Large-scale mRNA analysis of female skeletal muscles during 60 days of bed rest with and without exercise or dietary protein supplementation as countermeasures.

Microgravity has a dramatic impact on human physiology, illustrated in particular, with skeletal muscle impairment. A thorough understanding of the mechanisms leading to loss of muscle mass and structural disorders is necessary for defining efficient clinical and spaceflight countermeasures. We investigated the effects of long-term bed rest on the transcriptome of soleus (SOL) and vastus lateralis (VL) muscles in healthy women (BRC group, n = 8), and the potential beneficial impact of protein supplementation (BRN group, n = 8) and of a combined resistance and aerobic training (BRE group, n = 8). Gene expression profiles were obtained using a customized microarray containing 6,681 muscles-relevant genes. A two-class statistical analysis was applied on 2,103 genes with consolidated expression in BRC, BRN, and BRE groups. We identified 472 and 207 mRNAs whose expression was modified in SOL and VL from BRC group, respectively. Further clustering analysis, identifying relevant biological mechanisms and pathways, reported five main subclusters. Three are composed of upregulated mRNAs involved mainly in nucleic acid and protein metabolism, and two made up of downregulated transcripts encoding components involved in energy metabolism. Exercise countermeasure demonstrated drastic compensatory effects, decreasing the number of differentially expressed mRNAs by 89 and 96% in SOL and VL, respectively. In contrast, nutrition countermeasure had moderate effects and decreased the number of differentially-expressed transcripts by 40 and 25% in SOL and VL. Together, these data present a systematic, global and comprehensive view of the adaptive response of female muscle to long-term atrophy.

Does antioxidant system adaptive response alleviate related oxidative damage with long term bed rest?

OBJECTIVES: The aim of the study was to evidence oxidative damage and erythrocyte antioxidant enzyme activities during long term bed rest (LTBR) and recovery, while verifying the prophylactic effects of resistance exercise on LTBR-induced oxidative damage. DESIGN AND METHODS: 11 healthy male participated in the study. Nutrient intakes were monitored. Assessments occurred during LTBR (60th and 90th day) and 90 days after the end. RESULTS: LTBR induced only a slight decrease in total thiol protein (SH) group concentrations. Glutathione peroxidase (GPx) activity was upregulated during LTBR and down regulated after recovery suggesting that hypokinesia induces an oxidative stress. These effects where not correlated to antioxidant intake as nutritional density is preserved. Lipoperoxidative markers stay unchanged. CONCLUSIONS: Exercise alleviates hypokinesia outcomes by preserving glutathione reductase activity with minor effect on hypokinesia-induced antioxidant response and oxidative stress which both exhibit a high magnitude inter-individual variability. Return to initial physical activity allows biomarkers to return to initial values marking the end of the stress. Hypokinetic situations should be considered as an oxidative stressful situation requiring exercise and nutritional strategies.

Structural organization of the perimysium in bovine skeletal muscle: Junctional plates and associated intracellular subdomains.

We analyzed the structural features of the perimysium collagen network in bovine Flexor carpi radialis muscle using various sample preparation methods and microscopy techniques. We first observed by scanning electron microscopy that perimysium formed a regular network of collagen fibers with three hierarchical levels including (i) a loose lattice of large interwoven fibers ramified in (ii) numerous collagen plexi attaching together adjacent myofibers at the level of (iii) specific structures that we call perimysial junctional plates. Second, we looked more closely at the intracellular organization underneath each plate using transmission electron microscopy, immunohistochemistry, and a three-dimensional reconstruction from serial sections. We observed the accumulation of myonuclei arranged in clusters surrounded by a high density of subsarcolemmal mitochondria and the proximity of capillary branches. Third, we analyzed the distribution of these perimysial junctional plates, subsarcolemmal mitochondria, and myonuclei clusters along the myofibers using a statistical analysis of the distances between these structures. This revealed a global colocalization and the existence of adhesion domains between endomysium and perimysium. Taken together, our observations give a better description of the perimysium organization in skeletal muscle, and provide evidence that perimysial junctional plates with associated intracellular subdomains may participate in the lateral transmission of contractile forces as well as mechanosensing.

Ubiquitin targeting of rat muscle proteins during short periods of unloading.

AIM: The ubiquitin-proteasome system is known to be involved in many situations leading to skeletal muscle atrophy. However, the cellular mechanisms triggering the atrophic process initiation are still poorly understood. For short periods of rat hindlimb unloading, we assessed the specific ubiquitin targeting of sarcoplasmic or myofibrillar proteins in slow and fast rat muscle types. METHODS: Adult Sprague Dawley rats were randomly assigned to three groups: control, hindlimb-unloaded for 4 days (HU4) and hindlimb-unloaded for 8 days (HU8). In fractionated extracts from soleus (SOL) and Extensor Digitorum Longus (EDL) muscles, the relative contents of free and conjugated ubiquitin were quantified by immunoblotting. RESULTS: Hindlimb unloading of short durations resulted in a preferential atrophy of slow-twitch fibres and bound ubiquitin levels were increased by 37 and 68% in the soleus myofibrillar fraction after respectively 4 and 8 days. The ubiquitin conjugation was shown to principally affect the high molecular weight proteins. Free and conjugated ubiquitin levels remained unchanged in sarcoplasmic fraction from SOL muscle after 8 days HU. For the fast muscle (EDL), ubiquitin contents were approximately twofold lower in control conditions, and did not significantly change during the hindlimb unloading periods considered. CONCLUSION: The postural SOL muscle was shown to contain higher constitutive sarcoplasmic ubiquitin levels than the phasic EDL. The high response to unloading of the slow twitch fibres rich SOL muscle was accompanied by a specific conjugation of its myofibrillar proteins that may participate in the initiation of skeletal muscle remodelling consequent to disuse.

Changes in dysferlin, proteins from dystrophin glycoprotein complex, costameres, and cytoskeleton in human soleus and vastus lateralis muscles after a long-term bedrest with or without exercise.

This study was designed to evaluate the effects of hypokinesia and hypodynamia on cytoskeletal and related protein contents in human skeletal muscles. Twelve proteins: dystrophin and its associated proteins (DGC), dysferlin, talin, vinculin and meta-vinculin, alpha-actinin, desmin, actin, and myosin, were quantitatively analyzed during an 84-day long-term bedrest (LTBR). The preventive or compensatory effects of maximal resistance exercise (MRE) as a countermeasure were evaluated. Most of these proteins are involved in several myopathies, and they play an important role in muscle structure, fiber cohesion, cell integrity maintenance, and force transmission. This is the first comparison of the cytoskeletal protein contents between slow postural soleus (SOL) and mixed poly-functional vastus lateralis (VL) human muscles. Protein contents were higher in VL than in SOL (from 12 to 94%). These differences could be mainly explained by the differential mechanical constraints imposed on the muscles, i.e., cytoskeletal protein contents increase with mechanical constraints. After LTBR, proteins belonging to the DGC, dysferlin, and proteins of the costamere exhibited large increases, higher in SOL (from 67 to 216%) than in VL (from 32 to 142%). Plasma membrane remodeling during muscle atrophy is probably one of the key points for interpreting these modifications, and mechanisms other than those involved in the resistance of the cytoskeleton to mechanical constraints may be implicated (membrane repair). MRE compensates the cytoskeletal changes induced by LTBR in SOL, except for gamma-sarcoglycan (+70%) and dysferlin (+108%). The exercise only partly compensated the DGC changes induced in VL, and, as for SOL, dysferlin remained largely increased (+132%). Moreover, vinculin and metavinculin, which exhibited no significant change in VL after LTBR, were increased with MRE during LTBR, reinforcing the pre-LTBR differences between SOL and VL. This knowledge will contribute to the development of efficient space flight countermeasures and rehabilitation methods in clinical situations where musculoskeletal unloading is a component.

Analysis of altered gene expression in rat soleus muscle atrophied by disuse.

The present study involved a global analysis of genes whose expression was modified in rat soleus muscle atrophied after hindlimb suspension (HS). HS muscle unloading is a common model for muscle disuse that especially affects antigravity slow-twitch muscles such as the soleus muscle. A cDNA cloning strategy, based on suppression subtractive hybridization technology, led to the construction of two normalized soleus muscle cDNA libraries that were subtracted in opposite directions, i.e., atrophied soleus muscle cDNAs subtracted by control cDNAs and vice versa. Differential screening of the two libraries revealed 34 genes with altered expression in HS soleus muscle, including 11 novel cDNAs, in addition to the 2X and 2B myosin heavy chain genes expressed only in soleus muscles after HS. Gene up- and down-regulations were quantified by reverse Northern blot and classical Northern blot analysis. The 25 genes with known functions fell into seven important functional categories. The homogeneity of gene alterations within each category gave several clues for unraveling the interplay of cellular events implied in the muscle atrophy phenotype. In particular, our results indicate that modulations in slow- and fast-twitch-muscle component balance, the protein synthesis/secretion pathway, and the extracellular matrix/cytoskeleton axis are likely to be key molecular mechanisms of muscle atrophy. In addition, the cloning of novel cDNAs underlined the efficiency of the chosen technical approach and gave novel possibilities to further decipher the molecular mechanisms of muscle atrophy.

Effects of locomotor training on hindlimb regeneration in the urodele amphibian Pleurodeles waltlii.

1. The effects of locomotor training on hindlimb regeneration were studied in the urodele amphibian Pleurodeles waltlii. 2. After amputation of one hindlimb at mid-femur, adult animals were subjected to regular training sessions (1 h daily, 5 days a week, over 8 months) of terrestrial stepping. 3. Eight months post-amputation, trained animals exhibited regenerated limbs of reduced size as compared to animals kept in their aquaria. Histological data showed an abnormal regeneration of both the femur and distal structures (e.g. digit muscles, metatarsi and phalanges) while medial structures (e.g. tibia and fibula) were totally re-formed. The study of the electromyographical activity in regenerated limbs during stepping and that of their reflex responsiveness to electrical stimulation showed that both motor and sensory innervations were functional in the limb stump of trained animals. 4. The regenerative capacity of the abnormal stumps was preserved since following a second amputation a quite normal hindlimb was regenerated in 3 months, provided the re-amputated animals were not trained to terrestrial stepping. 5. The stress due to handling, change in locomotor medium (aquatic vs. terrestrial) and the friction of the wound epidermis with the ground were not involved in the disruption of limb regeneration. 6. The locomotor pattern, the reflex responsiveness and the muscle fibre composition were similar in supernumerary forelimbs grafted on the back and in normal forelimbs. However, the supernumerary forelimbs regenerated normally even in animals subjected to locomotor training while the hindlimb did not. It is concluded that the disrupting effects of locomotor training on limb regeneration were localized to the the limb directly involved in locomotion. 7. The mechanisms underlying abnormal limb regeneration in animals subjected to locomotor training are discussed.

Cytoskeletal protein contents before and after hindlimb suspension in a fast and slow rat skeletal muscle.

Transversal cytoskeletal organization of muscle fibers is well described, although very few data are available concerning protein content. Measurements of desmin, alpha-actinin, and actin contents in soleus and extensor digitorum longus (EDL) rat skeletal muscles, taken with the results previously reported for several dystrophin-glycoprotein complex (DGC) components, indicate that the contents of most cytoskeletal proteins are higher in slow-type fibers than in fast ones. The effects of hypokinesia and unloading on the cytoskeleton were also investigated, using hindlimb suspension. First, this resulted in a decrease in contractile protein contents, only after 6 wk, in the soleus. Dystrophin and associated proteins were shown to be reduced for soleus at 3 wk, whereas only the dystrophin-associated proteins were found to increase after 6 wk. On the other hand, the contents of DGC components were increased for EDL for the two durations. Desmin and alpha-actinin levels were unchanged in the same conditions. Consequently, it can be concluded that the cytoskeletal protein expression levels could largely contribute to muscle fiber adaptation induced by modified functional demands.

Fatigue of elbow flexors during repeated flexion-extension cycles: effect of movement strategy.

The objective of this work was to study the influence of movement strategy on fatigue of elbow flexors during repeated flexion-extension cycles in order to enlighten the fatigue process occurring during "pumping" in boardsailing. This dynamic exercise was performed by six high-level Olympic boardsailors according to two movement strategies with different amplitude and frequency. The parameters of the exercise were chosen to result in similar mechanical work at every instant for both modalities. Isometric evaluation was performed before and after dynamic exercise to quantify muscle fatigue. Analysis of physical parameters (maximum voluntary contraction, endurance time) and electromyographic parameters (in both temporal and frequency domains) emphasized the peripheral origin of muscle fatigue at the level of the biceps brachii and the brachioradialis. After considering the limitations of this type of study, the results are discussed in terms of synergy and differentiation between muscles, peripheral fatigue and movement strategy. Although the mechanical work and total physiological demands were similar for the two movement modalities, analysis of electromyographic parameters suggests that muscle fatigue mainly involves the biceps brachii when movements were slow and wide, and the brachioradialis when they were rapid and short. This study makes it possible to take these specificities into account in order to adjust the physical training program.

Changes in electrophysiological properties of tibial motoneurones in the rat following 4 weeks of tetrodotoxin-induced paralysis.

In this study, we test the hypothesis that 4 weeks tetrodotoxin (TTX) paralysis altered the passive membrane properties of rat tibial motoneurones. Impulse activity along the sciatic nerve was blocked for 4 weeks using TTX delivered by an osmotic minipump to a Silastic cuff placed around the nerve. That portion of the sample exhibiting the 20% slowest After-hyperpolarization (AHP) decay time (AHPd), and which therefore included presumptive type S motoneurons, demonstrated responses (reduced AHPd, increased rheobase and rheobase voltage), which were not evident in the rest of the sample (presumptive fast motoneurons), in which an increased AHPd, in fact, was found. The results thus support the hypothesis that retrograde signals from inactive slow and fast muscle fibers have different effects on their innervating motoneurones.

Quantitative analysis of relative protein contents by Western blotting: comparison of three members of the dystrophin-glycoprotein complex in slow and fast rat skeletal muscle.

We have developed a method for accurate quantitative analysis and statistical comparison of the relative contents of the dystrophin-glycoprotein complex (DGC) in skeletal muscle. This method was applied to compare DGC contents in slow (soleus) and in fast (extensor digitorum longus, EDL) rat skeletal muscles. The quantitative analysis combines a modified bicinchoninic acid (BCA) assay with Western blotting and enhanced chemiluminescence (ECL). This combination allows the use of high levels of detergents and reducing reagents essential for extracting DGC. In addition, the evaluation of the total amount of proteins in each sample makes it possible to have a reference and to accurately compare relative protein levels without using a specific standard. With a large gradient gel, we could concomitantly compare two groups (n = 9) and quantify all protein contents differing highly in their molecular masses (from 35 kDa to 427 kDa). Each experiment was triplicated and normalized; the two muscles were compared using the Mann-Whitney test (P<0.001) to establish their protein content. The DGC relative levels for the slow muscle soleus and the fast muscle EDL differed significantly: dystrophin, beta-dystroglycan, and gamma-sarcoglycan levels were 130%, 110% and 120% higher in the soleus, respectively. The differences observed in the expression level of cytoskeletal associated protein (dystrophin) and transmembranous anchorage components may correspond to a physiological response of the muscle fibers to duration, magnitude, and frequency of the imposed mechanical loading.

Myosin heavy chains in fibers of TTX-paralyzed rat soleus and medial gastrocnemius muscles.

The expression of five myosin heavy chain (MHC) isoforms was analyzed in the rat soleus (Sol) and the deep and superficial medial gastrocnemius (dGM, sGM) muscle after 2 and 4 wk of TTX paralysis by using immunohistochemical techniques. In Sol, after 4 wk of paralysis, fibers containing type I MHC were either pure type I (14%) or also contained developmental (D; 76%), IIa (26%), or IIx (18%) MHC. Values for corresponding fibers in dGM were 8.5, 65, 38, and 22%. Also, by 4 wk an increase was seen in the proportions of fibers expressing IIa MHC in Sol (from 16 to 38%) and dGM (from 24 to 74%). In a region of sGM in control muscles containing pure IIb fibers, a major proportion (86%) remained pure after 4 wk of paralysis, with the remainder coexpressing IIb and IIx. The results indicate that TTX-induced muscle paralysis results in an increase in fibers containing multiple MHC isoforms and that the D isoform appears in a major proportion of these hybrid fibers.

Effects of 14-day spaceflight on myosin heavy chain expression in biceps and triceps muscles of the rhesus monkey.

In rats, changes in myosin expression are induced by the chronic elimination of weight-bearing activity, particularly in the postural muscles. This occurs during spaceflight and hindlimb suspension. Myosin heavy chain (MHC) changes affect fast and slow fiber types differently depending on muscle function. An increase in co-expression of different MHC within the same fiber will signal early changes in muscle fibers. In the rat soleus muscles, the spaceflight-induced increase in fast MHC expression appears to be essentially due to the enhanced or de novo synthesis of IID or IIX MHC. In response to microgravity, the expression of slow-type myosin decreases, while that of fast-type increases. There is scarce information concerning the effect of microgravity on rhesus monkeys (Macaca mulatta), especially on their upper limbs. We investigated the expression of MHC using an immunocytochemical approach to determine the nature and magnitude of the changes in biceps and triceps muscles of rhesus monkeys during the Bion 11 14-day mission.

Exercise-induced muscle damage: absence of adaptive effect after a single session of eccentric isokinetic heavy resistance exercise.

BACKGROUND: Unaccustomed eccentric exercise induces muscle damage. A single session of eccentric exercise can induce an "adaptive effect" protecting exercised muscles during several weeks. Our aim was to verify this phenomenon in isokinetic exercise. Tested hypothesis was: the progressive muscle rise in tension due to isokinetic eccentric actions would be insufficient to induce the adaptive effect. EXPERIMENTAL DESIGN: prospective study. SETTING: general community. PARTICIPANTS: six healthy and moderately active (untrained) males (29.1 yr +/- 1.5 SEM). INTERVENTIONS: subjects performed two isokinetic eccentric exercises (EE1 and EE2) of the quadriceps femoris of both legs (120 degrees.s-1; 8 sets of 15 repetitions) separated by 4 weeks. MEASURES: type I serum myosin heavy chains (MHC) and creatine kinase concentrations (CK), and rate of perceived soreness (DOMS) were collected before each exercise and on days 1, 2, 4, 6 and 9. RESULTS: Both exercises induced significant (p < 0.01) increases in MHC and CK concentrations, and DOMS score. There was no significant difference between EE1 and EE2, at any measurement time for any parameter. Mean peak values (SEM) were respectively (EE1; EE2): MHC (microU.l-1): 308 (192); 285 (191). CK (U.l-1): 1217 (760); 1297 (1039). DOMS score: 2.67 (0.52); 2.33 (0.52). CONCLUSIONS: The first session of eccentric isokinetic exercise (EE1) had no adaptive effect against muscle damage when an identical session was performed 4 weeks later (EE2). Muscle adaptation could have resulted in increased work production (+10.2%; p < 0.05; from EE1 to EE2).

Heart rate and blood lactate responses during competitive Olympic boardsailing.

The rules of competitive boardsailing events were changed before the Atlanta Olympic Games. Pumping the sail (pulling repeatedly on the rig) is now allowed and the duration of races has been shortened. Eight members of the French national team (mean age 23+/-2.7 years) participated in this study. Their cardiac and metabolic responses were assessed by measuring heart rate and blood lactate concentration during various competitive events in two strengths of wind (light vs. moderate). Heart rate was higher in light (87.4+/-4.3% HRmax; mean racing time 37 min) than in moderate wind conditions (82.9+/-5.3% HRmax; mean racing time 33 min). The mean post-race blood lactate concentration (5.2+/-1.0 mmol x l(-1)) was not affected by the wind conditions. Mean heart rate was highest during downwind legs (88.0+/-3.1% HRmax; duration 7-10 min). The races consisted of two laps, the first of which induced significantly higher cardiac demands than the second. We conclude that the changes to the rules of competitive boardsailing have increased the cardiac and metabolic efforts involved.

Upregulation of M-creatine kinase and glyceraldehyde3-phosphate dehydrogenase: two markers of muscle disuse.

Muscle disuse induces substantial alterations in the highly plastic skeletal muscle tissues, which occur especially in antigravity slow muscles. We differentially screened a muscle cDNA array to identify modifications in gene profile expression induced in slow rat soleus muscle mechanically unloaded by hindlimb suspension as a model for muscle disuse. This study focused on muscle creatine kinase mRNA and protein and glyceraldehyde-3-phosphate dehydrogenase mRNA, which were found to be upregulated in unweighted muscles. These upregulations were analyzed over a 4-wk time course of hindlimb suspension and compared with variations in myosin heavy chain (MHC) isoforms while specifically focusing on type IIx MHC mRNA and protein. The two metabolic marker upregulations clearly preceded IIx MHC contractile protein upregulation. Muscle creatine kinase upregulation was shown to be an excellent, and the earliest, marker of muscle disuse at mRNA and protein levels.

Variability of some SEMG parameter estimates with electrode location.

Muscular action potential conduction velocity (CV) and mean power frequency (MPF) are commonly used parameters to describe the surface electromyographic signal (SEMG). The discrepancies concerning the behavior and interpretation of these main parameters in the literature have motivated this work. Our objective was to evaluate within- and between-individual reproducibility, sensitivity and variation of CV and MPF depending on the electrode location with respect to various contraction modalities. The results present evidence for significant influence of electrode location on CV and MPF, not only in their initial values but also in their changes during fatiguing efforts. This influence appears to be subject-dependent. This variability seems to be essentially due to the relative displacements of myotendinous and neuromuscular junctions with respect to the electrode set. Moreover, this study shows that MPF can be seen as force-dependent under certain conditions and that the CV-MPF relationship is strongly influenced by methodological factors. In conclusion, it seems irrelevant to derive reliable SEMG parameter estimates without considering electrode location. There is a strong need for proper standardization based on anatomical and methological aspects before attempting any individual characterization. Finally, we suggest a procedure for assessment of measurement quality.

Increase in rat soleus myotendinous interface after a 14-d spaceflight.

Myotendinous junctions (MTJs) transmit contractile force from skeletal muscles to tendons. The effects of a 14-d spaceflight on MTJ were studied in the soleus muscle of male adult Sprague Dawley rats by transmission electron microscopy and histomorphometric techniques. We showed that the length of the junctional membrane relative to the muscle fiber diameter increased by 58% after 14 d of spaceflight. This increase accompanies morphological changes at MTJs. The flight MTJs appeared more shredded. The ends of the muscle fibers exhibited T tubule dilatation, swollen mitochondria, Z-disk streaming, loss of myofilaments, a thinning down of subplasmalemmal densitites, multivesicular bodies and signs of junctional membrane and basal lamina remodelling. The ultrastructural observations suggest that the increase in myotendinous interface could result from the extracellular matrix spreading into remodelling muscle fiber, whereas the constraints related to unloading were reduced by spaceflight conditions.

Effect of a 14-day spaceflight on dystrophin associated proteins complex in rat soleus muscle.

One of the most obvious effects of hypokinesia and hypodynamia is muscular atrophy. Changes in myosin expression are induced by the chronic elimination of the weight-bearing activity, particularly in the postural muscles, which occur during spaceflight and hindlimb suspension. Other morphological changes, such as the remodeling of myotendinous junction, are also induced by this reduction in mechanical stress. Moreover, the transversal interface between the cytoskeleton and the extracellular matrix of the muscle fiber can also be modified by the functional demand imposed on muscle. Dystrophin and its associated proteins appear to be essential for the stability of this interface and the deletion of one of these proteins results in a pathological phenotype. Changes in their expression appears to be induced by muscle disuse. We have quantified the changes in the expression of dystrophin and associated proteins induced by a 14-day spaceflight.