Fast Extensor Digitorum Longus 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.

MicroRNA-129-5p inhibits C2C12 myogenesis and represses slow fiber gene expression in vitro

The miR-129 family is widely reported as tumor repressors, although their roles in skeletal muscle have not been fully investigated. Here, the function and mechanism of miR-129-5p in skeletal muscle, a member of the miR-129 family, were explored using C2C12 cell line. Our study showed that miR-129-5p was widely detected in mouse tissues, with the highest expression in skeletal muscle. Gain- and loss-of-function study showed that miR-129-5p could negatively regulate myogenic differentiation, indicated by reduced ratio of MyHC-positive myofibers and repressed expression of myogenic genes, such as MyoD, MyoG, and MyHC. Furthermore, miR-129-5p was more enriched in fast extensor digitorum longus (EDL) than in slow soleus (SOL). Enhanced miR-129-5p could significantly reduce the expression of mitochondrial cox family, together with that of MyHC I, and knockdown of miR-129-5p conversely increased the expression of cox genes and MyHC I. Mechanistically, miR-129-5p directly targeted the 3′-UTR of Mef2a, which was suppressed by miR-129-5p agomir at both mRNA and protein levels in C2C12 cells. Moreover, overexpression of Mef2a could rescue the inhibitory effects of miR-129-5p on the expression of myogenic factors and MyHC I. Collectively, our data revealed that miR-129-5p is a negative regulator of myogenic differentiation and slow fiber gene expression, thus affecting body metabolic homeostasis.

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.

Treatment of Dystrophic mdx Mice with an ADAMTS-5 Specific Monoclonal Antibody Increases the Ex Vivo Strength of Isolated Fast Twitch Hindlimb Muscles

Aberrant extracellular matrix synthesis and remodeling contributes to muscle degeneration and weakness in Duchenne muscular dystrophy (DMD). ADAMTS-5, a secreted metalloproteinase with catalytic activity against versican, is implicated in myogenesis and inflammation. Here, using the mdx mouse model of DMD, we report increased ADAMTS-5 expression in dystrophic hindlimb muscles, localized to regions of regeneration and inflammation. To investigate the pathophysiological significance of this, 4-week-old mdx mice were treated with an ADAMTS-5 monoclonal antibody (mAb) or IgG2c (IgG) isotype control for 3 weeks. ADAMTS-5 mAb treatment did not reduce versican processing, as protein levels of the cleaved versikine fragment did not differ between hindlimb muscles from ADAMTS-5 mAb or IgG treated mdx mice. Nonetheless, ADAMTS-5 blockade improved ex vivo strength of isolated fast extensor digitorum longus, but not slow soleus, muscles. The underpinning mechanism may include modulation of regenerative myogenesis, as ADAMTS-5 blockade reduced the number of recently repaired desmin positive myofibers without affecting the number of desmin positive muscle progenitor cells. Treatment with the ADAMTS-5 mAb did not significantly affect markers of muscle damage, inflammation, nor fiber size. Altogether, the positive effects of ADAMTS-5 blockade in dystrophic muscles are fiber-type-specific and independent of versican processing.

Effect of high-fat mixed lipid diet and swimming on fibre types in skeletal muscles of rats with colon tumours

Skeletal muscle fibre types, whose characteristics are determined by myosin heavy chain (MyHC) isoforms, can adapt to changed physiological demands with changed MyHC isoform expression resulting in the fibre type transitions. The endurance training is known to induce fastto- slow transitions and has beneficial effect in carcinogenesis, whereas the effect of an excessive fat intake and its interaction with the effect of swimming are less conclusive. Therefore, we studied the effect of high-fat mixed lipid (HFML) diet and long-term (21-week) swimming on fibre type transitions and their average diameters by immunohistochemical demonstration of MyHC isoforms in slow soleus (SOL), fast extensor digitorum longus (EDL), and mixed gastrocnemius medialis and lateralis (GM, GL) muscles, divided to deep and superficial portions (GMd, GMs, GLd, GLs), of sedentary and swimming Wistar rats with experimentally (dimethylhydrazine) induced colon tumours and fed either with HFML or low-fat corn oil (LFCO) diet. HFML diet induced only a trend for fast-to-slow transitions in SOL and in the opposite direction in GMd. Swimming triggered significant transitions in unexpected slow-to-fast direction in SOL, whereas in GMs the transitions had tendency to proceed in the expected fast-toslow direction. The average diameters of fibre types were mostly unaffected. Hence, it can be concluded that if present, the effects of HFML diet and swimming on fibre type transitions were counteractive and muscle-specific implying that each muscle possesses its own adaptive range of response to changed physiological conditions.

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.

TheCompactMutation of Myostatin Causes a Glycolytic Shift in the Phenotype of Fast Skeletal Muscles

Myostatin is an important negative regulator of skeletal muscle growth. The hypermuscular Compact (Cmpt) mice carry a 12-bp natural mutation in the myostatin propeptide, with additional modifier genes being responsible for the phenotype. Muscle cellularity of the fast-type tibialis anterior (TA) and extensor digitorum longus (EDL) as well as the mixed-type soleus (SOL) muscles of Cmpt and wild-type mice was examined by immunohistochemical staining of the myosin heavy chain (MHC) proteins. In addition, transcript levels of MHC isoforms were quantified by qPCR. Based on our results, all investigated muscles of Cmpt mice were significantly larger compared with that of wild-type mice, as characterized by fiber hyperplasia of different grades. Fiber hypertrophy was not present in TA; however, EDL muscles showed specific IIB fiber hypertrophy while the (I and IIA) fibers of SOL muscles were generally hypertrophied. Both the fast TA and EDL muscles of Cmpt mice contained significantly more glycolytic IIB fibers accompanied by a decreased number of IIX and IIA fibers; however, this was not the case for SOL muscles. In summary, despite the variances found in muscle cellularity between the different myostatin mutant mice, similar glycolytic shifts were observed in Cmpt fast muscles as in muscles from myostatin knockout mice.

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.

Effects of Chronic Tenotomy on the Post-Tetanic Responses of Fast and Slow Muscles in Rats

The effects of tenotomy present for two weeks on the post-tetanic responses of isolated fast (extensor digitorum longus) and slow (soleus) muscles were studied by isometric recording of muscle contraction in rats. After tetanic stimulation with bursts of 100 impulses at a frequency of 20 impulses/sec, the force of single fast muscle contractions exceeded the pretetanic level by 20‐25% (p < 0.05) in both intact and tenotomized muscles. In similar conditions, the slow muscle soleus showed a decrease in the amplitudes of single contractions, i.e., post-tetanic depression. Post-tetanic depression was not seen after tenotomy. Caffeine (4 mM) increased the force of single and tetanic contractions in control and tenotomized fast muscles, though no corresponding increase in post-tetanic responses were seen. In slow muscles, caffeine had no effect on the nature of post-tetanic responses. Dantrolene (10 μM) decreased the force of single and tetanic contractions but did not prevent the appearance of post-tetanic potentiation in fast muscles and depression in intact slow muscles. The fact that the phenomenon of post-tetanic potentiation disappeared after denervation of fast muscles within two weeks while it was present after tenotomy for as long as 40 days after surgery provides grounds for suggesting the existence of a potentiating factor which reaches muscles by axonal transport and increases single contractions.

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.

Contractile properties and movement behaviour in neonatal rats with axotomy, treated with the NMDA antagonist DAP5.

BackgroundIt is well known that axotomy in the neonatal period causes massive loss of motoneurons, which is reflected in the reduction of the number of motor units and the alteration in muscle properties. This type of neuronal death is attributed to the excessive activation of the ionotropic glutamate receptors (glutamate excitotoxicity). In the present study we investigated the effect of the NMDA antagonist DAP5 [D-2-amino-5-phosphonopentanoic acid] in systemic administration, on muscle properties and on behavioural aspects following peripheral nerve injury.MethodsWistar rats were subjected to sciatic nerve crush on the second postnatal day. Four experimental groups were included in this study: a) controls (injection of 0.9% NaCl solution) b) crush c) DAP5 treated and d) crush and DAP5 treated. Animals were examined with isometric tension recordings of the fast extensor digitorum longus and the slow soleus muscles, as well as with locomotor tests at four time points, at P14, P21, P28 and adulthood (2 months).Results1. Administration of DAP5 alone provoked no apparent adverse effects. 2. In all age groups, animals with crush developed significantly less tension than the controls in both muscles and had a worse performance in locomotor tests (p < 0.01). Crush animals injected with DAP5 were definitely improved as their tension recordings and their locomotor behaviour were significantly improved compared to axotomized ones (p < 0.01). 3. The time course of soleus contraction was not altered by axotomy and the muscle remained slow-contracting in all developmental stages in all experimental groups. EDL, on the other hand, became slower after the crush (p < 0.05). DAP5 administration restored the contraction velocity, even up to the level of control animals 4. Following crush, EDL becomes fatigue resistant after P21 (p < 0.01). Soleus, on the other hand, becomes less fatigue resistant. DAP5 restored the profile in both muscles.ConclusionsOur results confirm that contractile properties and locomotor behaviour of animals are severely affected by axotomy, with a differential impact on fast contracting muscles. Administration of DAP5 reverses these devastating effects, without any observable side-effects. This agent could possibly show a therapeutic potential in other models of excitotoxic injury as well.

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.