Publications by authors named "Romeo Betto"

28 Publications

  • Page 1 of 1

Reduction of circulating sphingosine-1-phosphate worsens mdx soleus muscle dystrophic phenotype.

Exp Physiol 2020 Sep 8. Epub 2020 Sep 8.

Department of Biomedical Sciences, University of Padova, Padova, Italy.

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.
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http://dx.doi.org/10.1113/EP088603DOI Listing
September 2020

Repairing folding-defective α-sarcoglycan mutants by CFTR correctors, a potential therapy for limb-girdle muscular dystrophy 2D.

Hum Mol Genet 2018 03;27(6):969-984

Department of Biomedical Sciences, University of Padova, 35131 Padova, Italy.

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, β-, 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.
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http://dx.doi.org/10.1093/hmg/ddy013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886177PMC
March 2018

Ablation of S1P receptor protects mouse soleus from age-related drop in muscle mass, force, and regenerative capacity.

Am J Physiol Cell Physiol 2017 Jul 26;313(1):C54-C67. Epub 2017 Apr 26.

Department of Biomedical Sciences, University of Padova, Padua, Italy;

We investigated the effects of S1P 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 S1P-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 S1P-null mice, neither force nor the contractile properties of soleus changed during aging. We also evaluated the regenerative capacity of soleus in old S1P-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 S1P-null mice was similar to that of muscle in adult mice. We conclude that in the absence of S1P, 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.
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http://dx.doi.org/10.1152/ajpcell.00027.2017DOI Listing
July 2017

S1P3 receptor influences key physiological properties of fast-twitch extensor digitorum longus muscle.

J Appl Physiol (1985) 2016 Jun 30;120(11):1288-300. Epub 2015 Dec 30.

Department of Biomedical Sciences, University of Padova, Padova, Italy; IIM, Interuniversity Institute of Myology, Italy;

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.
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http://dx.doi.org/10.1152/japplphysiol.00345.2015DOI Listing
June 2016

Inhibition of ubiquitin proteasome system rescues the defective sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1) protein causing Chianina cattle pseudomyotonia.

J Biol Chem 2014 Nov 6;289(48):33073-82. Epub 2014 Oct 6.

Comparative Biomedicine and Food Science, University of Padova,35020 Legnaro (Padova), Italy,

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.
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http://dx.doi.org/10.1074/jbc.M114.576157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246067PMC
November 2014

Unveiling the degradative route of the V247M α-sarcoglycan mutant responsible for LGMD-2D.

Hum Mol Genet 2014 Jul 23;23(14):3746-58. Epub 2014 Feb 23.

Department of Biomedical Sciences and

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, β, 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.
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http://dx.doi.org/10.1093/hmg/ddu088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4065151PMC
July 2014

Effects of pleiotrophin overexpression on mouse skeletal muscles in normal loading and in actual and simulated microgravity.

PLoS One 2013 28;8(8):e72028. Epub 2013 Aug 28.

Section of Pharmacology, Department of Pharmacy & Drug Sciences, University of Bari - Aldo Moro, Bari, Italy.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0072028PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3756024PMC
May 2014

Paracrine effects of IGF-1 overexpression on the functional decline due to skeletal muscle disuse: molecular and functional evaluation in hindlimb unloaded MLC/mIgf-1 transgenic mice.

PLoS One 2014 3;8(6):e65167. Epub 2013 Jun 3.

Section of Pharmacology, Department of Pharmacy & Drug Sciences, University of Bari Aldo Moro, Bari, Italy.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0065167PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3670938PMC
January 2015

S1P2 receptor promotes mouse skeletal muscle regeneration.

J Appl Physiol (1985) 2012 Sep 28;113(5):707-13. Epub 2012 Jun 28.

Department of Biomedical Sciences, University of Padova, Padova, Italy.

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.
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http://dx.doi.org/10.1152/japplphysiol.00300.2012DOI Listing
September 2012

Adaptation of mouse skeletal muscle to long-term microgravity in the MDS mission.

PLoS One 2012 28;7(3):e33232. Epub 2012 Mar 28.

Department of Biomedical Sciences, University of Padova, Padova, Italy.

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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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0033232PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3314659PMC
August 2012

Extracellular ATP signaling during differentiation of C2C12 skeletal muscle cells: role in proliferation.

Mol Cell Biochem 2011 May 10;351(1-2):183-96. Epub 2011 Feb 10.

Department of Biomedical Sciences, University of Padova, Viale G. Colombo 3, 35121 Padova, Italy.

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.
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http://dx.doi.org/10.1007/s11010-011-0726-4DOI Listing
May 2011

Sorting receptor Rer1 controls surface expression of muscle acetylcholine receptors by ER retention of unassembled alpha-subunits.

Proc Natl Acad Sci U S A 2011 Jan 27;108(2):621-5. Epub 2010 Dec 27.

Leibniz Institut für Altersforschung-Fritz Lipmann Institut, 07743 Jena, Germany.

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.
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http://dx.doi.org/10.1073/pnas.1001624108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3021032PMC
January 2011

Sphingosine 1-phosphate signaling is involved in skeletal muscle regeneration.

Am J Physiol Cell Physiol 2010 Mar 30;298(3):C550-8. Epub 2009 Dec 30.

Dept. of Human Anatomy and Physiology, Univ. of Padova, Via Marzolo 3, 35131 Padua, Italy.

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.
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http://dx.doi.org/10.1152/ajpcell.00072.2009DOI Listing
March 2010

Eccentric contractions lead to myofibrillar dysfunction in muscular dystrophy.

J Appl Physiol (1985) 2010 Jan 12;108(1):105-11. Epub 2009 Nov 12.

Dept. of Human Anatomy and Physiology, Univ. of Padua, Padua, Italy.

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.
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http://dx.doi.org/10.1152/japplphysiol.00803.2009DOI Listing
January 2010

Sarcoglycanopathies: molecular pathogenesis and therapeutic prospects.

Expert Rev Mol Med 2009 Sep 28;11:e28. Epub 2009 Sep 28.

Department of Biomedical Sciences, University of Padova, 35121 Padova, Italy.

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.
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http://dx.doi.org/10.1017/S1462399409001203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3279956PMC
September 2009

High-frequency fatigue of skeletal muscle: role of extracellular Ca(2+).

Eur J Appl Physiol 2008 Oct 17;104(3):445-53. Epub 2008 Jun 17.

Department of Human Anatomy and Physiology, University of Padova, Via Marzolo 3, 35131 Padova, Italy.

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.
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http://dx.doi.org/10.1007/s00421-008-0796-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2969177PMC
October 2008

Inhibition of proteasome activity promotes the correct localization of disease-causing alpha-sarcoglycan mutants in HEK-293 cells constitutively expressing beta-, gamma-, and delta-sarcoglycan.

Am J Pathol 2008 Jul 5;173(1):170-81. Epub 2008 Jun 5.

Department of Biomedical Sciences, University of Padova, Padova, Italy.

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.
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http://dx.doi.org/10.2353/ajpath.2008.071146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2438295PMC
July 2008

Trophic action of sphingosine 1-phosphate in denervated rat soleus muscle.

Am J Physiol Cell Physiol 2008 Jan 17;294(1):C36-46. Epub 2007 Oct 17.

Department of Human Anatomy and Physiology, University of Padua, Padua, Italy.

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.
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http://dx.doi.org/10.1152/ajpcell.00164.2007DOI Listing
January 2008

Isoform switching in myofibrillar and excitation-contraction coupling proteins contributes to diminished contractile function in regenerating rat soleus muscle.

J Appl Physiol (1985) 2007 Apr 18;102(4):1640-8. Epub 2007 Jan 18.

Department of Human Anatomy and Physiology, University of Padova, Padova, Italy.

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.
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http://dx.doi.org/10.1152/japplphysiol.01397.2006DOI Listing
April 2007

Deficiency of alpha-sarcoglycan differently affects fast- and slow-twitch skeletal muscles.

Am J Physiol Regul Integr Comp Physiol 2005 Nov 7;289(5):R1328-37. Epub 2005 Jul 7.

Department of Human Anatomy and Physiology, University of Padova, Italy.

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.
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http://dx.doi.org/10.1152/ajpregu.00673.2004DOI Listing
November 2005

Ultrastructure of diaphragm from dystrophic alpha-sarcoglycan-null mice.

Acta Biochim Pol 2005 ;52(2):453-60

Nencki Institute of Experimental Biology, Warszawa, Poland.

alpha-Sarcoglycan is a 50 kDa single-pass transmembrane glycoprotein exclusively expressed in striated muscle that, together with beta-, gamma-, and delta-sarcoglycan, forms a sub-complex at the muscle fibre cell membrane. The sarcoglycans are components of the dystrophin-associated glycoprotein (DAG) complex which forms a mechanical link between the intracellular cytoskeleton and extracellular matrix. The DAG complex function is to protect the muscle membrane from the stress of contractile activity and as a structure for the docking of signalling proteins. Genetic defects of DAG components cause muscular dystrophies. A lack or defects of alpha-sarcoglycan causes the severe type 2D limb girdle muscular dystrophy. alpha-Sarcoglycan-null (Sgca-null) mice develop progressive muscular dystrophy similar to the human disorder. This animal model was used in the present work for an ultrastructural study of diaphragm muscle. Diaphragm from Sgca-null mouse presents a clear dystrophic phenotype, with necrosis, regeneration, fibre hypertrophy and splitting, excess of collagen and fatty infiltration. Some abnormalities were also observed, such as centrally located nuclei of abnormal shape, fibres containing inclusion bodies within the contractile structure, and fibres with electron-dense material dispersed over almost the entire cell. Additionally, unusual interstitial cells of uncertain identity were detected within muscle fibres. The abnormal ultrastructure of the diaphragm from Sgca-null mice is discussed.
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August 2009

The T-tubule membrane ATP-operated P2X4 receptor influences contractility of skeletal muscle.

FASEB J 2005 Jul 26;19(9):1184-6. Epub 2005 Apr 26.

Department of Biomedical and Experimental Sciences, University of Padova, Padova, Italy.

Evidence indicates that extracellular ATP may have relevant functions in skeletal muscle, even though the physiological role and distribution of specific signaling pathway elements are not well known. The present work shows that P2X4 receptor, an extracellular ATP-regulated cell membrane channel permeable to Ca2+, is expressed in several tissues of the rat, including skeletal muscle. A specific antibody detected a protein band of approximately 60 kDa. Immunofluorescence demonstrated that P2X4 has an intracellular localization, and confocal analysis revealed that the receptor colocalizes with the T-tubule membrane DHP receptor. Considering that the natural agonist of P2X4 is ATP, we explored if changes of extracellular ATP levels could occur in contracting skeletal muscle to regulate the channel. In vitro experiments showed that substantial ATP is released and rapidly hydrolyzed after electrical stimulation of rat muscle fibers. Results show that the presence of ATP-degrading enzymes (hexokinase/apyrase), inhibitors of P2X receptors or Ca2+-free conditions, all abolished the progressive twitch tension potentiation produced in soleus muscle by low-frequency (0.05 Hz) stimulation. These data reveal that ATP-mediated Ca2+ entry, most likely through P2X4 receptor, may play an important role in modulating the contractility of skeletal muscle.
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http://dx.doi.org/10.1096/fj.04-3333fjeDOI Listing
July 2005

Sphingosine 1-phosphate protects mouse extensor digitorum longus skeletal muscle during fatigue.

Am J Physiol Cell Physiol 2005 Jun 19;288(6):C1367-73. Epub 2005 Jan 19.

Department of Human Anatomy and Physiology, University of Padua, Via Marzolo 3, 35131 Padua, Italy.

Sphingomyelin derivatives exert various second messenger actions in numerous tissues. Sphingosine (SPH) and sphingosine 1-phosphate (S1P) are two major sphingomyelin derivatives present at high levels in blood. The aim of the present work was to investigate whether S1P and SPH exert relevant actions in mouse skeletal muscle contractility and fatigue. Exogenous S1P and SPH administration caused a significant reduction of tension decline during fatigue of extensor digitorum longus muscle. Final tension after the fatiguing protocol was 40% higher than in untreated muscle. Interestingly, N,N-dimethylsphingosine, an inhibitor of SPH kinase (SK), abolished the effect of supplemented SPH but not that of S1P, suggesting that SPH acts through its conversion to S1P. Moreover, SPH was not effective in Ca(2+)-free solutions, in agreement with the hypothesis that SPH action is dependent on its conversion to S1P by the Ca(2+)-requiring enzyme SK. In contrast to SPH, S1P produced its positive effects on fatigue in Ca(2+)-free conditions, indicating that S1P action does not require Ca(2+) entry and most likely is receptor mediated. The effects of S1P could be ascribed in part to its ability to prevent the reduction (-20 mV) of action potential amplitude caused by fatigue. In conclusion, these results indicate that extracellular S1P has protective effects during the development of muscle fatigue and that the extracellular conversion of SPH to S1P may represent a rheostat mechanism to protect skeletal muscle from possible cytotoxic actions of SPH.
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http://dx.doi.org/10.1152/ajpcell.00246.2004DOI Listing
June 2005

Characterization of the ATP-hydrolysing activity of alpha-sarcoglycan.

Biochem J 2004 Jul;381(Pt 1):105-12

Department of Biomedical Sciences, University of Padova, Viale G. Colombo, 3, 35121 Padova, Italy.

Alpha-Sarcoglycan is a glycoprotein associated with the dystrophin complex at sarcolemma of skeletal and cardiac muscles. Gene defects in alpha-sarcoglycan lead to a severe muscular dystrophy whose molecular mechanisms are not yet clear. A first insight into the function of alpha-sarcoglycan was obtained by finding that it is an ATP-binding protein and that it probably confers ability to hydrolyse ATP to the purified dystrophin complex [Betto, Senter, Ceoldo, Tarricone, Biral and Salviati (1999) J. Biol. Chem. 274, 7907-7912]. In the present study, we present definitive evidence showing that alpha-sarcoglycan is an ATP-hydrolysing enzyme. The appearance of alpha-sarcoglycan protein expression was correlated with the increase in ecto-nucleotidase activity during differentiation of C2C12 cells. Approx. 25% of ecto-nucleotidase activity displayed by the C2C12 myotubes was inhibited by preincubating cells with an antibody specific for the ATP-binding motif of alpha-sarcoglycan. This demonstrates that alpha-sarcoglycan substantially contributes to total ecto-nucleotidase activity of C2C12 myotubes. To characterize further this activity, human embryonic kidney 293 cells were transfected with expression plasmids containing alpha-sarcoglycan cDNA. Transfected cells exhibited a significant increase in the ATP-hydrolysing activity that was abolished by the anti-alpha-sarcoglycan antibody. The enzyme had a substrate specificity for ATP and ADP, did not hydrolyse other triphosphonucleosides, and the affinity for ATP was in the low mM range. The ATPase activity strictly required the presence of both Mg2+ and Ca2+ and was completely inhibited by suramin and reactive blue-2. These results show that alpha-sarcoglycan is a Ca2+, Mg2+-ecto-ATPDase. The possible consequences of the absence of alpha-sarcoglycan activity in the pathogenesis of muscular dystrophy are discussed.
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http://dx.doi.org/10.1042/BJ20031644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1133767PMC
July 2004

The novel skeletal muscle sarcoplasmic reticulum JP-45 protein. Molecular cloning, tissue distribution, developmental expression, and interaction with alpha 1.1 subunit of the voltage-gated calcium channel.

J Biol Chem 2003 Oct 17;278(41):39987-92. Epub 2003 Jul 17.

Department of Anesthesia, Zentrum Für Lehr und Forschung Kantonsspital Basel, Hebelstrasse 20, 4031 Basel, Switzerland.

JP-45 is a novel integral protein constituent of the skeletal muscle sarcoplasmic reticulum junctional face membrane. We identified its primary structure from a cDNA clone isolated from a mouse skeletal muscle cDNA library. Mouse skeletal muscle JP-45 displays over 86 and 50% identity with two hypothetical NCBI data base protein sequences from mouse tongue and human muscle, respectively. JP-45 is predicted to have a cytoplasmic domain, a single transmembrane segment followed by an intralumenal domain enriched in positively charged amino acids. Northern and Western blot analyses reveal that the protein is mainly expressed in skeletal muscle. The mRNA encoding JP-45 appears in 17-day-old mouse embryos; expression of the protein peaks during the second month of postnatal development and then decreases approximately 3-fold during aging. Double immunofluorescence of adult skeletal muscle fibers demonstrates that JP-45 co-localizes with the sarcoplasmic reticulum calcium release channel. Co-immunoprecipitation experiments with a monoclonal antibody against JP-45 show that JP-45 interacts with the alpha1.1 subunit voltage-gated calcium channel and calsequestrin. These results are consistent with the localization of JP-45 in the junctional sarcoplasmic reticulum and with its involvement in the molecular mechanism underlying skeletal muscle excitation-contraction coupling.
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http://dx.doi.org/10.1074/jbc.M305016200DOI Listing
October 2003

Expression and functional characterization of SCaMPER: a sphingolipid-modulated calcium channel of cardiomyocytes.

Am J Physiol Cell Physiol 2003 Mar 6;284(3):C780-90. Epub 2002 Nov 6.

SDSU Heart Institute and Department of Biology, San Diego State University, California 92182-4614, USA.

Calcium channels are important in a variety of cellular events including muscle contraction, signaling, proliferation, and apoptosis. Sphingolipids have been recognized as mediators of intracellular calcium release through their actions on a calcium channel, sphingolipid calcium release-mediating protein of the endoplasmic reticulum (SCaMPER). The current study investigates the expression and function of SCaMPER in cardiomyocytes. Northern analyses and RT-PCR cloning and sequencing revealed SCaMPER expression in both human and rat cardiac tissue. Immunofluorescence and Western blot analyses demonstrated that SCaMPER is abundant in cardiac tissue and is localized to the sarcotubular junction. This was confirmed by the colocalization of SCaMPER with dihydropyridine and ryanodine receptors by confocal microscopy. Purified T tubules were shown to contain SCaMPER and immunoelectron micrographs suggested that SCaMPER is located to the junctional T tubules, but a junctional SR localization cannot be ruled out. The sphingolipid ligand for SCaMPER, sphingosylphosphorylcholine (SPC), initiated calcium release from the cardiomyocyte SR. Importantly, antisense knockdown of SCaMPER mRNA produced a substantial reduction of sphingolipid-induced calcium release, suggesting that SCaMPER is a potentially important calcium channel of cardiomyocytes.
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http://dx.doi.org/10.1152/ajpcell.00382.2002DOI Listing
March 2003

Early changes of type 2B fibers after denervation of rat EDL skeletal muscle.

J Appl Physiol (1985) 2002 May;92(5):2045-52

Dipartimento di Anatomia e Fisiologia Umana, Centro di Studio per la Biologia e la Fisiopatologia Muscolare, I-35131 Padova, Italy.

Skeletal muscle type 2B fibers normally receive a moderate level of motoneuron discharge. As a consequence, we hypothesize that type 2B fiber properties should be less sensitive to the absence of the nerve. Therefore, we have investigated the response of sarcoplasmic reticulum and myofibrillar proteins of type 2B fibers isolated from rat extensor digitorum longus muscle after denervation (2 and 7 days). Single fibers were identified by SDS-PAGE of myosin heavy chain isoforms. Electrophysiological and isometric contractile properties of the whole muscle were also analyzed. The pCa-tension relationship of type 2B single fibers was shifted to the left at 2 days and to right at 7 days after denervation, with significant differences in the Hill coefficients and pCa threshold values in 2- vs. 7-day-denervated fibers. The sarcoplasmic reticulum Ca2+ uptake capacity and rate significantly decreased after 2 days of denervation, whereas both increased at 7 days. Caffeine sensitivity of sarcoplasmic reticulum Ca2+ release was transitory and markedly increased in 2-day-denervated fibers. Our results indicate that type 2B fiber functional properties are highly sensitive to the interruption of nerve supply. Moreover, most of 2-day-denervated changes were reverted at 7 days.
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http://dx.doi.org/10.1152/japplphysiol.00673.2001DOI Listing
May 2002

Bupivacaine myotoxicity is mediated by mitochondria.

J Biol Chem 2002 Apr 14;277(14):12221-7. Epub 2002 Jan 14.

Department of Biomedical Sciences, University of Padova and the Consiglio Nazionale delle Ricerche Units for the Study of Muscle Pathophysiology and Biomembranes, Viale Giuseppe Colombo 3, I-35121 Padova, Italy.

We have investigated the effects of the myotoxic local anesthetic bupivacaine on rat skeletal muscle mitochondria and isolated myofibers from flexor digitorum brevis, extensor digitorum longus, soleus, and from the proximal, striated portion of the esophagus. In isolated mitochondria, bupivacaine caused a concentration-dependent mitochondrial depolarization and pyridine nucleotide oxidation, which were matched by an increased oxygen consumption at bupivacaine concentrations of 1.5 mm or less at pH 7.4, whereas respiration was inhibited at higher concentrations. As a consequence of depolarization, bupivacaine caused the opening of the permeability transition pore (PTP), a cyclosporin A-sensitive inner membrane channel that plays a key role in many forms of cell death. In intact flexor digitorum brevis fibers bupivacaine caused mitochondrial depolarization and pyridine nucleotides oxidation that were matched by increased concentrations of cytosolic free Ca(2+), release of cytochrome c, and eventually, hypercontracture. Both mitochondrial depolarization and cytochrome c release were inhibited by cyclosporin A, indicating that PTP opening rather than bupivacaine as such was responsible for these events. Similar responses to bupivacaine were observed in the soleus, which is highly oxidative. In contrast, fibers from the esophagus (which we show to be more fatigable than flexor digitorum brevis fibers) and from the highly glycolytic extensor digitorum longus didn't undergo pyridine nucleotide oxidation upon the addition of bupivacaine and were resistant to bupivacaine toxicity. These results suggest that active oxidative metabolism is a key determinant in bupivacaine toxicity, that bupivacaine myotoxicity is a relevant model of mitochondrial dysfunction involving the PTP and Ca(2+) dysregulation, and that it represents a promising system to test new PTP inhibitors that may prove relevant in spontaneous myopathies where mitochondria have long been suspected to play a role.
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http://dx.doi.org/10.1074/jbc.M108938200DOI Listing
April 2002