Compared with that of MUFA, a high dietary intake of n-3 PUFA does not reduce the degree of pathology in mdx mice

Duchenne muscular dystrophy (DMD) is a severe muscle disease that affects afflicted males from a young age, and the mdx mouse is an animal model of this disease. Although new drugs are in development, it is also essential to assess potential dietary therapies that could assist in the management of DMD. In the present study, we compared two diets, high-MUFA diet v. high-PUFA diet, in mdx mice. To generate the high-PUFA diet, a portion of dietary MUFA (oleic acid) was replaced with the dietary essential n-3 PUFA α-linolenic acid (ALA). We sought to determine whether ALA, compared with oleic acid, was beneficial in mdx mice. Consumption of the high-PUFA diet resulted in significantly higher n-3 PUFA content and reduced arachidonic acid content in skeletal muscle phospholipids (PL), while the high-MUFA diet led to higher oleate content in PL. Mdx mice on the high-MUFA diet exhibited 2-fold lower serum creatine kinase activity than those on the high-PUFA diet (P< 0·05) as well as a lower body fat percentage (P< 0·05), but no significant difference in skeletal muscle histopathology results. There was no significant difference between the dietary groups with regard to phosphorylated p65 (an inflammatory marker) in skeletal muscle. In conclusion, alteration of PL fatty acid (FA) composition by the high-PUFA diet made mdx muscle more susceptible to sarcolemmal leakiness, while the high-MUFA diet exhibited a more favourable impact. These results may be important for designing dietary treatments for DMD patients, and future work on dietary FA profiles, such as comparing other FA classes and dose effects, is needed.

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ProNGF/p75NTR Axis Drives Fiber Type Specification by Inducing the Fast-Glycolytic Phenotype in Mouse Skeletal Muscle Cells

Cells ◽

10.3390/cells9102232 ◽

2020 ◽

Vol 9 (10) ◽

pp. 2232

Author(s):

Valentina Pallottini ◽

Mayra Colardo ◽

Claudia Tonini ◽

Noemi Martella ◽

Georgios Strimpakos ◽

...

Keyword(s):

Skeletal Muscle ◽

Myosin Heavy Chain ◽

Heavy Chain ◽

Myogenic Differentiation ◽

Fiber Type ◽

Pcr Analysis ◽

Mdx Mouse ◽

Mdx Mice ◽

Muscle Physiology

Despite its undisputable role in the homeostatic regulation of the nervous system, the nerve growth factor (NGF) also governs the relevant cellular processes in other tissues and organs. In this study, we aimed at assessing the expression and the putative involvement of NGF signaling in skeletal muscle physiology. To reach this objective, we employed satellite cell-derived myoblasts as an in vitro culture model. In vivo experiments were performed on Tibialis anterior from wild-type mice and an mdx mouse model of Duchenne muscular dystrophy. Targets of interest were mainly assessed by means of morphological, Western blot and qRT-PCR analysis. The results show that proNGF is involved in myogenic differentiation. Importantly, the proNGF/p75NTR pathway orchestrates a slow-to-fast fiber type transition by counteracting the expression of slow myosin heavy chain and that of oxidative markers. Concurrently, proNGF/p75NTR activation facilitates the induction of fast myosin heavy chain and of fast/glycolytic markers. Furthermore, we also provided evidence that the oxidative metabolism is impaired in mdx mice, and that these alterations are paralleled by a prominent buildup of proNGF and p75NTR. These findings underline that the proNGF/p75NTR pathway may play a crucial role in fiber type determination and suggest its prospective modulation as an innovative therapeutic approach to counteract muscle disorders.

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In vitro and in vivo analysis of human fibroblast reprogramming and multipotency

Cellular & Molecular Biology Letters ◽

10.1515/cmble-2015-0024 ◽

2015 ◽

Vol 20 (3) ◽

Cited By ~ 1

Author(s):

Rongqing Pang ◽

Xiangqing Zhu ◽

Jia Geng ◽

Yongyun Zhang ◽

Qiang Wang ◽

...

Keyword(s):

Skeletal Muscle ◽

Human Fibroblasts ◽

Mdx Mouse ◽

Mdx Mice ◽

Therapeutic Effects ◽

Tail Vein ◽

Tail Vein Injection ◽

Activity Measurements ◽

Human Dystrophin

AbstractMultipotent stem cells have potential therapeutic roles in the treatment of Duchenne muscular dystrophy (DMD). However, the limited access to stem cell sources restricts their clinical application. To address this issue, we established a simple in vitro epigenetic reprogramming technique in which skin fibroblasts are induced to dedifferentiate into multipotent cells. In this study, human fibroblasts were isolated from circumcised adult foreskin and were reprogrammed by co-culture for 72 h with fish oocyte extract (FOE) in serum-free medium. The cells were then observed and analyzed by immunofluorescence staining, flow cytometry and in vitro differentiation assays. Then FOE-treated human fibroblasts were transplanted by tail vein injection into irradiated mdx mice, an animal model of DMD. Two months after injection, the therapeutic effects of FOE-treated fibroblasts on mdx skeletal muscle were evaluated by serum creatine kinase (CK) activity measurements and by immunostaining and RT-PCR of human dystrophin expression. The results indicated that the reprogrammed fibroblasts expressed higher levels of the pluripotent antigen markers SSEA-4, Nanog and Oct-4, and were able to differentiate in vitro into adipogenic cells, osteoblastic cells, and myotube-like cells. Tail vein injection of FOE-treated fibroblasts into irradiated mdx mice slightly reduced serum CK activity and the percentage of centrally nucleated myofibers two months after cell transplantation. Furthermore, we confirmed human dystrophin protein and mRNA expression in mdx mouse skeletal muscle. These data demonstrated that FOE-treated fibroblasts were multipotent and could integrate into mdx mouse myofibers through the vasculature.

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Clonal Isolation of Muscle-Derived Cells Capable of Enhancing Muscle Regeneration and Bone Healing

The Journal of Cell Biology ◽

10.1083/jcb.150.5.1085 ◽

2000 ◽

Vol 150 (5) ◽

pp. 1085-1100 ◽

Cited By ~ 449

Author(s):

Joon Yung Lee ◽

Zhuqing Qu-Petersen ◽

Baohong Cao ◽

Shigemi Kimura ◽

Ron Jankowski ◽

...

Keyword(s):

Skeletal Muscle ◽

Stem Cells ◽

Stem Cell ◽

Bone Healing ◽

Muscle Regeneration ◽

Mdx Mouse ◽

Mdx Mice ◽

Hematopoietic Stem ◽

Isolation And Characterization ◽

Osteogenic Lineage

Several recent studies suggest the isolation of stem cells in skeletal muscle, but the functional properties of these muscle-derived stem cells is still unclear. In the present study, we report the purification of muscle-derived stem cells from the mdx mouse, an animal model for Duchenne muscular dystrophy. We show that enrichment of desmin+ cells using the preplate technique from mouse primary muscle cell culture also enriches a cell population expressing CD34 and Bcl-2. The CD34+ cells and Bcl-2+ cells were found to reside within the basal lamina, where satellite cells are normally found. Clonal isolation and characterization from this CD34+Bcl-2+ enriched population yielded a putative muscle-derived stem cell, mc13, that is capable of differentiating into both myogenic and osteogenic lineage in vitro and in vivo. The mc13 cells are c-kit and CD45 negative and express: desmin, c-met and MNF, three markers expressed in early myogenic progenitors; Flk-1, a mouse homologue of KDR recently identified in humans as a key marker in hematopoietic cells with stem cell-like characteristics; and Sca-1, a marker for both skeletal muscle and hematopoietic stem cells. Intramuscular, and more importantly, intravenous injection of mc13 cells result in muscle regeneration and partial restoration of dystrophin in mdx mice. Transplantation of mc13 cells engineered to secrete osteogenic protein differentiate in osteogenic lineage and accelerate healing of a skull defect in SCID mice. Taken together, these results suggest the isolation of a population of muscle-derived stem cells capable of improving both muscle regeneration and bone healing.

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Extensive but Coordinated Reorganization of the Membrane Skeleton in Myofibers of Dystrophic (mdx) Mice

The Journal of Cell Biology ◽

10.1083/jcb.144.6.1259 ◽

1999 ◽

Vol 144 (6) ◽

pp. 1259-1270 ◽

Cited By ~ 65

Author(s):

McRae W. Williams ◽

Robert J. Bloch

Keyword(s):

Skeletal Muscle ◽

Membrane Proteins ◽

Muscle Fibers ◽

Membrane Skeleton ◽

Confocal Imaging ◽

Mdx Mouse ◽

Mdx Mice ◽

Intracellular Membrane ◽

Dystrophic Muscle ◽

Dihydropyridine Receptors

We used immunofluorescence techniques and confocal imaging to study the organization of the membrane skeleton of skeletal muscle fibers of mdx mice, which lack dystrophin. β-Spectrin is normally found at the sarcolemma in costameres, a rectilinear array of longitudinal strands and elements overlying Z and M lines. However, in the skeletal muscle of mdx mice, β-spectrin tends to be absent from the sarcolemma over M lines and the longitudinal strands may be disrupted or missing. Other proteins of the membrane and associated cytoskeleton, including syntrophin, β-dystroglycan, vinculin, and Na,K-ATPase are also concentrated in costameres, in control myofibers, and mdx muscle. They also distribute into the same altered sarcolemmal arrays that contain β-spectrin. Utrophin, which is expressed in mdx muscle, also codistributes with β-spectrin at the mutant sarcolemma. By contrast, the distribution of structural and intracellular membrane proteins, including α-actinin, the Ca-ATPase and dihydropyridine receptors, is not affected, even at sites close to the sarcolemma. Our results suggest that in myofibers of the mdx mouse, the membrane- associated cytoskeleton, but not the nearby myoplasm, undergoes widespread coordinated changes in organization. These changes may contribute to the fragility of the sarcolemma of dystrophic muscle.

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Long-term administration of antisense oligonucleotides into the paraspinal muscles of mdx mice reduces kyphosis

Journal of Applied Physiology ◽

10.1152/japplphysiol.00068.2008 ◽

2008 ◽

Vol 105 (2) ◽

pp. 662-668 ◽

Cited By ~ 8

Author(s):

Nicola Laws ◽

Renée A. Cornford-Nairn ◽

Nicole Irwin ◽

Russell Johnsen ◽

Susan Fletcher ◽

...

Keyword(s):

Skeletal Muscle ◽

Rna Splicing ◽

Antisense Oligonucleotides ◽

Latissimus Dorsi ◽

Stop Codon ◽

Premature Stop Codon ◽

Mdx Mouse ◽

Mdx Mice ◽

Paraspinal Muscles ◽

Reading Frame

The mdx mouse model of muscular dystrophy has a premature stop codon preventing production of dystrophin. This results in a progressive phenotype causing centronucleation of skeletal muscle fibers, muscle weakness, and fibrosis and kyphosis. Antisense oligonucleotides alter RNA splicing to exclude the nonsense mutation, while still maintaining the open reading frame to produce a shorter, but partially functional dystrophin protein that should ameliorate the extent of pathology. The present study investigated the benefits of chronic treatment of mdx mice by once-monthly deep intramuscular injections of antisense oligonucleotides into paraspinal muscles. After 8 mo of treatment, mdx mice had reduced development of kyphosis relative to untreated mdx mice, a benefit that was retained until completion of the study at 18 mo of age (16 mo of treatment). This was accompanied by reduced centronucleation in the latissimus dorsi and intercostals muscles and reduced fibrosis in the diaphragm and latissimus dorsi. These benefits were accompanied by a significant increase in dystrophin production. In conclusion, chronic antisense oligonucleotide treatment provides clear and ongoing benefits to paralumbar skeletal muscle, with associated marked reduction in kyphosis.

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Lmo7 is dispensable for skeletal muscle and cardiac function

AJP Cell Physiology ◽

10.1152/ajpcell.00177.2015 ◽

2015 ◽

Vol 309 (7) ◽

pp. C470-C479 ◽

Cited By ~ 6

Author(s):

Dieu Hung Lao ◽

Mary C. Esparza ◽

Shannon N. Bremner ◽

Indroneal Banerjee ◽

Jianlin Zhang ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscular Dystrophy ◽

Cardiac Function ◽

Cardiac Muscle ◽

Mapk Pathway ◽

Mdx Mouse ◽

Mdx Mice ◽

Mouse Line

Emery-Dreifuss muscular dystrophy (EDMD) is a degenerative disease primarily affecting skeletal muscles in early childhood as well as cardiac muscle at later stages. EDMD is caused by a number of mutations in genes encoding proteins associated with the nuclear envelope (e.g., Emerin, Lamin A/C, and Nesprin). Recently, a novel protein, Lim-domain only 7 ( lmo7) has been reported to play a role in the molecular pathogenesis of EDMD. Prior in vitro and in vivo studies suggested the intriguing possibility that Lmo7 plays a role in skeletal or cardiac muscle pathophysiology. To further understand the in vivo role of Lmo7 in striated muscles, we generated a novel Lmo7-null ( lmo7−/−) mouse line. Using this mouse line, we examined skeletal and cardiac muscle physiology, as well as the role of Lmo7 in a model of muscular dystrophy and regeneration using the dystrophin-deficient mdx mouse model. Our results demonstrated that lmo7−/− mice had no abnormalities in skeletal muscle morphology, physiological function, or regeneration. Cardiac function was also unaffected. Moreover, we found that ablation of lmo7 in mdx mice had no effect on the observed myopathy and muscular regeneration exhibited by mdx mice. Molecular analyses also showed no changes in dystrophin complex factors, MAPK pathway components, and Emerin levels in lmo7 knockout mice. Taken together, we conclude that Lmo7 is dispensable for skeletal muscle and cardiac physiology and pathophysiology.

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Diaphragm tension reduced in dystrophic mice by an oxidant, hypochlorous acid

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y09-117 ◽

2010 ◽

Vol 88 (2) ◽

pp. 130-140

Author(s):

Aude Lafoux ◽

Alexandra Divet ◽

Pascal Gervier ◽

Corinne Huchet-Cadiou

Keyword(s):

Skeletal Muscle ◽

Sarcoplasmic Reticulum ◽

Hypochlorous Acid ◽

Muscle Cells ◽

Skeletal Muscle Cells ◽

Diaphragm Muscle ◽

Mdx Mouse ◽

Mdx Mice ◽

Skinned Fibres ◽

Dystrophic Mice

In dystrophin-deficient skeletal muscle cells, in which Ca2+ homeostasis is disrupted and reactive oxygen species production is increased, we hypothesized that hypochlorous acid (HOCl), a strong H2O2-related free radical, damages contractile proteins and the sarcoplasmic reticulum. The aim of the present study was to investigate the effects of exposure to oxidative stress, generated by applying HOCl (100 µmol/L and 1 mmol/L), on the contractile function and sarcoplasmic reticulum properties of dystrophic mice. Experiments were performed on diaphragm muscle, which is severely affected in the mdx mouse, and the results were compared with those obtained in healthy (non-dystrophic) mice. In Triton-skinned fibres from C57BL/10 and mdx mice, 1 mmol/L HOCl increased myofibrillar Ca2+ sensitivity, but decreased maximal Ca2+-activated tension. In the presence of HOCl, higher concentrations of MgATP were required to produce rigor tensions. The interaction between HOCl and the Ca2+ uptake mechanisms was demonstrated using saponin-skinned fibres and sarcoplasmic reticulum vesicles. The results showed that HOCl, at micromolar or millimolar concentrations, can modify sarcoplasmic reticulum Ca2+ uptake and that this effect was more pronounced in diaphragm muscle from mdx mice. We conclude that in dystrophic diaphragm skeletal muscle cells, HOCl activates a cellular pathway that leads to an increase in the intracellular concentration of Ca2+.

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Involvement of TRPC in the abnormal calcium influx observed in dystrophic (mdx) mouse skeletal muscle fibers

The Journal of Cell Biology ◽

10.1083/jcb.200203091 ◽

2002 ◽

Vol 158 (6) ◽

pp. 1089-1096 ◽

Cited By ~ 245

Author(s):

Clarisse Vandebrouck ◽

Dominique Martin ◽

Monique Colson-Van Schoor ◽

Huguette Debaix ◽

Philippe Gailly

Keyword(s):

Skeletal Muscle ◽

Transient Receptor Potential ◽

Calcium Influx ◽

Surface Membrane ◽

Muscle Fibers ◽

Mdx Mouse ◽

Mdx Mice ◽

Patch Clamp Technique ◽

Adult Skeletal Muscle ◽

Skeletal Muscle Fibers

Duchenne muscular dystrophy results from the lack of dystrophin, a cytoskeletal protein associated with the inner surface membrane, in skeletal muscle. The absence of dystrophin induces an abnormal increase of sarcolemmal calcium influx through cationic channels in adult skeletal muscle fibers from dystrophic (mdx) mice. We observed that the activity of these channels was increased after depletion of the stores of calcium with thapsigargin or caffeine. By analogy with the situation observed in nonexcitable cells, we therefore hypothesized that these store-operated channels could belong to the transient receptor potential channel (TRPC) family. We measured the expression of TRPC isoforms in normal and mdx adult skeletal muscles fibers, and among the seven known isoforms, five were detected (TRPC1, 2, 3, 4, and 6) by RT-PCR. Western blot analysis and immunocytochemistry of normal and mdx muscle fibers demonstrated the localization of TRPC1, 4, and 6 proteins at the plasma membrane. Therefore, an antisense strategy was used to repress these TRPC isoforms. In parallel with the repression of the TRPCs, we observed that the occurrence of calcium leak channels was decreased to one tenth of its control value (patch-clamp technique), showing the involvement of TRPC in the abnormal calcium influx observed in dystrophic fibers.

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Dystrophin-associated proteins are greatly reduced in skeletal muscle from mdx mice.

The Journal of Cell Biology ◽

10.1083/jcb.115.6.1685 ◽

1991 ◽

Vol 115 (6) ◽

pp. 1685-1694 ◽

Cited By ~ 291

Author(s):

K Ohlendieck ◽

K P Campbell

Keyword(s):

Skeletal Muscle ◽

Immunoblot Analysis ◽

Protein Product ◽

Mdx Mouse ◽

Mdx Mice ◽

Normal Density ◽

Mouse Muscle ◽

Glycoprotein Complex ◽

Associated Proteins ◽

Dy Mouse

Dystrophin, the protein product of the human Duchenne muscular dystrophy gene, exists in skeletal muscle as a large oligomeric complex that contains four glycoproteins of 156, 50, 43, and 35 kD and a protein of 59 kD. Here, we investigated the relative abundance of each of the components of the dystrophin-glycoprotein complex in skeletal muscle from normal and mdx mice, which are missing dystrophin. Immunoblot analysis using total muscle membranes from control and mdx mice of ages 1 d to 30 wk found that all of the dystrophin-associated proteins were greatly reduced (80-90%) in mdx mouse skeletal muscle. The specificity of the loss of the dystrophin-associated glycoproteins was demonstrated by the finding that the major glycoprotein composition of skeletal muscle membranes from normal and mdx mice was identical. Furthermore, skeletal muscle membranes from the dystrophic dy/dy mouse exhibited a normal density of dystrophin and dystrophin-associated proteins. Immunofluorescence microscopy confirmed the results from the immunoblot analysis and showed a drastically reduced density of dystrophin-associated proteins in mdx muscle cryosections compared with normal and dy/dy mouse muscle. Therefore, our results demonstrate that all of the dystrophin-associated proteins are significantly reduced in mdx skeletal muscle and suggest that the loss of dystrophin-associated proteins is due to the absence of dystrophin and not due to secondary effects of muscle fiber degradation.

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Hindlimb immobilization applied to 21-day-oldmdx mice prevents the occurrence of muscle degeneration

Journal of Applied Physiology ◽

10.1152/jappl.1999.86.3.924 ◽

1999 ◽

Vol 86 (3) ◽

pp. 924-931 ◽

Cited By ~ 39

Author(s):

Asghar Mokhtarian ◽

Jean Pascal Lefaucheur ◽

Patrick C. Even ◽

Alain Sebille

Keyword(s):

Skeletal Muscle ◽

Motor Activity ◽

Skeletal Muscles ◽

Extensor Digitorum Longus ◽

Contralateral Side ◽

Mdx Mouse ◽

Mdx Mice ◽

Muscle Degeneration ◽

Limb Immobilization ◽

Old Mice

Dystrophin-deficient skeletal muscles of mdx mice undergo their first rounds of degeneration-regeneration at the age of 14–28 days. This feature is thought to result from an increase in motor activity at weaning. In this study, we hypothesize that if the muscle is prevented from contracting, it will avoid the degenerative changes that normally occur. For this purpose, we developed a procedure of mechanical hindlimb immobilization in 3-wk-old mice to restrain soleus (Sol) and extensor digitorum longus (EDL) muscles in the stretched or shortened position. After a 14-day period of immobilization, the striking feature was the low percentage of regenerated (centronucleated) myofibers in Sol and EDL muscles, regardless of the length at which they were fixed, compared with those on the contralateral side (stretched Sol: 8.4 ± 6.5 vs. 46.6 ± 10.3%, P = 0.0008; shortened Sol: 1.2 ± 1.6 vs. 50.4 ± 16.4%, P = 0.0008; stretched EDL: 05 ± 0.5 vs. 32.9 ± 17.5%, P = 0.002; shortened EDL: 3.3 ± 3.1 vs. 34.7 ± 11.1%, P = 0.002). Total numbers of myofibers did not change with immobilization. This study shows that limb immobilization prevents the occurrence of the first round of myofiber necrosis in mdx mice and suggests that muscle contractions play a role in the skeletal muscle degeneration of dystrophin-deficient mdx mouse muscles.

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