Publications by authors named "Daniela Danieli-Betto"

24 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

Loss of melusin is a novel, neuronal NO synthase/FoxO3-independent master switch of unloading-induced muscle atrophy.

J Cachexia Sarcopenia Muscle 2020 06 10;11(3):802-819. Epub 2020 Mar 10.

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

Background: Unloading/disuse induces skeletal muscle atrophy in bedridden patients and aged people, who cannot prevent it by means of exercise. Because interventions against known atrophy initiators, such as oxidative stress and neuronal NO synthase (nNOS) redistribution, are only partially effective, we investigated the involvement of melusin, a muscle-specific integrin-associated protein and a recognized regulator of protein kinases and mechanotransduction in cardiomyocytes.

Methods: Muscle atrophy was induced in the rat soleus by tail suspension and in the human vastus lateralis by bed rest. Melusin expression was investigated at the protein and transcript level and after treatment of tail-suspended rats with atrophy initiator inhibitors. Myofiber size, sarcolemmal nNOS activity, FoxO3 myonuclear localization, and myofiber carbonylation of the unloaded rat soleus were studied after in vivo melusin replacement by cDNA electroporation, and muscle force, myofiber size, and atrogene expression after adeno-associated virus infection. In vivo interference of exogenous melusin with dominant-negative kinases and other atrophy attenuators (Grp94 cDNA; 7-nitroindazole) on size of unloaded rat myofibers was also explored.

Results: Unloading/disuse reduced muscle melusin protein levels to about 50%, already after 6 h in the tail-suspended rat (P < 0.001), and to about 35% after 8 day bed rest in humans (P < 0.05). In the unloaded rat, melusin loss occurred despite of the maintenance of β1D integrin levels and was not abolished by treatments inhibiting mitochondrial oxidative stress, or nNOS activity and redistribution. Expression of exogenous melusin by cDNA transfection attenuated atrophy of 7 day unloaded rat myofibers (-31%), compared with controls (-48%, P = 0.001), without hampering the decrease in sarcolemmal nNOS activity and the increase in myonuclear FoxO3 and carbonylated myofibers. Infection with melusin-expressing adeno-associated virus ameliorated contractile properties of 7 day unloaded muscles (P ≤ 0.05) and relieved myofiber atrophy (-33%) by reducing Atrogin-1 and MurF-1 transcripts (P ≤ 0.002), despite of a two-fold increase in FoxO3 protein levels (P = 0.03). Atrophy attenuation by exogenous melusin did not result from rescue of Akt, ERK, or focal adhesion kinase activity, because it persisted after co-transfection with dominant-negative kinase forms (P < 0.01). Conversely, melusin cDNA transfection, combined with 7-nitroindazole treatment or with cDNA transfection of the nNOS-interacting chaperone Grp94, abolished 7 day unloaded myofiber atrophy.

Conclusions: Disuse/unloading-induced loss of melusin is an early event in muscle atrophy which occurs independently from mitochondrial oxidative stress, nNOS redistribution, and FoxO3 activation. Only preservation of melusin levels and sarcolemmal nNOS localization fully prevented muscle mass loss, demonstrating that both of them act as independent, but complementary, master switches of muscle disuse atrophy.
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http://dx.doi.org/10.1002/jcsm.12546DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7296270PMC
June 2020

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

Curcumin counteracts loss of force and atrophy of hindlimb unloaded rat soleus by hampering neuronal nitric oxide synthase untethering from sarcolemma.

J Physiol 2014 Jun 7;592(12):2637-52. Epub 2014 Apr 7.

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

Antioxidant administration aimed to antagonize the development and progression of disuse muscle atrophy provided controversial results. Here we investigated the effects of curcumin, a vegetal polyphenol with pleiotropic biological activity, because of its ability to upregulate glucose-regulated protein 94 kDa (Grp94) expression in myogenic cells. Grp94 is a sarco-endoplasmic reticulum chaperone, the levels of which decrease significantly in unloaded muscle. Rats were injected intraperitoneally with curcumin and soleus muscle was analysed after 7 days of hindlimb unloading or standard caging. Curcumin administration increased Grp94 protein levels about twofold in muscles of ambulatory rats (P < 0.05) and antagonized its decrease in unloaded ones. Treatment countered loss of soleus mass and myofibre cross-sectional area by approximately 30% (P ≤ 0.02) and maintained a force-frequency relationship closer to ambulatory levels. Indexes of muscle protein and lipid oxidation, such as protein carbonylation, revealed by Oxyblot, and malondialdehyde, measured with HPLC, were significantly blunted in unloaded treated rats compared to untreated ones (P = 0.01). Mechanistic involvement of Grp94 was suggested by the disruption of curcumin-induced attenuation of myofibre atrophy after transfection with antisense grp94 cDNA and by the drug-positive effect on the maintenance of the subsarcolemmal localization of active neuronal nitric oxide synthase molecules, which were displaced to the sarcoplasm by unloading. The absence of additive effects after combined administration of a neuronal nitric oxide synthase inhibitor further supported curcumin interference with this pro-atrophic pathway. In conclusion, curcumin represents an effective and safe tool to upregulate Grp94 muscle levels and to maintain muscle function during unweighting.
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http://dx.doi.org/10.1113/jphysiol.2013.268672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4080943PMC
June 2014

Altered Tnnt3 characterizes selective weakness of fast fibers in mice overexpressing FSHD region gene 1 (FRG1).

Am J Physiol Regul Integr Comp Physiol 2014 Jan 4;306(2):R124-37. Epub 2013 Dec 4.

Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy;

Facioscapulohumeral muscular dystrophy (FSHD), a common hereditary myopathy, is characterized by atrophy and weakness of selective muscle groups. FSHD is considered an autosomal dominant disease with incomplete penetrance and unpredictable variability of clinical expression within families. Mice overexpressing FRG1 (FSHD region gene 1), a candidate gene for this disease, develop a progressive myopathy with features of the human disorder. Here, we show that in FRG1-overexpressing mice, fast muscles, which are the most affected by the dystrophic process, display anomalous fast skeletal troponin T (fTnT) isoform, resulting from the aberrant splicing of the Tnnt3 mRNA that precedes the appearance of dystrophic signs. We determine that muscles of FRG1 mice develop less strength due to impaired contractile properties of fast-twitch fibers associated with an anomalous MyHC-actin ratio and a reduced sensitivity to Ca(2+). We demonstrate that the decrease of Ca(2+) sensitivity of fast-twitch fibers depends on the anomalous troponin complex and can be rescued by the substitution with the wild-type proteins. Finally, we find that the presence of aberrant splicing isoforms of TNNT3 characterizes dystrophic muscles in FSHD patients. Collectively, our results suggest that anomalous TNNT3 profile correlates with the muscle impairment in both humans and mice. On the basis of these results, we propose that aberrant fTnT represents a biological marker of muscle phenotype severity and disease progression.
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http://dx.doi.org/10.1152/ajpregu.00379.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3921317PMC
January 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

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

A subpopulation of rat muscle fibers maintains an assessable excitation-contraction coupling mechanism after long-standing denervation despite lost contractility.

J Neuropathol Exp Neurol 2009 Dec;68(12):1256-68

Interuniversitary Institute of Myology, Chieti, Italy.

To define the time course and potential effects of electrical stimulation on permanently denervated muscle, we evaluated excitation-contraction coupling (ECC) of rat leg muscles during progression to long-term denervation by ultrastructural analysis, specific binding to dihydropyridine receptors, ryanodine receptor 1 (RYR-1), Ca channels and extrusion Ca pumps, gene transcription and translation of Ca-handling proteins, and in vitro mechanical properties and electrophysiological analyses of sarcolemmal passive properties and L-type Ca current (ICa) parameters. We found that in response to long-term denervation: 1) isolated muscle that is unable to twitch in vitro by electrical stimulation has very small myofibers but may show a slow caffeine contracture; 2) only roughly half of the muscle fibers with "voltage-dependent Ca channel activity" are able to contract; 3) the ECC mechanisms are still present and, in part, functional; 4)ECC-related gene expression is upregulated; and 5) at any time point, there are muscle fibers that are more resistant than others to denervation atrophy and disorganization of the ECC apparatus. These results support the hypothesis that prolonged "resting" [Ca] may drive progression of muscle atrophy to degeneration and that electrical stimulation-induced [Ca] modulation may mimic the lost nerve influence, playing a key role in modifying the gene expression of denervated muscle. Hence, these data provide a potential molecular explanation for the muscle recovery that occurs in response to rehabilitation strategies developed based on empirical clinical observations.
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http://dx.doi.org/10.1097/NEN.0b013e3181c18416DOI Listing
December 2009

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

Skeletal muscle proteins oxidation in chronic right heart failure in rats: can different beta-blockers prevent it to the same degree?

Int J Cardiol 2010 Aug 16;143(2):192-9. Epub 2009 Mar 16.

CNR Institute of Neurosciences, Unit for Neuromuscular Biology and Pathophysiology, Department of Biomedical Sciences, University of Padova, Padova, Italy.

Background: Skeletal muscle atrophy and decreased expression of slow fibers contribute to exercise capacity limitation in Chronic Heart Failure (CHF). Pro-inflammatory cytokines and free radicals worsen muscle damage. In CHF sarcomeric proteins are oxidized with reduction of muscle twitch efficiency, and VO(2)-max. Beta-blockers with anti-oxidative capacity such as carvedilol have been shown to prevent contractile protein oxidation in CHF rats. Recently a new class of beta-blockers with NO donor activity has been introduced and approved for the treatment of CHF. Since a clinical clear superiority of a beta-blocker has never been shown, we compared nebivolol, that possesses NO donor activity, with bisoprolol, looking at possible differences in skeletal muscle that may have an impact on muscle function and exercise capacity in humans. We therefore studied skeletal muscle apoptosis and wastage, sarcomeric protein composition and oxidation, and muscle efficiency.

Methods And Results: In the monocrotaline rat model of CHF we compared nebivolol a beta-blocker with vasodilative properties mediated by NO production, with bisoprolol. Nebivolol prevented protein oxidation, while bisoprolol did it only partially, as demonstrated by the oxyblot analysis (Oxy/RP values) (0.90+/-0.14 Controls.; 1.7+/-0.14 CHF; 1.1+/-0.05 bisoprolol; 0.82+/-0.17 nebivolol low; 0.62+/-0.10 nebivolol high). Only nebivolol improved twitch force production and relaxation. Nebivolol prevented fibers shift towards fast isoforms, atrophy, decreased apoptosis and sphingosine levels.

Conclusions: Nebivolol seems better than bisoprolol in CHF by decreasing apoptosis and cytokines induced muscle wastage, preventing fibers shift and protein oxidation. Nebivolol by stimulating NO generation may have prevented protein oxidation. It could be speculated that ROS release, pro-inflammatory cytokines production and NF-kappa-B activation may play a key role. These positive changes could produce a favorable impact on exercise capacity in man.
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http://dx.doi.org/10.1016/j.ijcard.2009.02.012DOI Listing
August 2010

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

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

Denervation in murine fast-twitch muscle: short-term physiological changes and temporal expression profiling.

Physiol Genomics 2006 Mar 27;25(1):60-74. Epub 2005 Dec 27.

Centro di Ricerca Interdipartimentale per le Biotecnologie Innovative Biotechnology Center, University of Padova, Padua, Italy.

Denervation deeply affects muscle structure and function, the alterations being different in slow and fast muscles. Because the effects of denervation on fast muscles are still controversial, and high-throughput studies on gene expression in denervated muscles are lacking, we studied gene expression during atrophy progression following denervation in mouse tibialis anterior (TA). The sciatic nerve was cut close to trochanter in adult CD1 mice. One, three, seven, and fourteen days after denervation, animals were killed and TA muscles were dissected out and utilized for physiological experiments and gene expression studies. Target cDNAs from TA muscles were hybridized on a dedicated cDNA microarray of muscle genes. Seventy-one genes were found differentially expressed. Microarray results were validated, and the expression of relevant genes not probed on our array was monitored by real-time quantitative PCR (RQ-PCR). Nuclear- and mitochondrial-encoded genes implicated in energy metabolism were consistently downregulated. Among genes implicated in muscle contraction (myofibrillar and sarcoplasmic reticulum), genes typical of fast fibers were downregulated, whereas those typical of slow fibers were upregulated. Electrophoresis and Western blot showed less pronounced changes in myofibrillar protein expression, partially confirming changes in gene expression. Isometric tension of skinned fibers was little affected by denervation, whereas calcium sensitivity decreased. Functional studies in mouse extensor digitorum longus muscle showed prolongation in twitch time parameters and shift to the left in force-frequency curves after denervation. We conclude that, if studied at the mRNA level, fast muscles appear not less responsive than slow muscles to the interruption of neural stimulation.
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http://dx.doi.org/10.1152/physiolgenomics.00051.2005DOI Listing
March 2006

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

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

Skeletal muscle myofibrillar protein oxidation in heart failure and the protective effect of Carvedilol.

J Mol Cell Cardiol 2005 May;38(5):803-7

CNR Institute of Neurosciences, Unit for Neuromuscular Biology and Pathophysiology, Department of Biomedical Sciences, University of Padova, Viale G. Colombo 3, 35100 Padova, Italy.

Heart failure is characterized by limited exercise tolerance and by a skeletal muscle myopathy with atrophy and shift toward fast fibres. An inflammatory status with elevated pro-inflammatory cytokines and exaggerated free radicals production, can worsen muscle damage. In a well established model of heart failure, the monocrotaline treated rat, we show that CHF is accompanied by oxidation of the skeletal muscle actin, tropomyosin and myosin, which further depresses muscle function and exercise capacity. We have also tested the efficacy of Carvedilol, a non-selective beta(1)-beta(2)-blocker, which has been widely used in clinical trials to improve exercise tolerance and reduce mortality in moderate and severe CHF, in preventing contractile protein oxidation in CHF rats. As comparison we used Bisoprolol a beta(1) selective agent, without known anti-oxidative properties. Carvedilol at the dose of 2 mg/kg per day was able to prevent the myofibrillar protein oxidation, while Bisoprolol (0.1 mg/kg) did it only partially, as demonstrated by the oxyblot analysis. While Carvedilol improved force production on isolated muscles, Bisoprolol did not. After the COMET trial, the anti-oxidative capacity of Carvedilol has been invoked as one of the mechanism that makes this drug superior to other selective beta-blockers in the treatment of CHF. One of the reason of Carvedilol superiority could be the effect on skeletal muscle with reduction of contractile protein peroxidation, amelioration of muscle function and improvement of exercise tolerance. Inhibition of reactive oxygen species (ROS) production, and of pro-inflammatory cytokines may also lead to a decreased muscle wastage, another factor contributing to worsening of exercise tolerance.
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http://dx.doi.org/10.1016/j.yjmcc.2005.02.023DOI Listing
May 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

Beneficial effects of GH/IGF-1 on skeletal muscle atrophy and function in experimental heart failure.

Am J Physiol Cell Physiol 2004 Jan 17;286(1):C138-44. Epub 2003 Sep 17.

Internal Medicine II, Ospedale S. Bortolo, Viale Rodolfi 37, 36100 Vicenza, Italy.

Muscle atrophy is a determinant of exercise capacity in heart failure (CHF). Myocyte apoptosis, triggered by tumor necrosis factor-alpha (TNF-alpha) or its second messenger sphingosine (SPH), is one of the causes of atrophy. Growth hormone (GH) improves hemodynamic and cardiac trophism in several experimental models of CHF, but its effect on skeletal muscle in CHF is not yet clear. We tested the hypothesis that GH can prevent skeletal muscle apoptosis in rats with CHF. CHF was induced by injecting monocrotaline. After 2 wk, 2 groups of rats were treated with GH (0.2 mg.kg(-1).day(-1) and 1.0 mg.kg(-1).day(-1)) subcutaneously. A third group of controls had saline. After 2 additional weeks, rats were killed. Tibialis anterior cross-sectional area, myosin heavy chain (MHC) composition, and a study on myocyte apoptosis and serum levels of TNF-alpha and SPH were carried out. The number of apoptotic nuclei, muscle atrophy, and serum levels of TNF-alpha and SPH were decreased with GH at high but not at low doses compared with CHF rats. Bcl-2 was increased, whereas activated caspases and bax were decreased. The MHC pattern in GH-treated animals was similar to that of controls. Monocrotaline slowed down both contraction and relaxation but did not affect specific tetanic force, whereas absolute force was decreased. GH treatment restored contraction and relaxation to control values and brought muscle mass and absolute twitch and tetanic tension to normal levels. These findings may provide an insight into the therapeutic strategy of GH given to patients with CHF to improve exercise capacity.
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http://dx.doi.org/10.1152/ajpcell.00114.2003DOI Listing
January 2004

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