Contraction and energy metabolism are functions of skeletal muscles co‐regulated by still largely unknown signals. To help elucidating these interconnecting pathways, we are developing new cellular models that will allow to control the switch from a neonatal to an adult slow‐oxidative or fast‐glycolytic phenotype of myofibers, during in vitro differentiation. Thus, our purpose was to direct the differentiation of the newly characterized WTt clone, from a mixed towards either fast or slow phenotype, by modifying amounts of two transcription factors respectively involved in control of glycolytic and oxidative energy metabolism, namely HIF‐1α and PPARδ. Our data support the idea that HIF‐1α protein stabilization would favor expression of fast phenotypic markers, accompanied or not by a decreased expression of slow markers, depending on treatment conditions. Conversely, PPARδ over‐expression appears to enhance the slow‐oxidative phenotype of WTt myotubes. Furthermore, we have observed that expression of PGC‐1α, a coregulator of PPAR, is also modified in this cell line upon conditions that stabilize HIF‐1α protein. This observation points to the existence of a regulatory link between pathways controlled by the two transcription factors HIF‐1α and PPARδ. Therefore, these cells should be useful to analyze the balance between oxidative and glycolytic energy production as a function of phenotypic transitions occurring during myogenic maturation. The newly characterized murine WTt clone will be a good tool to investigate molecular mechanisms implicating HIF‐1α and PPARδ in the coordinated metabolic and contractile regulations involved in myogenesis. J. Cell. Biochem. 111: 82–93, 2010. © 2010 Wiley‐Liss, Inc.

We have investigated whether the phenotype of myogenic clones derived from satellite cells of different muscles from the transgenic immortomouse depended on muscle type origin. Clones derived from neonatal, or 6- to 12-week-old fast and slow muscles, were analyzed for myosin and enolase isoforms as phenotypic markers. All clones derived from slow-oxidative muscles differentiated into myotubes with a preferentially slow contractile phenotype, whereas some clones derived from rapid-glycolytic or neonatal muscles expressed both fast and slow myosin isoforms. Thus, muscle origin appears to bias myosin isoform expression in myotubes. The neonatal clone (WTt) was cultivated in various medium and substrate conditions, allowing us to determine optimized conditions for their differentiation. Matrigel allowed expressions of adult myosin isoforms, and an isozymic switch from embryonic alpha- toward muscle-specific beta-enolase, never previously observed in vitro. These cells will be a useful model for in vitro studies of muscle fiber maturation and plasticity.

It is of common knowledge that diabetes decreases skeletal muscle contractility and induces atrophy. However, how hyperglycemia and insulin deficiency modify muscle mass and neuromuscular recovery after muscle injury is not well known. We have analyzed two models of diabetes: streptozotocin (STZ)-treated Swiss mice and Akita mice that spontaneously develop diabetes. A fast muscle, the tibialis anterior, was injured following injection of a myotoxic agent (cardiotoxin). Neuromuscular function was evaluated by examining in situ isometric contractile properties of regenerating muscles in response to nerve stimulation 14, 28 and 56 days after myotoxic injury. We found that STZ-induced diabetes reduces muscle weight and absolute maximal tetanic force in both regenerating and uninjured muscles (p = 0.0001). Moreover, it increases specific maximal tetanic force and tetanic fusion in regenerating and uninjured muscles (p = 0.04). In the Akita mice, diabetes decreases muscle weight and absolute maximal tetanic force, and increases tetanic fusion in both regenerating and uninjured muscles (p ≤ 0.003). Interestingly, STZ-induced diabetes exerts more marked effects than diabetes of genetic origin, in particular on muscle weight. This reduction in muscle mass was not due to an increased expression of the atrogenes MuRF1 and atrogin-1 during STZ-induced diabetes. The present study in mice demonstrates that both models of diabetes impair regenerating muscles as well as uninjured muscles. Regenerating fast muscles are weaker, lighter and slower in diabetic compared with nondiabetic mice.

Enolase is a glycolytic enzyme, expressed as cell-type specific isoforms in higher vertebrates. Herein we demonstrated for the first time that enolase isoforms interact with microtubules during muscle satellite cell differentiation. While in undifferentiated myoblasts the ubiquitous alphaalpha enolase isoform, expressed at high level, exhibited extensive co-localization with microtubules, the muscle-specific betabeta isoform, expressed at low level, did not. During differentiation, the level of beta subunit increased significantly; the alpha and beta enolase immunoreactivities were detected both in cytosol and along the microtubules. We identified tubulin from muscle extract as an interacting protein for immobilized betabeta enolase. ELISA and surface plasmon resonance measurements demonstrated the direct binding of enolase isoforms to tubulin with an apparent KD below the micromolar range, and indicated that the presence of 0.8 mM 2-phosphoglycerate abolished the interaction. Our data showed that, at various stages of myogenic differentiation, microtubules were decorated by different enolase isoforms, which was controlled by the abundance of both partners. We suggest that the binding of enolase to microtubules could contribute to the regulation of the dynamism of the cytoskeletal filaments known to occur during the transition from myoblast to myotubes.

Transforming growth factor-beta1 (TGF-beta1) is known to be expressed in the environment of developing fast muscle fibres during ontogenesis. In the present study, we have examined effects of administration of either TGF-beta1 or neutralizing TGF-beta1 antibody on the induction of fast type phenotype in regenerating skeletal muscles in rats. Expressions of fast and slow myosin heavy chain (MHC) isoforms were studied using protein electrophoresis, at 3 and 6 weeks after myotoxic treatment. Muscle contractile properties were also measured in situ. The results have shown that a single injection of TGF-beta1 into the regenerating slow soleus muscle increased the expression of fast MHC-2x/d and MHC-2a and decreases that of slow MHC-1 (P<0.05). Moreover, it reduced the degree of tetanic fusion during contraction (P<0.05). Conversely, injection of neutralizing antibody against TGF-beta1 into the regenerating fast EDL muscle increased the expression of MHC-2a and MHC-1 (P<0.05). In conclusion, when the slow muscle was regenerating in the presence of an increased level of TGF-beta1, it induced a shift to a less slow MHC phenotype and contractile characteristics. Conversely, neutralization of TGF-beta1 in the regenerating fast muscle induced a shift to a less fast MHC expression. Together these results suggest that TGF-beta1 influences some aspects of fast muscle-type patterning during skeletal muscle regeneration.

Peripheral vascular occlusive diseases are frequently observed in humans, and studies with animal models have been largely used. However the effects of sustained lower limb ischemia on normal and regenerating hindlimb skeletal muscles are not well known in the mouse model. Therefore prolonged unilateral hindlimb ligation was generated by femoral artery ligation. Normal (myotoxic-untreated) and regenerating (myotoxic-reated) ischemic muscles were studied by analyses of the in situ contractile properties and histological parameters. Concerning normal mouse muscles, we found that femoral artery ligation reduced hindlimb perfusion and altered muscle structure and function. Thus 7 days after ligation, maximal tetanic force was reduced by about 70%, (p < 0.05). By 56 days after ligation, muscle weights and cross-section areas of muscle fibers were still reduced (p < 0.05). Concerning myotoxic treated muscles, we report that ligation reduced the recovery of muscle weight and maximal tetanic force and increased fatigue resistance at 56 days (p < 0.05). In conclusion, our results demonstrate that sustained peripheral arterial insufficiency in mice induces long-term as well as acute detrimental effects in both normal and regenerating muscles.

Background Gender influences the cardiac response to prolonged increases in workload, with differences at structural, functional, and molecular levels. However, it is unknown if post-ischemic function or metabolism of female hypertrophied hearts differ from male hypertrophied hearts. Thus, we tested the hypothesis that gender influences post-ischemic function of pressure-overload hypertrophied hearts and determined if the effect of gender on post-ischemic outcome could be explained by differences in metabolism, especially the catabolic fate of glucose. Methods Function and metabolism of isolated working hearts from sham-operated and aortic-constricted male and female Sprague-Dawley rats before and after 20 min of no-flow ischemia (N = 17 to 27 per group) were compared. Parallel series of hearts were perfused with Krebs-Henseleit solution containing 5.5 mM [5- 3 H/U- 14 C]-glucose, 1.2 mM [1- 14 C]-palmitate, 0.5 mM [U- 14 C]-lactate, and 100 mU/L insulin to measure glycolysis and glucose oxidation in one series and oxidation of palmitate and lactate in the second. Statistical analysis was performed using two-way analysis of variance. The sequential rejective Bonferroni procedure was used to correct for multiple comparisons and tests. Results Female gender negatively influenced post-ischemic function of non-hypertrophied hearts, but did not significantly influence function of hypertrophied hearts after ischemia such that mass-corrected hypertrophied heart function did not differ between genders. Before ischemia, glycolysis was accelerated in hypertrophied hearts, but to a greater extent in males, and did not differ between male and female non-hypertrophied hearts. Glycolysis fell in all groups after ischemia, except in non-hypertrophied female hearts, with the reduction in glycolysis after ischemia being greatest in males. Post-ischemic glycolytic rates were, therefore, similarly accelerated in hypertrophied male and female hearts and higher in female than male non-hypertrophied hearts. Glucose oxidation was lower in female than male hearts and was unaffected by hypertrophy or ischemia. Consequently, non-oxidative catabolism of glucose after ischemia was lowest in male non-hypertrophied hearts and comparably elevated in hypertrophied hearts of both sexes. These differences in non-oxidative glucose catabolism were inversely related to post-ischemic functional recovery. Conclusion Gender does not significantly influence post-ischemic function of hypertrophied hearts, even though female sex is detrimental to post-ischemic function in non-hypertrophied hearts. Differences in glucose catabolism may contribute to hypertrophy-induced and gender-related differences in post-ischemic function.

We have analysed the rate and ultimate extent of muscle functional recovery after snake venom-induced myotoxicity, as well as the relationships between functional, biochemical and structural indices of recovery. We also compared the effects of various injuries leading to muscle necrosis, loss of innervation/vasculature and/or precursors of muscle cells (pmc). We found that several parameters of rat soleus muscle such as maximal isometric force, slow myosin heavy chain, and citrate synthase, were fully and rapidly restored within 6 weeks after treatment with snake Notechis scutatus venom (im, 2 μg/muscle). In contrast, some muscle contractile properties (degree of tetanic fusion, fatigue resistance…) were not fully recovered even by 12 weeks after venom treatment. However, when compared to other injuries, recovery 3 weeks after venom treatment, was better than that observed after severing the terminal nerve and accompanying vessels and after cryodamage known to kill pmc. In conclusion, our studies demonstrate that—contrary to what is commonly believed - muscle treated by myotoxic agent does not recover rapidly and fully. However, the degree or rate of muscle recovery after snake venom treatment was much better when compared to other types of injury. In addition, histological and biochemical parameters cannot be used as such to easily predict functional recovery following injury.

Adaptation of myocardial energy substrate utilization may contribute to the cardioprotective effects of regular exercise, a possibility supported by evidence showing that pharmacological metabolic modulation is beneficial to ischemic hearts during reperfusion. Thus we tested the hypothesis that the beneficial effect of regular physical exercise on recovery from ischemia-reperfusion is associated with a protective metabolic phenotype. Function, glycolysis, and oxidation of glucose, lactate, and palmitate were measured in isolated working hearts from sedentary control ( C) and treadmill-trained ( T: 10 wk, 4 days/wk) female Sprague-Dawley rats submitted to 20 min ischemia and 40 min reperfusion. Training resulted in myocardial hypertrophy ( 1.65 +/- 0.05 vs. 1.30 +/- 0.03 g heart wet wt, P < 0.001) and improved recovery of function after ischemia by nearly 50% ( P < 0.05). Glycolysis was 25 - 30% lower in T hearts before and after ischemia ( P < 0.05), whereas rates of glucose oxidation were 45% higher before ischemia ( P < 0.01). As a result, the fraction of glucose oxidized before and after ischemia was, respectively, twofold and 25% greater in T hearts ( P < 0.05). Palmitate oxidation was 50 - 65% greater in T than in C before and after ischemia ( P < 0.05), whereas lactate oxidation did not differ between groups. Alteration in content of selected enzymes and proteins, as assessed by immunoblot analysis, could not account for the reduction in glycolysis or increase in glucose and palmitate oxidation observed. Combined with the studies on the beneficial effect of pharmacological modulation of energy metabolism, the present results provide support for a role of metabolic adaptations in protecting the trained heart against ischemia-reperfusion injury.

In higher vertebrates, the glycolytic enzyme enolase (2-phospho-D-glycerate hydrolase; EC 4.2.1.11) is active as a dimer formed from three different subunits, α, β and γ, encoded by separate genes. The expression of these genes is developmentally regulated in a tissue-specific manner. A shift occurs during development, from the unique embryonic isoform αα, towards specific isoforms in two tissues with high energy demands: αβ and γγ in the nervous system, αβ and ββ in striated muscles. The αα remains widely distributed in adult tissues. Here we report the results of the first extensive study of β enolase expression during human development. Indeed, the β subunit is specifically expressed at early stages of human myogenesis. Immunocytochemical analyses demonstrated that it is first detected in the heart of 3-week-old embryos and in the myotomal compartment of somites from 4-week-old embryos. At this stage, the muscle-specific sarcomeric protein titin is expressed in this structure, which will give rise to all body skeletal muscles, but embryonic myosin heavy chain is not yet present. Analyses at the protein level show that, during human ontogenesis, myogenesis is accompanied by an increase in β enolase expression and by a decrease in the expression of the two other α and γ subunits. Furthermore, β enolase subunit is expressed in proliferating myoblasts from both embryonic and post-natal muscles. In addition, clonal analysis of primary cell cultures, obtained from the leg muscle of a 7-week-old human embryo, revealed that the β subunit is present in the dividing myoblasts of all four types, according to the classification of Edom-Vovard et al . [(1999) J Cell Sci 112: 191–199], but not in cells of the non-myogenic lineage. Myoblast fusion is accompanied by a large increase in β enolase expression. Our results demonstrate that this muscle-specific isoform of a glycolytic enzyme (β enolase) is among the earliest markers of myogenic differentiation in humans.

Nothing is known about the expression of the glycolytic enzyme enolase in skeletal muscle alterations such as myofiber degeneration and regeneration. Enolase is a dimeric enzyme which exhibits cell type specific isoforms. The embryonic form, alphaalpha, remains expressed in most adult tissues, whereas a transition towards specific isoforms occurs during ontogenesis in two cell types with high energy requirements: alphagamma and gammagamma in neurons, alphabeta and betabeta in striated muscle cells. During murine myogenesis, beta enolase transcripts are detected early in the forming muscles, and the beta gene is further upregulated at specific stages of muscle development. The alpha and beta subunits exhibit characteristic developmental microheterogeneity patterns. High levels of beta enolase subunits characterize the glycolytic fast-twitch fibers of adult muscles. We have investigated the expression of enolase subunits in a mouse experimental model of muscle regeneration. Following a single intramuscular injection of the necrotic agent cardiotoxin, we observed a rapid decrease in the level of the major muscle enolase subunit beta, accounting for the drop in total enolase activity that correlated with the degeneration of myofibers. Concomitant with the regeneration of new fibers, beta subunit levels began to increase, reaching normal values by 30 days after injury. Changes in the embryonic and ubiquitous subunit, alpha, mimicked those occurring during development by two aspects: modifications in electrophoretic variants and redistribution between soluble and insoluble compartments of muscle extracts. Imunocytochemical analyses of alpha and beta enolase subunits first revealed a homogeneous labeling within myofibers. Striations characteristic of normal adult muscle tissue were visible again by day 14 after injury. A perinuclear alpha and beta immunoreactivity was often observed in regenerating myofibers but its functional significance remains to be elucidated. Double labeling experiments with anti-gamma enolase and FITC-alpha bungarotoxin allowed us to follow the neuromuscular junction remodeling that occurs during muscle regeneration despite the absence of nerve injury.

During muscle development, an isozymic transition of the glycolytic enzyme enolase occurs from the embryonic and ubiquitous alphaalpha-isoform to the muscle-specific betabeta-isoform. Here, we demonstrate a stimulatory role of thyroid hormones on these two enolase genes during rat development in hindlimb muscles and an inhibitory effect on the muscle-specific enolase gene in cardiac muscle. In hindlimb muscles the ubiquitous alpha-transcript level is diminished by hypothyroidism, starting at birth. On the contrary, the more abundant muscle-specific beta-transcript is insensitive to hypothyroidism before establishment of the functional diversification of fibers and is greatly decreased thereafter. Our data support the hypothesis of a role of thyroid hormones in coordinating the expressions of contractile proteins and metabolic enzymes during muscle development. The subcellular localization of isoenolases, established here, is not modified by hypothyroidism. Our results underline the specificity of action of thyroid hormones, which modulate differentially two isozymes in the same muscle and regulate, in opposite directions, the expression of the same gene in two different muscles.

Enolase is a dimeric glycolytic enzyme exhibiting tissue specific isoforms. During ontogenesis, a transition occurs from the embryonic alphaalpha towards the specific alphabeta, and betabeta isoforms in striated muscle. Immunocytochemical analyses on transverse sections of adult mouse gastrocnemius muscle, allowed us to compare the expression of alpha and beta subunits to that of myosin heavy chain (MHC) isoforms. Levels of beta immunoreactivity followed the order IIB > IIX > IIA > I. This gradient parallels the ATPase activity associated to MHC isoforms, indicating that the expression of beta enolase in myofibres is finely regulated as a function of energetic requirements. By contrast, variations in alpha immunolabelling intensity appeared independent of fibre types. Longitudinal muscle sections exhibited a striated pattern of alpha immunoreactivity. Confocal microscopy analyses demonstrated that alpha was localised at the M band. Most beta immunoreactivity was diffuse all over the sarcoplasm. However, some beta immunoreactivity was striated and localized at both Z and M bands. Thus, betabeta enolase could participate to multi-enzyme complexes present at the I band, and involved with local ATP production. Our results support the notion that isozymes differ in their ability to interact with other macromolecules, thus segregating to different subcellular sites where they would respond to specific functional demands.

We report here, for the first time, the expression of the muscle-specific isoform of the glycolytic enzyme, enolase (EC 4.2.1. 11) (beta enolase), in rabbit skeletal muscles. We have analysed the fast-twitch gastrocnemius and the slow-twitch soleus muscles during normal postnatal development and following denervation. We show that, in rabbit, as already described in rodents, beta enolase gene expression behaves as a good marker of the fast-twitch fibers. In soleus muscle, the beta enolase transcript level is 10-20% of that found in gastrocnemius. Denervation, performed at 8 postnatal days, induces an important drop of beta enolase transcript levels in both developing soleus and gastrocnemius muscles, with a 80% decrease observed 1 week after denervation in the operated muscles, as compared to the corresponding contralateral muscles. Thereafter, the beta enolase transcript level continues to decrease in the fast-twitch muscle, with the beta enolase subunit being detectable only in the atrophic fast-twitch fibers. In contrast, the beta transcript level tends to increase in the denervated slow-twitch muscle, reaching about 50% of that in contralateral soleus, at 7 weeks after surgery. The level of beta enolase transcripts still expressed after denervation seems to stabilize at the same low level in both types of inactive muscles. This suggests that the small fraction of beta enolase expression which is not controlled by the nerve, or by the contractile activity imposed by it, is independent of the muscle phenotype.

Glycerol-skinned skeletal muscle fibres retain the defined sarcomeric structure of the myofibrils. We show here that a small fraction of two enzymes important for energy metabolism, the cytosolic muscle isoform of creatine kinase (EC 2.7.3.2), MM-creatine kinase (MM-CK), and enolase (EC 4.2.1.11), remains bound to skinned fibres. CK is slowly exchangeable, whereas enolase is firmly bound. Two-dimensional gel electrophoresis followed by Western blot analyses demonstrates that both alpha (ubiquitous) and beta (muscle-specific) subunits of enolase are present in these preparations. Enolase and CK were co-localized at the M-band of the sarcomeres, as observed by indirect immunofluorescence and confocal microscopy. Cross-linking experiments were performed on skinned fibres with three bifunctional succinimidyl esters of different lengths and yielded a protein complex of 150 kDa that reacted with antibodies directed against either M-CK or beta-enolase. The cross-linking efficiency was greatest for the longest reagent and zero for the shortest one. The length of the cross-linker giving a covalent complex between the two enzymes does not support the notion of a direct interaction between M-CK and enolase. This is the first demonstration of the presence of an enzyme of energy metabolism other than CK at the M-band of myofibres.

In higher vertebrates, the glycolytic enzyme enolase (2-phospho-d- glycerate hydrolyase; EC 4.2.1.11) is active as a dimeric protein formed from three subunits – α: ubiquitous, β: muscle specific, and γ: neuron specific – encoded by different genes. In the present study, we have shown that an antiserum previously produced against the mouse ββ enolase is also a specific reagent for the muscle specific human enolase. Using this antiserum to study human muscles, we demonstrated novel patterns of the β subunit microheterogeneity which are distinctive from those observed previously in rodents and which appear to be independent of age, gender and muscular activity. Two variants of the beta subunit differing by their size have been detected: one heavy form of 46 kDa (βH) and one light form of 45 kDa (βL). Muscle biopsies expressed either βH or βL or βH + βL, and all muscles of an individual expressed the same variants. The products of in vitro translation of RNA prepared from human muscle displayed β subunit variants identical to those of the protein present in the biopsy. Therefore the differences observed between individuals reveal a difference already present at the level of the RNA transcripts. These observations suggest the existence of an yet undescribed polymorphism of the human β enolase gene which could affect the coding sequence. Comparative immunocytochemical and histochemical analyses of biopsies demonstrated that the β subunit was expressed in all fast fibres (type II), but not in slow fibres (type I). No difference was observed in the intensity of β enolase immunolabelling between the various types (IIA, IIAB, IIB) of fast fibres. No significant difference in fibre type composition and histological appearance was visible between muscles presenting either one of the three patterns of microheterogeneity.

Biology of the CellVolume 90, Issue 1 p. 127-127 Subcellular localization of enolase isoforms expressed in striated muscle Angelica Keller, Angelica Keller INSERM U469, 141 rue de la Cardonille, 34094 Montpellier, FranceSearch for more papers by this authorTatyana Merkulova, Tatyana Merkulova INSERM U469, 141 rue de la Cardonille, 34094 Montpellier, FranceSearch for more papers by this author Angelica Keller, Angelica Keller INSERM U469, 141 rue de la Cardonille, 34094 Montpellier, FranceSearch for more papers by this authorTatyana Merkulova, Tatyana Merkulova INSERM U469, 141 rue de la Cardonille, 34094 Montpellier, FranceSearch for more papers by this author First published: 01 February 2012 https://doi.org/10.1016/S0248-4900(98)80323-7AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume90, Issue1January 1998Pages 127-127 RelatedInformation

The glycolytic enzyme enolase (EC 4.2.1.11) is active as dimers formed from three subunits encoded by different genes. The embryonic alphaalpha isoform remains distributed in many adult cell types, whereas a transition towards betabeta and gammagamma isoforms occurs in striated muscle cells and neurons respectively. It is not understood why enolase exhibits tissue-specific isoforms with very close functional properties. We approached this problem by the purification of native betabeta-enolase from mouse hindlimb muscles and by raising specific antibodies of high titre against this protein. These reagents have been useful in revealing a heterogeneity of the beta-enolase subunit that changes with in vivo and in vitro maturation. A basic carboxypeptidase appears to be involved in generating an acidic beta-enolase variant, and may regulate plasminogen binding by this subunit. We show for the first time that pure betabeta-enolase binds with high affinity the adjacent enzymes in the glycolytic pathway (pyruvate kinase and phosphoglycerate mutase), favouring the hypothesis that these three enzymes form a functional glycolytic segment. betabeta-Enolase binds with high affinity sarcomeric troponin but not actin and tropomyosin. Some of these binding properties are shared by the alphaalpha-isoenolase, which is also expressed in striated muscle, but not by the neuron-specific gammagamma-enolase. These results support the idea that specific interactions with macromolecules will address muscle enolase isoforms at the subcellular site where ATP, produced through glycolysis, is most needed for contraction. Such a specific targeting could be modulated by post-translational modifications.