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1.
Exp Physiol ; 105(11): 1895-1906, 2020 11.
Article in English | MEDLINE | ID: mdl-32897592

ABSTRACT

NEW FINDINGS: What is the central question of the study? What are the consequences of reducing circulating sphingosine-1-phosphate (S1P) for muscle physiology in the murine model of Duchenne muscular dystrophy (DMD)? What is the main result and its importance? Reduction of the circulating S1P level in mdx mice aggravates the dystrophic phenotype, as seen by an increase in fibre atrophy, fibrosis and loss of specific force, suggesting that S1P signalling is a potential therapeutic target in DMD. Although further studies are needed, plasma S1P levels have the intriguing possibility of being used as a biomarker for disease severity, an important issue in DMD. ABSTRACT: Sphingosine-1-phosphate (S1P) is an important regulator of skeletal muscle properties. The dystrophin-deficient mdx mouse possesses low levels of S1P (∼50%) compared with wild type. Increased S1P availability was demonstrated to ameliorate the dystrophic phenotype in Drosophila and in mdx mice. Here, we analysed the effects produced by further reduction of S1P availability on the mass, force and regenerative capacity of dystrophic mdx soleus. Circulating S1P was neutralized by a specific anti-S1P antibody (S1P-Ab) known to lower the extracellular concentration of this signalling lipid. The S1P-Ab was administered intraperitoneally in adult mdx mice every 2 days for the duration of experiments. Soleus muscle properties were analysed 7 or 14 days after the first injection. The decreased availability of circulating S1P after the 14 day treatment reduced mdx soleus fibre cross-sectional area (-16%, P < 0.05), an effect that was associated with an increase in markers of proteolytic (MuRF1 and atrogin-1) and autophagic (p62 and LC3-II/LC3-I ratio) pathways. Moreover, an increase of fibrosis was also observed (+26%, P < 0.05). Notably, the treatment also caused a reduction of specific tetanic tension (-29%, P < 0.05). The mdx soleus regenerative capacity was only slightly influenced by reduced S1P. In conclusion, neutralization of circulating S1P reduces the mass and specific force and increases fibrosis of mdx soleus muscle, thus worsening the dystrophic phenotype. The results confirm that active, functional S1P signalling might counteract the progression of soleus mdx pathology and validate the pathway as a potential therapeutic target for muscular dystrophies.


Subject(s)
Dystrophin , Muscular Dystrophy, Duchenne , Animals , Disease Models, Animal , Dystrophin/metabolism , Lysophospholipids/metabolism , Lysophospholipids/therapeutic use , Mice , Mice, Inbred mdx , Muscle, Skeletal/metabolism , Muscular Dystrophy, Duchenne/metabolism , Phenotype , Sphingosine/analogs & derivatives
2.
Hum Mol Genet ; 27(6): 969-984, 2018 03 15.
Article in English | MEDLINE | ID: mdl-29351619

ABSTRACT

Limb-girdle muscular dystrophy type 2D (LGMD2D) is a rare autosomal-recessive disease, affecting striated muscle, due to mutation of SGCA, the gene coding for α-sarcoglycan. Nowadays, more than 50 different SGCA missense mutations have been reported. They are supposed to impact folding and trafficking of α-sarcoglycan because the defective polypeptide, although potentially functional, is recognized and disposed of by the quality control of the cell. The secondary reduction of α-sarcoglycan partners, ß-, γ- and δ-sarcoglycan, disrupts a key membrane complex that, associated to dystrophin, contributes to assure sarcolemma stability during muscle contraction. The complex deficiency is responsible for muscle wasting and the development of a severe form of dystrophy. Here, we show that the application of small molecules developed to rescue ΔF508-CFTR trafficking, and known as CFTR correctors, also improved the maturation of several α-sarcoglycan mutants that were consequently rescued at the plasma membrane. Remarkably, in myotubes from a patient with LGMD2D, treatment with CFTR correctors induced the proper re-localization of the whole sarcoglycan complex, with a consequent reduction of sarcolemma fragility. Although the mechanism of action of CFTR correctors on defective α-sarcoglycan needs further investigation, this is the first report showing a quantitative and functional recovery of the sarcoglycan-complex in human pathologic samples, upon small molecule treatment. It represents the proof of principle of a pharmacological strategy that acts on the sarcoglycan maturation process and we believe it has a great potential to develop as a cure for most of the patients with LGMD2D.


Subject(s)
Sarcoglycanopathies/drug therapy , Sarcoglycans/metabolism , Cell Line, Tumor , Cell Membrane/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , HEK293 Cells , Humans , Muscle Contraction , Muscle, Skeletal/metabolism , Muscle, Striated/metabolism , Mutation, Missense , Proof of Concept Study , Sarcoglycanopathies/genetics , Sarcoglycanopathies/metabolism , Sarcoglycans/genetics
3.
Am J Physiol Cell Physiol ; 313(1): C54-C67, 2017 Jul 01.
Article in English | MEDLINE | ID: mdl-28446426

ABSTRACT

We investigated the effects of S1P3 deficiency on the age-related atrophy, decline in force, and regenerative capacity of soleus muscle from 23-mo-old male (old) mice. Compared with muscle from 5-mo-old (adult) mice, soleus mass and muscle fiber cross-sectional area (CSA) in old wild-type mice were reduced by ~26% and 24%, respectively. By contrast, the mass and fiber CSA of soleus muscle in old S1P3-null mice were comparable to those of adult muscle. Moreover, in soleus muscle of wild-type mice, twitch and tetanic tensions diminished from adulthood to old age. A slowing of contractile properties was also observed in soleus from old wild-type mice. In S1P3-null mice, neither force nor the contractile properties of soleus changed during aging. We also evaluated the regenerative capacity of soleus in old S1P3-null mice by stimulating muscle regeneration through myotoxic injury. After 10 days of regeneration, the mean fiber CSA of soleus in old wild-type mice was significantly smaller (-28%) compared with that of regenerated muscle in adult mice. On the contrary, the mean fiber CSA of regenerated soleus in old S1P3-null mice was similar to that of muscle in adult mice. We conclude that in the absence of S1P3, soleus muscle is protected from the decrease in muscle mass and force, and the attenuation of regenerative capacity, all of which are typical characteristics of aging.


Subject(s)
Aging/genetics , Muscle, Skeletal/metabolism , Receptors, Lysosphingolipid/genetics , Sarcopenia/genetics , Aging/metabolism , Animals , Gene Expression , Male , Mice , Mice, Knockout , Muscle Contraction/physiology , Muscle Fibers, Slow-Twitch/metabolism , Muscle Fibers, Slow-Twitch/pathology , Muscle Strength/physiology , Muscle, Skeletal/physiopathology , Receptors, Lysosphingolipid/deficiency , Regeneration/physiology , Sarcopenia/metabolism , Sarcopenia/physiopathology , Sphingosine-1-Phosphate Receptors
4.
J Appl Physiol (1985) ; 120(11): 1288-300, 2016 Jun 01.
Article in English | MEDLINE | ID: mdl-26718782

ABSTRACT

To examine the role of sphingosine 1-phosphate (S1P) receptor 3 (S1P3) in modulating muscle properties, we utilized transgenic mice depleted of the receptor. Morphological analyses of extensor digitorum longus (EDL) muscle did not show evident differences between wild-type and S1P3-null mice. The body weight of 3-mo-old S1P3-null mice and the mean cross-sectional area of transgenic EDL muscle fibers were similar to those of wild-type. S1P3 deficiency enhanced the expression level of S1P1 and S1P2 receptors mRNA in S1P3-null EDL muscle. The contractile properties of S1P3-null EDL diverge from those of wild-type, largely more fatigable and less able to recover. The absence of S1P3 appears responsible for a lower availability of calcium during fatigue. S1P supplementation, expected to stimulate residual S1P receptors and signaling, reduced fatigue development of S1P3-null muscle. Moreover, in the absence of S1P3, denervated EDL atrophies less than wild-type. The analysis of atrophy-related proteins in S1P3-null EDL evidences high levels of the endogenous regulator of mitochondria biogenesis peroxisome proliferative-activated receptor-γ coactivator 1α (PGC-1α); preserving mitochondria could protect the muscle from disuse atrophy. In conclusion, the absence of S1P3 makes the muscle more sensitive to fatigue and slows down atrophy development after denervation, indicating that S1P3 is involved in the modulation of key physiological properties of the fast-twitch EDL muscle.


Subject(s)
Muscle Fibers, Fast-Twitch/metabolism , Muscle Fibers, Fast-Twitch/physiology , Receptors, Lysosphingolipid/metabolism , Animals , Atrophy/metabolism , Atrophy/physiopathology , Calcium/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic/metabolism , Mice, Transgenic/physiology , Mitochondria/metabolism , Mitochondria/physiology , Muscle Fatigue/physiology , Muscular Diseases/metabolism , Muscular Diseases/physiopathology , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism , RNA, Messenger/metabolism , Sphingosine-1-Phosphate Receptors
5.
J Biol Chem ; 289(48): 33073-82, 2014 Nov 28.
Article in English | MEDLINE | ID: mdl-25288803

ABSTRACT

A missense mutation in ATP2A1 gene, encoding sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1) protein, causes Chianina cattle congenital pseudomyotonia, an exercise-induced impairment of muscle relaxation. Skeletal muscles of affected cattle are characterized by a selective reduction of SERCA1 in sarcoplasmic reticulum membranes. In this study, we provide evidence that the ubiquitin proteasome system is involved in the reduced density of mutated SERCA1. The treatment with MG132, an inhibitor of ubiquitin proteasome system, rescues the expression level and membrane localization of the SERCA1 mutant in a heterologous cellular model. Cells co-transfected with the Ca(2+)-sensitive probe aequorin show that the rescued SERCA1 mutant exhibits the same ability of wild type to maintain Ca(2+) homeostasis within cells. These data have been confirmed by those obtained ex vivo on adult skeletal muscle fibers from a biopsy from a pseudomyotonia-affected subject. Our data show that the mutation generates a protein most likely corrupted in proper folding but not in catalytic activity. Rescue of mutated SERCA1 to sarcoplasmic reticulum membrane can re-establish resting cytosolic Ca(2+) concentration and prevent the appearance of pathological signs of cattle pseudomyotonia.


Subject(s)
Cattle Diseases/enzymology , Isaacs Syndrome/enzymology , Isaacs Syndrome/veterinary , Muscle Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Sarcoplasmic Reticulum/enzymology , Ubiquitin/metabolism , Animals , Calcium/metabolism , Cattle , Cattle Diseases/genetics , Cattle Diseases/pathology , Cricetinae , HEK293 Cells , Humans , Isaacs Syndrome/genetics , Isaacs Syndrome/pathology , Leupeptins/pharmacology , Muscle Proteins/genetics , Mutation , Proteasome Endopeptidase Complex/genetics , Proteasome Inhibitors/pharmacology , Protein Folding/drug effects , Sarcoplasmic Reticulum/genetics , Sarcoplasmic Reticulum/pathology , Sarcoplasmic Reticulum Calcium-Transporting ATPases/genetics , Ubiquitin/genetics
6.
Hum Mol Genet ; 23(14): 3746-58, 2014 Jul 15.
Article in English | MEDLINE | ID: mdl-24565866

ABSTRACT

Many membrane and secretory proteins that fail to pass quality control in the endoplasmic reticulum (ER) are dislocated into the cytosol and degraded by the proteasome. In applying rigid rules, however, quality control sometimes discharges proteins that, even though defective, retain their function. The unnecessary removal of such proteins represents the pathogenetic hallmark of diverse genetic diseases, in the case of ΔF508 mutant of cystic fibrosis transmembrane conductance regulator being probably the best known example. Recently, the inappropriate proteasomal degradation of skeletal muscle sarcoglycans (α, ß, γ and δ) with missense mutation has been proposed to be at the bases of mild-to-severe forms of limb girdle muscular dystrophy (LGMD) known as type 2D, 2E, 2C and 2F, respectively. The quality control pathway responsible for sarcoglycan mutant disposal, however, is so far unexplored. Here we reveal key components of the degradative route of V247M α-sarcoglycan mutant, the second most frequently reported mutation in LGMD-2D. The disclosure of the pathway, which is led by the E3 ligases HRD1 and RFP2, permits to identify new potential druggable targets of a disease for which no effective therapy is at present available. Notably, we show that the pharmacological inhibition of HRD1 activity rescues the expression of V247-α-sarcoglycan both in a heterologous cell model and in myotubes derived from a LGMD-2D patient carrying the L31P/V247M mutations. This represents the first evidence that the activity of E3 ligases, the enzymes in charge of mutant fate, can be eligible for drug interventions to treat sarcoglycanopathy.


Subject(s)
DNA-Binding Proteins/metabolism , Sarcoglycanopathies/metabolism , Sarcoglycans/genetics , Sarcoglycans/metabolism , Tumor Suppressor Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Cells, Cultured , Endoplasmic Reticulum/metabolism , Enzyme Inhibitors/pharmacology , HEK293 Cells , Humans , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/pathology , Mutation, Missense , Sarcoglycanopathies/genetics , Sarcoglycanopathies/pathology , Signal Transduction/drug effects , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitination
7.
PLoS One ; 8(6): e65167, 2014.
Article in English | MEDLINE | ID: mdl-23755187

ABSTRACT

Slow-twitch muscles, devoted to postural maintenance, experience atrophy and weakness during muscle disuse due to bed-rest, aging or spaceflight. These conditions impair motion activities and can have survival implications. Human and animal studies demonstrate the anabolic role of IGF-1 on skeletal muscle suggesting its interest as a muscle disuse countermeasure. Thus, we tested the role of IGF-1 overexpression on skeletal muscle alteration due to hindlimb unloading (HU) by using MLC/mIgf-1 transgenic mice expressing IGF-1 under the transcriptional control of MLC promoter, selectively activated in skeletal muscle. HU produced atrophy in soleus muscle, in terms of muscle weight and fiber cross-sectional area (CSA) reduction, and up-regulation of atrophy gene MuRF1. In parallel, the disuse-induced slow-to-fast fiber transition was confirmed by an increase of the fast-type of the Myosin Heavy Chain (MHC), a decrease of PGC-1α expression and an increase of histone deacetylase-5 (HDAC5). Consistently, functional parameters such as the resting chloride conductance (gCl) together with ClC-1 chloride channel expression were increased and the contractile parameters were modified in soleus muscle of HU mice. Surprisingly, IGF-1 overexpression in HU mice was unable to counteract the loss of muscle weight and the decrease of fiber CSA. However, the expression of MuRF1 was recovered, suggesting early effects on muscle atrophy. Although the expression of PGC-1α and MHC were not improved in IGF-1-HU mice, the expression of HDAC5 was recovered. Importantly, the HU-induced increase of gCl was fully contrasted in IGF-1 transgenic mice, as well as the changes in contractile parameters. These results indicate that, even if local expression does not seem to attenuate HU-induced atrophy and slow-to-fast phenotype transition, it exerts early molecular effects on gene expression which can counteract the HU-induced modification of electrical and contractile properties. MuRF1 and HDAC5 can be attractive therapeutic targets for pharmacological countermeasures and then deserve further investigations.


Subject(s)
Hindlimb/physiopathology , Insulin-Like Growth Factor I/metabolism , Muscle, Skeletal/physiopathology , Muscular Atrophy/physiopathology , Myosin Light Chains/metabolism , Paracrine Communication/drug effects , Animals , Behavior, Animal , Biochemical Phenomena , Body Weight , Calcium/metabolism , Chloride Channels/metabolism , Cytosol/metabolism , Gene Expression Regulation , Humans , Mice, Transgenic , Muscle Contraction , Muscle Fibers, Fast-Twitch/metabolism , Muscle Fibers, Slow-Twitch/metabolism , Muscle, Skeletal/pathology , Muscular Atrophy/pathology , Rats , Rest , Weight-Bearing
8.
PLoS One ; 8(8): e72028, 2013.
Article in English | MEDLINE | ID: mdl-24015201

ABSTRACT

Pleiotrophin (PTN) is a widespread cytokine involved in bone formation, neurite outgrowth, and angiogenesis. In skeletal muscle, PTN is upregulated during myogenesis, post-synaptic induction, and regeneration after crushing, but little is known regarding its effects on muscle function. Here, we describe the effects of PTN on the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles in mice over-expressing PTN under the control of a bone promoter. The mice were maintained in normal loading or disuse condition, induced by hindlimb unloading (HU) for 14 days. Effects of exposition to near-zero gravity during a 3-months spaceflight (SF) into the Mice Drawer System are also reported. In normal loading, PTN overexpression had no effect on muscle fiber cross-sectional area, but shifted soleus muscle toward a slower phenotype, as shown by an increased number of oxidative type 1 fibers, and increased gene expression of cytochrome c oxidase subunit IV and citrate synthase. The cytokine increased soleus and EDL capillary-to-fiber ratio. PTN overexpression did not prevent soleus muscle atrophy, slow-to-fast transition, and capillary regression induced by SF and HU. Nevertheless, PTN exerted various effects on sarcolemma ion channel expression/function and resting cytosolic Ca(2+) concentration in soleus and EDL muscles, in normal loading and after HU. In conclusion, the results show very similar effects of HU and SF on mouse soleus muscle, including activation of specific gene programs. The EDL muscle is able to counterbalance this latter, probably by activating compensatory mechanisms. The numerous effects of PTN on muscle gene expression and functional parameters demonstrate the sensitivity of muscle fibers to the cytokine. Although little benefit was found in HU muscle disuse, PTN may emerge useful in various muscle diseases, because it exerts synergetic actions on muscle fibers and vessels, which could enforce oxidative metabolism and ameliorate muscle performance.


Subject(s)
Carrier Proteins/metabolism , Cytokines/metabolism , Muscle, Skeletal/metabolism , Animals , Calcium/metabolism , Carrier Proteins/genetics , Citrate (si)-Synthase/genetics , Citrate (si)-Synthase/metabolism , Cytokines/genetics , Electron Transport Complex IV/genetics , Electron Transport Complex IV/metabolism , Gene Expression , Hindlimb Suspension , Humans , Ion Channels/genetics , Ion Channels/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Muscle Fibers, Skeletal , Muscle Proteins/genetics , Muscle Proteins/metabolism , Muscle, Skeletal/pathology , Muscular Atrophy/metabolism , Sarcolemma/metabolism , Space Flight
9.
J Appl Physiol (1985) ; 113(5): 707-13, 2012 Sep 01.
Article in English | MEDLINE | ID: mdl-22744969

ABSTRACT

Sphingosine 1-phosphate is a bioactive lipid that modulates skeletal muscle growth through its interaction with specific receptors localized in the cell membrane of muscle fibers and satellite cells. This study analyzes the role of S1P(2) receptor during in vivo regeneration of soleus muscle in two models of S1P(2) deficiency: the S1P(2)-null mouse and wild-type mice systemically treated with the S1P(2) receptor antagonist JTE-013. To stimulate regeneration, muscle degeneration was induced by injecting into soleus muscle the myotoxic drug notexin. Both ablation of S1P(2) receptor and its functional inactivation delayed regeneration of soleus muscle. The exogenous supplementation of S1P or its removal, by a specific antibody, two conditions known to stimulate or inhibit, respectively, soleus muscle regeneration, were without effects when the S1P(2) receptor was absent or inactive. The delayed regeneration was associated with a lower level of myogenin, a muscle differentiation marker, and reduced phosphorylation of Akt, a key marker of muscle growth. Consistently, silencing of S1P(2) receptor abrogated the pro-myogenic action of S1P in satellite cells, paralleled by low levels of the myogenic transcription factor myogenin. The study indicates that S1P(2) receptor plays a key role in the early phases of muscle regeneration by sustaining differentiation and growth of new-forming myofibers.


Subject(s)
Muscle, Skeletal/physiology , Receptors, Lysosphingolipid/physiology , Regeneration/physiology , Animals , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cells, Cultured , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle, Skeletal/drug effects , Pyrazoles/pharmacology , Pyridines/pharmacology , Receptors, Lysosphingolipid/antagonists & inhibitors , Regeneration/drug effects
10.
PLoS One ; 7(3): e33232, 2012.
Article in English | MEDLINE | ID: mdl-22470446

ABSTRACT

The effect of microgravity on skeletal muscles has so far been examined in rat and mice only after short-term (5-20 day) spaceflights. The mice drawer system (MDS) program, sponsored by Italian Space Agency, for the first time aimed to investigate the consequences of long-term (91 days) exposure to microgravity in mice within the International Space Station. Muscle atrophy was present indistinctly in all fiber types of the slow-twitch soleus muscle, but was only slightly greater than that observed after 20 days of spaceflight. Myosin heavy chain analysis indicated a concomitant slow-to-fast transition of soleus. In addition, spaceflight induced translocation of sarcolemmal nitric oxide synthase-1 (NOS1) into the cytosol in soleus but not in the fast-twitch extensor digitorum longus (EDL) muscle. Most of the sarcolemmal ion channel subunits were up-regulated, more in soleus than EDL, whereas Ca(2+)-activated K(+) channels were down-regulated, consistent with the phenotype transition. Gene expression of the atrophy-related ubiquitin-ligases was up-regulated in both spaceflown soleus and EDL muscles, whereas autophagy genes were in the control range. Muscle-specific IGF-1 and interleukin-6 were down-regulated in soleus but up-regulated in EDL. Also, various stress-related genes were up-regulated in spaceflown EDL, not in soleus. Altogether, these results suggest that EDL muscle may resist to microgravity-induced atrophy by activating compensatory and protective pathways. Our study shows the extended sensitivity of antigravity soleus muscle after prolonged exposition to microgravity, suggests possible mechanisms accounting for the resistance of EDL, and individuates some molecular targets for the development of countermeasures.


Subject(s)
Adaptation, Physiological , Muscle, Skeletal/metabolism , Weightlessness , Animals , Down-Regulation , Immunohistochemistry , Insulin-Like Growth Factor I/metabolism , Interleukin-6/metabolism , Male , Mice , Mice, Inbred C57BL , Myosin Heavy Chains/metabolism , Nitric Oxide Synthase Type I/metabolism , Potassium Channels, Calcium-Activated/metabolism , Rats , Space Flight , Ubiquitin-Protein Ligases/metabolism , Up-Regulation
11.
Mol Cell Biochem ; 351(1-2): 183-96, 2011 May.
Article in English | MEDLINE | ID: mdl-21308481

ABSTRACT

Evidence shows that extracellular ATP signals influence myogenesis, regeneration and physiology of skeletal muscle. Present work was aimed at characterizing the extracellular ATP signaling system of skeletal muscle C2C12 cells during differentiation. We show that mechanical and electrical stimulation produces substantial release of ATP from differentiated myotubes, but not from proliferating myoblasts. Extracellular ATP-hydrolyzing activity is low in myoblasts and high in myotubes, consistent with the increased expression of extracellular enzymes during differentiation. Stimulation of cells with extracellular nucleotides produces substantial Ca(2+) transients, whose amplitude and shape changed during differentiation. Consistently, C2C12 cells express several P2X and P2Y receptors, whose level changes along with maturation stages. Supplementation with either ATP or UTP stimulates proliferation of C2C12 myoblasts, whereas excessive doses were cytotoxic. The data indicate that skeletal muscle development is accompanied by major functional changes in extracellular ATP signaling.


Subject(s)
Adenosine Triphosphate/metabolism , Cell Differentiation , Cell Proliferation , Muscle, Skeletal/metabolism , Signal Transduction , Animals , Base Sequence , Blotting, Western , Cell Line , DNA Primers , Mice , Muscle, Skeletal/cytology , Reverse Transcriptase Polymerase Chain Reaction
12.
Proc Natl Acad Sci U S A ; 108(2): 621-5, 2011 Jan 11.
Article in English | MEDLINE | ID: mdl-21187406

ABSTRACT

The nicotinic acetylcholine receptor of skeletal muscle is composed of five subunits that are assembled in a stepwise manner. Quality control mechanisms ensure that only fully assembled receptors reach the cell surface. Here, we show that Rer1, a putative Golgi-ER retrieval receptor, is involved in the biogenesis of acetylcholine receptors. Rer1 is expressed in the early secretory pathway in the myoblast line C2C12 and in mouse skeletal muscle, and up-regulated during myogenesis. Upon down-regulation of Rer1 in C2C12 cells, unassembled acetylcholine receptor α-subunits escape from the ER and are transported to the plasma membrane and lysosomes, where they are degraded. As a result, the amount of fully assembled receptor at the cell surface is reduced. In vivo Rer1 knockdown and genetic inactivation of one Rer1 allele lead to significantly smaller neuromuscular junctions in mice. Our data show that Rer1 is a functionally important unique factor that controls surface expression of muscle acetylcholine receptors by localizing unassembled α-subunits to the early secretory pathway.


Subject(s)
Cell Membrane/metabolism , Endoplasmic Reticulum/metabolism , Membrane Glycoproteins/physiology , Muscles/metabolism , Receptors, Cholinergic/metabolism , Receptors, Cytoplasmic and Nuclear/physiology , Adaptor Proteins, Vesicular Transport , Alleles , Animals , Down-Regulation , Lysosomes/metabolism , Mice , Mice, Transgenic , Muscle, Skeletal/metabolism , Protein Transport , Receptors, Cytoplasmic and Nuclear/genetics , Reverse Transcriptase Polymerase Chain Reaction , Up-Regulation
13.
Am J Physiol Cell Physiol ; 298(3): C550-8, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20042733

ABSTRACT

Sphingosine 1-phosphate (S1P) is a bioactive lipid known to control cell growth that was recently shown to act as a trophic factor for skeletal muscle, reducing the progress of denervation atrophy. The aim of this work was to investigate whether S1P is involved in skeletal muscle fiber recovery (regeneration) after myotoxic injury induced by bupivacaine. The postnatal ability of skeletal muscle to grow and regenerate is dependent on resident stem cells called satellite cells. Immunofluorescence analysis demonstrated that S1P-specific receptors S1P(1) and S1P(3) are expressed by quiescent satellite cells. Soleus muscles undergoing regeneration following injury induced by intramuscular injection of bupivacaine exhibited enhanced expression of S1P(1) receptor, while S1P(3) expression progressively decreased to adult levels. S1P(2) receptor was absent in quiescent cells but was transiently expressed in the early regenerating phases only. Administration of S1P (50 microM) at the moment of myotoxic injury caused a significant increase of the mean cross-sectional area of regenerating fibers in both rat and mouse. In separate experiments designed to test the trophic effects of S1P, neutralization of endogenous circulating S1P by intraperitoneal administration of anti-S1P antibody attenuated fiber growth. Use of selective modulators of S1P receptors indicated that S1P(1) receptor negatively and S1P(3) receptor positively modulate the early phases of regeneration, whereas S1P(2) receptor appears to be less important. The present results show that S1P signaling participates in the regenerative processes of skeletal muscle.


Subject(s)
Lysophospholipids/metabolism , Muscle Development , Muscle, Skeletal/metabolism , Muscular Diseases/metabolism , Regeneration , Satellite Cells, Skeletal Muscle/metabolism , Signal Transduction , Sphingosine/analogs & derivatives , Animals , Bupivacaine , Cell Membrane/metabolism , Cell Proliferation , Cells, Cultured , Disease Models, Animal , Injections, Intramuscular , Lysophospholipids/administration & dosage , Male , Mice , Mice, Inbred C57BL , Muscle Development/drug effects , Muscle, Skeletal/drug effects , Muscle, Skeletal/physiopathology , Muscular Diseases/chemically induced , Muscular Diseases/physiopathology , Rats , Rats, Wistar , Receptors, Lysosphingolipid/drug effects , Receptors, Lysosphingolipid/metabolism , Regeneration/drug effects , Satellite Cells, Skeletal Muscle/drug effects , Signal Transduction/drug effects , Sphingosine/administration & dosage , Sphingosine/metabolism , Time Factors
14.
J Appl Physiol (1985) ; 108(1): 105-11, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19910334

ABSTRACT

It is commonly accepted that skeletal muscles from dystrophin-deficient mdx mice are more susceptible than those from wild-type mice to damage from eccentric contractions. However, the downstream mechanisms involved in this enhanced force drop remain controversial. We studied the reduction of contractile force induced by eccentric contractions elicited in vivo in the gastrocnemius muscle of wild-type mice and three distinct models of muscle dystrophy: mdx, alpha-sarcoglycan (Sgca)-null, and collagen 6A1 (Col6a1)-null mice. In mdx and Sgca-null mice, force decreased 35% compared with 14% in wild-type mice. Drop of force in Col6a1-null mice was comparable to that in wild-type mice. To identify the determinants of the force drop, we measured force generation in permeabilized fibers dissected from gastrocnemius muscle that had been exposed in vivo to eccentric contractions and from the contralateral unstimulated muscle. A force loss in skinned fibers after in vivo eccentric contractions was detectable in fibers from mdx and Sgca-null, but not wild-type and Col6a1-null, mice. The enhanced force reduction in mdx and Sgca-null mice was observed only when eccentric contractions were elicited in vivo, since eccentric contractions elicited in vitro had identical effects in wild-type and dystrophic skinned fibers. These results suggest that 1) the enhanced force loss is due to a myofibrillar impairment that is present in all fibers, and not to individual fiber degeneration, and 2) the mechanism causing the enhanced force reduction is active in vivo and is lost after fiber permeabilization.


Subject(s)
Muscle Contraction , Muscle Fibers, Skeletal , Muscle, Skeletal/pathology , Muscle, Skeletal/physiopathology , Muscular Dystrophies/physiopathology , Animals , Mice , Mice, Inbred C57BL , Mice, Knockout
15.
Expert Rev Mol Med ; 11: e28, 2009 Sep 28.
Article in English | MEDLINE | ID: mdl-19781108

ABSTRACT

Sarcoglycanopathies are a group of autosomal recessive muscle-wasting disorders caused by genetic defects in one of four cell membrane glycoproteins, alpha-, beta-, gamma- or delta-sarcoglycan. These four sarcoglycans form a subcomplex that is closely linked to the major dystrophin-associated protein complex, which is essential for membrane integrity during muscle contraction and provides a scaffold for important signalling molecules. Proper assembly, trafficking and targeting of the sarcoglycan complex is of vital importance, and mutations that severely perturb tetramer formation and localisation result in sarcoglycanopathy. Gene defects in one sarcoglycan cause the absence or reduced concentration of the other subunits. Most genetic defects generate mutated proteins that are degraded through the cell's quality control system; however, in many cases, conformational modifications do not affect the function of the protein, yet it is recognised as misfolded and prematurely degraded. Recent evidence shows that misfolded sarcoglycans could be rescued to the cell membrane by assisting their maturation along the ER secretory pathway. This review summarises the etiopathogenesis of sarcoglycanopathies and highlights the quality control machinery as a potential pharmacological target for therapy of these genetic disorders.


Subject(s)
Dystrophin-Associated Protein Complex/metabolism , Endoplasmic Reticulum/metabolism , Muscle, Skeletal/metabolism , Muscular Dystrophies/metabolism , Sarcoglycans/metabolism , Amino Acid Sequence , Animals , Cell Membrane/metabolism , Dystrophin-Associated Protein Complex/chemistry , Humans , Molecular Sequence Data , Muscular Dystrophies/genetics , Muscular Dystrophies/therapy , Mutation, Missense/genetics , Mutation, Missense/physiology , Protein Transport/physiology , Sarcoglycans/genetics
16.
Am J Pathol ; 173(1): 170-81, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18535179

ABSTRACT

Sarcoglycanopathies are progressive muscle-wasting disorders caused by genetic defects of four proteins, alpha-, beta-, gamma-, and delta-sarcoglycan, which are elements of a key transmembrane complex of striated muscle. The proper assembly of the sarcoglycan complex represents a critical issue of sarcoglycanopathies, as several mutations severely perturb tetramer formation. Misfolded proteins are generally degraded through the cell's quality-control system; however, this can also lead to the removal of some functional polypeptides. To explore whether it is possible to rescue sarcoglycan mutants by preventing their degradation, we generated a heterologous cell system, based on human embryonic kidney (HEK) 293 cells, constitutively expressing three (beta, gamma, and delta) of the four sarcoglycans. In these betagammadelta-HEK cells, the lack of alpha-sarcoglycan prevented complex formation and cell surface localization, wheras the presence of alpha-sarcoglycan allowed maturation and targeting of the tetramer. As in muscles of sarcoglycanopathy patients, transfection of betagammadelta-HEK cells with disease-causing alpha-sarcoglycan mutants led to dramatic reduction of the mutated proteins and the absence of the complex from the cell surface. Proteasomal inhibition reduced the degradation of mutants and facilitated the assembly and targeting of the sarcoglycan complex to the plasma membrane. These data provide important insights for the potential development of pharmacological therapies for sarcoglycanopathies.


Subject(s)
Proteasome Endopeptidase Complex/metabolism , Protein Isoforms/metabolism , Sarcoglycans/metabolism , Blotting, Western , Cell Line , Cysteine Proteinase Inhibitors/pharmacology , Electrophoresis, Polyacrylamide Gel , Fluorescent Antibody Technique , Humans , Immunoprecipitation , Leupeptins/pharmacology , Microscopy, Confocal , Mutagenesis, Site-Directed , Mutation , Protein Folding , Protein Isoforms/chemistry , Protein Transport/drug effects , Protein Transport/physiology , Sarcoglycans/chemistry , Sarcoglycans/genetics , Transfection
17.
Eur J Appl Physiol ; 104(3): 445-53, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18560877

ABSTRACT

The present study evaluated whether Ca(2+) entry operates during fatigue of skeletal muscle. The involvement of different skeletal muscle membrane calcium channels and of the Na(+)/Ca(2+) exchanger (NCX) has been examined. The decline of force was analysed in vitro in mouse soleus and EDL muscles submitted to 60 and 110 Hz continuous stimulation, respectively. Stimulation with this high-frequency fatigue (HFF) protocol, in Ca(2+)-free conditions, caused in soleus muscle a dramatic increase of fatigue, while in the presence of high Ca(2+) fatigue was reduced. In EDL muscle, HFF was not affected by external Ca(2+) levels either way, suggesting that external Ca(2+) plays a general protective role only in soleus. Calciseptine, a specific antagonist of the cardiac isoform (alpha1C) of the dihydropyridine receptor, gadolinium, a blocker of both stretch-activated and store-operated Ca(2+) channels, as well as inhibitors of P2X receptors did not affect the development of HFF. Conversely, the Ca(2+) ionophore A23187 increased the protective action of extracellular Ca(2+). KB-R7943, a selective inhibitor of the reverse mode of NCX, produced an effect similar to that of Ca(2+)-free solution. These results indicate that a transmembrane Ca(2+) influx, mainly through NCX, may play a protective role during HFF development in soleus muscle.


Subject(s)
Calcium Signaling , Calcium/metabolism , Extracellular Fluid/metabolism , Muscle Contraction , Muscle Fatigue , Muscle Strength , Muscle, Skeletal/metabolism , Animals , Calcimycin/pharmacology , Calcium Channel Blockers/pharmacology , Calcium Channels/drug effects , Calcium Channels/metabolism , Calcium Signaling/drug effects , Cell Membrane/metabolism , Elapid Venoms/pharmacology , Electric Stimulation , Gadolinium/pharmacology , In Vitro Techniques , Ionophores/pharmacology , Mice , Muscle Contraction/drug effects , Muscle Fatigue/drug effects , Muscle Strength/drug effects , Muscle, Skeletal/drug effects , Purinergic P2 Receptor Antagonists , Pyridoxal Phosphate/analogs & derivatives , Pyridoxal Phosphate/pharmacology , Receptors, Purinergic P2/metabolism , Sodium-Calcium Exchanger/antagonists & inhibitors , Sodium-Calcium Exchanger/metabolism , Suramin/pharmacology , Thiourea/analogs & derivatives , Thiourea/pharmacology , Time Factors , Triazines/pharmacology
18.
Am J Physiol Cell Physiol ; 294(1): C36-46, 2008 Jan.
Article in English | MEDLINE | ID: mdl-17942639

ABSTRACT

Sphingosine 1-phosphate (S1P) mediates a number of cellular responses, including growth and proliferation. Skeletal muscle possesses the full enzymatic machinery to generate S1P and expresses the transcripts of S1P receptors. The aim of this work was to localize S1P receptors in rat skeletal muscle and to investigate whether S1P exerts a trophic action on muscle fibers. RT-PCR and Western blot analyses demonstrated the expression of S1P(1) and S1P(3) receptors by soleus muscle. Immunofluorescence revealed that S1P(1) and S1P(3) receptors are localized at the cell membrane of muscle fibers and in the T-tubule membranes. The receptors also decorate the nuclear membrane. S1P(1) receptors were also present at the neuromuscular junction. The possible trophic action of S1P was investigated by utilizing the denervation atrophy model. Rat soleus muscle was analyzed 7 and 14 days after motor nerve cut. During denervation, S1P was continuously delivered to the muscle through a mini osmotic pump. S1P and its precursor, sphingosine (Sph), significantly attenuated the progress of denervation-induced muscle atrophy. The trophic effect of Sph was prevented by N,N-dimethylsphingosine, an inhibitor of Sph kinase, the enzyme that converts Sph into S1P. Neutralization of circulating S1P by a specific antibody further demonstrated that S1P was responsible for the trophic effects of S1P during denervation atrophy. Denervation produced the down regulation of S1P(1) and S1P(3) receptors, regardless of the presence of the receptor agonist. In conclusion, the results suggest that S1P acts as a trophic factor of skeletal muscle.


Subject(s)
Lysophospholipids/metabolism , Muscle, Skeletal/metabolism , Muscular Atrophy/metabolism , Receptors, Lysosphingolipid/metabolism , Sphingosine/analogs & derivatives , Animals , Antibodies , Cell Enlargement , Cell Membrane/metabolism , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Hypertrophy , Infusion Pumps, Implantable , Lysophospholipids/administration & dosage , Male , Muscle Denervation , Muscle, Skeletal/enzymology , Muscle, Skeletal/innervation , Muscle, Skeletal/pathology , Muscular Atrophy/pathology , Muscular Atrophy/prevention & control , MyoD Protein/metabolism , Myogenin/metabolism , Myosin Heavy Chains/metabolism , Neuromuscular Junction/metabolism , Nuclear Envelope/metabolism , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Phosphotransferases (Alcohol Group Acceptor)/metabolism , RNA, Messenger/metabolism , Rats , Rats, Wistar , Receptors, Lysosphingolipid/genetics , Receptors, Lysosphingolipid/immunology , Sciatic Nerve/surgery , Sphingosine/administration & dosage , Sphingosine/metabolism , Sphingosine/pharmacology , Time Factors
19.
J Appl Physiol (1985) ; 102(4): 1640-8, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17234797

ABSTRACT

Postnatal development of skeletal muscle occurs through the progressive transformation of diverse biochemical, metabolic, morphological, and functional characteristics from the embryonic to the adult phenotype. Since muscle regeneration recapitulates postnatal development of muscle fiber, it offers an appropriate experimental model to investigate the existing relationships between diverse muscle functions and the expression of key protein isoforms, particularly at the single-fiber level. This study was carried out in regenerating soleus muscle 14 days after injury. At this intermediate stage, the regenerating muscle exhibited a recovery of mass greater than its force generation capacity. The lower specific tension of regenerating muscle suggested intrinsic defective excitation-contraction coupling and/or contractility processes. The presence of developmental isoforms of both the voltage-gated Ca(2+) channel (alpha(1)C) and of ryanodine receptor 3, paralleled by an abnormal caffeine contracture development, confirms the immature excitation-contraction coupling of the regenerating muscle. The defective Ca(2+) handling could also be confirmed by the lower sarcoplasmic reticulum caffeine sensitivity of regenerating single fibers. Also, regenerating single fibers revealed a lower maximal specific tension, which was associated with the residual presence of embryonic myosin heavy chains. Moreover, the fibers showed a reduced Ca(2+) sensitivity of myofibrillar proteins, particularly those simultaneously expressing the slow and fast isoforms of troponin C. The present results indicate that the expression of developmental proteins determines the incomplete functional recovery of regenerating soleus.


Subject(s)
Isometric Contraction/physiology , Molecular Motor Proteins/metabolism , Muscle Proteins/metabolism , Muscle, Skeletal/physiology , Myofibrils/physiology , Sarcoplasmic Reticulum/physiology , Animals , Cells, Cultured , Male , Protein Isoforms/metabolism , Rats , Rats, Wistar
20.
Am J Physiol Regul Integr Comp Physiol ; 289(5): R1328-37, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16002556

ABSTRACT

Alpha-sarcoglycan (Sgca) is a transmembrane glycoprotein of the dystrophin complex located at skeletal and cardiac muscle sarcolemma. Defects in the alpha-sarcoglycan gene (Sgca) cause the severe human-type 2D limb girdle muscular dystrophy. Because Sgca-null mice develop progressive muscular dystrophy similar to human disorder they are a valuable animal model for investigating the physiopathology of the disorder. In this study, biochemical and functional properties of fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus muscles of the Sgca-null mice were analyzed. EDL muscle of Sgca-null mice showed twitch and tetanic kinetics comparable with those of wild-type controls. In contrast, soleus muscle showed reduction of twitch half-relaxation time, prolongation of tetanic half-relaxation time, and increase of maximal rate of rise of tetanus. EDL muscle of Sgca-null mice demonstrated a marked reduction of specific twitch and tetanic tensions and a higher resistance to fatigue compared with controls, changes that were not evident in dystrophic soleus. Contrary to EDL fibers, soleus muscle fibers of Sgca-null mice distinctively showed right shift of the pCa-tension (pCa is the negative log of Ca2+ concentration) relationships and reduced sensitivity to caffeine of sarcoplasmic reticulum. Both EDL and soleus muscles showed striking changes in myosin heavy-chain (MHC) isoform composition, whereas EDL showed a larger number of hybrid fibers than soleus. In contrast to the EDL, soleus muscle of Sgca-null mice contained a higher number of regenerating fibers and thus higher levels of embryonic MHC. In conclusion, this study revealed profound distinctive biochemical and physiological modifications in fast- and slow-twitch muscles resulting from alpha-sarcoglycan deficiency.


Subject(s)
Muscle Fibers, Fast-Twitch/physiology , Muscle Fibers, Slow-Twitch/physiology , Muscle, Skeletal/physiology , Sarcoglycans/deficiency , Sarcoplasmic Reticulum/metabolism , Animals , Caffeine/pharmacology , Calcium/metabolism , Calcium/pharmacology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle Contraction/physiology , Muscle Fibers, Fast-Twitch/drug effects , Muscle Fibers, Slow-Twitch/drug effects , Muscle, Skeletal/drug effects , Myosin Heavy Chains/genetics , Myosin Heavy Chains/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , Sarcoglycans/genetics , Sarcoplasmic Reticulum/drug effects
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