Skeletal muscle repair by adult human mesenchymal stem cells from synovial membrane

We have demonstrated previously that adult human synovial membrane-derived mesenchymal stem cells (hSM-MSCs) have myogenic potential in vitro (De Bari, C., F. Dell'Accio, P. Tylzanowski, and F.P. Luyten. 2001. Arthritis Rheum. 44:1928–1942). In the present study, we have characterized their myogenic differentiation in a nude mouse model of skeletal muscle regeneration and provide proof of principle of their potential use for muscle repair in the mdx mouse model of Duchenne muscular dystrophy. When implanted into regenerating nude mouse muscle, hSM-MSCs contributed to myofibers and to long term persisting functional satellite cells. No nuclear fusion hybrids were observed between donor human cells and host mouse muscle cells. Myogenic differentiation proceeded through a molecular cascade resembling embryonic muscle development. Differentiation was sensitive to environmental cues, since hSM-MSCs injected into the bloodstream engrafted in several tissues, but acquired the muscle phenotype only within skeletal muscle. When administered into dystrophic muscles of immunosuppressed mdx mice, hSM-MSCs restored sarcolemmal expression of dystrophin, reduced central nucleation, and rescued the expression of mouse mechano growth factor

Pathogenetic mechanisms in animal models of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy is the most frequent inherited neuromuscular disease. Its incidence is high: around 1 affected boy out 3500 male births. Genetic counselling is inefficient in case of new mutations which account for 1/3 of the cases. The disease progress so that the DMD boys are wheelchair bound in their first decade and they die in their twenties. No curative therapy is available yet. The defective gene in DMD and its encoded protein, dystrophin, have been known for more than fifteen years, but the mechanisms leading from the absence of dystrophin to the muscle cell death is still poorly understood. Dystrophin binds to the cytoskeleton via subsarcolemmal F-actin and to the extracellular matrix via dystroglycans and laminin; besides, this scaffolding is stabilized by a complex of transmembranous proteins, the function of which remains uncertain.Animals sharing similar genetic defects affecting dystrophin were discovered. CXMD dogs are the most affected, but the mdx mice are definitely the most studied model. The latter show indisputable signs of muscle dystrophy, although they look little affected in vivo despite an absence of dystrophin. The different severity of the disease between the species is a puzzling observation. To better understand the role of dystrophin, an inventory of the defects related to its absence in mdx mice is necessary. In vivo, they show altered force and endurance as well as an raised serum creatine kinase level, as observed in human DMD boys. The improper muscular function is better characterized on isolated muscles. Muscles lacking dystrophin develop less tension. The hallmark of dystrophinopathy is the inability of the affected muscle to sustain the high mechanical stress produced during "eccentric" contractions (in which the muscle is forcibly lengthened while in full contracted state). Contractions following a series of such eccentric contractions show a marked reduction of force. This is correlated with the intracellular accumulation of vital dye, normally excluded from the fibres, witnessing membrane damages. Therefore, an important role of dystrophin is thought to be related to the maintenance of the mechanical integrity of the membrane during the high strains that occur in muscle contractions. In mdx muscle, calcium accumulates with age, as in muscles from DMD boys. The precise localisation of the accumulation is unresolved. [...]La dystrophie musculaire de Duchenne est la myopathie héréditaire la plus fréquente. Elle affecte un enfant toutes les 3500 naissances mâles ; le conseil génétique en est difficile. La perte de la marche survient vers 10 ans et les patients dépassent exceptionnellement 30 ans. Cette maladie est actuellement incurable. Bien que la protéine (la dystrophine) qui fait défaut et son gène soient connus depuis quinze ans, le rôle de cette protéine et la cascade d'évènements s'achevant par la mort de la fibre musculaire restent mystérieux. La dystrophine est une longue protéine située sous le sarcolemme, qui entretient des rapports étroits avec d'autres protéines du cytosquelette ou transmembranaires. Sa fonction est inconnue, mais des rôles de stabilisation membranaire ou dans la transduction du signal sont proposés. Plusieurs modèles animaux de dystrophie musculaire existent. Le chien dystrophique semble plus affecté, à première vue, que la souris mdx. Cependant cette dernière présente des signes indubitables de dystrophie musculaire. Elle constitue d'ailleurs un modèle largement utilisé. La souris mdx présente une élévation plasmatique de certaines enzymes musculaires. Elle développe une tension diminuée et, surtout, ses muscles sont particulièrement sensibles aux contractions avec étirement forcé (" excentriques "). Après quelques contractions de ce type, le muscle des souris mdx a perdu une grande partie de sa force. Ceci est corrélé avec l'accumulation intracellulaire d'un colorant vital, normalement exclu de la fibre. Ceci suggère que la dystrophine joue un rôle dans le maintien de l'intégrité membranaire. Par ailleurs, il a été démontré qu'avec l'âge, le calcium s'accumule dans le muscle de la souris mdx, sa localisation précise restant incertaine. En collaboration avec deux laboratoires européens, nous avons modifié le modèle mdx vers l'aggravation ou vers l'amélioration en utilisant des techniques de transgenèse. Leur évaluation fit appel à une batterie de tests fonctionnels. (1) Le premier modèle a été obtenu en croisant des souris mdx avec des souris déficientes en parvalbumine, un tampon calcique cytosolique. Cette protéine est surexprimée dans le muscle mdx mais n'est présente qu'à l'état de traces dans le muscle humain. Elle pourrait jouer un rôle protecteur en complexant le calcium lors d'influx trop importants. Les signes de dégénérescence et de pseudohypertrophie sont plus marqués dans les muscles du double mutant. Cependant, d'excellentes capacités de régénération permettent à ce modèle murin, dépourvu de parvalbumine et de dystrophine d'être, en apparence, peu différent du modèle mdx. L'absence de parvalbumine peut donc partiellement expliquer le phénotype plus marqué présenté par le patient atteint de DMD, mais cet effet est modeste. (2) Des souris ont été crées à Oxford. Sur un fond mdx, elles expriment de façon conditionnelle de l'utrophine. Cette protéine est fort semblable à la dystrophine et il existe des preuves qu'elle en possède les capacités fonctionnelles. Cependant, il importait de savoir si une expression importante débutant après la naissance ou le sevrage permettait également une récupération fonctionnelle chez la souris ...(SBIM 3)--UCL, 200

EFFECT OF PROTEASOME INHIBITOR MG-132 ON MUSCLE DISUSE ATROPHY

The purpose of this study was to evaluate whether administration of a proteasome inhibitor (MG-132) in vivo is able to prevent muscle atrophy caused by hindlimb unloading (HU). Twenty-seven NMRI mice were assigned to a weight-bearing control, a 6-day HU or a 6-day HU+MG-132 (1mg/kg/48h) treatment group. Soleus (SOL), gastrocnemius (GAS) and tibialis anterior (TA) muscles were removed and weighed. After HU muscle wasting was 20% in SOL, 7% in TA and 13% in GAS (P<0.05). MG-132 treatment prevented 50% of atrophy induced by HU in GAS only (P<0.05). In this muscle, HU was associated with an increased expression of MuRF-1 (P<0.05), Atrogin-1 (P<0.05) and myostatin mRNA (P<0.055), whereas E3α, Nedd-4 and IL-6 remained unchanged. A 19% increase in chymotrypsin-like proteasomal activity was observed in HU (P<0.05) but not in HU+MG-132 GAS. Interestingly, a repression of MurF-1 and myostatin mRNA was also observed with MG-132. In TA, pro-caspase-3 cytosolic expression was decreased by 60% with HU (P<0.05) and only by 20% with HU+MG-132 (P<0.05). These results suggest that in vivo administration of MG-132 can prevent partially muscle atrophy associated with disuse, but that this effect is not specific of a proteasome activity inhibition

Activation of ER stress by hydrogen peroxide in C2C12 myotubes

The purpose of this study was to examine the link between oxidative stress and endoplasmic reticulum (ER) stress in myogenic cells. C2C12 myotubes were incubated with hydrogen peroxide (H2O2, 200 μM) and harvested 4h or 17 h after the induction of this oxidative stress. A massive upregulation of binding immunoglobulin protein (BiP) was found, indicating the presence of ER stress. Nevertheless, the three branches of the unfolded protein response (UPR) were not activated to the same extent. The double-stranded RNA-dependent protein kinase (PKR)-like ER kinase (PERK) branch was the most activated as shown by the increase of phospho-eukaryotic translation-initiation factor 2α (eIF2α, Ser51) and the mRNA levels of activating transcription factor 4 (ATF4), C/EBP homologous (CHOP) and tribbles homolog 3 (TRB3). The slight increase in the spliced form of X-box binding protein 1 (XBP1s) together with the decrease of the unspliced form (XBP1u) indicated a higher endoribonuclease activity of inositol-requiring 1α (IRE1α). The transcriptional activity of activating transcription factor 6 (ATF6) remained unchanged after incubation with H2O2. The mechanisms by which the three branches of UPR can be specifically regulated by oxidative stress are currently unresolved and need further investigations.status: publishe

TLR2 and TLR4 Activate p38 MAPK and JNK during Endurance Exercise in Skeletal Muscle

PURPOSE: Toll-like receptors 2 and 4 (TLR2, TLR4) are found in the membrane of skeletal muscle cells. A variety of molecular components can activate TLR2 and TLR4, among others, long-chain fatty acids. The subsequent downstream signaling triggers the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways. Therefore, the purpose of this study was to test whether an elevation of extracellular nonesterified fatty acids (NEFA) observed during endurance exercise may activate the MAPK and NF-κB pathways via TLR2 and TLR4. METHODS: tlr2 and tlr4 mice and wild-type C57BL/6J animals (WT) were submitted to a standardized endurance exercise. RESULTS: Immediately after exercise, the phosphorylation state of p38 MAPK, c-Jun NH2-terminal kinase (JNK), and c-Jun was increased in the tibialis anterior (TA) and soleus (SOL) muscles of WT (P < 0.05). The phosphorylation state of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and IκB kinase α/β and the DNA-binding of NF-κB remained unchanged. The activation of p38 MAPK, JNK, and c-Jun was completely blunted in TA of tlr2 and tlr4 mice, whereas in SOL, it represented only 25% of the increase observed in WT mice. The causal relationship between NEFA concentration and MAPK activation was evaluated by injecting mice with heparin. A similar increase in plasma NEFA was observed after heparin injection than after endurance exercise. JNK and p38 MAPK were activated under heparin in TA and SOL of WT (P < 0.05) but not in muscles of tlr2 and tlr4 mice. CONCLUSIONS: The present study supports the idea that during endurance exercise, TLR2 and TLR4 mediate a signal linking the elevated plasma NEFA concentration to the activation of p38 MAPK and JNK.status: publishe

Prevention of muscle disuse atrophy by MG132 proteasome inhibitor.

Introduction: Our goal was to determine whether in vivo administration of the proteasome inhibitor MG132 can prevent muscle atrophy caused by hindlimb unloading (HU). Methods: Twenty-seven NMRI mice were assigned to a weight-bearing control, a 6-day HU, or a HU+MG132 (1 mg/kg/48 h) treatment group. Results: Gastrocnemius wasting was significantly less in HU+MG132 mice (-6.7 ± 2.0%) compared with HU animals (-12.6 ± 1.1%, P = 0.011). HU was also associated with an increased expression of MuRF-1 (P = 0.006), MAFbx (P = 0.001), and USP28 (P = 0.027) mRNA, whereas Nedd4, E3α, USP19, and UBP45 mRNA did not change significantly. Increases in MuRF-1, MAFbx, and USP28 mRNA were largely repressed after MG132 administration. β5 proteasome activity tended to increase in HU (+16.7 ± 6.1%, P = 0.086). Neither β1 and β2 proteasome activities nor ubiquitin-conjugated proteins were changed by HU. Conclusions: Our results indicate that in vivo administration of MG132 partially prevents muscle atrophy associated with disuse and highlight an unexpected regulation of MG132 proteasome inhibitor on ubiquitin-ligases. Muscle Nerve, 2011

Prevention of muscle disuse atrophy by MG132 proteasome inhibitor

INTRODUCTION: Our goal was to determine whether in vivo administration of the proteasome inhibitor MG132 can prevent muscle atrophy caused by hindlimb unloading (HU). METHODS: Twenty-seven NMRI mice were assigned to a weight-bearing control, a 6-day HU, or a HU+MG132 (1 mg/kg/48 h) treatment group. RESULTS: Gastrocnemius wasting was significantly less in HU+MG132 mice (-6.7 ± 2.0%) compared with HU animals (-12.6 ± 1.1%, P = 0.011). HU was also associated with an increased expression of MuRF-1 (P = 0.006), MAFbx (P = 0.001), and USP28 (P = 0.027) mRNA, whereas Nedd4, E3α, USP19, and UBP45 mRNA did not change significantly. Increases in MuRF-1, MAFbx, and USP28 mRNA were largely repressed after MG132 administration. β5 proteasome activity tended to increase in HU (+16.7 ± 6.1%, P = 0.086). Neither β1 and β2 proteasome activities nor ubiquitin-conjugated proteins were changed by HU. CONCLUSIONS: Our results indicate that in vivo administration of MG132 partially prevents muscle atrophy associated with disuse and highlight an unexpected regulation of MG132 proteasome inhibitor on ubiquitin-ligases.status: publishe

TLR2 and TLR4 Activate p38 MAPK and JNK during Endurance Exercise in Skeletal Muscle.

The present study supports the idea that during endurance exercise, TLR2 and TLR4 mediate a signal linking the elevated plasma NEFA concentration to the activation of p38 MAPK and JNK

TLR2 and TLR4 activation induces p38 MAPK-dependent phosphorylation of S6 kinase 1 in C2C12 myotubes

Toll-like receptors 2 (TLR2) and 4 (TLR4) are present in the plasma membrane of skeletal muscle cells where their functions remain incompletely resolved. They are able to bind various extra-cellular ligands, such as FSL-1, lipopolysaccharide (LPS) and/or palmitic acid (PA). The purpose of this study was to investigate the link between PA, TLR2/4, and ribosomal S6 kinase 1 (S6K1) in C2C12 myotubes. Incubation with agonists of either TLR2 or TLR4, as well as incubation with high concentration of PA, led to an increase in S6K1 phosphorylation level. Canonical upstream kinases of S6K1, protein kinase B (PKB) and mammalian target of rapamycin complex 1 (mTORC1), were regulated in the opposite way by PA, indicating that those kinases were probably not involved. By using the SB202190 inhibitor, we evidenced that p38 MAPK was a key mediator of PA-induced phosphorylation of S6K1. Down-regulation of either tlr2 or tlr4 gene expression by small interfering RNAs prevented the activation of both p38 MAPK and S6K1 by FSL-1, LPS or PA. In summary, our results showed that TLR2 and TLR4 agonists are able to increase the level of S6K1 phosphorylation in a p38 MAPK dependent way in C2C12 myotubes. As PA induced the same intracellular signaling, we evidenced for the first time an atypical pathway for PA that is induced at the cellular membrane level and results in a higher phosphorylation state of S6K1.status: publishe