Expression In Skeletal Muscle Research Articles

Tectorigenin targets PKACα to promote GLUT4 expression in skeletal muscle and improve insulin resistance in vitro and in vivo.

The decreased expression and dysfunction of glucose transporter 4 (GLUT4), the insulin-responsive glucose transporter, are closely related to the occurrence of insulin resistance (IR). To improve the expression of GLUT4 may represent a promising strategy to prevent and treat IR and type 2 diabetes (T2DM). Here, we demonstrate that the natural compound tectorigenin (TG) enhances GLUT4 expression, glucose uptake and insulin responsiveness via activating AMP-activated protein kinase (AMPK)/myocyte enhancer factor 2 (MEF2) signaling in both normal and IR skeletal muscle cells and tissues. Accordingly, prophylactic and therapeutic uses of TG can significantly ameliorate IR and hyperglycemia in T2DM mice. Mechanistically, we identify protein kinase A catalytic subunit α (PKACα) as the target of TG to increase GLUT4 expression and TG-PKACα binding promotes the dissociation of PKACα from the regulatory subunits, leading to the activation of PKA/AMPK signaling. PKACα knockdown in local quadriceps muscles almost completely abolished the therapeutic effects of TG on IR and T2DM, as well as the enhancement on AMPK signaling and GLUT4 expression in skeletal muscle. This study supports TG as a new drug candidate to treat IR and its related diseases, but also enriches our knowledge of PKA signaling in glucose metabolism in skeletal muscle.

Mitophagy-mediated inflammation and oxidative stress contribute to muscle wasting in cancer cachexia.

Cancer cachexia is commonly seen in patients with malignant tumors, which usually leads to poor life quality and negatively affects long-term prognosis and survival. Mitochondria dysfunction and enhanced autophagy are well-established to play an important role in skeletal muscle wasting. However, whether mitophagy is engaged in the pathogenesis of cancer cachexia requires further investigation. This study comprised a clinical study and animal experimentation. Clinical data such as CT images and laboratory results were obtained and analyzed. Then mice model of cancer cachexia and mitophagy inhibition were established. Data including skeletal muscle mass and function, mitochondria structure and function, inflammatory factors as well as ROS concentration. Mitophagy was enhanced in cancer cachexia patients with increased inflammatory factors. Greater disruption of skeletal muscle fiber and mitochondria structure were seen in cancer cachexia, with a higher level of inflammatory factors and ROS expression in skeletal muscle. Meanwhile, ATP production was undermined, indicating a close relationship with mitophagy, inflammation, and oxidative stress in the skeletal muscle of cancer cachexia mice models. In conclusion, mitophagy is activated in cancer cachexia and may play a role in skeletal muscle atrophy, and inflammation and oxidative stress might participate in mitophagy-related skeletal muscle injury.

Identification of C-C motif chemokine ligand 5 as a heat-dependent myokine.

The skeletal muscle is an endocrine organ that produces proteins and peptides, collectively termed as myokines. The temperature of skeletal muscles varies during exercise and/or with changes in ambient temperature. However, whether myokine secretion is regulated by heat stimulation is unclear. Thus, we aimed to explore the effects of environmental heat stimulation on myokine secretion. We initially investigated the secretome of C2C12 myotubes and identified several novel heat-responsive myokines. The concentration of C-C motif chemokine ligand 5 (CCL5) dramatically decreased by 0.3-fold in response to heat stress. After 3 h heat stimulation of C2C12 cells, the expression of heat shock protein 70 was induced, and the gene expression and secretion of CCL5 was significantly attenuated in C2C12 cells. We then examined the effects of acute heat stress on serum CCL5 levels in mice and Ccl5 gene expression in skeletal muscles. Mice were maintained at 23°C, exposed to 45°C for 30 min, and then returned to the 23°C chamber for recovery. The expression of Ccl5 in the skeletal muscle significantly decreased after 3 h of recovery. Serum CCL5 levels increased by approximately 1.9-fold after 30 min of heat exposure and then significantly decreased by approximately 0.7-fold after 23 h of recovery. This study suggests that heat stimulation decreases CCL5 secretion from the skeletal muscle in vitro and in vivo. Given its fundamental role in inflammation by recruiting several immune cells, CCL5 has a potential role in controlling inflammatory responses in the body after heat stimulation.

Effects of low-protein diet and feed restriction on mRNA expression of cationic amino acid transporters in porcine skeletal muscles.

We investigated the effects of a low-protein diet and feed restriction on the mRNA expression of cationic amino acid transporters (CATs) in the longissimus dorsi (LD), rhomboideus (RH), and biceps femoris (BF) muscles of pigs. Eighteen piglets were divided into three groups: a control (CP21%), low-protein diet (LP, CP16%), and feed-restricted diet (FR, CP21%, 76% feed intake of control pigs) groups. The expression levels of CAT-1 in the LD and BF muscles of LP pigs were higher than that of control pigs, whereas that of FR pigs showed no difference. The CAT-2A expression levels in the RH muscle of FR pigs were higher than that of control pigs. The free lysine concentrations in all muscles of LP and FR pigs were lower than that of control pigs. To examine the factors that affect CATs mRNA expression, we evaluated the effects of lysine, arginine, insulin-like growth factor-I, and dexamethasone on the expression of CATs in C2C12 myotubes. CAT-1 expression levels increased in lysine and/or arginine deprivation. We show that CAT-1 and CAT-2A expression levels in skeletal muscles differ in response to dietary treatments and CAT-1 expression in skeletal muscles appears to increase in response to low free lysine concentrations.

A Cross-Sectional Analysis of Receptor for Advanced Glycation End Products in Human Skeletal Muscle: A Comparative Analysis between Patients with and without Sarcopenia

Introduction: Animal studies suggest that advanced glycation end products (AGEs) and their interaction with receptor for AGEs (RAGE) are involved in sarcopenia, but their relationship in human skeletal muscles has yet to be elucidated. We aimed to determine whether RAGE expression in human skeletal muscle is associated with serum AGE levels and sarcopenia-related changes. Methods: We retrospectively reviewed 33 consecutive women (mean age, 65 years) with distal radius fracture who had consented to donate a sample of forearm muscle for research purposes, which was taken during surgical fracture repair. The muscle RAGE expression was measured with immunohistochemistry staining and serum AGE levels using ELISA method. We compared RAGE expression and AGE levels in patients with and without sarcopenia. We also correlated RAGE expression with such clinical parameters as demographic factors, as well as sarcopenia-related changes, including grip strength, appendicular skeletal muscle mass, and muscle cross-sectional area (CSA) ratios. Results: Twelve patients (36%) were diagnosed with sarcopenia. They had a significantly higher RAGE expression (p = 0.044) and AGE level (p < 0.001) than those without sarcopenia. The RAGE expression correlated significantly with a high AGE level (r = 0.510, p = 0.011) and correlated inversely with a muscle CSA ratio (r = −0.696, p < 0.001). Discussion: This study shows that RAGE expression increases in sarcopenic patient skeletal muscles. This expression also correlates positively with serum AGE levels and inversely with muscle CSA ratios. Further studies are necessary to determine whether targeting RAGEs can be a therapeutic option for sarcopenia.

PDIA4, a new endoplasmic reticulum stress protein, modulates insulin resistance and inflammation in skeletal muscle.

Endoplasmic reticulum (ER) stress has emerged as a key player in insulin resistance (IR) progression in skeletal muscle. Recent reports revealed that ER stress-induced the expression of protein disulfide isomerase family a member 4 (PDIA4), which may be involved in IR-related diseases. A previous study showed that metformin modulated ER stress-induced IR. However, it remained unclear whether metformin alleviated IR by regulating PDIA4 expression in skeletal muscle. Herein, we used palmitate-induced IR in C2C12 cells and a high-fat diet-induced IR mouse model to document the relations between metformin, IR, and PDIA4. In C2C12 cells, palmitate-induced IR increased inflammatory cytokines and PDIA4 expression. Besides, knocking down PDIA4 decreased palmitate-induced IR and inflammation in C2C12 cells. Furthermore, metformin modulated PDIA4 expression and alleviated IR both in vitro and in vivo. In addition, serum PDIA4 concentrations are associated with IR and inflammatory cytokines levels in human subjects. Thus, this study is the first to demonstrate that PDIA4 participates in the metformin-induced effects on skeletal muscle IR and indicates that PDIA4 is a potential novel therapeutic target for directly alleviating IR.

Sex and HDAC4 Differently Affect the Pathophysiology of Amyotrophic Lateral Sclerosis in SOD1-G93A Mice.

Amyotrophic Lateral Sclerosis (ALS) is a devastating adult-onset neurodegenerative disease, with ineffective therapeutic options. ALS incidence and prevalence depend on the sex of the patient. Histone deacetylase 4 (HDAC4) expression in skeletal muscle directly correlates with the progression of ALS, pointing to the use of HDAC4 inhibitors for its treatment. Contrarily, we have found that deletion of HDAC4 in skeletal muscle worsened the pathological features of ALS, accelerating and exacerbating skeletal muscle loss and negatively affecting muscle innervations in male SOD1-G93A (SOD1) mice. In the present work, we compared SOD1 mice of both sexes with the aim to characterize ALS onset and progression as a function of sex differences. We found a global sex-dependent effects on disease onset and mouse lifespan. We further investigated the role of HDAC4 in SOD1 females with a genetic approach, and discovered morpho-functional effects on skeletal muscle, even in the early phase of the diseases. The deletion of HDAC4 decreased muscle function and exacerbated muscle atrophy in SOD1 females, and had an even more dramatic effect in males. Therefore, the two sexes must be considered separately when studying ALS.

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.

Differential expression of myosin heavy chain isoforms type II in skeletal muscles of polar and black bears.

In this study, the pattern of myosin heavy chain (MHC) isoforms expression in skeletal muscles of the trunk, forelimb and hindlimb in Polar Bear (PB) Ursus maritimus; American Black Bear (AmBB), Ursus americanus and Asian Black Bear (AsBB), Ursus thibetanus was analysed by immunohistochemistry and SDS-PAGE. Results showed that slow (MHC-I) and fast (MHC-II) isoforms exist in muscles of bears. Type II fibres were classified further into Type IIa and IIx in PB but not in AsBB and AmBB. The distribution of Type I and Type II fibres in the trunk, forelimb and hindlimb varied based on muscle type and animal species. The proportions of Type I fibres formed approximately one-third of muscle composition in PB (trunk, 32.0%; forelimb, 34.7%; hindlimb, 34.5%) and a half in both AsBB and AmBB whereas Type IIa and IIx formed approximately two-third in PB (trunk, 68.0%; forelimb, 65.3%; hindlimb, 65.5%) and a half of Type II in both AmBB and AsBB. PB is a good swimmer, lives in Arctic Ocean on slippery ice catching aquatic mammals such as seals and is larger in size compared to the medium sized AmBB (living in forest) and AsBB (arboreal). The results suggest that in bears, there is greater diversity in MHC isoforms II, being expressed in selected fast contracting skeletal muscles in response to variety of environments, weight bearing and locomotion.

Dandelion (Taraxacum mongolicum Hand.-Mazz.) suppresses the decrease in physical strength and exercise capacity caused by insufficient liver function

In this study, D-galactose was used to establish a model of liver dysfunction caused by oxidative stress in mice, and the effect of dandelion on improving the exercise capacity of mice with liver dysfunction was observed and its mechanism was expounded. This study examined the role and mechanism of dandelion in improving running ability, swimming endurance, blood biochemical indices, histopathological changes, and tissue mRNA expression changes. The animal results showed that dandelion extended the running and swimming time to exhaustion in liver dysfunctional mice, reduced the serum levels of blood urea nitrogen (BUN), blood lactic acid (BLA) and malondialdehyde (MDA) in the liver, and increased hepatic glycogen (HG) and muscle glycogen (MG) levels as well as uperoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities. Histopathological observations suggested that dandelion alleviated lesions in the liver. The quantitative polymerase chain reaction (qPCR) analysis results showed that dandelion downregulated inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-α) mRNA expression and neuronal nitric oxide synthase (nNOS), copper/zinc-superoxide dismutase (Cu/Zn-SOD), manganese-superoxide dismutase (Mn-SOD), and catalase (CAT) expression in the liver and skeletal muscle of the liver-dysfunctional mice. In contrast, dandelion downregulated syncytin-1 mRNA expression in skeletal muscle of mice with a dysregulated liver. The positional analysis showed that the main components of dandelion were gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid, p-coumaric acid, rutin, myricitrin, isoquercitrin, isochlorogenic acid A, and luteolin.

Peroxisome proliferator-activated receptor γ coactivator 1-α overexpression improves angiogenic signalling potential of skeletal muscle-derived extracellular vesicles.

What is the central question of this study? Skeletal muscle extracellular vesicles likely act as pro-angiogenic signalling factors: does overexpression of peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) alter skeletal muscle myotube extracellular vesicle release, contents and angiogenic potential? What is the main finding and its importance? Overexpression of PGC-1α results in secretion of extracellular vesicles that elevate measures of angiogenesis and protect against acute oxidative stress in vitro. Skeletal muscle with high levels of PGC-1α expression, commonly associated with exercise induced angiogenesis and high basal capillarization, may secrete extracellular vesicles that support capillary growth and maintenance. Skeletal muscle capillarization is proportional to muscle fibre mitochondrial content and oxidative capacity. Skeletal muscle cells secrete many factors that regulate neighbouring capillary endothelial cells (ECs), including extracellular vesicles (SkM-EVs). Peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) regulates mitochondrial biogenesis and the oxidative phenotype in skeletal muscle. Skeletal muscle PGC-1α also regulates secretion of multiple angiogenic factors, but it is unknown whether PGC-1α regulates SkM-EV release, contents and angiogenic signalling potential. PGC-1α was overexpressed via adenovirus in primary human myotubes. EVs were collected from PGC-1α-overexpressing myotubes (PGC-EVs) as well as from green fluorescent protein-overexpressing myotubes (GFP-EVs), and from untreated myotubes. EV release and select mRNA contents were measured from EVs. Additionally, ECs were treated with EVs to measure angiogenic potential of EVs in normal conditions and following an oxidative stress challenge. PGC-1α overexpression did not impact EV release but did elevate EV content of mRNAs for several antioxidant proteins (nuclear factor erythroid 2-related factor 2, superoxide dismutase 2, glutathione peroxidase). PGC-EV treatment of cultured human umbilical vein endothelial cells (HUVECs) increased their proliferation (+36.6%), tube formation (length: +28.1%; number: +25.7%) and cellular viability (+52.9%), and reduced reactive oxygen species levels (-41%) compared to GFP-EVs. Additionally, PGC-EV treatment protected against tube formation impairments and induction of cellular senescence following acute oxidative stress. Overexpression of PGC-1α in human myotubes increases the angiogenic potential of SkM-EVs. These angiogenic benefits coincided with increased anti-oxidative capacity of recipient HUVECs. High PGC-1α expression in skeletal muscle may prompt the release of SkM-EVs that support vascular redox homeostasis and angiogenesis.

Plasma proteomic profiling in postural orthostatic tachycardia syndrome (POTS) reveals new disease pathways

Postural orthostatic tachycardia syndrome (POTS) is a cardiovascular autonomic disorder characterized by excessive heart rate increase on standing, leading to debilitating symptoms with limited therapeutic possibilities. Proteomics is a large-scale study of proteins that enables a systematic unbiased view on disease and health, allowing stratification of patients based on their protein background. The aim of the present study was to determine plasma protein biomarkers of POTS and to reveal proteomic pathways differentially regulated in POTS. We performed an age- and sex-matched, case–control study in 130 individuals (case–control ratio 1:1) including POTS and healthy controls. Mean age in POTS was 30 ± 9.8 years (84.6% women) versus controls 31 ± 9.8 years (80.0% women). We analyzed plasma proteins using data-independent acquisition (DIA) mass spectrometry. Pathway analysis of significantly differently expressed proteins was executed using a cutoff log2 fold change set to 1.2 and false discovery rate (p-value) of < 0.05. A total of 393 differential plasma proteins were identified. Label-free quantification of DIA-data identified 30 differentially expressed proteins in POTS compared with healthy controls. Pathway analysis identified the strongest network interactions particularly for proteins involved in thrombogenicity and enhanced platelet activity, but also inflammation, cardiac contractility and hypertrophy, and increased adrenergic activity. Our observations generated by the first use a label-free unbiased quantification reveal the proteomic footprint of POTS in terms of a hypercoagulable state, proinflammatory state, enhanced cardiac contractility and hypertrophy, skeletal muscle expression, and adrenergic activity. These findings support the hypothesis that POTS may be an autoimmune, inflammatory and hyperadrenergic disorder.

The Effects of Dietary Supplements, Nutraceutical Agents, and Physical Exercise on Myostatin Levels: Hope or Hype?

Myostatin, a secreted growth factor belonging to the transforming growth factor β (TGF-β) family, performs a role in hindering muscle growth by inhibiting protein kinase B (Akt) phosphorylation and the associated activation of hypertrophy pathways (e.g., IGF-1/PI3K/Akt/mTOR pathway). In addition to pharmacological agents, some supplements and nutraceutical agents have demonstrated modulatory effects on myostatin levels; however, the clinical magnitude must be appraised with skepticism before translating the mechanistic effects into muscle hypertrophy outcomes. Here, we review the effects of dietary supplements, nutraceutical agents, and physical exercise on myostatin levels, addressing the promise and pitfalls of relevant randomized clinical trials (RCTs) to draw clinical conclusions. RCTs involving both clinical and sports populations were considered, along with wasting muscle disorders (e.g., sarcopenia) and resistance training-induced muscle hypertrophy, irrespective of disease status. Animal models were considered only to expand the mechanisms of action, and observational data were consulted to elucidate potential cutoff values. Collectively, the effects of dietary supplements, nutraceutical agents, and physical exercise on myostatin mRNA expression in skeletal muscle and serum myostatin levels are not uniform, and there may be reductions, increases, or neutral effects. Large amounts of research using resistance protocols shows that supplements or functional foods do not clearly outperform placebo for modulating myostatin levels. Thus, despite some biological hope in using supplements or certain functional foods to decrease myostatin levels, caution must be exercised not to propagate the hope of the food supplement market, select health professionals, and laypeople.

Muscular Swedish mutant APP-to-Brain axis in the development of Alzheimer’s disease

Alzheimer’s disease (AD) is the most common form of dementia. Notably, patients with AD often suffer from severe sarcopenia. However, their direct link and relationship remain poorly understood. Here, we generated a mouse line, TgAPPsweHSA, by crossing LSL (LoxP-STOP-LoxP)-APPswe with HSA-Cre mice, which express APPswe (Swedish mutant APP) selectively in skeletal muscles. Examining phenotypes in TgAPPsweHSA mice showed not only sarcopenia-like deficit, but also AD-relevant hippocampal inflammation, impairments in adult hippocampal neurogenesis and blood brain barrier (BBB), and depression-like behaviors. Further studies suggest that APPswe expression in skeletal muscles induces senescence and expressions of senescence-associated secretory phenotypes (SASPs), which include inflammatory cytokines and chemokines; but decreases growth factors, such as PDGF-BB and BDNF. These changes likely contribute to the systemic and hippocampal inflammation, deficits in neurogenesis and BBB, and depression-like behaviors, revealing a link of sarcopenia with AD, and uncovering an axis of muscular APPswe to brain in AD development.

Effects of Moxibustion at CV8 on Prohibitin-1 Expression and Mitochondrial Function in Skeletal Muscles of Rats With Long-Term Exercise-Induced fatigue

Background: Physiological exercise-induced fatigue is a potential risk factor to human body in motion which can restrict the effectiveness of training and resulting muscle and joint strain. Both ancient texts and modern clinical reports have shown that moxibustion at CV8 alleviates exercise-induced fatigue. However, the pathophysiological mechanism of action is still unclear. Objective: To investigate the effect of moxibustion on mitochondrial energy metabolism in skeletal muscle of fatigued rats. Materials and Methods: In this study, rats treated with moxibustion for 8 weeks to assess its efficacy in enhancing energy metabolism in skeletal muscle. Succinate dehydrogenase (SDH), malate dehydrogenase (MDH), adenosine triphosphate (ATP), prohibitin-1 (PHB1), FoF1-ATPase and mitochondrial morphology of the biceps femoris were analyzed to explore evidence of moxibustion in long-term exercise. Results: Our research indicated that moxibustion could reduce the levels of biceps MDH, PHB1 and FoF1-ATPase in fatigued rats, and increased SDH and ATP levels. The electron microscopy showed that the mitochondrial morphology in the untreated and non-acupoint groups swollen, the cristae either decreased or fractured, but the degree of CV8 group was significantly lower than the other two groups. Conclusions: Moxibustion at CV8 relieves motor fatigue by regulating the energy metabolism of skeletal muscle and is a safe and effective non-drug treatment.

A deep redox proteome profiling workflow and its application to skeletal muscle of a Duchenne Muscular Dystrophy model.

Perturbation to the redox state accompanies many diseases and its effects are viewed through oxidation of biomolecules, including proteins, lipids, and nucleic acids. The thiol groups of protein cysteine residues undergo an array of redox post-translational modifications (PTMs) that are important for regulation of protein and pathway function. To better understand what proteins are redox regulated following a perturbation, it is important to be able to comprehensively profile protein thiol oxidation at the proteome level. Herein, we report a deep redox proteome profiling workflow and demonstrate its application in measuring the changes in thiol oxidation along with global protein expression in skeletal muscle from mdx mice, a model of Duchenne Muscular Dystrophy (DMD). In-depth coverage of the thiol proteome was achieved with >18,000 Cys sites from 5,608 proteins in muscle being quantified. Compared to the control group, mdx mice exhibit markedly increased thiol oxidation, where a ∼2% shift in the median oxidation occupancy was observed. Pathway analysis for the redox data revealed that coagulation system and immune-related pathways were among the most susceptible to increased thiol oxidation in mdx mice, whereas protein abundance changes were more enriched in pathways associated with bioenergetics. This study illustrates the importance of deep redox profiling in gaining greater insight into oxidative stress regulation and pathways/processes that are perturbed in an oxidizing environment.

Irisin and ALCAT1 mediated aerobic exercise-alleviated oxidative stress and apoptosis in skeletal muscle of mice with myocardial infarction.

Skeletal muscle in patients with heart failure (HF) exhibits altered structure, function and metabolism. Myocardial infarction (MI) is the most common cause of HF. Oxidative stress and cell apoptosis are involved in the pathophysiology of MI/HF-induced skeletal muscle atrophy. It is well recognized that aerobic exercise (AE) could prevent skeletal muscle atrophy after MI, but the underlying mechanism and molecular targets are still not fully clarified. In this study, Fndc5-/- and Alcat1-/- mice were used to establish the MI model and subjected to six weeks of moderate-intensity AE. C2C12cells were treated with H2O2 and recombinant human Irisin (rhIrisin), or transduced with a lentiviral vector to mediate the overexpression of ALCAT1 (LV-Alcat1). Results showed that MI reduced Irisin expression and antioxidant capacity of skeletal muscle, increased ALCAT1 expression, induced protein degradation and cell apoptosis, which were partly reversed by AE; Knockout of Fndc5 further aggravated MI-induced oxidative stress and cell apoptosis in skeletal muscle, and partly weakened the beneficial effects of AE. In contrast, knockout of Alcat1 reduced MI-induced oxidative stress and cell apoptosis and strengthened the beneficial effects of AE. rhIrisin and AICAR intervention inhibited ALCAT1 expression, oxidative stress and cell apoptosis, which induced by H2O2 or LV-Alcat1 in C2C12cells. These findings reveal that AE could alleviate the levels of oxidative stress and apoptosis in skeletal muscle following MI, partly via up-regulating Irisin and inhibiting ALCAT1 expression.

Efficient Downregulation of Alk4 in Skeletal Muscle After Systemic Treatment with Conjugated siRNAs in a Mouse Model for Duchenne Muscular Dystrophy.

Downregulation of genes involved in the secondary pathology of Duchenne muscular dystrophy, for example, inflammation, fibrosis, and adiposis, is an interesting approach to ameliorate degeneration of muscle and replacement by fibrotic and adiposis tissue. Small interfering RNAs (siRNAs) are able to downregulate target genes, however, delivery of siRNAs to skeletal muscle still remains a challenge. We investigated delivery of fully chemically modified, cholesterol-conjugated siRNAs targeting Alk4, a nontherapeutic target that is expressed highly in muscle. We observed that a single intravenous or intraperitoneal (IP) injection of 10 mg/kg resulted in significant downregulation of Alk4 mRNA expression in skeletal muscles in both wild-type and mdx mice. Treatment with multiple IP injections of 10 mg/kg led to an overall reduction of Alk4 expression, reaching significance in tibialis anterior (39.7% ± 6.2%), diaphragm (32.7% ± 5.8%), and liver (41.3% ± 29.9%) in mdx mice. Doubling of the siRNA dose did not further increase mRNA silencing in muscles of mdx mice. The chemically modified conjugated siRNAs used in this study are very promising for delivery to both nondystrophic and dystrophic muscles and could have major implications for treatment of muscular dystrophy pathology.

Resistance exercise enhances long-term mTORC1 sensitivity to leucine

ObjectiveExercise enhances the sensitivity of mammalian target of rapamycin complex 1 (mTORC1) to amino acids, in particular leucine. How long this enhanced sensitivity lasts, and which mechanisms control enhanced leucine-mediated mTORC1 activation following exercise is currently unknown. MethodsC57BL/6J mice were exercised for one night in a resistance-braked running wheel after a 12-day acclimatization period. Mice were gavaged with a submaximal dose of l-leucine or saline acutely or 48 h after exercise cessation, following 3 h food withdrawal. Muscles were excised 30 min after leucine administration. To study the contribution of mTORC1, we repeated those experiments but blocked mTORC1 activation using rapamycin immediately before the overnight running bout and one hour before the first dose of leucine. mTORC1 signaling, muscle protein synthesis and amino acid sensing machinery were assessed using immunoblot and qPCR. Leucine uptake was measured using L-[14C(U)]-leucine tracer labeling. ResultsWhen compared to sedentary conditions, leucine supplementation more potently activated mTORC1 and protein synthesis in acutely exercised muscle. This effect was observed in m. soleus but not in m. tibialis anterior nor m. plantaris. The synergistic effect in m. soleus was long-lasting as key downstream markers of mTORC1 as well as protein synthesis remained higher when leucine was administered 48 h after exercise. We found that exercise enhanced the expression of amino acid transporters and promoted uptake of leucine into the muscle, leading to higher free intramuscular leucine levels. This coincided with increased expression of activating transcription factor 4 (ATF4), a main transcriptional regulator of amino acid uptake and metabolism, and downstream activation of amino acid genes as well as leucyl-tRNA synthetase (LARS), a putative leucine sensor. Finally, blocking mTORC1 using rapamycin did not reduce expression and activation of ATF4, suggesting that the latter does not act downstream of mTORC1. Rather, we found a robust increase in eukaryotic initiation factor 2α (eIF2α) phosphorylation, suggesting that the integrated stress response pathway, rather than exercise-induced mTORC1 activation, drives long-term ATF4 expression in skeletal muscle after exercise. ConclusionsThe enhanced sensitivity of mTORC1 to leucine is maintained at least 48 h after exercise. This shows that the anabolic window of opportunity for protein ingestion is not restricted to the first hours immediately following exercise. Increased mTORC1 sensitivity to leucine coincided with enhanced leucine influx into muscle and higher expression of genes involved in leucine sensing and amino acid metabolism. Also, exercise induced an increase in ATF4 protein expression. Altogether, these data suggest that muscular contractions switch on a coordinated program to enhance amino acid uptake as well as intramuscular sensing of key amino acids involved in mTORC1 activation and the stimulation of muscle protein synthesis.

PGC-1α and MEF2 Regulate the Transcription of the Carnitine Transporter OCTN2 Gene in C2C12 Cells and in Mouse Skeletal Muscle.

OCTN2 (SLC22A5) is a carnitine transporter whose main function is the active transport of carnitine into cells. In skeletal muscle and other organs, the regulation of the SLC22A5 gene transcription has been shown to depend on the nuclear transcription factor PPAR-α. Due to the observation that the muscle OCTN2 mRNA level is maintained in PPAR-α knock-out mice and that PGC-1α overexpression in C2C12 myoblasts increases OCTN2 mRNA expression, we suspected additional regulatory pathways for SLC22A5 gene transcription. Indeed, we detected several binding sites of the myocyte-enhancing factor MEF2 in the upstream region of the SLC22A5 gene, and MEF2C/MEF2D stimulated the activity of the OCTN2 promoter in gene reporter assays. This stimulation was increased by PGC-1α and was blunted for a SLC22A5 promoter fragment with a mutated MEF2 binding site. Further, we demonstrated the specific binding of MEF2 to the SLC22A5 gene promoter, and a supershift of the MEF2/DNA complex in electrophoretic mobility shift assays. In immunoprecipitation experiments, we could demonstrate the interaction between PGC-1α and MEF2. In addition, SB203580, a specific inhibitor of p38 MAPK, blocked and interferon-γ stimulated the transcriptional activity of the SLC22A5 gene promoter. Finally, mice with muscle-specific overexpression of OCTN2 showed an increase in OCTN2 mRNA and protein expression in skeletal muscle. In conclusion, we detected and characterized a second stimulatory pathway of SLC22A5 gene transcription in skeletal muscle, which involves the nuclear transcription factor MEF2 and co-stimulation by PGC-1α and which is controlled by the p38 MAPK signaling cascade.