Isolated Extensor Digitorum Longus Muscles Research Articles

Unacylated Ghrelin Protects Against Age-Related Loss of Muscle Mass and Contractile Dysfunction in Skeletal Muscle.

Sarcopenia, the progressive loss of muscle mass and function, universally affects older adults and is closely associated with frailty and reduced quality of life. Despite the inevitable consequences of sarcopenia and its relevance to healthspan, no pharmacological therapies are currently available. Ghrelin is a gut-released hormone that increases appetite and body weight through acylation. Acylated ghrelin activates its receptor, growth hormone secretagogue receptor 1a (GHSR1a), in the brain by binding to it. Studies have demonstrated that acyl and unacylated ghrelin (UnAG) both have protective effects against acute pathological conditions independent of receptor activation. Here, we investigated the long-term effects of UnAG in age-associated muscle atrophy and contractile dysfunction in mice. Four-month-old and 18-month-old mice were subjected to either UnAG or control treatment for 10 months. UnAG did not affect food consumption or body weight. Gastrocnemius and quadriceps muscle weights were reduced by 20%-30% with age, which was partially protected against by UnAG. Specific force, force per cross-sectional area, measured in isolated extensor digitorum longus muscle was diminished by 30% in old mice; however, UnAG prevented the loss of specific force. UnAG also protected from decreases in mitochondrial respiration and increases in hydrogen peroxide generation of skeletal muscle of old mice. Results of bulk mRNA-seq analysis and our contractile function data show that UnAG reversed neuromuscular junction impairment that occurs with age. Collectively, our data revealed the direct role of UnAG in mitigating sarcopenia in mice, independent of food consumption or body weight, implicating UnAG treatment as a potential therapy against sarcopenia.

Searching for Mechanisms Underlying the Assembly of Calcium Entry Units: The Role of Temperature and pH

Store-operated Ca2+ entry (SOCE) is a mechanism that allows muscle fibers to recover external Ca2+, which first enters the cytoplasm and then, via SERCA pump, also refills the depleted intracellular stores (i.e., the sarcoplasmic reticulum, SR). We recently discovered that SOCE is mediated by Calcium Entry Units (CEUs), intracellular junctions formed by: (i) SR stacks containing STIM1; and (ii) I-band extensions of the transverse tubule (TT) containing Orai1. The number and size of CEUs increase during prolonged muscle activity, though the mechanisms underlying exercise-dependent formation of new CEUs remain to be elucidated. Here, we first subjected isolated extensor digitorum longus (EDL) muscles from wild type mice to an ex vivo exercise protocol and verified that functional CEUs can assemble also in the absence of blood supply and innervation. Then, we evaluated whether parameters that are influenced by exercise, such as temperature and pH, may influence the assembly of CEUs. Results collected indicate that higher temperature (36 °C vs. 25 °C) and lower pH (7.2 vs. 7.4) increase the percentage of fibers containing SR stacks, the n. of SR stacks/area, and the elongation of TTs at the I band. Functionally, assembly of CEUs at higher temperature (36 °C) or at lower pH (7.2) correlates with increased fatigue resistance of EDL muscles in the presence of extracellular Ca2+. Taken together, these results indicate that CEUs can assemble in isolated EDL muscles and that temperature and pH are two of the possible regulators of CEU formation.

Short communication: Leucine, but not muscle contractions, stimulates protein synthesis in isolated EDL muscles from golden geckos

Resistance exercise and protein ingestion stimulate muscle protein synthesis in mammals and the combination of both stimuli exert an additive effect. However, mechanisms regulating muscle mass may be different in ectothermic vertebrates because these animals are adapted to low energy consumption, short bouts of physical activity, and prolonged periods of inactivity. Here, we investigated the effects of administration of leucine and simulated resistance exercise induced by electrical stimulation (ES) on protein synthesis rate in isolated extensor digitorum longus muscle from golden geckos (Gekko badenii). Muscles were placed in Krebs-Ringer buffer equilibrated with O2 (97%) and CO2 (3%) at 30 °C. One muscle from each animal was subjected to one of three interventions: 1) administration of leucine (0.5 mM) at rest, 2) isometric contractions evoked by ES, or 3) a combination of contractions and leucine, while the contralateral muscle served as untreated control. The rate of protein synthesis was measured through pyromycin-labeling. Administration of leucine led to a 2.75 (±1.88)-fold rise in protein synthesis rate in inactive muscles, whereas isometric contractions had no effect (0.67 ± 0.37-fold). The combination of isometric contractions and leucine did not affect protein synthesis rate (1.02 ± 0.34-fold), suggesting that muscle contractions attenuated the positive influence of leucine. Our study identifies leucine as a potent positive regulator of muscle protein synthesis in golden geckos, but also demonstrates that muscle contraction is not. More studies should be conducted in other taxonomic groups of ectothermic vertebrates to identify whether this is a general pattern.

Effects of HOCl oxidation on excitation–contraction coupling: Implications for the pathophysiology of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a fatal X-linked genetic disease characterized by progressive loss of skeletal muscle. The mechanisms underlying the DMD pathology likely involve the complex interaction between reactive oxygen species (ROS) impaired Ca2+ handling and chronic inflammation, characterized by the presence of immune cells such as neutrophils. Hypochlorous acid (HOCl) is a highly reactive form of ROS produced endogenously via the actions of myeloperoxidase, an enzyme secreted by neutrophils. Myeloperoxidase activity is significantly elevated in dystrophic muscle. This study aimed to determine the effect of HOCl exposure on excitation-contraction coupling and its potential contribution to the dystrophic pathology. Isolated extensor digitorum longus (EDL) muscles and single fibers from C57 (wild type) and mdx (dystrophic) mice were used to investigate the effects of HOCl on whole muscle function, intracellular Ca2+ handling, and myofilament force production. HOCl exposure significantly decreased maximum specific force in isolated EDL muscles by 26% and 49%, respectively, in C57 and mdx mice (P < 0.0001). In single interosseous fibers, HOCl exposure significantly increased resting intracellular Ca2+ concentration by ∼17-19% (P < 0.05) and decreased the amplitude of electrically induced Ca2+ transients by ∼45% and 50%, respectively, in C57 and mdx fibers (C57, P < 0.05; mdx, P < 0.01). These effects of HOCl on resting Ca2+ could be blocked via application of tetracaine (ryanodine receptor blocker) or Gd3+ (stretch-activated channel blocker; C57, P < 0.01; mdx, P < 0.01 for both). The effect of HOCl on Ca2+ transient amplitude was significantly reduced by Gd3+ (C57, P < 0.05; mdx, P < 0.01). In chemically skinned EDL fibers, HOCl exposure decreased maximum Ca2+-activated force by ∼40% in both C57 and mdx fibers (P < 0.001). These results indicate that HOCl potently affects excitation-contraction coupling via impaired Ca2+ handling and myofilament force production. Hence, HOCl potentially links the chronic inflammation, oxidative stress, and impaired Ca2+ handling that underlies the dystrophic pathology.

Rat skeletal muscle-nerve preparation to teach skeletal muscle physiology.

This sourcebook update describes a variation of a previous sourcebook experiment that used isolated extensor digitorum longus muscle from mouse to teach skeletal muscle properties (Head SI, Arber MS. Adv Physiol Educ 37: 405-414, 2013). Gastrocnemius-sciatic nerve preparation in an anaesthetized rat was developed and muscle contractions were recorded in a computerized data acquisition system using an isometric force transducer. Teachers and students in physiology or biology can use this preparation to demonstrate skeletal muscle properties like simple muscle twitch, quantal summation, wave summation, superposition, incomplete tetanus, complete tetanus, treppe, fatigue, and length-tension relationship.

Unacylated ghrelin ameliorates muscle atrophy and contractile dysfunction in age‐associated loss of muscle mass and function

Sarcopenia, the progressive loss of muscle mass and dysfunction, universally affects the elderly and is closely associated with frailty and reduced quality of life. Despite the inevitable consequences of sarcopenia and its relevance to healthspan, no pharmacological therapies are currently available. Ghrelin is a gut-released hormone that increases appetite and body weight through acylation (acyl ghrelin, AG), which binds to its receptors (GHSR1a) in the brain. Growing studies demonstrate that AG and “unacyl” ghrelin (UnAG) both have direct impacts on proliferation independent of GHSR1a. Neuronal precursor cells and myoblast incubated with UnAG increases proliferation and protein synthesis rate by promoting differentiation and fusion. Thus, we hypothesized that UnAG ameliorates neurogenic atrophy and contractile dysfunction in aged skeletal muscle. Young (4 mo.) and old (18 mo.) female mice were treated with saline or UnAG. UnAG (100 µg/kg/day) via osmotic pump for 9-10 months. We assessed in vitro contractile properties of isolated extensor digitorum longus (EDL). UnAG treatment significantly increased terminal serum UnAG levels in the old animals. In contrast to previous studies using AG, UnAG did not affect food consumption or body weight. Muscle mass of gastrocnemius and quadriceps were ~20-30% decreased in old mice, but UnAG ameliorated the atrophy by 15-30% for both muscles. Specific force (force per cross-sectional are) of isolated EDL muscle was diminished by ~30% in Sod1KO, but UnAG restored the force deficit by ~80% in the KO mice. We find that upregulation of protein kinase A (PKA) and hyper-phosphorylation of FoxO3a signaling pathways may contribute to the protection. RNA-seq analysis further revealed that UnAG shifted mRNA expression of 366 genes towards younger states that are differentially regulated in old mice. Our data show a direct role of UnAG on sarcopenia independent of changes of food consumption or body weight, implicating UnAG as a potential therapeutic strategy that can enhance quality of life and healthspan of rapidly growing elderly population.

Isolated extensor digitorum longus muscles from old mdx dystrophic mice show little force recovery 120 minutes after eccentric damage

Duchenne muscular dystrophy is characterized by progressive wasting and cycles of regeneration in skeletal muscle. Our laboratory work suggests, branched fibres, could be responsible for the terminal phase of muscle damage in old (58–112 weeks) dystrophic mice. A recent study in 12 week old dystrophic mice reported that the majority of force loss produced by a series of eccentric contractions (EC) in extensor digitorum longus (EDL) muscles recovers (65%) within 120 minutes, concluding this is incompatible with the assumption that EC force loss is due to mechanical damage.The aim of this project was to assess the recovery post EC damage in adult (~16 weeks) and old (~88 weeks) dystrophic muscles. Male mdx mice and littermate controls were euthanized, EDL muscles dissected from the hind limbs and maintained in Krebs solution at room temperature. The muscles were maximally stimulated, and a series of 6× EC performed to assess force loss. Muscles were left to rest for up to 120 minutes whilst measuring recovery. Single muscle fibres were isolated enzymatically to assess the degree of fibre branching.Our findings replicated force recovery in young mdx mice with simple fibre branches. However, minimal recovery (~23%) in muscles of 88 week old mdx mice at 120 minutes post EC was observed. This data supports our “tipping point” hypothesis and shows a distinct pathophysiology in aged mdx mice with EC force loss due to acute fibre rupture at branch nodes that occurs in “old” dystrophic EDL muscles with &gt;70% complex branched fibres. These findings have important implications for pre‐clinical drug studies that use protection from EC damage in young mdx mice as a marker for drug efficacy.A better understanding of the pathology of muscle damage in the mdx mice will improve our ability to test and interpret pre‐clinical drug studies using this model.

Isolated Extensor Digitorum Longus Muscles from Old mdx Dystrophic Mice Show Little Force Recovery 120 Minutes after Eccentric Damage

Duchenne muscular dystrophy is characterized by progressive wasting and cycles of regeneration in skeletal muscle. Our laboratory work suggests, branched fibres, could be responsible for the terminal phase of muscle damage in old (58-112 weeks) dystrophic mice. A recent study in 12 week old dystrophic mice reported that the majority of force loss produced by a series of eccentric contractions (EC) in extensor digitorum longus (EDL) muscles recovers (65%) within 120 minutes, concluding this is incompatible with the assumption that EC force loss is due to mechanical damage (Olthoff et al. 2018). The aim of this project was to assess the recovery post EC damage in adult (∼16 weeks) and old (∼88 weeks) dystrophic muscles. Male mdx mice and littermate controls were euthanized, EDL muscles dissected from the hind limbs and maintained in Krebs solution at room temperature. The muscles were maximally stimulated, and a series of 6x EC performed to assess force loss. Muscles were left to rest for up to 120 minutes whilst measuring recovery. Single muscle fibres were isolated enzymatically to assess the degree of fibre branching. Our findings replicated force recovery in young mdx mice with simple fibre branches. However, minimal recovery (∼23%) in muscles of 88 week old mdx mice at 120 minutes post EC was observed. This data supports our “tipping point” hypothesis and shows a distinct pathophysiology in aged mdx mice with EC force loss due to acute fibre rupture at branch nodes that occurs in “old” dystrophic EDL muscles with >70% complex branched fibres. These findings have important implications for pre-clinical drug studies that use protection from EC damage in young mdx mice as a marker for drug efficacy.

\u03b2-arrestin 1 regulates \u03b22-adrenergic receptor-mediated skeletal muscle hypertrophy and contractility

Backgroundβ2-adrenergic receptors (β2ARs) are the target of catecholamines and play fundamental roles in cardiovascular, pulmonary, and skeletal muscle physiology. An important action of β2AR stimulation on skeletal muscle is anabolic growth, which has led to the use of agonists such as clenbuterol by athletes to enhance muscle performance. While previous work has demonstrated that β2ARs can engage distinct signaling and functional cascades mediated by either G proteins or the multifunctional adaptor protein, β-arrestin, the precise role of β-arrestin in skeletal muscle physiology is not known. Here, we tested the hypothesis that agonist activation of the β2AR by clenbuterol would engage β-arrestin as a key transducer of anabolic skeletal muscle growth.MethodsThe contractile force of isolated extensor digitorum longus muscle (EDL) and calcium signaling in isolated flexor digitorum brevis (FDB) fibers were examined from the wild-type (WT) and β-arrestin 1 knockout mice (βarr1KO) followed by chronic administration of clenbuterol (1 mg/kg/d). Hypertrophic responses including fiber composition and fiber size were examined by immunohistochemical imaging. We performed a targeted phosphoproteomic analysis on clenbuterol stimulated primary cultured myoblasts from WT and βarr1KO mice. Statistical significance was determined by using a two-way analysis with Sidak’s or Tukey’s multiple comparison test and the Student’s t test.ResultsChronic administration of clenbuterol to WT mice enhanced the contractile force of EDL muscle and calcium signaling in isolated FDB fibers. In contrast, when administered to βarr1KO mice, the effect of clenbuterol on contractile force and calcium influx was blunted. While clenbuterol-induced hypertrophic responses were observed in WT mice, this response was abrogated in mice lacking β-arrestin 1. In primary cultured myoblasts, clenbuterol-stimulated phosphorylation of multiple pro-hypertrophy proteins required the presence of β-arrestin 1.ConclusionsWe have identified a previously unappreciated role for β-arrestin 1 in mediating β2AR-stimulated skeletal muscle growth and strength. We propose these findings could have important implications in the design of future pharmacologic agents aimed at reversing pathological conditions associated with skeletal muscle wasting.

Passive stretch regulates skeletal muscle glucose uptake independent of nitric oxide synthase.

Skeletal muscle contraction increases glucose uptake via an insulin-independent mechanism. Signaling pathways arising from mechanical strain are activated during muscle contractions, and mechanical strain in the form of passive stretching stimulates glucose uptake. However, the exact mechanisms regulating stretch-stimulated glucose uptake are not known. Since nitric oxide synthase (NOS) has been implicated in the regulation of glucose uptake during ex vivo and in situ muscle contractions and during exercise, and NO is increased with stretch, we examined whether the increase in muscle glucose uptake during stretching involves NOS. We passively stretched isolated extensor digitorum longus muscles (15 min at ~100-130 mN) from control mice and mice lacking either neuronal NOSµ (nNOSµ) or endothelial NOS (eNOS) isoforms, as well as used pharmacological inhibitors of NOS. Stretch significantly increased muscle glucose uptake appoximately twofold ( P < 0.05), and this was unaffected by the presence of the NOS inhibitors NG-monomethyl-l-arginine (100 µM) or NG-nitro-l-arginine methyl ester (100 µM). Similarly, stretch-stimulated glucose uptake was not attenuated by deletion of either eNOS or nNOSµ isoforms. Furthermore, stretching failed to increase skeletal muscle NOS enzymatic activity above resting levels. These data clearly demonstrate that stretch-stimulated skeletal muscle glucose uptake is not dependent on NOS. NEW & NOTEWORTHY Passive stretching is known to activate muscle glucose uptake through mechanisms that partially overlap with contraction. We report that genetic knockout of endothelial nitric oxide synthase (NOS) or neuronal NOS or pharmacological NOS inhibition does not affect stretch-stimulated glucose uptake. Passive stretch failed to increase NOS activity above resting levels. This information is important for the study of signaling pathways that regulate stretch-stimulated glucose uptake and indicate that NOS should be excluded as a potential signaling factor in this regard.

Submaximal Level Single Twitch Kinetics Dependent on Disease State in Duchenne Muscular Dystrophy Mouse Model

In muscular dystrophy research, it is pertinent to analyze the force of the muscles affected by the disease to assess pathology and to assess potential effectiveness of interventions. Maximal tetanic contractions are the most commonly used protocol to assess muscle function. However, the vast majority of in vivo muscle function is at submaximal contraction. Moreover, although force of contraction is assessed, the kinetics of the contraction are rarely reported. To investigate the effect of muscular dystrophy on twitch contraction kinetics, isolated diaphragm and extensor digitorum longus (EDL) of 20‐week untreated “het” (dystrophin deficient and utrophin haploinsufficient) and wild‐type C57 mice were analyzed. In isolated diaphragm strips, Time‐to‐Peak (TTP) is slower in “het” mice (14.2 ± 0.3 ms) than in C57 (11.5 ± 0.3 ms) mice, while no significant difference was identified for Time for 50% Relaxation (RT50) of the twitch contraction. In isolated EDL muscles, TTP was also slower in “het” mice (24.0 ± 0.4 ms) compared to C57 mice (20.7 ± 0.3 ms). Unlike the diaphragm, it was found that RT50 of EDL muscles is also disease‐dependent. The RT50 of “het” mice (15.3 ± 0.6 ms) was significantly slower than the C57 model (11.8 ± 0.2 ms). These findings suggest that not only is the maximal force of the muscles important, but also the kinetics of the muscles are impacted by disease.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Deletion of estrogen receptor α in skeletal muscle results in impaired contractility in female mice.

Estradiol deficiency in females can result in skeletal muscle strength loss, and treatment with estradiol mitigates the loss. There are three primary estrogen receptors (ERs), and estradiol elicits effects through these receptors in various tissues. Ubiquitous ERα-knockout mice exhibit numerous biological disorders, but little is known regarding the specific role of ERα in skeletal muscle contractile function. The purpose of this study was to determine the impact of skeletal muscle-specific ERα deletion on contractile function, hypothesizing that ERα is a main receptor through which estradiol affects muscle strength in females. Deletion of ERα specifically in skeletal muscle (skmERαKO) did not affect body mass compared with wild-type littermates (skmERαWT) until 26 wk of age, at which time body mass of skmERαKO mice began to increase disproportionally. Overall, skmERαKO mice had low strength demonstrated in multiple muscles and by several contractile parameters. Isolated extensor digitorum longus muscles from skmERαKO mice produced 16% less eccentric and 16-26% less submaximal and maximal isometric force, and isolated soleus muscles were more fatigable, with impaired force recovery relative to skmERαWT mice. In vivo maximal torque productions by plantarflexors and dorsiflexors were 16% and 12% lower in skmERαKO than skmERαWT mice, and skmERαKO muscles had low phosphorylation of myosin regulatory light chain. Plantarflexors also generated 21-32% less power, submaximal isometric and peak concentric torques. Data support the hypothesis that ablation of ERα in skeletal muscle results in muscle weakness, suggesting that the beneficial effects of estradiol on muscle strength are receptor mediated through ERα. NEW & NOTEWORTHY We comprehensively measured in vitro and in vivo skeletal muscle contractility in female estrogen receptor α (ERα) skeletal muscle-specific knockout mice and report that force generation is impaired across multiple parameters. These results support the hypothesis that a primary mechanism through which estradiol elicits its effects on strength is mediated by ERα. Evidence is presented that estradiol signaling through ERα appears to modulate force at the molecular level via posttranslational modifications of myosin regulatory light chain.

G.P.356 - Preclinical data for a novel Toll-like receptor antagonist in mdx mice support its clinical development as a potential treatment for disease-related muscle inflammation in Duchenne muscular dystrophy

Immunologically triggered inflammatory processes play a central role in the pathogenesis of Duchenne muscular dystrophy (DMD). Toll-like receptors (TLRs) are key components of the innate immune system involved in regulating inflammation and adaptive immunity. Data have shown that TLR 7 is significantly over-expressed in DMD patients at all stages of the disease, suggesting TLR activation is an early trigger of muscle inflammation. Targeting of TLRs is a novel therapeutic approach for DMD that has the potential to reduce chronic muscle inflammation and slow disease progression across a broad patient population regardless of dystrophin mutation type. IMO-8400 is a novel oligonucleotide-based antagonist of TLRs 7, 8 and 9 that was generally well tolerated and demonstrated anti-inflammatory activity in patients with autoimmune disease. To evaluate in vivo activity in a DMD model, we conducted multiple studies in mdx mice. Across studies, treated mice received one of several TLR antagonist regimens by i.p. injection for 5 weeks, and were assessed on measures of muscle function, muscle histology, biochemical response, and cytokine gene expression. Treatment resulted in significant reductions in muscle inflammation, serum creatine kinase, and gene expression of IL-6. An increase in the specific force of isolated extensor digitorum longus muscle was also observed. Detailed results from these preclinical studies will be presented. DMD is a genetic neuromuscular disorder in which immunologically triggered inflammatory processes play a central role in disease pathogenesis. TLRs are key drivers of inflammation and are over-activated in DMD patients at all stages of the disease. A TLR antagonist improved disease-associated measures in mdx mice. Collectively, these data support advancing the TLR antagonist drug candidate IMO-8400 into clinical development as a potential non-steroidal anti-inflammatory treatment for DMD patients.

Acute Lonomia obliqua caterpillar envenomation-induced physiopathological alterations in rats: Evidence of new toxic venom activities and the efficacy of serum therapy to counteract systemic tissue damage

The clinical manifestations of Lonomia obliqua caterpillar envenomation are systemic hemorrhage and acute kidney injury. In an effort to better understand the physiopathological mechanisms of envenomation, a rat model was established to study systemic tissue damage during L. obliqua envenomation. An array of acute venom effects was characterized, including biochemical, hematological, histopathological, myotoxic and genotoxic alterations. Rapid increases in serum alanine and aspartate transaminases, γ-glutamyl transferase, lactate dehydrogenase, hemoglobin, bilirubin, creatinine, urea and uric acid were observed, indicating that intravascular hemolysis and liver and kidney damage had occurred. Treatment with a specific antivenom (antilonomic serum) for up to 2 h post-venom injection neutralized the biochemical alterations. However, treatment after 6 h post-venom injection failed to normalize all biochemical parameters, despite its efficacy in reversing coagulation dysfunction. The hematological findings were consistent with hemolytic anemia and neutrophilic leukocytosis. The histopathological alterations were mainly related to hemorrhage and inflammation in the subcutaneous tissue, lung, heart and kidneys. Signs of congestion and hemosiderosis were evident in the spleen, and hemoglobin and/or myoglobin casts were also detected in the renal tubules. Increased levels of creatine kinase and creatine kinase-MB were correlated with the myocardial necrosis observed in vivo and confirmed the myotoxicity detected in vitro in isolated extensor digitorum longus muscles. Significant DNA damage was observed in the kidneys, heart, lung, liver and lymphocytes. The majority of the DNA lesions in the kidney were due to oxidative damage. The results presented here will aid in understanding the pathology underlying Lonomia's envenomation.

Elevation of muscle temperature stimulates muscle glucose uptake in vivo and in vitro

The purpose of this study was to examine whether elevation of muscle temperature per se might be a stimulatory factor to increase muscle glucose uptake. Heat stimulation to rat hindlimbs increased glucose uptake measured in vivo in the extensor digitorum longus (EDL) and soleus muscles with a significant increase in muscle temperature. This thermal effect was observed again when glucose uptake was measured in vitro in both isolated muscles immediately after the heat stimulation in vivo. When heat stimulation was imposed on isolated EDL muscles, glucose uptake was facilitated in proportion to the increase in muscle temperature. The heat stimulation led to a significant amplification in the phosphorylation of AMP-activated protein kinase (AMPK) and Akt, and treatment with compound C, wortmannin, or LY294002 partially blocked the thermal effect on muscle glucose uptake. We provide evidence that elevation of muscle temperature per se can directly stimulate muscle glucose uptake and that this thermal effect is compound C-, wortmannin-, and LY294002-inhibitable.

Dexamethasone antagonizes the in vivo myotoxic and inflammatory effects of Bothrops venoms

In the present work we investigated the toxic activities of two Bothrops snake venoms using in vivo and in vitro experimental protocols in mice and tested the protective effect of dexamethasone (DEXA) in different conditions, comparing it with the polyvalent antivenom. We also expanded the investigations on the antiophidic effect of the Eclipta prostrata (EP) crude extract.The administration of Bothrops jararaca and Bothrops jararacussu snake venoms induced muscle damage demonstrated in vivo by the elevation on plasma creatine kinase (CK) activity in mice and by the decrease in CK content in the extensor digitorum longus (EDL) muscle of these animals, and in vitro by the increase in the rate of CK release from the isolated EDL muscle. We also observed inflammatory response following perimuscular injection of B. jararacussu venom (1.0 mg/kg). Treatment with DEXA (1.0 mg/kg) preserved over 50% of the EDL muscle CK content in vivo when evaluated 24 and 72 h after the injection of B. jararacussu venom in mice, and likewise reduced about 20% of the edema induced by this venom. DEXA reduced in 50% the presence of inflammatory cells and their activity in EDL muscle. The EP extract (50 mg/kg) showed similar ability in preventing the induction of edema and the decrease in muscle CK content, and its association with DEXA showed additive effect. EP reduced over 77% of the plasma CK activity induced by the B. jararacussu venom. In the in vitro experiments, DEXA was not able to change the rate of CK release from EDL muscles exposed to 25 μg/mL of B. jararacussu venom, neither to prevent the fall in the amplitude of the indirectly evoked twitch at the phrenic-diaphragm preparation. EP extract showed otherwise a protective effect on these protocols, reaching up to 100% of protection when concentrations of 50.0 and 100.0 μg/mL were used.Altogether our results show that inflammation is at least in part responsible for the tissue damage induced by Bothrops snake venoms, once the steroidal anti-inflammatory drug dexamethasone was able to decrease the myotoxic effects of these venoms, by reducing the inflammatory response to the venom injection.

Loss of muscle strength during sepsis is in part regulated by glucocorticoids and is associated with reduced muscle fiber stiffness

Sepsis is associated with impaired muscle function but the role of glucocorticoids in sepsis-induced muscle weakness is not known. We tested the role of glucocorticoids in sepsis-induced muscle weakness by treating septic rats with the glucocorticoid receptor antagonist RU38486. In addition, normal rats were treated with dexamethasone to further examine the role of glucocorticoids in the regulation of muscle strength. Sepsis was induced in rats by cecal ligation and puncture, and muscle force generation (peak twitch and tetanic tension) was determined in lower extremity muscles. In other experiments, absolute and specific force as well as stiffness (reflecting the function of actomyosin cross bridges) were determined in isolated skinned muscle fibers from control and septic rats. Sepsis and treatment with dexamethasone resulted in reduced maximal twitch and tetanic force in intact isolated extensor digitorum longus muscles. The absolute and specific maximal force in isolated muscle fibers was reduced during sepsis together with decreased fiber stiffness. These effects of sepsis were blunted (but not abolished) by RU38486. The results suggest that muscle weakness during sepsis is at least in part regulated by glucocorticoids and reflects loss of contractility at the cellular (individual muscle fiber) level. In addition, the results suggest that reduced function of the cross bridges between actin and myosin (documented as reduced muscle fiber stiffness) may be involved in sepsis-induced muscle weakness. An increased understanding of mechanisms involved in loss of muscle strength will be important for the development of new treatment strategies in patients with this debilitating consequence of sepsis.

Inhibition of acetyl-CoA carboxylase 2 enhances skeletal muscle fatty acid oxidation and improves whole-body glucose homeostasis in db/db mice

Excessive ectopic lipid deposition contributes to impaired insulin action in peripheral tissues and is considered an important link between obesity and type 2 diabetes mellitus. Acetyl-CoA carboxylase 2 (ACC2) is a key regulatory enzyme controlling skeletal muscle mitochondrial fatty acid oxidation; inhibition of ACC2 results in enhanced oxidation of lipids. Several mouse models lacking functional ACC2 have been reported in the literature. However, the phenotypes of the different models are inconclusive with respect to glucose homeostasis and protection from diet-induced obesity. Here, we studied the effects of pharmacological inhibition of ACC2 using as a selective inhibitor the S enantiomer of compound 9c ([S]-9c). Selectivity was confirmed in biochemical assays using purified human ACC1 and ACC2. (S)-9c significantly increased fatty acid oxidation in isolated extensor digitorum longus muscle from different mouse models (EC(50) 226 nmol/l). Accordingly, short-term treatment of mice with (S)-9c decreased malonyl-CoA levels in skeletal muscle and concomitantly reduced intramyocellular lipid levels. Treatment of db/db mice for 70 days with (S)-9c (10 and 30 mg/kg, by oral gavage) resulted in improved oral glucose tolerance (AUC -36%, p < 0.05), enhanced skeletal muscle 2-deoxy-2-[(18)F]fluoro-D-glucose (FDG) uptake, as well as lowered prandial glucose (-31%, p < 0.01) and HbA(1c) (-0.7%, p < 0.05). Body weight, liver triacylglycerol, plasma insulin and pancreatic insulin content were unaffected by the treatment. In conclusion, the ACC2-selective inhibitor (S)-9c revealed glucose-lowering effects in a mouse model of diabetes mellitus.

Contractile properties of mice deficient in dystrophin and the NADPH subunit p47phox

Duchenne muscular dystrophy is associated with a mutation in the gene encoding dystrophin. Dystrophin‐deficient muscle is highly susceptible to contraction‐induced injury. A report by N.P. Whitehead et al (2010) regarding mdx mice, described the efficacy of pharmacological inhibition of NADPH oxidase at decreasing contraction‐induced force deficits. This indicated that during lengthening contractions, NADPH oxidase may be a source of reactive oxygen species which contributes to the opening of stretch activated channels and the deleterious influx of extracellular calcium. To evaluate this proposal further and the role of p47phox – a subunit of NADPH oxidase – in particular, we tested the hypothesis that a stretch activated channel inhibitor (streptomycin), antioxidant (N‐acetylcysteine), calcium‐free buffer, and p47phox deficiency would decrease contraction‐induced injury for muscles of mdx mice. Mdx mice were crossed with p47phox null mice and isolated extensor digitorum longus muscles were exposed to the various conditions. Specific force was unaffected in all cases. Force deficits were reduced with the addition of streptomycin, N‐aceytcysteine, or calcium‐free buffer confirming earlier reports. A deficiency in p47phox diminished the increased muscle mass typical of mdx mice. However, lacking p47phox had no effect on force deficit. The results are consistent with reactive oxygen species and the influx of extracellular calcium via stretch activated channels mediating contraction‐induced injury in dystrophin deficient muscle. However, the findings indicate that the absence of p47phox does not reduce muscle dysfunction associated with dystrophin deficiency.

The effect of N‐acetylcysteine on contractile function and protein‐thiol oxidation in skeletal muscles of mdx mice

Duchenne Muscular Dystrophy (DMD) is a fatal X‐linked recessive disease characterized by severe muscle weakness. We hypothesized that oxidation of skeletal muscle proteins such as myosin contributes to dystrophic muscle weakness seen in DMD boys and dystrophic mdx mice and that this muscle weakness will be attenuated by treatment with the antioxidant N‐acetylcysteine (NAC).Six week old mdx mice and non‐dystrophic, C57 mice were treated with 2% NAC in drinking water for six weeks and compared to untreated mdx and C57 mice. Grip strength and body weight were measured weekly during the treatment period. After six weeks of treatment, the 12 week old mice were anaesthetized (sodium pentobarbitone; 40 mg/kg; IP) and the extensor digitorum longus (EDL) muscles were excised for analysis of contractile function and protein thiol‐oxidation.In mdx mice, NAC treatment significantly increased normalized grip strength and maximum specific force in isolated EDL muscles (NAC = 13.1 ± 1.2 N/cm2; Untreated = 9.8 ± 0.8 N/cm2, p&lt;0.05), and significantly reduced myosin protein‐thiol oxidation (NAC = 10.6 ± 0.8 %; Untreated = 13.7 ± 0.8 %, p&lt;0.05). In non‐dystrophic C57 mice, NAC treatment significantly increased normalized grip strength by 36%, but had no significant effect on maximum specific force or myosin protein‐thiol oxidation in EDL muscles.These data suggest that oxidative stress contributes to contractile dysfunction in dystrophic muscle via modification of thiol groups on the contractile protein myosin. Furthermore, this muscle weakness may be ameliorated with the anti‐oxidant, NAC.