A missense mutation in ATP2A1 gene, encoding 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 paper 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 cotransfected with the Ca2+ sensitive probe aequorin, show that the rescued SERCA1 mutant exhibits the same ability of wild-type to maintain Ca2+ 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 Ca2+ concentration and prevent the appearance of pathological signs of cattle pseudomyotonia.
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 ?F508 mutant of cystic fibrosis transmembrane conductance regulator being probably the best known example. Recently, the inappropriate proteasomal degradation of skeletal muscle sarcoglycans (?, ?, ? and ?) 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 ?-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-?-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.
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.
Cell-penetrating peptides are short cationic peptides with the property of translocating across the plasma membrane and transferring macromolecules otherwise unable to permeate cell membranes. We investigated the potential ability of the protein transduction domain derived from amino acids 47-57 of the human immunodeficiency virus type 1 (HIV-1) TAT (transactivator of transcription) protein to be used as a nanocarrier for the delivery of aequorin, a Ca(2+)-sensitive photoprotein widely used as a reliable Ca(2+) reporter in cell populations. The TAT peptide, either covalently linked to apoaequorin or ionically bound to plasmids encoding differentially targeted aequorin, was supplied to plant suspension-cultured cells. The TAT-aequorin fusion protein was found to be rapidly and effectively translocated into plant cells. The chimeric molecule was internalized in fully active biological form and at levels suitable to monitor intracellular Ca(2+) concentrations. Plant cells incubated for just 5 min with TAT-aequorin responded to different environmental stimuli with the expected Ca(2+) signatures. On the other hand, TAT-mediated plasmid internalization did not provide the necessary level of transformation efficiency to allow calibration of luminescence signals into Ca(2+) concentration values. These results indicate that TAT-mediated aequorin transduction is a promising alternative to traditional plant transformation methods to monitor intracellular Ca(2+) dynamics rapidly and effectively in plant cells.
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.
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.
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 cells 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.
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.
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