Sandra Furlan - Academia.edu (original) (raw)

Papers by Sandra Furlan

International Journal of Molecular Sciences

The molecular mechanisms of skeletal muscle atrophy under extended periods of either disuse or mi... more The molecular mechanisms of skeletal muscle atrophy under extended periods of either disuse or microgravity are not yet fully understood. The transition of Homer isoforms may play a key role during neuromuscular junction (NMJ) imbalance/plasticity in space. Here, we investigated the expression pattern of Homer short and long isoforms by gene array, qPCR, biochemistry, and laser confocal microscopy in skeletal muscles from male C57Bl/N6 mice (n = 5) housed for 30 days in space (Bion-flight = BF) compared to muscles from Bion biosatellite on the ground-housed animals (Bion ground = BG) and from standard cage housed animals (Flight control = FC). A comparison study was carried out with muscles of rats subjected to hindlimb unloading (HU). Gene array and qPCR results showed an increase in Homer1a transcripts, the short dominant negative isoform, in soleus (SOL) muscle after 30 days in microgravity, whereas it was only transiently increased after four days of HU. Conversely, Homer2 long-...

Journal of General Physiology

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are enriched at postsynaptic membrane compartments... more Inositol 1,4,5-trisphosphate receptors (IP3Rs) are enriched at postsynaptic membrane compartments of the neuromuscular junction (NMJ), surrounding the subsynaptic nuclei and close to nicotinic acetylcholine receptors (nAChRs) of the motor endplate. At the endplate level, it has been proposed that nerve-dependent electrical activity might trigger IP3-associated, local Ca2+ signals not only involved in excitation–transcription (ET) coupling but also crucial to the development and stabilization of the NMJ itself. The present study was undertaken to examine whether denervation affects the subsynaptic IP3R distribution in skeletal muscles and which are the underlying mechanisms. Fluorescence microscopy, carried out on in vivo denervated muscles (following sciatectomy) and in vitro denervated skeletal muscle fibers from flexor digitorum brevis (FDB), indicates that denervation causes a reduction in the subsynaptic IP3R1-stained region, and such a decrease appears to be determined by the l...

Antioxidants

Increased oxidative stress by reactive oxygen species (ROS) and reactive nitrogen species (RNS) i... more Increased oxidative stress by reactive oxygen species (ROS) and reactive nitrogen species (RNS) is a major determinant of disuse-induced muscle atrophy. Muscle biopsies (thigh vastus lateralis, VL) obtained from healthy male subjects enrolled in the Toulouse Cocktail bedrest (BR) study were used to assess efficacy of an antioxidant cocktail (polyphenols, omega-3, vitamin E, and selenium) to counteract the increased redox homeostasis and enhance the antioxidant defense response by using label-free LC–MS/MS and NITRO-DIGE (nitrosated proteins), qPCR, and laser confocal microscopy. Label-free LC–MS/MS indicated that treatment prevented the redox homeostasis dysregulation and promoted structural remodeling (TPM3, MYH7, MYBPC, MYH1, MYL1, HRC, and LUM), increment of RyR1, myogenesis (CSRP3), and skeletal muscle development (MUSTN1, LMNA, AHNAK). These changes were absent in the Placebo group. Glycolysis, tricarboxylic acid cycle (TCA), oxidative phosphorylation, fatty acid beta-oxidation...

Loss of skeletal muscle mass and force occurs in si tuations of altered use such as denervation, ... more Loss of skeletal muscle mass and force occurs in si tuations of altered use such as denervation, immobilization, aging and microgravity . The atrophy program induced under these conditions leads to an enhanced muscle protei n degradation mainly via the ubiquitinproteasome and the autophagy-lysosome systems. The aim of our work is the definition of signaling pathways involved in muscle atrophy durin g space flights, that were never explored so far. As cellular model, we used culture d single skeletal muscle fibers isolated from adult mice. On September 2007, we had the uniq ue opportunity to expose skeletal muscle fibers to microgravity condition during the unmanned Russian Foton-M3 mission, sponsored by Italian Space Agency (ASI) LIFE progra m. We utilized six pre-existing STROMA bioreactors (produced by Kayser Italia), fou r of which were for molecular analyses and two for morphology. Here we report the results of this space flight experience whose specific objective was to determi...

Metabolites, 2021

Homer represents a diversified family of scaffold and transduction proteins made up of several is... more Homer represents a diversified family of scaffold and transduction proteins made up of several isoforms. Here, we present preliminary observations on skeletal muscle adaptation and plasticity in a transgenic model of Homer 2-/- mouse using a multifaceted approach entailing morphometry, quantitative RT-PCR (Reverse Transcription PCR), confocal immunofluorescence, and electrophysiology. Morphometry shows that Soleus muscle (SOL), at variance with Extensor digitorum longus muscle (EDL) and Flexor digitorum brevis muscle (FDB), displays sizable reduction of fibre cross-sectional area compared to the WT counterparts. In SOL of Homer 2-/- mice, quantitative RT-PCR indicated the upregulation of Atrogin-1 and Muscle ring finger protein 1 (MuRF1) genes, and confocal immunofluorescence showed the decrease of neuromuscular junction (NMJ) Homer content. Electrophysiological measurements of isolated FDB fibres from Homer 2-/- mice detected the exclusive presence of the adult ε-nAChR isoform excl...

Frontiers in Neuroanatomy, 2020

Calcium stores in neurons are heterogeneous in compartmentalization and molecular composition. Da... more Calcium stores in neurons are heterogeneous in compartmentalization and molecular composition. Danio rerio (zebrafish) is an animal model with a simply folded cerebellum similar in cellular organization to that of mammals. The aim of the study was to identify new endoplasmic reticulum (ER) calcium store markers in zebrafish adult brain with emphasis on cerebellum and optic tectum. By quantitative polymerase chain reaction, we found three RNA transcripts coding for the intra-ER calcium binding protein calsequestrin: casq1a, casq1b, and casq2. In brain homogenates, two isoforms were detected by mass spectrometry and western blotting. Fractionation experiments of whole brain revealed that Casq1a and Casq2 were enriched in a heavy fraction containing ER microsomes and synaptic membranes. By in situ hybridization, we found the heterogeneous expression of casq1a and casq2 mRNA to be compatible with the cellular localization of calsequestrins investigated by immunofluorescence. Casq1 was expressed in neurogenic differentiation 1 expressing the granule cells of the cerebellum and the periventricular zone of the optic tectum. Casq2 was concentrated in parvalbumin expressing Purkinje cells. At a subcellular level, Casq1 was restricted to granular cell bodies, and Casq2 was localized in cell bodies, dendrites, and axons. Data are discussed in relation to the differential cellular and subcellular distribution of other cerebellum calcium store markers and are evaluated with respect to the putative relevance of calsequestrins in the neuron-specific functional activity.

Gerontology & geriatric medicine

Physical activity plays an important role in preventing muscle atrophy and chronic diseases in ad... more Physical activity plays an important role in preventing muscle atrophy and chronic diseases in adults and in the elderly. Calcium (Ca) cycling and activation of specific molecular pathways are essential in contraction-induced muscle adaptation. This study attains human muscle sections and total homogenates prepared from biopsies obtained before (control) and after 9 weeks of training by electrical stimulation (ES) on a group of volunteers. The aim of the study was to investigate about the molecular mechanisms that support functional muscle improvement by ES. Evidences of kinase/phosphatase pathways activation after ES were obtained. Moreover, expression of Sarcalumenin, Calsequestrin and sarco/endoplasmic reticulum Ca-ATPase (Serca) isoforms was regulated by training. In conclusion, this work shows that neuromuscular ES applied to vastus lateralis muscle of sedentary seniors combines fiber remodeling with activation of Ca-Calmodulin molecular pathways and modulation of key Ca-handli...

Biochemical Journal, 1990

Junctional transverse tubules (TT) isolated from triads of rabbit skeletal muscle by centrifugati... more Junctional transverse tubules (TT) isolated from triads of rabbit skeletal muscle by centrifugation in an ion-free sucrose gradient were compared with membrane subfractions, predominantly derived from the free portion of TT, that had been purified from sarcoplasmic reticulum membrane contaminants by three different methods. The markers used were diagnostic membrane markers and the dihydropyridine (DHP) receptor, which is a specific marker of the junctional membrane of TT. Junctional TT have a high membrane density (Bmax. 60 pmol/mg of protein) of high-affinity (Kd 0.25 nM) DHP-binding sites using [3H]PN200-110 as the specific ligand. When analysed by SDS/PAGE under reducing conditions and by Western blot techniques, the TT were found to contain a concanavalin A-binding 150 kDa glycoprotein which probably corresponds to the alpha 2-subunit of the DHP receptor. This conclusion was supported by correlative immunoblot experiments with a specific antibody. Junctional TT are further disti...

Frontiers in physiology, 2017

Microgravity as well as chronic muscle disuse are two causes of low back pain originated at least... more Microgravity as well as chronic muscle disuse are two causes of low back pain originated at least in part from paraspinal muscle deconditioning. At present no study investigated the complexity of the molecular changes in human or mouse paraspinal muscles exposed to microgravity. The aim of this study was to evaluate longissimus dorsi adaptation to microgravity at both morphological and global gene expression level. C57BL/N6 male mice were flown aboard the BION-M1 biosatellite for 30 days (BF) or housed in a replicate flight habitat on ground (BG). Myofiber cross sectional area and myosin heavy chain subtype patterns were respectively not or slightly altered in longissimus dorsi of BF mice. Global gene expression analysis identified 89 transcripts differentially regulated in longissimus dorsi of BF vs. BG mice. Microgravity-induced gene expression changes of lipocalin 2 (Lcn2), sestrin 1(Sesn1), phosphatidylinositol 3-kinase, regulatory subunit polypeptide 1 (p85 alpha) (Pik3r1), v-m...

Journal of muscle research and cell motility, Apr 20, 2015

Calsequestrin (Casq) is a high capacity, low affinity Ca(2+)-binding protein, critical for Ca(2+)... more Calsequestrin (Casq) is a high capacity, low affinity Ca(2+)-binding protein, critical for Ca(2+)-buffering in cardiac and skeletal muscle sarcoplasmic reticulum. All vertebrates have multiple genes encoding for different Casq isoforms. Increasing interest has been focused on mammalian and human Casq genes since mutations of both cardiac (Casq2) and skeletal muscle (Casq1) isoforms cause different, and sometime severe, human pathologies. Danio rerio (zebrafish) is a powerful model for studying function and mutations of human proteins. In this work, expression, biochemical properties cellular and sub-cellular localization of D. rerio native Casq isoforms are investigated. By quantitative PCR, three mRNAs were detected in skeletal muscle and heart with different abundances. Three zebrafish Casqs: Casq1a, Casq1b and Casq2 were identified by mass spectrometry (Data are available via ProteomeXchange with identifier PXD002455). Skeletal and cardiac zebrafish calsequestrins share propertie...

Biochemical Journal, 1993

Dystrophin, the protein coded by the gene missing in Duchenne muscular dystrophy, is assumed to b... more Dystrophin, the protein coded by the gene missing in Duchenne muscular dystrophy, is assumed to be a component of the membrane cytoskeleton of skeletal muscle. Like other cytoskeletal proteins in different cell types, dystrophin bound to sarcolemma membranes was found to be phosphorylated by endogenous protein kinases. The phosphorylation of dystrophin was activated by cyclic AMP, cyclic GMP, calcium and calmodulin, and was inhibited by cyclic AMP-dependent protein kinase peptide inhibitor, mastoparan and heparin. These results suggest that membrane-bound dystrophin is a substrate of endogenous cyclic AMP- and cyclic GMP-dependent protein kinases, calcium/calmodulin-dependent kinase and casein kinase II. The possibility that dystrophin could be phosphorylated by protein kinase C is suggested by the inhibition of phosphorylation by staurosporin. On the other hand dystrophin seems not to be a substrate for protein tyrosine kinases, as shown by the lack of reaction of phosphorylated dy...

Basic and applied myology: BAM

An RNA-mediated toxic gain-of-function has been indicated as the pathogenic mechanism underlying ... more An RNA-mediated toxic gain-of-function has been indicated as the pathogenic mechanism underlying Myotonic dystrophies type 1 and type 2 (DM1 and DM2), two dominantly inherited myopathies with complex phenotypes and involvement of multiple organs. A number of gene transcripts are aberrantly spliced in DM1 and DM2 leading to the expression of foetal isoforms during the adult life. In particular, mutated mRNAs derived from the Troponin T (TnT) genes in striated muscles have been reported. The aim of this work is to demonstrate the abnormal expression of foetal TnT isoforms in adult DM2 human biopsies. PCR-amplified products have been analysed by agarose-gel electrophoresis, and amplicons abnormally migrating, with respect to the controls, have been sequenced. We show here that at least two isoforms derived from the alternative splicing within the hypervariable region of the fast TNNT3 gene still contain the foetal exon. The persistence of the foetal exon has been found both within a full-length isoform not previously described in adults and in association with a shorter sequence derived from alternative splicing in the hypervariable region.

Basic and applied myology: BAM

An RNA-mediated toxic gain-of-function has been indicated as the pathogenic mechanism underlying ... more An RNA-mediated toxic gain-of-function has been indicated as the pathogenic mechanism underlying Myotonic dystrophies type 1 and type 2 (DM1 and DM2), two dominantly inherited myopathies with complex phenotypes and involvement of multiple organs. A number of gene transcripts are aberrantly spliced in DM1 and DM2 leading to the expression of foetal isoforms during the adult life. In particular, mutated mRNAs derived from the Troponin T (TnT) genes in striated muscles have been reported. The aim of this work is to demonstrate the abnormal expression of foetal TnT isoforms in adult DM2 human biopsies. PCR-amplified products have been analysed by agarose-gel electrophoresis, and amplicons abnormally migrating, with respect to the controls, have been sequenced. We show here that at least two isoforms derived from the alternative splicing within the hypervariable region of the fast TNNT3 gene still contain the foetal exon. The persistence of the foetal exon has been found both within a fu...

Myotonic dystrophy is a multisystem disorder associated to a trinucleotide repeat expansion in th... more Myotonic dystrophy is a multisystem disorder associated to a trinucleotide repeat expansion in the 3'-untranslated region of a gene encoding a protein kinase, DMPK. The physiologi-cal role of the DMPK is largely unknown. DMPK and other closely related protein kinases interact with members of the Rho family of small GTPases suggesting that they may be in-volved in signal transduction. On the other hand, the discovered interaction between DMPK and the small heat shock protein MKBP suggests that DMPK might be involved in the re-sponse against external stimuli. The possibility exists that DMPK might act as a switch between different intracellular pathways, depending on distinct external signals. We report here that heat shock does not appear to change phenotypic expression of DMPK neither in the whole tissue nor in cultured cells. Dystrophia myotonica (DM) was initially described as myotonia atrophica by Steinert [17] and Batten and Gibb [2]. This myotonic muscular dystrophy is a di...

Journal of Muscle Research and Cell Motility, 1997

Myotonic dystrophy is an autosomal dominant multisystem disease primarily affecting skeletal musc... more Myotonic dystrophy is an autosomal dominant multisystem disease primarily affecting skeletal muscle and is characterized by the presence of an amplified trinucleotide repeat in the 3–2; untranslated region of the myotonic dystrophy protein kinase gene. In this study, the subcellular localization of the myotonic dystrophy protein kinase in muscle tissues has been investigated at both morphological and biochemical level, by

Neurological Sciences, 2005

Myotonic dystrophy type 1 (DM1) is an autosomal dominant disease caused by a trinucleotide repeat... more Myotonic dystrophy type 1 (DM1) is an autosomal dominant disease caused by a trinucleotide repeatexpansion, cytosine-thymine-guanine (CTG)n, in the 3 ı untranslated region of a gene encoding the myotonic dystrophy protein kinase (DMPK). To correlate CTG expansion and protein expression, we studied muscle specimens from 16 adult DM1 patients using three anti-DMPK antibodies for immunoblotting. We estimated the amount of the full-length DMPK (85 kDa) in muscle biopsies from normal controls and from DM1 patients carrying different (CTG) n expansions. We found that DMPK concentration was decreased to about 50% in DM patients' muscles; the protein decrease did not seem correlated with the CTG repeat length. However, the fibre type composition in skeletal muscle seemed somehow to affect DMPK decrease, as the lowest level of the enzyme was found in patients with the lowest content of type 1 fibre.

International Journal of Molecular Sciences

The molecular mechanisms of skeletal muscle atrophy under extended periods of either disuse or mi... more The molecular mechanisms of skeletal muscle atrophy under extended periods of either disuse or microgravity are not yet fully understood. The transition of Homer isoforms may play a key role during neuromuscular junction (NMJ) imbalance/plasticity in space. Here, we investigated the expression pattern of Homer short and long isoforms by gene array, qPCR, biochemistry, and laser confocal microscopy in skeletal muscles from male C57Bl/N6 mice (n = 5) housed for 30 days in space (Bion-flight = BF) compared to muscles from Bion biosatellite on the ground-housed animals (Bion ground = BG) and from standard cage housed animals (Flight control = FC). A comparison study was carried out with muscles of rats subjected to hindlimb unloading (HU). Gene array and qPCR results showed an increase in Homer1a transcripts, the short dominant negative isoform, in soleus (SOL) muscle after 30 days in microgravity, whereas it was only transiently increased after four days of HU. Conversely, Homer2 long-...

Journal of General Physiology

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are enriched at postsynaptic membrane compartments... more Inositol 1,4,5-trisphosphate receptors (IP3Rs) are enriched at postsynaptic membrane compartments of the neuromuscular junction (NMJ), surrounding the subsynaptic nuclei and close to nicotinic acetylcholine receptors (nAChRs) of the motor endplate. At the endplate level, it has been proposed that nerve-dependent electrical activity might trigger IP3-associated, local Ca2+ signals not only involved in excitation–transcription (ET) coupling but also crucial to the development and stabilization of the NMJ itself. The present study was undertaken to examine whether denervation affects the subsynaptic IP3R distribution in skeletal muscles and which are the underlying mechanisms. Fluorescence microscopy, carried out on in vivo denervated muscles (following sciatectomy) and in vitro denervated skeletal muscle fibers from flexor digitorum brevis (FDB), indicates that denervation causes a reduction in the subsynaptic IP3R1-stained region, and such a decrease appears to be determined by the l...

Antioxidants

Increased oxidative stress by reactive oxygen species (ROS) and reactive nitrogen species (RNS) i... more Increased oxidative stress by reactive oxygen species (ROS) and reactive nitrogen species (RNS) is a major determinant of disuse-induced muscle atrophy. Muscle biopsies (thigh vastus lateralis, VL) obtained from healthy male subjects enrolled in the Toulouse Cocktail bedrest (BR) study were used to assess efficacy of an antioxidant cocktail (polyphenols, omega-3, vitamin E, and selenium) to counteract the increased redox homeostasis and enhance the antioxidant defense response by using label-free LC–MS/MS and NITRO-DIGE (nitrosated proteins), qPCR, and laser confocal microscopy. Label-free LC–MS/MS indicated that treatment prevented the redox homeostasis dysregulation and promoted structural remodeling (TPM3, MYH7, MYBPC, MYH1, MYL1, HRC, and LUM), increment of RyR1, myogenesis (CSRP3), and skeletal muscle development (MUSTN1, LMNA, AHNAK). These changes were absent in the Placebo group. Glycolysis, tricarboxylic acid cycle (TCA), oxidative phosphorylation, fatty acid beta-oxidation...

Loss of skeletal muscle mass and force occurs in si tuations of altered use such as denervation, ... more Loss of skeletal muscle mass and force occurs in si tuations of altered use such as denervation, immobilization, aging and microgravity . The atrophy program induced under these conditions leads to an enhanced muscle protei n degradation mainly via the ubiquitinproteasome and the autophagy-lysosome systems. The aim of our work is the definition of signaling pathways involved in muscle atrophy durin g space flights, that were never explored so far. As cellular model, we used culture d single skeletal muscle fibers isolated from adult mice. On September 2007, we had the uniq ue opportunity to expose skeletal muscle fibers to microgravity condition during the unmanned Russian Foton-M3 mission, sponsored by Italian Space Agency (ASI) LIFE progra m. We utilized six pre-existing STROMA bioreactors (produced by Kayser Italia), fou r of which were for molecular analyses and two for morphology. Here we report the results of this space flight experience whose specific objective was to determi...

Metabolites, 2021

Homer represents a diversified family of scaffold and transduction proteins made up of several is... more Homer represents a diversified family of scaffold and transduction proteins made up of several isoforms. Here, we present preliminary observations on skeletal muscle adaptation and plasticity in a transgenic model of Homer 2-/- mouse using a multifaceted approach entailing morphometry, quantitative RT-PCR (Reverse Transcription PCR), confocal immunofluorescence, and electrophysiology. Morphometry shows that Soleus muscle (SOL), at variance with Extensor digitorum longus muscle (EDL) and Flexor digitorum brevis muscle (FDB), displays sizable reduction of fibre cross-sectional area compared to the WT counterparts. In SOL of Homer 2-/- mice, quantitative RT-PCR indicated the upregulation of Atrogin-1 and Muscle ring finger protein 1 (MuRF1) genes, and confocal immunofluorescence showed the decrease of neuromuscular junction (NMJ) Homer content. Electrophysiological measurements of isolated FDB fibres from Homer 2-/- mice detected the exclusive presence of the adult ε-nAChR isoform excl...

Frontiers in Neuroanatomy, 2020

Calcium stores in neurons are heterogeneous in compartmentalization and molecular composition. Da... more Calcium stores in neurons are heterogeneous in compartmentalization and molecular composition. Danio rerio (zebrafish) is an animal model with a simply folded cerebellum similar in cellular organization to that of mammals. The aim of the study was to identify new endoplasmic reticulum (ER) calcium store markers in zebrafish adult brain with emphasis on cerebellum and optic tectum. By quantitative polymerase chain reaction, we found three RNA transcripts coding for the intra-ER calcium binding protein calsequestrin: casq1a, casq1b, and casq2. In brain homogenates, two isoforms were detected by mass spectrometry and western blotting. Fractionation experiments of whole brain revealed that Casq1a and Casq2 were enriched in a heavy fraction containing ER microsomes and synaptic membranes. By in situ hybridization, we found the heterogeneous expression of casq1a and casq2 mRNA to be compatible with the cellular localization of calsequestrins investigated by immunofluorescence. Casq1 was expressed in neurogenic differentiation 1 expressing the granule cells of the cerebellum and the periventricular zone of the optic tectum. Casq2 was concentrated in parvalbumin expressing Purkinje cells. At a subcellular level, Casq1 was restricted to granular cell bodies, and Casq2 was localized in cell bodies, dendrites, and axons. Data are discussed in relation to the differential cellular and subcellular distribution of other cerebellum calcium store markers and are evaluated with respect to the putative relevance of calsequestrins in the neuron-specific functional activity.

Gerontology & geriatric medicine

Physical activity plays an important role in preventing muscle atrophy and chronic diseases in ad... more Physical activity plays an important role in preventing muscle atrophy and chronic diseases in adults and in the elderly. Calcium (Ca) cycling and activation of specific molecular pathways are essential in contraction-induced muscle adaptation. This study attains human muscle sections and total homogenates prepared from biopsies obtained before (control) and after 9 weeks of training by electrical stimulation (ES) on a group of volunteers. The aim of the study was to investigate about the molecular mechanisms that support functional muscle improvement by ES. Evidences of kinase/phosphatase pathways activation after ES were obtained. Moreover, expression of Sarcalumenin, Calsequestrin and sarco/endoplasmic reticulum Ca-ATPase (Serca) isoforms was regulated by training. In conclusion, this work shows that neuromuscular ES applied to vastus lateralis muscle of sedentary seniors combines fiber remodeling with activation of Ca-Calmodulin molecular pathways and modulation of key Ca-handli...

Biochemical Journal, 1990

Junctional transverse tubules (TT) isolated from triads of rabbit skeletal muscle by centrifugati... more Junctional transverse tubules (TT) isolated from triads of rabbit skeletal muscle by centrifugation in an ion-free sucrose gradient were compared with membrane subfractions, predominantly derived from the free portion of TT, that had been purified from sarcoplasmic reticulum membrane contaminants by three different methods. The markers used were diagnostic membrane markers and the dihydropyridine (DHP) receptor, which is a specific marker of the junctional membrane of TT. Junctional TT have a high membrane density (Bmax. 60 pmol/mg of protein) of high-affinity (Kd 0.25 nM) DHP-binding sites using [3H]PN200-110 as the specific ligand. When analysed by SDS/PAGE under reducing conditions and by Western blot techniques, the TT were found to contain a concanavalin A-binding 150 kDa glycoprotein which probably corresponds to the alpha 2-subunit of the DHP receptor. This conclusion was supported by correlative immunoblot experiments with a specific antibody. Junctional TT are further disti...

Frontiers in physiology, 2017

Microgravity as well as chronic muscle disuse are two causes of low back pain originated at least... more Microgravity as well as chronic muscle disuse are two causes of low back pain originated at least in part from paraspinal muscle deconditioning. At present no study investigated the complexity of the molecular changes in human or mouse paraspinal muscles exposed to microgravity. The aim of this study was to evaluate longissimus dorsi adaptation to microgravity at both morphological and global gene expression level. C57BL/N6 male mice were flown aboard the BION-M1 biosatellite for 30 days (BF) or housed in a replicate flight habitat on ground (BG). Myofiber cross sectional area and myosin heavy chain subtype patterns were respectively not or slightly altered in longissimus dorsi of BF mice. Global gene expression analysis identified 89 transcripts differentially regulated in longissimus dorsi of BF vs. BG mice. Microgravity-induced gene expression changes of lipocalin 2 (Lcn2), sestrin 1(Sesn1), phosphatidylinositol 3-kinase, regulatory subunit polypeptide 1 (p85 alpha) (Pik3r1), v-m...

Journal of muscle research and cell motility, Apr 20, 2015

Calsequestrin (Casq) is a high capacity, low affinity Ca(2+)-binding protein, critical for Ca(2+)... more Calsequestrin (Casq) is a high capacity, low affinity Ca(2+)-binding protein, critical for Ca(2+)-buffering in cardiac and skeletal muscle sarcoplasmic reticulum. All vertebrates have multiple genes encoding for different Casq isoforms. Increasing interest has been focused on mammalian and human Casq genes since mutations of both cardiac (Casq2) and skeletal muscle (Casq1) isoforms cause different, and sometime severe, human pathologies. Danio rerio (zebrafish) is a powerful model for studying function and mutations of human proteins. In this work, expression, biochemical properties cellular and sub-cellular localization of D. rerio native Casq isoforms are investigated. By quantitative PCR, three mRNAs were detected in skeletal muscle and heart with different abundances. Three zebrafish Casqs: Casq1a, Casq1b and Casq2 were identified by mass spectrometry (Data are available via ProteomeXchange with identifier PXD002455). Skeletal and cardiac zebrafish calsequestrins share propertie...

Biochemical Journal, 1993

Dystrophin, the protein coded by the gene missing in Duchenne muscular dystrophy, is assumed to b... more Dystrophin, the protein coded by the gene missing in Duchenne muscular dystrophy, is assumed to be a component of the membrane cytoskeleton of skeletal muscle. Like other cytoskeletal proteins in different cell types, dystrophin bound to sarcolemma membranes was found to be phosphorylated by endogenous protein kinases. The phosphorylation of dystrophin was activated by cyclic AMP, cyclic GMP, calcium and calmodulin, and was inhibited by cyclic AMP-dependent protein kinase peptide inhibitor, mastoparan and heparin. These results suggest that membrane-bound dystrophin is a substrate of endogenous cyclic AMP- and cyclic GMP-dependent protein kinases, calcium/calmodulin-dependent kinase and casein kinase II. The possibility that dystrophin could be phosphorylated by protein kinase C is suggested by the inhibition of phosphorylation by staurosporin. On the other hand dystrophin seems not to be a substrate for protein tyrosine kinases, as shown by the lack of reaction of phosphorylated dy...

Basic and applied myology: BAM

An RNA-mediated toxic gain-of-function has been indicated as the pathogenic mechanism underlying ... more An RNA-mediated toxic gain-of-function has been indicated as the pathogenic mechanism underlying Myotonic dystrophies type 1 and type 2 (DM1 and DM2), two dominantly inherited myopathies with complex phenotypes and involvement of multiple organs. A number of gene transcripts are aberrantly spliced in DM1 and DM2 leading to the expression of foetal isoforms during the adult life. In particular, mutated mRNAs derived from the Troponin T (TnT) genes in striated muscles have been reported. The aim of this work is to demonstrate the abnormal expression of foetal TnT isoforms in adult DM2 human biopsies. PCR-amplified products have been analysed by agarose-gel electrophoresis, and amplicons abnormally migrating, with respect to the controls, have been sequenced. We show here that at least two isoforms derived from the alternative splicing within the hypervariable region of the fast TNNT3 gene still contain the foetal exon. The persistence of the foetal exon has been found both within a full-length isoform not previously described in adults and in association with a shorter sequence derived from alternative splicing in the hypervariable region.

Basic and applied myology: BAM

An RNA-mediated toxic gain-of-function has been indicated as the pathogenic mechanism underlying ... more An RNA-mediated toxic gain-of-function has been indicated as the pathogenic mechanism underlying Myotonic dystrophies type 1 and type 2 (DM1 and DM2), two dominantly inherited myopathies with complex phenotypes and involvement of multiple organs. A number of gene transcripts are aberrantly spliced in DM1 and DM2 leading to the expression of foetal isoforms during the adult life. In particular, mutated mRNAs derived from the Troponin T (TnT) genes in striated muscles have been reported. The aim of this work is to demonstrate the abnormal expression of foetal TnT isoforms in adult DM2 human biopsies. PCR-amplified products have been analysed by agarose-gel electrophoresis, and amplicons abnormally migrating, with respect to the controls, have been sequenced. We show here that at least two isoforms derived from the alternative splicing within the hypervariable region of the fast TNNT3 gene still contain the foetal exon. The persistence of the foetal exon has been found both within a fu...

Myotonic dystrophy is a multisystem disorder associated to a trinucleotide repeat expansion in th... more Myotonic dystrophy is a multisystem disorder associated to a trinucleotide repeat expansion in the 3'-untranslated region of a gene encoding a protein kinase, DMPK. The physiologi-cal role of the DMPK is largely unknown. DMPK and other closely related protein kinases interact with members of the Rho family of small GTPases suggesting that they may be in-volved in signal transduction. On the other hand, the discovered interaction between DMPK and the small heat shock protein MKBP suggests that DMPK might be involved in the re-sponse against external stimuli. The possibility exists that DMPK might act as a switch between different intracellular pathways, depending on distinct external signals. We report here that heat shock does not appear to change phenotypic expression of DMPK neither in the whole tissue nor in cultured cells. Dystrophia myotonica (DM) was initially described as myotonia atrophica by Steinert [17] and Batten and Gibb [2]. This myotonic muscular dystrophy is a di...

Journal of Muscle Research and Cell Motility, 1997

Myotonic dystrophy is an autosomal dominant multisystem disease primarily affecting skeletal musc... more Myotonic dystrophy is an autosomal dominant multisystem disease primarily affecting skeletal muscle and is characterized by the presence of an amplified trinucleotide repeat in the 3–2; untranslated region of the myotonic dystrophy protein kinase gene. In this study, the subcellular localization of the myotonic dystrophy protein kinase in muscle tissues has been investigated at both morphological and biochemical level, by

Neurological Sciences, 2005

Myotonic dystrophy type 1 (DM1) is an autosomal dominant disease caused by a trinucleotide repeat... more Myotonic dystrophy type 1 (DM1) is an autosomal dominant disease caused by a trinucleotide repeatexpansion, cytosine-thymine-guanine (CTG)n, in the 3 ı untranslated region of a gene encoding the myotonic dystrophy protein kinase (DMPK). To correlate CTG expansion and protein expression, we studied muscle specimens from 16 adult DM1 patients using three anti-DMPK antibodies for immunoblotting. We estimated the amount of the full-length DMPK (85 kDa) in muscle biopsies from normal controls and from DM1 patients carrying different (CTG) n expansions. We found that DMPK concentration was decreased to about 50% in DM patients' muscles; the protein decrease did not seem correlated with the CTG repeat length. However, the fibre type composition in skeletal muscle seemed somehow to affect DMPK decrease, as the lowest level of the enzyme was found in patients with the lowest content of type 1 fibre.