Fast Extensor Digitorum Longus Muscles Research Articles

Differences in thick filament activation in fast rodent skeletal muscle and slow porcine cardiac muscle.

There is a growing appreciation that regulation of muscle contraction requires both thin filament and thick filament activation in order to fully activate the sarcomere. The prevailing mechano-sensing model for thick filament activation was derived from experiments on fast-twitch muscle. We address the question whether, or to what extent, this mechanism can be extrapolated to the slow muscle in the hearts of large mammals, including humans. We investigated the similarities and differences in structural signatures of thick filament activation in porcine myocardium as compared to fast rat extensor digitorum longus (EDL) skeletal muscle under relaxed conditions and sub-maximal contraction using small angle X-ray diffraction. Thick and thin filaments were found to adopt different structural configurations under relaxing conditions, and myosin heads showed different changes in configuration upon sub-maximal activation, when comparing the two muscle types. Titin was found to have an X-ray diffraction signature distinct from those of the overall thick filament backbone, and its spacing change appeared to be positively correlated to the force exerted on the thick filament. Structural changes in fast EDL muscle were found to be consistent with the mechano-sensing model. In porcine myocardium, however, the structural basis of mechano-sensing is blunted suggesting the need for additional activation mechanism(s) in slow cardiac muscle. These differences in thick filament regulation can be related to their different physiological roles where fast muscle is optimized for rapid, burst-like, contractions, and the slow cardiac muscle in large mammalian hearts adopts a more finely tuned, graded response to allow for their substantial functional reserve. KEY POINTS: Both thin filament and thick filament activation are required to fully activate the sarcomere. Thick and thin filaments adopt different structural configurations under relaxing conditions, and myosin heads show different changes in configuration upon sub-maximal activation in fast extensor digitorum longus muscle and slow porcine cardiac muscle. Titin has an X-ray diffraction signature distinct from those of the overall thick filament backbone and this titin reflection spacing change appeared to be directly proportional to the force exerted on the thick filament. Mechano-sensing is blunted in porcine myocardium suggesting the need for additional activation mechanism(s) in slow cardiac muscle. Fast skeletal muscle is optimized for rapid, burst-like contractions, and the slow cardiac muscle in large mammalian hearts adopts a more finely tuned graded response to allow for their substantial functional reserve.

Dietary oleic acid intake increases the proportion of type 1 and 2X muscle fibers in mice

Skeletal muscle is one of the largest metabolic tissues in mammals and is composed of four different types of muscle fibers (types 1, 2A, 2X, and 2B); however, type 2B is absent in humans. Given that slow-twitch fibers are superior to fast-twitch fibers in terms of oxidative metabolism and are rich in mitochondria, shift of muscle fiber types in direction towards slower fiber types improves metabolic disorders and endurance capacity. We previously had reported that oleic acid supplementation increases type 1 fiber formation in C2C12 myotubes; however, its function still remains unclear. This study aimed to determine the effect of oleic acid on the muscle fiber types and endurance capacity. An in vivo mouse model was used, and mice were fed a 10% oleic acid diet for 4 weeks. Two different skeletal muscles, slow soleus muscle with the predominance of slow-twitch fibers and fast extensor digitorum longus (EDL) muscle with the predominance of fast-twitch fibers, were used. We found that dietary oleic acid intake improved running endurance and altered fiber type composition of muscles, the proportion of type 1 and 2X fibers increased in the soleus muscle and type 2X increased in the EDL muscle. The fiber type shift in the EDL muscle was accompanied by an increased muscle TAG content. In addition, blood triacylglycerol (TAG) and non-esterified fatty acid levels decreased during exercise. These changes suggested that lipid utilization as an energy substrate was enhanced by oleic acid. Increased proliferator-activated receptor γ coactivator-1β protein levels were observed in the EDL muscle, which potentially enhanced the fiber type transitions towards type 2X and muscle TAG content. In conclusion, dietary oleic acid intake improved running endurance with the changes of muscle fiber type shares in mice. This study elucidated a novel functionality of oleic acid in skeletal muscle fiber types. Further studies are required to elucidate the underlying mechanisms. Our findings have the potential to contribute to the field of health and sports science through nutritional approaches, such as the development of supplements aimed at improving muscle function.

Thick filament activation is different in fast- and slow-twitch skeletal muscle.

The contractile properties of fast-twitch and slow-twitch skeletal muscles are primarily determined by the myosin isoform content and modulated by a variety of sarcomere proteins. X-ray diffraction studies of regulatory mechanisms in muscle contraction have focused predominately on fast- or mixed-fibre muscle with slow muscle being much less studied. Here, we used time-resolved X-ray diffraction to investigate the dynamic behaviour of the myofilament proteins in relatively pure slow-twitch-fibre rat soleus (SOL) and pure fast-twitch-fibre rat extensor digitorum longus (EDL) muscle during twitch and tetanic contractions at optimal length. During twitch contractions the diffraction signatures indicating a transition in the myosin heads from ordered OFF states, where heads are held close to the thick filament backbone, to disordered ON states, where heads are free to bind to thin filaments, were found in EDL and not in SOL muscle. During tetanic contraction, changes in the disposition of myosin heads as active tension develops is a quasi-stepwise process in EDL muscle whereas in SOL muscle this relationship appears to be linear. The observed reduced extensibility of the thick filaments in SOL muscle as compared to EDL muscles indicates a molecular basis for this behaviour. These data indicate that for the EDL, thick filament activation is a cooperative strain-induced mechano-sensing mechanism, whereas for the SOL, thick filament activation has a more graded response. These different approaches to thick filament regulation in fast- and slow-twitch muscles may be adaptations for short-duration, strong contractions versus sustained, finely controlled contractions, respectively. KEY POINTS: Fast-twitch muscle and slow-twitch muscle are optimized for strong, short-duration contractions and for tonic postural activity, respectively. Structural events (OFF to ON transitions) in the myosin-containing thick filaments in fast muscle help determine the timing and strength of contractions, but these have not been studied in slow-twitch muscle. The X-ray diffraction signatures of structural OFF to ON transitions are different in fast extensor digitorum longus (EDL) and slow soleus (SOL) muscle, being completely absent during twitches in soleus muscle and blunted during tetanic contractions SOL as compared to EDL Quasi-stepwise thick filament structural OFF to ON transitions in fast twitch muscle may be an adaptation for rapid, ballistic movements, whereas more graded OFF to ON structural transitions in slow-twitch muscle may be an adaptation for slower, finer motions.

Reduce Muscle Fibrosis through Exercise via NRG1/ErbB2 Modification in Diabetic Rats.

Diabetic myopathy refers to the manifestations in the skeletal muscle as a result of altered glucose homeostasis which reflects as fibrosis. Since physical exercise has been indicated a protective strategy for improving glucose metabolism in skeletal muscle, we tested a hypothesis under which the endurance exercise training could reverse the produced skeletal muscle fibrosis by diabetes. Eight-week-old male Wistar rats were randomly assigned into four groups including healthy control (HC), healthy trained (HT), diabetic control (DC), and diabetic trained (DT) groups. Diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ; 45 mg/kg). Rats in the HT and DT groups carried out an exercise program on a motorized treadmill for five days a week over six weeks. Skeletal muscle levels of NRG1and ErbB2 were measured by the Western blot method. Exercise training decreased blood glucose levels in the DT group. Induction of diabetes increased skeletal muscle fibrosis in both the fast extensor digitorum longus (EDL) and slow soleus muscles, while endurance training modified it in diabetic trained rats. Moreover, muscle NRG1and ErbB2 levels were increased in diabetic rats, while training modified muscle NRG1and ErbB2 levels in diabetic trained rats. Our study provides novel evidence that endurance training could modify skeletal muscle fibrosis through NRG1/ErbB2 modification in STZ-induced diabetic rats.

Effect of PGC1-beta ablation on myonuclear organisation

Skeletal muscle fibres are large, elongated multinucleated cells. Each nucleus within a myofibre is responsible for generating gene products for a finite volume of cytoplasm—the myonuclear domain (MND). Variation in MND sizes during atrophy, hypertrophy and disease states, are common. The factors that contribute to definitive MND sizes are not yet fully understood. Previous work has shown that peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1-α) modulates MND volume, presumably to support increased biogenesis of mitochondria. The transcriptional co-regulator peroxisome proliferator-activated receptor gamma coactivator 1β (PGC1-β) is a homologue of PGC1-α with overlapping functions. To investigate the role of this protein in MND size regulation, we studied a mouse skeletal muscle specific knockout (cKO). Myofibres were isolated from the fast twitch extensor digitorum longus (EDL) muscle, membrane-permeabilised and analysed in 3 dimensions using confocal microscopy. PGC1-β ablation resulted in no significant difference in MND size between cKO and wild type (WT) mice, however, subtle differences in nuclear morphology were observed. To determine whether these nuclear shape changes were associated with alterations in global transcriptional activity, acetyl histone H3 immunostaining was carried out. We found there was no significant difference in nuclear fluorescence intensity between the two genotypes. Overall, the results suggest that PGC-1α and PGC-1β play different roles in regulating nuclear organisation in skeletal muscle; however, further work is required to pinpoint their exact functions.

Cachexia does not induce loss of myonuclei or muscle fibres during xenografted prostate cancer in mice

Cachexia is a severe wasting disorder involving loss of body- and muscle mass reducing survival and quality of life in cancer patients. We aim at determining if cachexia is a mere perturbation of the protein balance or if the condition also involves a degenerative loss of myonuclei within the fibre syncytia or loss of whole muscle fibres. We induced cachexia by xenografting PC3 prostate cancer cells in nu/nu mice. Six weeks later, we counted myonuclei by in vivo microscopic imaging of single live fibres in the extensor digitorum longus muscle (EDL), and the EDL, soleus and tibialis anterior muscles were also harvested for ex vivo histology. The mice lost on average 15% of the whole-body wt. The muscle wet weight of the glycolytic, fast EDL was reduced by 14%, the tibialis anterior by 17%, and the slow, oxidative soleus by 6%. The fibre cross-sectional area in the EDL was reduced by 21% with no loss of myonuclei or any significant reduction in the number of muscle fibres. TUNEL-positive nuclei or fibres with embryonic myosin were rare both in cachectic and control muscles, and haematoxylin-eosin staining revealed no clear signs of muscle pathology. The data suggest that the cachexia induced by xenografted prostate tumours induces a pronounced atrophy not accompanied by a loss of myonuclei or a loss of muscle fibres. Thus, stem cell related treatment might be redundant, and the quest for treatment options should rather focus on intervening with intracellular pathways regulating muscle fibre size.

Phenotype-Specific Response of Circulating miRNAs Provides New Biomarkers of Slow or Fast Muscle Damage.

Skeletal muscle is a heterogeneous tissue composed of a continuum of contracting fibers ranging from slow-type to fast-type fibers. Muscle damage is a frequent event and a susceptibility of fast-fibers to exercise-induced damage (EIMD) or statins toxicity has been reported. Biological markers of muscle damage such as creatine kinase (CK) are not fiber-type specific and new biomarkers are needed. Some microRNAs (miRNAs) are specific to the muscle tissue, can be found in the extracellular compartment and can rise in the plasma following muscle damage. Our aim was to identify whether a set of circulating miRNAs can be used as fiber-type specific biomarkers of muscle damage in a model of traumatic (crush) injuries induced either in the slow soleus (SOL) or in the fast extensor digitorum longus (EDL) muscles of rats. A subset of miRNAs composed of miR-1-3p, -133a-3p, -133b-3p, 206-3p, -208b-3p, 378a-3p, -434-3p, and -499-5p were measured by RT-PCR in non-injured SOL or EDL muscle and in the plasma of rats 12 h after damage induced to SOL or EDL. MiR-133b-3p, -378a-3p, and -434-3p were equally expressed both in SOL and EDL muscles. MiR-1-3-p and -133a-3p levels were higher in EDL compared to SOL (1.3- and 1.1-fold, respectively). Conversely, miR-206-3p, -208b-3p, and -499-5p were mainly expressed in SOL compared to EDL (7.4-, 35.4-, and 10.7-fold, respectively). In the plasma, miR-1-3p and -133a-3p were elevated following muscle damage compared to a control group, with no difference between SOL and EDL. MiR-133b-3p and -434-3p plasma levels were significantly higher in EDL compared to SOL (1.8- and 2.4-fold, respectively), while miR-378a-3p rose only in the EDL group. MiR-206-3p levels were elevated in SOL only (fourfold compared to EDL). Our results show that plasma miR-133b-3p and -434 are fast-fiber specific biomarkers, while miR-206-3p is a robust indicator of slow-fiber damage, opening new perspectives to monitor fiber-type selective muscle damage in research and clinic.

Proteomic analysis reveals the distinct energy and protein metabolism characteristics involved in myofiber type conversion and resistance of atrophy in the extensor digitorum longus muscle of hibernating Daurian ground squirrels

Previous hibernation studies demonstrated that such a natural model of skeletal muscle disuse causes limited muscle atrophy and a significant fast-to-slow fiber type shift. However, the underlying mechanism as defined in a large-scale analysis remains unclarified. Isobaric tags for relative and absolute quantification (iTRAQ) based quantitative analysis were used to examine proteomic changes in the fast extensor digitorum longus muscles (EDL) of Daurian ground squirrels (Spermophilus dauricus). Although the wet weights and fiber cross-sectional area of the EDL muscle showed no significant decrease, the percentage of slow type fiber was 61% greater (P < 0.01) in the hibernation group. Proteomics analysis identified 264 proteins that were significantly changed (ratio < 0.83 or >1.2-fold and P < 0.05) in the hibernation group, of which 23 proteins were categorized into energy production and conversion and translation and 22 proteins were categorized into ribosomal structure and biogenesis. Along with the validation by western blot, MAPKAP kinase 2, ATP5D, ACADSB, calcineurin, CSTB and EIF2S were up-regulated in the hibernation group, whereas PDK4, COX II and EIF3C were down-regulated in the hibernation group. MAPKAP kinase 2 and PDK4 were associated with glycolysis, COX II and ATP5D were associated with oxidative phosphorylation, ACADSB was associated with fatty acid metabolism, calcineurin and CSTB were associated with catabolism, and EIF2S and EIF3C were associated with anabolism. Moreover, the total proteolysis rate of EDL in the hibernation group was significantly inhibited compared with that in the pre-hibernation group. These distinct energy and protein metabolism characteristics may be involved in myofiber type conversion and resistance to atrophy in the EDL of hibernating Daurian ground squirrels.

Proportions of Myosin Heavy Chain mRNAs, Protein Isoforms and Fiber Types in the Slow and Fast Skeletal Muscles Are Maintained After Alterations of Thyroid Status in Rats

Recently, we have established that slow soleus (SOL) and fast extensor digitorum longus (EDL) muscles of euthyroid (EU) Lewis rats posses the same proportions between their four myosin heavy chain (MyHC) mRNAs, protein isoforms and fiber types as determined by real time RT-PCR, SDS-PAGE and 2-D stereological fiber type analysis, respectively. In the present paper we investigated if these proportions are maintained in adult Lewis rats with hyperthyroid (HT) and hypothyroid (HY) status. Although HT and HY states change MyHC isoform expression, results from all three methods showed that proportion between MyHC mRNA-1, 2a, -2x/d, -2b, protein isoforms MyHC-1, -2a, -2x/d, -2b and to lesser extent also fiber types 1, 2A, 2X/D, 2B were preserved in both SOL and EDL muscles. Furthermore, in the SOL muscle mRNA expression of slow MyHC-1 remained up to three orders higher compared to fast MyHC transcripts, which explains the predominance of MyHC-1 isoform and fiber type 1 even in HT rats. Although HT status led in the SOL to increased expression of MyHC-2a mRNA, MyHC-2a isoform and 2A fibers, it preserved extremely low expression of MyHC-2x and -2b mRNA and protein isoforms, which explains the absence of pure 2X/D and 2B fibers. HY status, on the other hand, almost completely abolished expression of all three fast MyHC mRNAs, MyHC protein isoforms and fast fiber types in the SOL muscle. Our data present evidence that a correlation between mRNA, protein content and fiber type composition found in EU status is also preserved in HT and HY rats.

Effect of porcine Akirin2 on skeletal myosin heavy chain isoform expression.

Akirin2 plays an important role in skeletal myogenesis. In this study, we found that porcine Akirin2 (pAkirin2) mRNA level was significantly higher in fast extensor digitorum longus (EDL) and longissimus lumborum (LL) muscles than in slow soleus (SOL) muscle of pigs. Overexpression of pAkirin2 increased the number of myosin heavy chain (MHC)-positive cells, indicating that pAkirin2 promoted myoblast differentiation. We also found that overexpression of pAkirin2 increased the mRNA expressions of MHCI and MHCIIa and decreased the mRNA expression of MHCIIb. Myocyte enhancer factor 2 (MEF2) and nuclear factor of activated T cells (NFAT) are the major downstream effectors of calcineurin. Here we also observed that the mRNA expressions of MEF2C and NFATc1 were notably elevated by pAkirin2 overexpression. Together, our data indicate that the role of pAkirin2 in modulating MHCI and MHCIIa expressions may be achieved through calcineurin/NFATc1 signaling pathway.

Altered contractile properties of fast and slow skeletal muscles from db/db mice (1102.16)

In the db/db mouse, leptin receptor mutation results in a model of metabolic syndrome. Initial hyperinsulinemia brought on by hyperphagia is followed by peripheral insulin resistance, elevated blood glucose and increased body weight. The purpose of this study was to determine the skeletal muscle contractile properties in the BKS.Cg‐Dock7m +/+Leprdb/J mouse model of metabolic syndrome (i.e. db/db mouse). Muscle contractile properties were determined ex vivo in the fast extensor digitorum longus (EDL) muscle and the slow soleus muscle in 17 week old mice. Body weight was 43% greater in db/db mice compared to wild type control mice. Soleus muscle mass was 49% lower and EDL muscle mass was 55% lower in the db/db mouse. EDL twitch force was 58% lower than control with an associated 20% longer contraction time and 25% longer 1/2 relaxation time. There were no differences in soleus twitch force. However, contraction time was 1.7‐fold longer and 1/2 relaxation time was 1.6‐fold longer than control. The force‐frequency relationship in the soleus muscle was significantly shifted to the left toward lower stimulation frequencies, with no differences in the EDL. The rate of force loss with repeated contractions was also consistent with a less oxidative profile in both muscle types. Collectively, these data demonstrate the effects of risk factors for metabolic syndrome on muscle contractile properties in the db/db mouse.

Influence of chronic food deprivation on structure–function relationship of juvenile rat fast muscles

In the present study, we analyze the influence of chronic undernutrition on protein expression, muscle fiber type composition, and fatigue resistance of the fast extensor digitorum longus (EDL) muscle of male juvenile rats (45 ± 3 days of life; n = 25 and 31 rats for control and undernourished groups, respectively). Using 2D gel electrophoresis and mass spectrometry, we identified in undernourished muscles 12 proteins up-regulated (8 proteins of the electron transport chain and the glycolytic pathway, 2 cross-bridge proteins, chaperone and signaling proteins that are related to the stress response). In contrast, one down-regulated protein related to the fast muscle contractile system and two other proteins with no changes in expression were used as charge controls. By means of COX and alkaline ATPase histochemical techniques and low-frequency fatigue protocols we determined that undernourished muscles showed a larger proportion (15% increase) of Type IIa/IId fibers (oxidative-glycolytic) at the expense of Type IIb (glycolytic) fibers (15.5% decrease) and increased fatigue resistance (55.3%). In addition, all fiber types showed a significant reduction in their cross-sectional area (slow: 64.4%; intermediate: 63.9% and fast: 61.2%). These results indicate that undernourished EDL muscles exhibit an increased expression of energy metabolic and myofibrillar proteins which are associated with the predominance of oxidative and Type IIa/IId fibers and to a higher resistance to fatigue. We propose that such alterations may act as protective and/or adaptive mechanisms that counterbalance the effect of chronic undernourishment.

Comparison of Myosin Heavy Chain mRNAs, Protein Isoforms and Fiber Type Proportions in the Rat Slow and Fast Muscles

We studied the expression of myosin heavy chain isoforms at mRNA and protein levels as well as fiber type composition in the fast extensor digitorum longus (EDL) and slow soleus (SOL) twitch muscles of adult inbred Lewis strain rats. Comparison of the results from Real Time RT-PCR, SDS-PAGE and fiber type analysis showed corresponding proportions of MyHC transcripts (MyHC-1, -2a, -2x/d, -2b), protein isoforms (MyHC-1, -2a, -2x/d, -2b) and fiber types (type 1, 2A, 2X/D, 2B) in both muscles. Furthermore, we found that slow MyHC-1 mRNA expression in the SOL was up to three orders higher than that of fast MyHC transcripts. This finding can explain the predominance of MyHC-1 isoform and fiber type 1 and the absence of pure 2X/D and 2B fibers in the SOL muscle. Based on our data presenting quantitative evidence of corresponding proportions between mRNA level, protein content and fiber type composition, we suggest that the Real Time RT-PCR technique can be used as a routine method for analysis of muscle composition changes and could be advantageous for the analysis of scant biological samples such as muscle biopsies in humans.

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.

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.

Riluzole Treatment Reduces Motoneuron Death Induced by Axotomy in Newborn Rats

Nerve injury in neonatal rats leads to considerable motoneuron death. We investigated whether treatment with riluzole (a presynaptic inhibitor of glutamate release) is able to enhance survival of motor units (MUs) in the slow soleus (Sol) and fast extensor digitorum longus (EDL) muscles after sciatic nerve crush in newborn rats. Examination of 3- to 4-month-old rats revealed a beneficial effect of riluzole treatment after injury carried out on the first day after birth. At this time increased MU survival occurred in both the Sol and EDL muscles. In rats with nerve injury carried out on the second day after birth, increased MU survival occurred only in the Sol. We conclude that although riluzole treatment can rescue motoneurons destined to die and improve muscle performance, its beneficial effect is age-dependent, and the difference between the rescue of Sol and EDL MUs may be due to the slower maturation of motoneurons to soleus muscle. These findings have important implications regarding the motoneuron properties required for riluzole's beneficial effect.

Characterization of the Role of PKC-Theta in the Modulation of ClC-1 Chloride Channel Function and Calcium Homeostasis in Fast- and Slow-Twitch Skeletal Muscle by using PKC-Theta Null Mice

In skeletal muscle the resting chloride conductance (gCl), sustained by the ClC-1 chloride channel, controls membrane electrical stability as its absence causes myotonia. The gCl is typically higher in fast-twitch than in slow-twitch muscle and is negatively modulated by Protein Kinase C (PKC). Different PKC isoforms are expressed in skeletal muscle, including the PKC-theta. In PKC-theta-null mice (Sun et al., Nature, 2000) we found a significant increase in gCl of slow-twitch soleus muscle with respect to wild-type, being 1876±53 μS/cm2 (n=41) and 1356±37 μS/cm2 (n=19), respectively. A minor 13% increase of gCl was found in the fast extensor digitorum longus (EDL) muscle. Muscle excitability was reduced accordingly. Chelerythrine, a non-specific PKC inhibitor, further increases gCl by 25% in transgenic soleus muscle showing that other PKC isoforms are involved in the control of gCl. Preliminary experiments suggest indeed an up-regulation of the PKC-alpha isoform in these mice. Minor effect of chelerythrine was found in EDL of PKC-theta null mice. Fluvastatin, known to activate PKC (Pierno et al., Br J Pharmacol, 2009) reduced gCl in EDL muscle more than in soleus muscle of transgenic mice confirming that other PKC isoforms contribute to ClC-1 modulation. No modification of ClC-1 expression was found in soleus and EDL muscle of PKC-theta-null mice compared to wild-type. In these mice we also found a significant reduction of resting calcium concentration in both muscle types due to decreased sarcolemmal permeability at rest. Our results indicate that PKC-theta contributes to the regulation of ClC-1 channel differently in the muscle types and that this isoform can also modulate calcium homeostasis likely by interacting with sarcolemma channels. (ASI-OSMA)

Dysferlin is Dispensable for Recovering Saponin-Induced Membrnane Damage but Essential for Recovering Lengthening-Contraction-Induced Injury in mdx Mice

Dysferlin plays an important role in repairing membrane damage and dysferlin deficiency causes muscular dystrophy. Proteins such as perforin, complement component C9, and bacteria-derived cytolysins, as well as the natural detergent saponin can form large pores on the cell membrane via complexation with cholesterol. However, it is not clear whether dysferlin plays a role in repairing membrane damage induced by pore-forming reagents. Here we showed that dysferlin-deficient muscles recovered the tetanic force production to the same extent as their WT counterparts following a 5-min saponin exposure (50 μg/ml). Interestingly, the slow soleus muscles recovered significantly better than the fast extensor digitorum longus (EDL) muscles. These data suggest that dysferlin is unlikely involved in repairing saponin-induced membrane damage, and that the slow muscle is more efficient than the fast muscle in repairing such damage. Furthermore, the mice deficient in both dystrophin and dysferlin (DD-null) exhibited more severe muscle pathology than either mdx or dysferlin-null mice. Although lengthening contractions (LCs) caused similar force deficits regardless of dysferlin expression, we observed that the recovery of force within 45 minutes following LCs was hampered in DD-null muscles compared to mdx muscles. These data suggest that dysferlin plays a role in recovering the initial LCs-induced muscle injury of the DGC-compromised muscles. Dystrophin deficiency unmasks the function of dysferlin in membrane repair during LCs, providing a useful assay to evaluate the effectiveness of therapies designed to treat dysferlin deficiency.

On Marathons and Sprints: An Integrated Quantitative Proteomics and Transcriptomics Analysis of Differences Between Slow and Fast Muscle Fibers

Skeletal muscle tissue contains slow as well as fast twitch muscle fibers that possess different metabolic and contractile properties. Although the distribution of individual proteins in fast and slow fibers has been investigated extensively, a comprehensive proteomic analysis, which is key for any systems biology approach to muscle tissues, is missing. Here, we compared the global protein levels and gene expression profiles of the predominantly slow soleus and fast extensor digitorum longus muscles using the principle of in vivo stable isotope labeling with amino acids based on a fully lysine-6 labeled SILAC-mouse. We identified 551 proteins with significant quantitative differences between slow soleus and fast extensor digitorum longus fibers out of >2000 quantified proteins, which greatly extends the repertoire of proteins differentially regulated between both muscle types. Most of the differentially regulated proteins mediate cellular contraction, ion homeostasis, glycolysis, and oxidation, which reflect the major functional differences between both muscle types. Comparison of proteomics and transcriptomics data uncovered the existence of fiber-type specific posttranscriptional regulatory mechanisms resulting in differential accumulation of Myosin-8 and α-protein kinase 3 proteins and mRNAs among others. Phosphoproteome analysis of soleus and extensor digitorum longus muscles identified 2573 phosphosites on 973 proteins including 1040 novel phosphosites. The in vivo stable isotope labeling with amino acids-mouse approach used in our study provides a comprehensive view into the protein networks that direct fiber-type specific functions and allows a detailed dissection of the molecular composition of slow and fast muscle tissues with unprecedented resolution.

Comparison of New ELISA Method With Established SDS-PAGE Method for Determination of Muscle Myosin Heavy Chain Isoforms

We developed a new method for the quantitative determination of myosin heavy chain (MyHC) isoforms taking advantage of immunochemical differences and based on the ELISA principle. In the present paper we compare analysis of MyHC isoforms using the SDS-PAGE and the ELISA methods in the same samples of adult female inbred Lewis strain euthyroid, hyperthyroid and hypothyroid rats. In all thyroid states, the same composition and corresponding changes of MyHC isoforms were determined using both methodological approaches in the slow soleus and the fast extensor digitorum longus muscles. Our results showed that ELISA can be used for a "semi-quantitative" or "comparative" measurement of MyHC isoforms in multiple muscle samples, but that it is neither more exact nor faster compared to SDS-PAGE.