Digitorum Longus Muscles Research Articles

Calcitonin pump improves nerve regeneration after transection injury and repair.

After nerve injury, excessive calcium impedes nerve regeneration. We previously showed that calcitonin improved nerve regeneration in crush injury. We aimed to validate the direct effect of calcitonin on transected and repaired nerve. Two rat groups (n = 8) underwent sciatic nerve transection followed by direct repair. In the calcitonin group, a calcitonin-filled mini-osmotic pump was implanted subcutaneously, with a catheter parallel to the repaired nerve. The control group underwent repair only, without a pump. Evaluation and comparison between the groups included: (1) compound muscle action potential recording of the extensor digitorum longus (EDL) muscle; (2) tetanic muscle force test of EDL; (3) nerve calcium concentration; and (4) nerve fiber count and calcified spot count. The calcitonin pump group showed superior recovery. Calcitonin affects injured and repaired peripheral nerve directly. The calcitonin-filled mini-osmotic pump improved nerve functional recovery by accelerating calcium absorption from the repaired nerve. This finding has potential clinical applications.

The expression and function of gelatinolytic activity at the rat neuromuscular junction upon physical exercise.

The gelatinases MMP-9 and MMP-2 have been implicated in skeletal muscle adaptation to training; however, their specific role(s) in the different muscle types are only beginning to be unraveled. Recently, we found that treadmill running increased the activity and/or expression of these enzymes in myonuclei and in activated satellite cells of the soleus (Sol), but not extensor digitorum longus (EDL) muscles on the fifth day of training of adult rats. Here, we asked whether the gelatinases can be involved in physical exercise-induced adaptation of the neuromuscular compartment. To determine the subcellular localization of the gelatinolytic activity, we used high-resolution in situ zymography and immunofluorescence techniques. In both control and trained muscles, strong gelatinolytic activity was associated with myelin sheaths within intramuscular nerve twigs. In EDL, but not Sol, there was an increase in the gelatinolytic activity at the postsynaptic domain of the neuromuscular junction (NMJ). The increased activity was found within punctate structures situated in the vicinity of synaptic cleft of the NMJ, colocalizing with a marker of endoplasmic reticulum. Our results support the hypothesis that the gelatinolytic activity at the NMJ may be involved in NMJ plasticity.

Quantification of muscle-derived signal interference during monopolar needle electromyography of a peripheral nerve interface in the rat hind limb.

High-fidelity signal acquisition is critical for the fundamental control of a neuroprosthesis. Our group has developed a bio-artificial interface consisting of a muscle graft neurotized by a severed nerve in a rat hind limb model. This regenerative peripheral nerve interface (RPNI) permits nerve signal transmission, amplification, and detection via in situ electromyography (EMG). Our study examined the magnitude of signal interference from simultaneously contracting muscles adjacent to our muscle of interest. In eighteen F344 rats, the extensor digitorum longus (EDL) muscle was used to fabricate simulated RPNI constructs of various sizes in which the neurovascular pedicle was preserved, obviating the need for reinnervation or revascularization. After 3 weeks of recovery, in situ EMG testing was performed using electrical stimulation of the common peroneal nerve. A recording needle was placed in the EDL muscle with a reference/ground electrode in the contralateral toe webspace, comprising a monopolar recording configuration. The superficial peroneal nerve was transected to further isolate stimulation of the anterior compartment. Recordings from the EDL were performed before and after excision of the tibialis anterior (TA) and extensor hallucis longus (EHL) muscles. After TA/EHL excision, EDL compound muscle action potential (CMAP) peak-to-peak amplitudes were significantly lower by an average of 7.4±5.6(SD) mV, or 32±18%, (paired t(17)=-5.7, p<;0.0001). A significant positive linear correlation was seen between CMAP amplitude and EDL mass both before TA/EHL excision (r=0.68, n=18, p<;0.01) and after TA/EHL excision (r=0.79, n=18, p<;0.0001). EDL mass did not correlate with differences in CMAP amplitude or area caused by TA/EHL excision. Monopolar needle EMG recordings from the EDL muscle are significantly, but predictively, contaminated by concomitant muscular contractions in the anterior compartment of the rat hind limb. Further investigation of strategies to reduce this signal interference, including electrode choice or configuration, use of bioelectrical insulators, and filtering methods, is warranted to promote high-fidelity signal acquisition for prosthetic control.

Slow to fast muscle transformation following heterochronous isotransplantation is influenced by host thyroid hormone status.

We studied the effect of regeneration, altered innervation and thyroid hormone (TH) levels on fiber type transitions in slow soleus (SOL) muscles grafted (GRAFT) into host extensor digitorum longus (EDLh) muscles of euthyroid (EU), hyperthyroid (HT) and hypothyroid (HY) Lewis strain rats. SOL muscles were excised from 3-week to 4-week-old inbred Lewis rats and intramuscularly transplanted into EDLh muscles of 2-month-old female rats of the same strain. The proportions of type 1, 2A, 2X and 2B fibers of GRAFT were determined by immunohistochemistry and compared with those of EDLh muscle and EDL and SOL muscles of the unoperated contralateral hind limb. After an average regeneration period of 6-7 months and after being reinnervated by the "fast" peroneal nerve of EDLh muscle, GRAFT was transformed into a fast muscle. However, the extent of GRAFT transformation varied with different TH states. In the EU rats, GRAFT contained about 95 % of fast fibers, among which type 2X and 2B fibers predominated (about 75 %). The transition toward fast muscle phenotype was more pronounced in HT status, where the fastest type 2B fibers predominated. On the contrary, in HY status, the slow to fast transformation was less pronounced, as GRAFT contained less type 2B and 2X but more type 2A and 1 fibers. We conclude that the type of innervation is the crucial factor for the slow to fast fiber type transitions in GRAFT, but the extent of muscle transformation is further modulated by altered TH status.

EFFECT OF ENDURANCE EXERCISE ON SKELETAL MUSCLE WITH CHRONIC ALCOHOL INGESTION IN RAT

The effect of endurance exercise training on mechanical properties of skeletal muscle with chronic ethanol ingestion was determined in this study. Three to four-week-old male Wistar rats were randomly assigned to four groups: control (CON), control with exercise (CON/EXE), ethanol (ETH), and ethanol with exercise (ETH/EXE). The CON/EXE and ETH/EXE groups were trained to run for 12 weeks on a motor driven treadmill. ETH and ETH/EXE groups were adapted to a liquid alcohol diet (Lieber–DeCarli). Tetanic, twitch force generation, specific force, fatigue time, and shortening velocity of extensor digitorum longus (EDL) and soleus muscles were tested by using an in vitro muscle testing system. Our study shows that exercise does not improve the contractile properties of skeletal muscle with chronic ethanol ingestion indicated by similar twitch force and fatigue time between ETH/EXE and ETH groups in Types I and II fibers, and by lowered tetanic and specific forces in Type I fibers in ETH/EXE group compared to ETH group, possibly due to damage induced by oxidative stress. Future studies on interaction of the biochemical changes and contractile properties of the skeletal muscle with chronic ethanol ingestion will be conducted to better understand mechanisms behind alterations in contractile properties.

Carbonic Anhydrase III Contributes to Fatigue Tolerance and Recovery of Skeletal Muscle

Carbonic anhydrase III (CA3) is a metabolic enzyme with a potential role in regulating intracellular pH. CA3 is highly expressed in slow twitch skeletal muscles. Here we demonstrated that mouse tibialis anterior (TA), a fast twitch muscle, also expresses a high level of CA3 while its myofilament protein contents were similar to that of CA3-negative extensor digitorum longus (EDL) muscle. To investigate the function of CA3 in muscle contractility and tolerance to fatigue, we studied skeletal muscles of CA3 knockout (Car3-/-) mice. Sciatic nerve stimulation-generated in situ contractility of EDL and TA muscles were examined in comparison with wild type controls. The results of isometric twitch and tetanic contractions showed no significant difference between TA and EDL muscle of wild type mice or between Car3-/- and wild type TA muscles. Nonetheless, intermittent fatigue treatment revealed faster fatigue and slower recovery of wild type TA muscle than that of wild type EDL muscle. Car3-/- TA muscle exhibited slower and less fatigue but also slower recovery than that of wild type TA muscle, whereas the ultimate level of force recovery was unchanged. It is suggested that CA3 increases the sensitivity of muscle to fatigue, which might serve as an acute physiological protection. In the meantime, Western-blot detected a low molecular weight fast troponin T (TnT) variant specifically in TA muscle of adult Car3-/- mice, suggesting a chronically adaptive response through alternative RNA splicing. The role of CA3 in fatigue tolerance and recovery of skeletal muscle suggests a molecular therapeutic target for functional enhancement.

Muscle nuclei remember to cheat death

Muscle nuclei remember to cheat death

Efficiency and cross-bridge work output of skeletal muscle is decreased at low levels of activation

The purpose of this study was to determine how the mechanical efficiency of skeletal muscle is affected by level of activation. Experiments were performed in vitro (35°C) using bundles of fibres from fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus muscles of mice. Measurements were made of the total work and heat produced in response to 10 brief contractions. Mechanical efficiency was the ratio of total work performed to (total heat produced + work performed). Level of activation was varied by altering stimulation frequency between 40 and 160Hz. Efficiency did not differ significantly between the two muscle types but was significantly lower using 40Hz stimulation (mean efficiency ± SEM, 0.092 ± 0.012, n = 12, averaged across EDL and soleus) than at any of the other frequencies (160Hz: 0.147 ± 0.007, n = 12). Measurements of the partitioning of energy output between force-dependent and force-independent components enabled calculation of the amount of Ca(2+) released and number of cross-bridge cycles performed during the contractions. At 40Hz stimulation frequency, less Ca(2+) was released than at higher frequencies and fewer cross-bridge cycles were performed. Furthermore, less work was performed in each cross-bridge cycle. It is concluded that skeletal muscles are less efficient at low levels of activation than when fully activated and this indicates that level of activation affects not only the number of cycling cross-bridges but also the ability of individual cross-bridges to perform work.

Myoblast expansion defects leads to muscle growth delay and subsequent compensatory adaptation in adult Atrx cKO skeletal muscle

Background The growth of muscle tissue and its regeneration from injury is crucially dependent on a self-renewing population of muscle progenitor cells called satellite cells. Activated satellite cells give rise to a rapidly expanding population of myoblasts that increase muscle mass by differentiating into new or adding into pre-existing muscle fibers. Patients with mutations in the chromatin remodeling gene ATRX are clinically characterised by severe cognitive disabilities and muscular hypotonia, thus dramatically compromising their independent locomotory ability. Materials and methods We generated a skeletal muscle specific knockout mouse model by interbreeding Myf5-Cre mice with mice harbouring the Atrx floxed conditional knockout allele (herein referred to as Atrx cKO). Body mass of Atrx cKO and control littermates were measured along with general morphometric analysis of select hindlimb muscles at 3weeks, 5-weeks, and greater than 8-weeks of age. Methods of analysis included single muscle fiber preparations from the extensor digitorum longus (EDL) and soleus muscles as well as RNA expression analysis from the EDL and soleus muscles. Primary myoblasts were also cultured from dissociated muscle tissue from the hindlimbs and analyzed via immuno-fluorescent microscopy. Neuromuscular acuity and endurance was assessed in Atrx cKO and control littermates by the roto-rod apparatus. Muscle strength was assessed by utilizing a digital grip strength measuring apparatus. Results Atrx cKO mice presented with telltale characteristics of weaker musculature, exemplified by spinal kyphosis and reduced body mass at 3-weeks of age. Satellite cell derived myoblasts from Atrx cKO mice were incapable of rapid expansion in culture but were fully capable of terminally differentiating. Atrx cKO myoblasts displayed delayed cell cycle progression through mid-late S-phase and rampant signs of genomic instability characterised by fragmented nuclei, g-H2AX foci, and telomeric aberrations. Despite inefficient myoblast proliferative capacity, Atrx cKO animals were able to re-establish normal body mass by adulthood. Muscular fitness and function in Atrx cKO mice was also age dependent, as younger 3-week old Atrx cKO mice had a reduced capacity to stay on the roto-rod and poorer gripping strength. However, differences in grip strength of adult Atrx cKO mice were almost indistinguishable from their control littermates. Data regarding pathways mediating the hypertrophic compensatory adaptation in Atrx cKO mice will also be presented. Conclusions Inefficient expansion of activated satellite cells in Atrx cKO mice results in delayed muscle development up to 3-weeks of age, when myonuclear accretion reaches its plateau. Reduced muscle mass at 3-weeks of age also correlated with poorer performance in physical tasks that require muscular force, endurance, and coordination. Compensatory mechanisms are triggered after 3-weeks of age in Atrx cKO mice that allow for the eventual recovery of body mass and muscle functionality in adults.

Isolation of Sarcolemmal Plasma Membranes by Mechanically Skinning Rat Skeletal Muscle Fibers for Phospholipid Analysis

Membrane phospholipid (PL) composition has been shown to affect cellular function by altering membrane physical structure. The sarcolemma plasma membrane (SLpm) is integral to skeletal muscle function and health. Previous studies assessing SLpm PL composition have demonstrated contamination from transverse (t)-tubule, sarcoplasmic reticulum, and nuclear membranes. This study assessed the possibility of isolating SL by mechanically skinning skeletal muscle fiber segments for the analysis of SLpm PL composition. Mechanically skinned SLpm from rat extensor digitorum longus (EDL) muscle fibers underwent Western blot analysis to assess contamination from t-tubule, sarcoplasmic reticulum, nuclear and mitochondrial membranes. The results indicate that isolated SLpm had minimal nuclear and mitochondrial membrane contamination and was void of contamination from sarcoplasmic reticulum and t-tubule membranes. After performing both high-performance thin layer chromatography and gas chromatography, we found that the SLpm obtained by mechanical skinning had higher sphingomyelin and total fatty acid saturation and lower phosphatidylcholine when compared to previous literature. Thus, by avoiding the use of various chemical treatments and membrane fractionation, we present data that may truly represent the SLpm and future studies can use this technique to assess potential changes under various perturbations and disease conditions such as insulin resistance and muscular dystrophy.

Investigation of the nerve distribution pattern of leg muscles in rat

Information about the distribution of intramuscular nerve fibres within the skeletal muscles will enhance the understanding of their morphological structure and functions. This study was designed to examine the entire intramuscular nerve distribution pattern in rat leg muscles. The tibialis cranialis, tibialis caudalis, extensor digitorum longus, flexor digitorum longus, gastrocnemius, peroneus longus and brevis muscles were dissected from origo and insertion points under a surgical microscope in seven rats. These skeletal muscles from right hindlimbs were stained with Sihler's stain. The diameter of extramuscular and major nerve branches, number of major and minor nerve branches and anastomoses were measured and photographed under a stereomicroscope. In addition, serial sections were obtained from the left hindlimb muscles with S100 immunohistochemical staining and transferred to the computer to reconstruct images. A significant difference was found between the gastrocnemius and tibialis caudalis (p<0.001), flexor digitorum longus and tibialis caudalis (p<0.003), and peroneus longus and tibialis caudalis (p<0.049) with regard to the diameter of major branches. The gastrocnemius was significantly different from the flexor digitorum longus, peroneus longus, extensor digitorum longus, tibialis caudalis and tibialis cranialis with regard to the number of minor nerve branches (p<0.001). Knowledge of the branching pattern and some key landmarks, such as the number and diameter of major and minor nerve branches and the number of anastomoses between the nerve branches of skeletal muscles, is helpful in surgical or therapeutic interventions and botulinum toxin injections in areas of high extramuscular and intramuscular nerve density.

Extracellular hyperosmotic stress stimulates glucose uptake in incubated fast-twitch rat skeletal muscle

The influence of hyperosmotic stress on glucose uptake, handling, and signaling processes remains unclear in mammalian skeletal muscle. Thus, the purpose of this study was to investigate alterations in glucose uptake and handling during extracellular hyperosmotic stress in isolated fast-twitch mammalian skeletal muscle. Using an established in vitro isolated whole-muscle model, extensor digitorum longus (EDL) muscles were dissected from male rats (4-6 weeks of age) and incubated (30-60 min) in an organ bath, containing Sigma Medium-199 with 8 mmol·L(-1) D-glucose, and mannitol was added to the targeted osmolalities (ISO, iso-osmotic, 290 mmol·kg(-1); HYPER, hyperosmotic, 400 mmol·kg(-1)). Results demonstrate that relative water content decreased in HYPER. HYPER resulted in significant alterations in muscle metabolite concentrations (lower glycogen, elevated lactate, and glucose-6-phosphate), suggesting a decrease in energy charge. Glucose uptake was also found to be higher in HYPER, and AS160 (implicated in insulin- and contraction-mediated glucose uptake) was found to be significantly more phosphorylated in HYPER than in ISO after 30 min. In conclusion, glucose uptake and handling is altered with hyperosmotic extracellular stress in the fast-twitch EDL. The increases in glucose uptake might be facilitated through alterations in AS160 signaling after 30 to 60 min of osmotic stress.

The effects of elevated levels of sodium bicarbonate (NaHCO3) on the acute power output and time to fatigue of maximally stimulated mouse soleus and EDL muscles

This study examined the effects of elevated buffer capacity [~32 mM HCO₃(-)] through administration of sodium bicarbonate (NaHCO₃) on maximally stimulated isolated mouse soleus (SOL) and extensor digitorum longus (EDL) muscles undergoing cyclical length changes at 37 °C. The elevated buffering capacity was of an equivalent level to that achieved in humans with acute oral supplementation. We evaluated the acute effects of elevated [HCO₃(-)] on (1) maximal acute power output (PO) and (2) time to fatigue to 60 % of maximum control PO (TLIM60), the level of decline in muscle PO observed in humans undertaking similar exercise, using the work loop technique. Acute PO was on average 7.0 ± 4.8 % greater for NaHCO₃-treated EDL muscles (P < 0.001; ES = 2.0) and 3.6 ± 1.8 % greater for NaHCO₃-treated SOL muscles (P < 0.001; ES = 2.3) compared to CON. Increases in PO were likely due to greater force production throughout shortening. The acute effects of NaHCO₃ on EDL were significantly greater (P < 0.001; ES = 0.9) than on SOL. Treatment of EDL (P = 0.22; ES = 0.6) and SOL (P = 0.19; ES = 0.9) with NaHCO₃ did not alter the pattern of fatigue. Although significant differences were not observed in whole group data, the fatigability of muscle performance was variable, suggesting that there might be inter-individual differences in response to NaHCO₃ supplementation. These results present the best indication to date that NaHCO₃ has direct peripheral effects on mammalian skeletal muscle resulting in increased acute power output.

Gender and Contractile Functions of Slow and Fast Skeletal Muscles in Streptozotocin Induced Diabetic Sprague Dawley Rats

AbstractObjectives: Diabetes mellitus has been linked with specific morphological andmetabolic abnormalities of skeletal muscle in a fiber specific manner. Aim: Thepresent study was designed to compare the contractile functions of slow and fastskeletal muscles in streptozotocin (STZ) induced diabetic male and female SpragueDawley rats. Material and methods: Thirty healthy Sprague Dawley rats (15 maleand 15 female) were divided into two groups and studied after four weeks followingdiabetes induction. The rats in group I (male diabetic; n = 15) and group II (femalediabetic; n = 15) were fed on normal pellet diet and water ad libitum and rendereddiabetic by single intraperitoneal injection of STZ 65 mg/kg body weight at the startof study (day 1). At the end of four weeks, the contractile parameters of slow soleusand fast extensor digitorum longus (EDL) muscles were recorded by iWorx advancedanimal/human physiology data acquisition unit (AHK/214). Results: At the end offour weeks, the weight of isolated soleus and EDL muscles in the male diabetic ratswas significantly higher (p &lt; 0.001) as compared to the female diabetic rats.However, no significant difference was found in any of the contractile functions ofisolated soleus and EDL muscles when compared between the male and femalediabetic rats. Conclusion: No gender differences exist in the contractile functions ofslow and fast skeletal muscles in streptozotocin induced diabetic Sprague Dawleyrats.

Both short intense and prolonged moderate in vitro stimulation reduce the mRNA expression of calcium-regulatory proteins in rat skeletal muscle

Sarcoplasmic and t-tubule membrane proteins regulating sarcoplasmic Ca(2+) concentration exhibit fibre-type-dependent isoform expression, and play central roles in muscle contraction and relaxation. The purpose of this study was to evaluate the effects of in vitro electrical stimulation on the mRNA expression of components involved in Ca(2+) regulation in oxidative and glycolytic skeletal muscle. The mRNA level of Ca(2+)-ATPase (SERCA1, 2), calsequestrin (CASQ1, 2), ryanodine receptor (RyR1), and dihydropyridine receptor (Cacna1) was assessed in rat extensor digitorum longus (EDL) and soleus (SOL) muscles at 4 h of recovery following in vitro stimulations (either short intensive (SHO) 60 Hz, 5 min, or prolonged moderate (PRO) 20 Hz, 40 min). Stimulation induced acute regulation of the mRNA level of Ca(2+)-regulating proteins in a manner that does not follow typical fibre-type-specific transitions. In general, stimulation decreased mRNA content of all proteins studied. Most prominent down-regulation was observed for Cacna1 (26 and 32 % after SHO and PRO, respectively, in SOL; 19 % after SHO in EDL). SERCA1, SERCA2, CASQ1, CASQ2, and RyR1 mRNA content also decreased significantly in both muscles relative to resting control. Of notice is that hexokinase II mRNA content was increased in EDL and unchanged in SOL underlining the specificity of the down-regulation of mRNA of Ca(2+) regulatory proteins. The results demonstrate contraction-induced down-regulation of mRNAs for the main components of Ca(2+)-regulating system in skeletal muscle. The down-regulation of both isoforms of SERCA and CASQ after a single electrical stimulation session suggests that adaptations to repeated stimulation involve further regulatory mechanisms in addition to acute mRNA responses.

G.P.118 Targeting sarcopenia: Molecular basis of denervation of aged muscles and benefits of life-long exercise

Abstract With ageing, the progressive loss of muscle mass and function (sarcopenia) results in frailty, loss of independence and is a major cause of increased falls and fractures. The precise reasons for sarcopenia are unclear. We have previously described the time course of sarcopenia in female C57Bl/6J mice with significant loss of muscle mass at 24 and 28 months (m) of age (Shavlakadze et al., 2010: Biogerontology). Muscle function and maintenance of muscle mass require electrical stimulation by a nerve, yet denervation as a primary cause for sarcopenia has barely been considered. We have described altered morphology and striking denervation of neuromuscular junctions (NMJs) for muscles in geriatric (28 m) compared to young (3 m) female C57Bl/6J mice, using immunostaining of whole intact fast extensor digitorum longus (EDL) muscle (Chai et al., 2011: PlosONE). Myofibre type changes indicative of muscle denervation and re-innervation were observed in all muscles examined. However, there was no loss of alpha-motoneuron cell bodies in the lumbar spinal cord of geriatric (28 m) mice. These data suggest that changes in the muscles per se are a primary cause of the age-related denervation. Establishing these baseline data for geriatric mice is essential in order to take full advantage of the wealth of genetically modified mice available to study sarcopenia, and for testing therapeutic interventions. Our molecular analyses of muscles from mice aged 3, 15, 24 and 28 months aim to identify the molecular basis for this age-relate denervation. We have already demonstrated that life-long exercise significantly reduces sarcopenia (measured by loss of muscle mass) at 28 months of age (unpublished) and we are investigating the molecular basis for the benefits of this exercise on ageing muscles with a focus on NMJ innervation.

G.P.128 The effect of non-severe dorsal burn injury on the contractile properties of fast-twitch hind limb skeletal muscle of the mouse

Abstract Loss of skeletal muscle function is cited as the most common reason preventing burn patients from returning to work. However, the effect of non-severe burn injury on muscle function has not been investigated, despite this type of burn accounting for >90% of all burn injuries. We examined the effect of non-severe burn injury on muscle mass and contractile function in the mouse extensor digitorum longus (EDL) muscle. Anaesthetised mice (4% isoflurane in O 2 ) received a full-thickness burn to 6% of total body surface area. Analgesics (buprenorphine (0.1 mg/kg) and paracetamol (0.01 mg/mL)) were administered post-injury. At 7, 28 or 84 days after burn injury, mice were anaesthetised with pentobarbitone (40 mg/kg) and EDL muscles were excised for assessment of contractile function. The effects of burn-injury on the contractile filaments were also determined using the skinned fibre technique. The muscle mass from burn-injured and control mice were not significantly different at any time point. The maximum specific force (N/cm 2 ) in EDL muscles from burn-injured mice was significantly higher than controls at day 7 (by 16%), but significantly lower at days 28 and 84 (by 18% and 14%, respectively). Skinned fibres from burn injured muscles exhibited significantly greater maximum force at day 7, but were not significantly different from controls at day 28 or day 84 post-injury. Non-severe burn injuries caused significant long-term contractile dysfunction in remotely located hind-limb muscles. These effects were independent of muscle atrophy or direct effects on the contractile apparatus. Therefore, non-severe burn injury-related muscle weakness may be an important factor impeding patient recovery. If similar effects occur in humans, non-severe burn injury-related muscle weakness may be an important factor impeding patient recovery.

ROS-Mediated Decline in Maximum Ca2+-Activated Force in Rat Skeletal Muscle Fibers following In Vitro and In Vivo Stimulation

We hypothesised that normal skeletal muscle stimulated intensely either in vitro or in situ would exhibit reactive oxygen species (ROS)-mediated contractile apparatus changes common to many pathophysiological conditions. Isolated soleus (SOL) and extensor digitorum longus (EDL) muscles of the rat were bubbled with 95% O2 and stimulated in vitro at 31°C to give isometric tetani (50 Hz for 0.5 s every 2 s) until maximum force declined to ≤30%. Skinned superficial slow-twitch fibers from the SOL muscles displayed a large reduction (∼41%) in maximum Ca2+-activated specific force (Fmax), with Ca2+-sensitivity unchanged. Fibers from EDL muscles were less affected. The decrease in Fmax in SOL fibers was evidently due to oxidation effects on cysteine residues because it was reversed if the reducing agent DTT was applied prior to activating the fiber. The GSH∶GSSG ratio was ∼3-fold lower in the cytoplasm of superficial fibers from stimulated muscle compared to control, confirming increased oxidant levels. The presence of Tempol and L-NAME during in vitro stimulation prevented reduction in Fmax. Skinned fibers from SOL muscles stimulated in vivo at 37°C with intact blood supply also displayed reduction in Fmax, though to a much smaller extent (∼12%). Thus, fibers from muscles stimulated even with putatively adequate O2 supply display a reversible oxidation-induced decrease in Fmax without change in Ca2+-sensitivity, consistent with action of peroxynitrite (or possibly superoxide) on cysteine residues of the contractile apparatus. Significantly, the changes closely resemble the contractile deficits observed in a range of pathophysiological conditions. These findings highlight how readily muscle experiences ROS-related deficits, and also point to potential difficulties when defining muscle performance and fatigue.

The Fast Skeletal Troponin Activator, CK-2017357, Increases Muscle Function and Survival in SOD1 (G93A) Mice; a Model of ALS (P05.169)

Objective: To examine the effects of CK-2017357 in a mouse model of ALS. Background CK-2017357 is a small molecule activator of fast skeletal troponin that sensitizes the sarcomere to calcium and amplifies the muscle9s response to motor neuron input at sub-tetanic forces. Design/Methods: The potential effects of CK-2017357 were examined on muscle force, respiratory function and survival in SOD1 G93A mutant transgenic mice, a model of amyotrophic lateral sclerosis (ALS) in humans. Results: Extensor digitorum longus (EDL) muscle was stimulated via the peroneal nerve over a range of frequencies (10-200 Hz, 350 ms trains) to establish the force-frequency relationship. EDL tetanic force (26.3 ± 4.4 N/cm 2 ) was reduced by 44% ± 3.9% in 90-100 day-old SOD1 vs. WT and 66% ± 7.7% in 100-110 day-old SOD1 vs. WT, reflecting progressive loss of muscle force with disease progression. Infusion of CK-2017357 resulted in dose-dependent increases (mean ± SEM) in force at 30 Hz compared to baseline in WT (206% ± 29%), 90-100 day-old (200% ± 52%) and 100-110 day-old (153% ± 55%) SOD1 G93A mice. In female SOD1 G93A mice fed chow containing CK-2017357 (400 ppm) beginning at 11 weeks of age, animals survived to a humane endpoint longer compared to control chow-fed mice (p=0.024; hazard ratio=0.66). Body weight was similar in the two groups. There were no differences in respiratory parameters (breathing frequency, tidal volume and ventilation during hypercapnic challenge) in CK-2017357 chow-fed SOD1 G93A mice compared to WT mice. SOD1 G93A mice fed 400 ppm CK-2017357 chow exhibited a significant increase in hindlimb grip strength compared to control chow fed SOD1 G93A mice, while forelimb grip strength was not altered. Conclusions: CK-2017357 increased isometric in situ muscle force and hindlimb grip strength in SOD1 G93A mice and improved survival. These results indicate that CK-2017357 has the potential to improve muscle function in patients suffering from ALS. Supported by: Cytokinetics, Inc. Disclosure: Dr. Malik has received personal compensation for activities with Cytokinetics, Inc. Dr. Malik holds stock and/or stock options in Cytokinetics, Inc. Dr. Malik has received research support from Cytokinetics, Inc. Mr. Russell holds stock and/or stock options in Former Cytokinetics, Inc. which sponsored research in which Mr. Russell was involved as an investigator. Dr. Pannirselvam holds stock and/or stock options in Cytokinetics, Inc., which sponsored research in which Dr. Pannirselvam was involved as an investigator.Dr. Pannirselvam has received research support from Cytokinetics, Inc. Dr. Hinken holds stock and/or stock options in Cytokinetics, Inc., which sponsored research in which Dr. Hinken was involved as an investigator.Dr. Hinken has received research support from Cytokinetics, Inc. Dr. Thomsen holds stock and/or stock options in Cytokinetics, Inc., which sponsored research in which Dr. Thomsen was involved as an investigator. Dr. Thomsen has received research support from Cytokinetics, Inc. Dr. Ardiana holds stock and/or stock options in Cytokinetics, Inc., which sponsored research in which Dr. Ardiana was involved as an investigator. Dr. Ardiana has received research support from Cytokinetics, Inc. as an employee. Dr. Godinez holds stock and/or stock options in Cytokinetics, Inc., which sponsored research in which Dr. Godinez was involved as an investigator. Dr. Jia holds stock and/or stock options in Cytokinetics, Inc., which sponsored research in which Dr. Jia was involved as an investigator. Dr. Saikali holds stock and/or stock options in Cytokinetics, Inc. Dr. Saikali has received research support from Cytokinetics, Inc. Dr. Chen holds stock and/or stock options in Cytokinetics, Inc., which sponsored research in which Dr. Chen was involved as an investigator. Dr. Chen has received research support from Cytokinetics, Inc. Dr. Morgans holds stock and/or stock options in Cytokinetics, Inc. Dr. Morgans has received research support from Cytokinetics, Inc. Dr. Jasper holds stock and/or stock options in Cytokinetics, Inc., which sponsored research in which Dr. Jasper was involved as an investigator.Dr. Jasper has received research support from Cytokinetics, Inc..

The dose-dependent effects of endotoxin on protein metabolism in two types of rat skeletal muscle

Endotoxin administration is frequently used as a model of systemic inflammatory response which is considered the important pathogenetic factor in muscle wasting development in severe illness, such as sepsis, cancer, injury, AIDS and others. The main purpose of this study was determining the effect of various doses of endotoxin on protein and amino acid metabolism in two types of rat skeletal muscle. Sepsis was induced by intraperitoneal administration of endotoxin in a dose of 1, 3 and 5 mg/kg body weight (bw); control animals received a corresponding volume of the saline solution. After 24 h, extensor digitorum longus (EDL) and soleus (SOL) muscles were isolated and used for determination of total and myofibrillar proteolysis, protein synthesis, activity of cathepsins B and L, chymotrypsin-like activity of proteasome and amino acid release. The endotoxemia induced the body weight loss, the rise of total cholesterol and triglyceride plasma concentration and the protein catabolic state in skeletal muscle, which was caused by a higher increase in protein breakdown (due to activation of the proteasome system) than protein synthesis. The more significant effect of endotoxin was seen in EDL than SOL. The dose of 5 mg of endotoxin/kg bw induced the most significant changes in parameters of the protein and amino acid metabolism measured and could be therefore considered appropriate for studies of protein catabolism in young rat skeletal muscle at 24 h after endotoxin treatment.