAT/enhancer binding protein homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) in gastrocnemius of mice were determined with Western blotting. (2) Cell experiments. Mice skeletal muscle precursor cells C2C12 were cultured in vitro, and cells of the second passage were selected for the experiments. The cells were divided into normal control group, burn serum group, and burn serum+ glutamine group, with 3 dishes in each group and 1×10(3) cells in each dish. Cells in normal control group were cultured with 1 mL Dulbecco's modified Eagle medium (DMEM) with fetal bovine serum of volume fraction 10%, cells in burn serum group were cultured with 1 mL DMEM with burn serum of volume fraction 10%, and cells in burn serum+ glutamine group were cultured with 1 mL DMEM with burn serum of volume fraction 10% and 4 μL glutamine with a final molar concentration of 8 mmol/L. After 24 hours of culturing, the repair function of myocyte membrane after differentiation of skeletal muscle precursor cells in mice was detected with the same method before. Another cells were grouped and cultured as before, with 3 wells in each group and 1×10(5) cells in each well. After 24 hours of culturing, the expressions of MG53 dimer and monomer and endoplasmic reticulum stress marker proteins in the cells were detected as before. Data were processed with analysis of variance of factorial design, one-way analysis of variance, least significant difference t test, and Student Newman Keuls test. Results: Animal experiments. (1) Compared with those in sham injury group, the mass and total protein content of gastrocnemius muscle of mice in burn group were significantly decreased on PBD 5, 10, and 14 (P 0.05). Compared with 58.5±1.8 in normal control group, the expression of MG53 dimer of cells in burn serum group was significantly decreased after 24 hours of culturing (14.1±1.4, P<0.05). Compared with that in burn serum group, the expression of MG53 dimer of cells in burn serum+ glutamine group was significantly increased after 24 hours of culturing (30.9±0.6, P<0.05). Compared with those in normal control group, the protein expressions of CHOP and GRP78 of cells were significantly elevated in burn serum group after 24 hours of culturing (P<0.05). Compared with those in burn serum group, the protein expressions of CHOP and GRP78 of cells were significantly reduced in burn serum+ glutamine group after 24 hours of culturing (P<0.05). Conclusions: Glutamine can promote MG53 dimerization by alleviating endoplasmic reticulum stress in severely burned mice. Thus it can accelerate skeletal muscle membrane repair, reduce the local inflammatory reaction of skeletal muscle and consumption of skeletal muscle.

Chronic Angiotensin 1-7 Infusion Prevents Angiotensin-II-Induced Cognitive Dysfunction and Skeletal Muscle Injury in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is increasingly viewed as a neurological disease accompanied by a systemic disorder. Accumulating evidence supports that angiotensin II and angiotensin 1-7 exert opposite effects on various organs including the brain. However, the interaction between angiotensin II and angiotensin 1-7 in AD remains to be defined. The present study was undertaken to examine the interaction between these peptides in AD. 5XFAD mice, a useful model of AD, were separated into three groups: 1) saline-infused, 2) angiotensin II-infused, and 3) angiotensin II-infused and angiotensin 1-7-co-infused. These peptides were systemically given to 5XFAD mice via osmotic minipump for 4 weeks. Systemic angiotensin II infusion for 4 weeks induced significant hypertension in both wild-type and 5XFAD mice. Angiotensin II induced cognitive abnormality in 5XFAD mice as estimated by the Morris water maze test and the nest building test, and this effect was associated with cerebral blood flow reduction, cortical arterial amyloid-β deposition, hippocampal inflammation, and neuron loss in 5XFAD mice. In addition, angiotensin II infusion led to gastrocnemius muscle atrophy in 5XFAD mice. Co-infusion of angiotensin 1-7 prevented the above mentioned detrimental effects of angiotensin II in the brain and gastrocnemius muscle in 5XFAD mice, without significant influence on blood pressure. The left ventricular hypertrophic response to angiotensin II was attenuated in 5XFAD mice compared with wild-type mice, which was not significantly altered by co-administration of angiotensin 1-7. Our results show that angiotensin 1-7 counteracts angiotensin II-induced cognitive impairment, brain injury, and skeletal muscle injury in AD mice.

Ectopic human muscle progenitor cells expression of neurotrophic factors improves recovery in a mouse model of sciatic nerve injury

Background & Aim The peripheral nervous system has an intrinsic ability to regenerate after injury. However, this process is slow, incomplete, and often accompanied by disturbing motor and sensory consequences. Sciatic nerve injury (SNI), which is the most common model for studying peripheral nerve injury, is characterized by damage to both motor and sensory fibers. The main goal of this study is to examine the feasibility of administration of human muscle progenitor cells (hMPC) overexpressing neurotrophic factor (NTF) genes, known to protect peripheral neurons and enhance axon regeneration and functional recovery, to ameliorate motoric and sensory deficits in SNI mouse model. Methods, Results & Conclusion To this end, human muscle progenitor cells (hMPCs) were isolated from a human muscle biopsy, and manipulated to ectopically express BDNF, GDNF, VEGF, and IGF-1. These hMPC-NTF were transplanted into the gastrocnemius muscle of mice after SNI, and motor and sensory functions of the mice were assessed using the CatWalk XT system and the hot plate test. ELISA analysis showed that genetically manipulated hMPC-NTF express significant amounts of BDNF, GDNF, VEGF, or IGF-1. Transplantation of 3 × 106 hMPC-NTF was shown to improve motor function and gait pattern in mice following SNI surgery, as indicated by the CatWalk XT system 7 days post-surgery. Moreover, using the hot-plate test, performed 6 days after surgery, the treated mice showed less sensory deficits, indicating a palliative effect of the treatment. ELISA analysis following transplantation demonstrated increased NTF expression levels in the gastrocnemius muscle of the treated mice, reinforcing the hypothesis that the observed positive effect was due to the transplantation of the genetically manipulated hMPC-NTF. These results show that genetically-modified hMPC can alleviate both motoric and sensory deficits of SNI. The use of hMPC-NTF demonstrates the feasibility of a treatment paradigm, which may lead to rapid, high-quality healing of damaged peripheral nerves due to administration of hMPC. Our approach suggests a possible clinical application for the treatment of peripheral nerve injury.

Tumor protein 53-induced nuclear protein 1 deficiency alters mouse gastrocnemius muscle function and bioenergetics invivo.

Tumor protein 53‐induced nuclear protein 1 (TP53INP1) deficiency leads to oxidative stress‐associated obesity and insulin resistance. Although skeletal muscle has a predominant role in the development of metabolic syndrome, the bioenergetics and functional consequences of TP53INP1 deficiency upon this tissue remain undocumented. To clarify this issue, gastrocnemius muscle mechanical performance, energy metabolism, and anatomy were investigated in TP53INP1‐deficient and wild‐type mice using a multidisciplinary approach implementing noninvasive multimodal‐NMR techniques. TP53INP1 deficiency increased body adiposity but did not affect cytosolic oxidative stress, lipid content, and mitochondrial pool and capacity in myocyte. During a fatiguing bout of exercise, the in vivo oxidative ATP synthesis capacity was dramatically reduced in TP53INP1‐deficient mice despite ADP level (the main in vivo stimulator of mitochondrial respiration) did not differ between both genotypes. Moreover, TP53INP1 deficiency did not alter fatigue resistance but paradoxically increased the contractile force, whereas there were no differences for muscle fiber‐type distribution and calcium homeostasis between both genotypes. In addition, muscle proton efflux was decreased in TP53INP1‐deficient mice, thereby indicating a reduced blood supply. In conclusion, TP53INP1 plays a role in muscle function and bioenergetics through oxidative capacity impairment possibly as the consequence of abnormal mitochondrial respiration regulation and/or defective blood supply.

Permittivity of ex vivo healthy and diseased murine skeletal muscle from 10\u2009kHz to 1\u2009MHz

A better understanding of the permittivity property of skeletal muscle is essential for the development of new diagnostic tools and approaches for neuromuscular evaluation. However, there remain important knowledge gaps in our understanding of this property in healthy and diseased skeletal muscle, which hinder its translation into clinical application. Here, we report the permittivity of gastrocnemius muscle in healthy wild type mice and murine models of spinal muscular atrophy, muscular dystrophy, diabetes, amyotrophic lateral sclerosis and in a model of myofiber hypertrophy. Data were measured ex vivo from 10 kHz to 1 MHz using the four-electrode impedance technique. Additional quantitative histology information were obtained. Ultimately, the normative data reported will offer the scientific community the opportunity to develop more accurate models for the validation and prediction of experimental observations in both pre-clinical and clinical neuromuscular disease research.

Inhibition of TLR9 attenuates skeletal muscle fibrosis in aged sarcopenic mice via the p53/SIRT1 pathway.

Sarcopenia is an age-related syndrome characterized by a gradual loss of muscle mass and function, but its pathophysiological mechanism remains unclear. Skeletal muscle extracellular matrix (ECM) remodeling is an important pathological change in sarcopenia, and fibrosis is the most obvious manifestation of this change. We found that the expression of the immunoreceptor Toll-like receptor 9 (TLR9) is significantly increased in skeletal muscle in aged mice and is positively related to muscle fibrosis. Moreover, in previous reports, the longevity gene Sirt1 was reported to attenuate ECM deposition and improve muscle function. In this study, we hypothesized that TLR9 modulated skeletal muscle fibrosis via Sirt1. We used TLR9 knockout (TLR9 KO) mice and C57 mice, and grip strength and body composition were compared at different ages. We found that TLR9 knockout significantly attenuated skeletal muscle fibrosis and improved muscle function in aged mice. Furthermore, silent information regulator 1 (Sirt1) activity in mice was inhibited by Ex527, which is a specific inhibitor of Sirt1. Negative Sirt1 regulation via the activation of TLR9-related signaling pathways participated in skeletal muscle fibrosis in the sarcopenic mice, and this process might mediated by the Sirt1/Smad signaling pathway. Our findings revealed that fibrosis changes in the gastrocnemius muscle in sarcopenic mice are closely related to TLR9 activation, and TLR9 modulation could be a therapeutic strategy for combating sarcopenia during aging.

Characterization of the Regulation and Function of Family with Sequence Similarity 83D (Fam83d) in Skeletal Muscle

Skeletal muscle atrophy is a serious condition that can arise due to aging, cancer, corticosteroid exposure, and denervation. To better characterize the molecular genetic events of neurogenic atrophy, a previous study isolated mouse gastrocnemius muscle from mice at 3 and 14 days following sciatic nerve denervation. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue to identify novel neurogenic atrophy‐induced genes. The microarray data revealed for the first time that Fam83d is expressed in skeletal muscle and is significantly induced in response to denervation. Quantitative PCR (qPCR) analysis revealed that Fam83d is more highly expressed in proliferating myoblasts compared to differentiated myotubes. Characterization of the transcriptional regulation of Fam83d was analyzed by cloning fragments of the proximal promoter region located immediately upstream from the start of transcription and fusing them with a reporter gene. The reporter plasmids were then transfected into C2C12 mouse muscle cells alone or in combination with myogenic regulatory factor expression plasmids, resulting in a differential effect on reporter activity. Analysis of the promoter of Fam83d revealed conserved E‐box elements in the proximal regulatory region, which are known myogenic regulatory factor (MRF) binding sites. To assess where Fam83d is localized in the cell, we fused Fam83d with green fluorescent protein (GFP), transfected the Fam83d‐GFP expression plasmid into C2C12 mouse myoblasts cells, visualized by confocal microscopy, and observed that Fam83d is localized exclusively to the cytoplasm. It has recently been determined that Fam83d contains a putative phospholipase domain, is mis‐regulated in several different cancer types, and may modulate MAP Kinase signaling. Therefore, Fam83d was ectopically expressed in cultured muscle cells and markers of muscle cell differentiation and MAP Kinase signaling were analyzed. Fam83d overexpression resulted in significantly repressed levels of myosin heavy chain and myogenin expression, while levels of phosphorylated ERK were dramatically repressed. The discovery that Fam83d is expressed in skeletal muscle combined with the observation that Fam83d, a potential modulator of MAP Kinase signaling, is induced in response to neurogenic atrophy helps further our understanding of the molecular and cellular events of skeletal muscle wasting.Support or Funding InformationThe work was support by University of North Florida Transformational Learning Opportunity grants and a University of North Florida Foundation Board Grant to D.W.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Rapid changes in the microvascular circulation of skeletal muscle impair insulin delivery during sepsis

IntroductionHyperglycemia is prevalent in hospitalized critically ill patients, even in those without a prior diagnosis of diabetes. In patients without pre‐existing diabetes, hyperglycemia is associated with increased morbidity and mortality. Hyperglycemia is one of the earliest clinical indications of sepsis, and clinical trials show that if the hyperglycemia is controlled, patient outcomes are improved. Previous investigations have focused on events in the myocyte (e.g. insulin signaling, glucose transport and subsequent metabolism) as the causes for this insulin resistant state. However, the delivery of insulin to the skeletal muscle (SkM) is an important determinant of insulin action.HypothesisReduced capillary blood flow to skeletal muscle belies the insulin resistance of sepsis by reducing insulin delivery to the myocyte and that protecting the endothelium from sepsis‐induced inflammation will prevent insulin resistance and reduced capillary blood flow.MethodIntravital microscopy was used to track fluorescent beads in mouse gastrocnemius muscle capillaries. Briefly, C57BL/6J mice with jugular vein catheters were anesthetized with isoflurane, and the gastrocnemius was exposed. Mice were then injected florescent beads. Images were subjected to a positional tracking. This data was used to build capillary flow speed maps for enhanced spatial visualization of flow data using MATLAB. Insulin delivery and insulin efflux out of the vessels were also examined using intravital microscopy and insulin conjugated with Alexa Flour 647.ResultsThis hypothesis was examined in mice treated with lipopolysaccharide (LPS) followed by intravital microscopy of the SkM microcirculation. We found that flowing capillary area was reduced by ~50% (Figure 1A) and velocity of blood flowing in the capillaries was slowed (Figure 1B) as well as more tortuous (Figure 1C) after LPS administration. We assessed capillary barrier permeability to insulin and surprisingly found no significant difference in insulin exchanges rates (iKT) for either control (0.25 Min−1) or LPS treated mice (0.21 Min−1). Next we determined insulin delivery to the SkM using two independent methods and found that sepsis rapidly reduces insulin delivery to the skeletal muscle by ~40%; this was driven by decreases in capillary flow velocity and number of perfused capillaries (Figure 2A&amp;B). Further, the changes in SkM microcirculation occur prior to changes in both cardiac output and arterial blood pressure. Finally, we created mice with an endothelial specific knockdown of NFκB signaling through the deletion of P65 (P65eko) and found they were protected from both LPS‐induced reductions in blood flow speed and increases in blood flow tortuosity (Figure 2C).ConclusionThese data suggest that a rapid reduction in skeletal muscle insulin delivery contributes to the induction of insulin resistance during sepsis. Additionally, we determined that protecting the endothelium from inflammation prevents altered microvascular flow during sepsis.Support or Funding InformationR01 DK043748 DK078188 T32DK101003This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Reduced lung cancer burden by selective immunomodulators elicits improvements in muscle proteolysis and strength in cachectic mice.

Identification of to what extent tumor burden influences muscle mass independently of specific treatments for cancer-cachexia remains to be elucidated. We hypothesized that reduced tumor burden by selective treatment of tumor with immunomodulators may exert beneficial effects on muscle wasting and function in mice. Body and muscle weight, grip strength, physical activity, muscle morphometry, apoptotic nuclei, troponin-I systemic levels, interleukin-6, proteolytic markers, and tyrosine release, and apoptosis markers were determined in diaphragm and gastrocnemius muscles of lung cancer (LP07 adenocarcinoma cells) mice (BALB/c) treated with monoclonal antibodies (mAbs), against immune check-points and pathways (CD-137, cytotoxic T-lymphocyte associated protein-4, programed cell death-1, and CD-19; N = 10/group). Nontreated lung cancer cachectic mice were the controls. T and B cell numbers and macrophages were counted in tumors of both mouse groups. Compared to nontreated cachectic mice, in the mAbs-treated animals, T cells increased, no differences in B cells or macrophages, the variables final body weight, body weight and grip strength gains significantly improved. In diaphragm and gastrocnemius of mAbs-treated cachectic mice, number of apoptotic nuclei, tyrosine release, proteolysis, and apoptosis markers significantly decreased compared to nontreated cachectic mice. Systemic levels of troponin-I significantly decreased in treated cachectic mice compared to nontreated animals. We conclude that reduced tumor burden as a result of selective treatment of the lung cancer cells with immunomodulators elicits per se beneficial effects on muscle mass loss through attenuation of several biological mechanisms that lead to increased protein breakdown and apoptosis, which translated into significant improvements in limb muscle strength but not in physical activity parameters.

Effects of Acute Aerobic Exercise on the Early Stage of Energy Metabolism Related Gene Expression in Mouse Gastrocnemius: Microarray Analysis

Effects of Acute Aerobic Exercise on the Early Stage of Energy Metabolism Related Gene Expression in Mouse Gastrocnemius: Microarray Analysis

Perm1 regulates CaMKII activation and shapes skeletal muscle responses to endurance exercise training

ObjectiveEndurance exercise training remodels skeletal muscle, leading to increased mitochondrial content and oxidative capacity. How exercise entrains skeletal muscle signaling pathways to induce adaptive responses remains unclear. In past studies, we identified Perm1 (PGC-1 and ERR induced regulator, muscle 1) as an exercise-induced gene and showed that Perm1 overexpression elicits similar muscle adaptations as endurance exercise training. The mechanism of action and the role of Perm1 in exercise-induced responses are not known. In this study, we aimed to determine the pathway by which Perm1 acts as well as the importance of Perm1 for acute and long-term responses to exercise. MethodsWe performed immunoprecipitation and mass spectrometry to identify Perm1 associated proteins, and validated Perm1 interactions with the Ca2+/calmodulin-dependent protein kinase II (CaMKII). We also knocked down Perm1 expression in gastrocnemius muscles of mice via AAV-mediated delivery of shRNA and assessed the impact of reduced Perm1 expression on both acute molecular responses to a single treadmill exercise bout and long-term adaptive responses to four weeks of voluntary wheel running training. Finally, we asked whether Perm1 levels are modulated by diet or diseases affecting skeletal muscle function. ResultsWe show that Perm1 associates with skeletal muscle CaMKII and promotes CaMKII activation. In response to an acute exercise bout, muscles with a knock down of Perm1 showed defects in the activation of CaMKII and p38 MAPK and blunted induction of regulators of oxidative metabolism. Following four weeks of voluntary training, Perm1 knockdown muscles had attenuated mitochondrial biogenesis. Finally, we found that Perm1 expression is reduced in diet-induced obese mice and in muscular dystrophy patients and mouse models. ConclusionsOur findings identify Perm1 as a muscle-specific regulator of exercise-induced signaling and Perm1 levels as tuners of the skeletal muscle response to exercise. The decreased Perm1 levels in states of obesity or muscle disease suggest that Perm1 may link pathological states to inefficient exercise responses.

Human Muscle Progenitor Cells Overexpressing Neurotrophic Factors Improve Neuronal Regeneration in a Sciatic Nerve Injury Mouse Model.

The peripheral nervous system has an intrinsic ability to regenerate after injury. However, this process is slow, incomplete, and often accompanied by disturbing motor and sensory consequences. Sciatic nerve injury (SNI), which is the most common model for studying peripheral nerve injury, is characterized by damage to both motor and sensory fibers. The main goal of this study is to examine the feasibility of administration of human muscle progenitor cells (hMPCs) overexpressing neurotrophic factor (NTF) genes, known to protect peripheral neurons and enhance axon regeneration and functional recovery, to ameliorate motoric and sensory deficits in SNI mouse model. To this end, hMPCs were isolated from a human muscle biopsy, and manipulated to ectopically express brain-derived neurotrophic factor (BDNF), glial-cell-line-derived neurotrophic factor (GDNF), vascular endothelial growth factor (VEGF), and insulin-like growth factor (IGF-1). These hMPC-NTF were transplanted into the gastrocnemius muscle of mice after SNI, and motor and sensory functions of the mice were assessed using the CatWalk XT system and the hot plate test. ELISA analysis showed that genetically manipulated hMPC-NTF express significant amounts of BDNF, GDNF, VEGF, or IGF-1. Transplantation of 3 × 106 hMPC-NTF was shown to improve motor function and gait pattern in mice following SNI surgery, as indicated by the CatWalk XT system 7 days post-surgery. Moreover, using the hot-plate test, performed 6 days after surgery, the treated mice showed less sensory deficits, indicating a palliative effect of the treatment. ELISA analysis following transplantation demonstrated increased NTF expression levels in the gastrocnemius muscle of the treated mice, reinforcing the hypothesis that the observed positive effect was due to the transplantation of the genetically manipulated hMPC-NTF. These results show that genetically modified hMPC can alleviate both motoric and sensory deficits of SNI. The use of hMPC-NTF demonstrates the feasibility of a treatment paradigm, which may lead to rapid, high-quality healing of damaged peripheral nerves due to administration of hMPC. Our approach suggests a possible clinical application for the treatment of peripheral nerve injury.