Fiber Nuclei Research Articles

Low-level laser therapy (LLLT) accelerates the sternomastoid muscle regeneration process after myonecrosis due to bupivacaine

BackgroundBecause of its long-lasting analgesic action, bupivacaine is an anesthetic used for peripheral nerve block and relief of postoperative pain. Muscle degeneration and neurotoxicity are its main limitations. There is strong evidence that low-level laser therapy (LLLT) assists in muscle and nerve repair. The authors evaluated the effects of a Gallium Arsenide laser (GaAs), on the regeneration of muscle fibers of the sternomastoid muscle and accessory nerve after injection of bupivacaine. MethodsIn total, 30 Wistar adult rats were divided into 2 groups: control group (C: n=15) and laser group (L: n=15). The groups were subdivided by antimere, with 0.5% bupivacaine injected on the right and 0.9% sodium chloride on the left. LLLT (GaAs 904nm, 0,05W, 2.8J per point) was administered for 5 consecutive days, starting 24h after injection of the solutions. Seven days after the trial period, blood samples were collected for determination of creatine kinase (CK). The sternomastoid nerve was removed for morphological and morphometric analyses; the surface portion of the sternomastoid muscle was used for histopathological and ultrastructural analyses. Muscle CK and TNFα protein levels were measured. ResultsThe anesthetic promoted myonecrosis and increased muscle CK without neurotoxic effects. The LLLT reduced myonecrosis, characterized by a decrease in muscle CK levels, inflammation, necrosis, and atrophy, as well as the number of central nuclei in the muscle fibers and the percentage of collagen. TNFα values remained constant. ConclusionsLLLT, at the dose used, reduced fibrosis and myonecrosis in the sternomastoid muscle triggered by bupivacaine, accelerating the muscle regeneration process.

Increased risk of coronary perforation during percutaneous intervention of myocardial bridge: What histopathology says.

Introduction: Myocardial bridge (MB) is a segment of a major epicardial coronary artery that goes intramurally under a bridge of overlying myocardium. Complications have been reported during or after stent implantation particularly coronary perforation. The aim of this study was to determine histological differences between proximal left anterior descending artery (LAD) and the tunneled segment that may have a possible role in increased risk of coronary artery perforation during percutaneous coronary intervention. Methods: Twenty specimens of MB were obtained from dissection of 45 cadavers. Sections were stained using hematoxylin and eosin (H&E), and trichrome methods. The proximal section and the tunneled artery were compared with a normal sample in terms of the characteristics of a muscle artery. Results: The findings of this study showed an MB prevalence of 51%, as 23 out of the 45 examined cadavers were discovered to be afflicted by the MB. The intima layer in the suffering artery had gone through significant hypertrophy, while it had remained thin in the tunneled artery section. The epithelial cells under the bridge were spindle-shaped, while they were polygonal in the proximal section. In the myocardium the nuclei of the muscle fibers in the MB section were smaller than the normal section. Adventitial layer was almost normal. Conclusion: The histopathological differences between MB and proximal part of vessel combined with small vessel diameter in the tunneled segment can explain the high incidence of the LAD rupture and perforation in the section under the bridge.

The Need for a Consensus on the Locution "Central Nuclei" in Striated Muscle Myopathies.

OPINION article Front. Physiol., 23 November 2016Sec. Striated Muscle Physiology Volume 7 - 2016 | https://doi.org/10.3389/fphys.2016.00577

Calcium homeostasis alterations in a mouse model of the Dynamin 2-related centronuclear myopathy.

ABSTRACTAutosomal dominant centronuclear myopathy (CNM) is a rare congenital myopathy characterized by centrally located nuclei in muscle fibers. CNM results from mutations in the gene encoding dynamin 2 (DNM2), a large GTPase involved in endocytosis, intracellular membrane trafficking, and cytoskeleton regulation. We developed a knock-in mouse model expressing the most frequent DNM2-CNM mutation; i.e. the KI-Dnm2R465W model. Heterozygous (HTZ) KI-Dnm2 mice progressively develop muscle atrophy, impairment of contractile properties, histopathological abnormalities, and elevated cytosolic calcium concentration. Here, we aim at better characterizing the calcium homeostasis impairment in extensor digitorum longus (EDL) and soleus muscles from adult HTZ KI-Dnm2 mice. We demonstrate abnormal contractile properties and cytosolic Ca2+ concentration in EDL but not soleus muscles showing that calcium impairment is correlated with muscle weakness and might be a determinant factor of the spatial muscle involvement. In addition, the elevated cytosolic Ca2+ concentration in EDL muscles is associated with an increased sarcolemmal permeability to Ca2+ and releasable Ca2+ content from the sarcoplasmic reticulum. However, amplitude and kinetics characteristics of the calcium transient appear unchanged. This suggests that calcium defect is probably not a primary cause of decreased force generation by compromised sarcomere shortening but may be involved in long-term deleterious consequences on muscle physiology. Our results highlight the first pathomechanism which may explain the spatial muscle involvement occurring in DNM2-related CNM and open the way toward development of a therapeutic approach to normalize calcium content.

Phenylbutazone induces expression of MBNL1 and suppresses formation of MBNL1-CUG RNA foci in a mouse model of myotonic dystrophy.

Myotonic dystrophy type 1 (DM1) is caused by abnormal expansion of CTG repeats in the 3′ untranslated region of the DMPK gene. Expanded CTG repeats are transcribed into RNA and make an aggregate with a splicing regulator, MBNL1, in the nucleus, which is called the nuclear foci. The nuclear foci sequestrates and downregulates availability of MBNL1. Symptomatic treatments are available for DM1, but no rational therapy is available. In this study, we found that a nonsteroidal anti-inflammatory drug (NSAID), phenylbutazone (PBZ), upregulated the expression of MBNL1 in C2C12 myoblasts as well as in the HSALR mouse model for DM1. In the DM1 mice model, PBZ ameliorated aberrant splicing of Clcn1, Nfix, and Rpn2. PBZ increased expression of skeletal muscle chloride channel, decreased abnormal central nuclei of muscle fibers, and improved wheel-running activity in HSALR mice. We found that the effect of PBZ was conferred by two distinct mechanisms. First, PBZ suppressed methylation of an enhancer region in Mbnl1 intron 1, and enhanced transcription of Mbnl1 mRNA. Second, PBZ attenuated binding of MBNL1 to abnormally expanded CUG repeats in cellulo and in vitro. Our studies suggest that PBZ is a potent therapeutic agent for DM1 that upregulates availability of MBNL1.

Molecular hydrogen alleviates motor deficits and muscle degeneration in mdx mice

Objective: Duchenne muscular dystrophy (DMD) is a devastating muscle disease caused by a mutation in DMD encoding dystrophin. Oxidative stress accounts for dystrophic muscle pathologies in DMD. We examined the effects of molecular hydrogen in mdx mice, a model animal for DMD.Methods: The pregnant mother started to take supersaturated hydrogen water (>5 ppm) ad libitum from E15.5 up to weaning of the offspring. The mdx mice took supersaturated hydrogen water from weaning until age 10 or 24 weeks when they were sacrificed.Results: Hydrogen water prevented abnormal body mass gain that is commonly observed in mdx mice. Hydrogen improved the spontaneous running distance that was estimated by a counter-equipped running-wheel, and extended the duration on the rota-rod. Plasma creatine kinase activities were decreased by hydrogen at ages 10 and 24 weeks. Hydrogen also decreased the number of central nuclei of muscle fibers at ages 10 and 24 weeks, and immunostaining for nitrotyrosine in gastrocnemius muscle at age 24 weeks. Additionally, hydrogen tended to increase protein expressions of antioxidant glutathione peroxidase 1, as well as anti-apoptotic Bcl-2, in skeletal muscle at age 10 weeks.Discussion: Although molecular mechanisms of the diverse effects of hydrogen remain to be elucidated, hydrogen potentially improves muscular dystrophy in DMD patients.

The zebrafish fast myosin light chain mylpfa:H2B-GFP transgene is a useful tool for in vivo imaging of myocyte fusion in the vertebrate embryo

BackgroundSkeletal muscle fibers are multinucleated syncytia that arise from the fusion of mononucleated precursors, the myocytes, during embryonic development, muscle hypertrophy in post-embryonic growth and muscle regeneration after injury. Even though myocyte fusion is central to skeletal muscle differentiation, our current knowledge of the molecular mechanism of myocyte fusion in the vertebrates is rather limited. Previous work, from our group and others, has shown that the zebrafish embryo is a very useful model for investigating the cell biology and genetics of vertebrate myocyte fusion in vivo. ResultsHere, we report the generation of a stable transgenic zebrafish strain that expresses the Histone 2B-GFP (H2B-GFP) fusion protein in the nuclei of all fast-twitch muscle fibers under the control of the fast-twitch muscle-specific myosin light chain, phosphorylatable, fast skeletal muscle a (mylpfa) gene promoter. By introducing this transgene into a mutant for junctional adhesion molecule 3b (jam3b), which encodes a cell adhesion protein previously implicated in myocyte fusion, we demonstrate the feasibility of using this transgene for the analysis of myocyte fusion during the differentiation of the trunk musculature of the zebrafish embryo. ConclusionsSince we know so little about the molecules regulating vertebrate myocyte fusion, we propose that the mylpfa:H2B-GFP transgene will be a very useful reporter for conducting forward and reverse genetic screens to identify new components regulating vertebrate myocyte fusion.

Activation of the wnt/β-Catenin Signaling Pathway in Polymyositis, Dermatomyositis and Duchenne Muscular Dystrophy.

Background and PurposeThe wnt/β-catenin signaling pathway plays a critical role in embryonic development and adult-tissue homeostasis. Recent investigations implicate the importance of wnt/β-catenin signaling in normal wound healing and its sustained activation being associated with fibrogenesis. We investigated the immunolocalization and activation of wnt/β-catenin in polymyositis (PM), dermatomyositis (DM), and Duchenne muscular dystrophy (DMD).MethodsImmunofluorescence staining and Western blot analysis of β-catenin were performed in muscle specimens from 6 PM, 8 DM, and 6 DMD subjects. The β-catenin/Tcf4 DNA-binding activity in muscle was studied using an electrophoretic mobility shift assay (EMSA), and serum wnt/β-catenin/Tcf transcriptional activity was measured using a luciferase reporter gene assay.ResultsImmunoreactivity for β-catenin was found in the cytoplasm and nuclei of muscle fibers in PM, DM, and DMD. The protein level of β-catenin was elevated, and EMSA analysis confirmed the activation of wnt/β-catenin signaling. The transcriptional activities of β-catenin/Tcf in the circulation were increased in patients with PM, DM, and DMD, especially in those with interstitial lung disease, and these transcriptional activities decreased when PM or DM patients exhibited obvious clinical improvements.ConclusionsOur findings indicate that wnt/β-catenin signaling is activated in PM, DM, and DMD. Its activation in muscle tissue and the circulation may play a role in modulating muscle regeneration and be at least partly involved in the process of muscle and pulmonary fibrosis.

Localized nuclear and perinuclear Ca(2+) signals in intact mouse skeletal muscle fibers.

Nuclear Ca2+ is important for the regulation of several nuclear processes such as gene expression. Localized Ca2+ signals (LCSs) in skeletal muscle fibers of mice have been mainly studied as Ca2+ release events from the sarcoplasmic reticulum. Their location with regard to cell nuclei has not been investigated. Our study is based on the hypothesis that LCSs associated with nuclei are present in skeletal muscle fibers of adult mice. Therefore, we carried out experiments addressing this question and we found novel Ca2+ signals associated with nuclei of skeletal muscle fibers (with possibly attached satellite cells). We measured localized nuclear and perinuclear Ca2+ signals (NLCSs and PLCSs) alongside cytosolic localized Ca2+ signals (CLCSs) during a hypertonic treatment. We also observed NLCSs under isotonic conditions. The NLCSs and PLCSs are Ca2+ signals in the range of micrometer [FWHM (full width at half maximum): 2.75 ± 0.27 μm (NLCSs) and 2.55 ± 0.17 μm (PLCSs), S.E.M.]. Additionally, global nuclear Ca2+ signals (NGCSs) were observed. To investigate which type of Ca2+ channels contribute to the Ca2+ signals associated with nuclei in skeletal muscle fibers, we performed measurements with the RyR blocker dantrolene, the DHPR blocker nifedipine or the IP3R blocker Xestospongin C. We observed Ca2+ signals associated with nuclei in the presence of each blocker. Nifedipine and dantrolene had an inhibitory effect on the fraction of fibers with PLCSs. The situation for the fraction of fibers with NLCSs is more complex indicating that RyR is less important for the generation of NLCSs compared to the generation of PLCSs. The fraction of fibers with NLCSs and PLCSs is not reduced in the presence of Xestospongin C. The localized perinuclear and intranuclear Ca2+ signals may be a powerful tool for the cell to regulate adaptive processes as gene expression. The intranuclear Ca2+ signals may be particularly interesting in this respect.

Effects of supplemental lysine and methionine with zilpaterol hydrochloride on feedlot performance, carcass merit, and skeletal muscle fiber characteristics in finishing feedlot cattle.

Feeding zilpaterol hydrochloride (ZH) with ruminally protected AA was evaluated in a small-pen feeding trial. Crossbred steers ( = 180; initial BW = 366 kg) were blocked by weight and then randomly assigned to treatments (45 pens; 9 pens/treatment). Treatment groups consisted of no ZH and no AA (Cont-), ZH and no AA (Cont+), ZH and a ruminally protected lysine supplement (Lys), ZH and a ruminally protected methionine supplement (Met), and ZH and ruminally protected lysine and methionine (Lys+Met). Zilpaterol hydrochloride (8.3 mg/kg DM) was fed for the last 20 d of the finishing period with a 3-d withdrawal period. Lysine and Met were top dressed daily for the 134-d feeding trial to provide 12 or 4 g·hd·d, respectively, to the small intestine. Carcass characteristics, striploins, and prerigor muscle samples were collected following harvest at a commercial facility. Steaks from each steer were aged for 7, 14, 21, and 28 d, and Warner-Bratzler shear force (WBSF) was determined as an indicator of tenderness. Prerigor muscle samples were used for immunohistological analysis. Cattle treated with Met and Lys+Met had increased final BW ( < 0.3) and ADG ( < 0.05) compared to Cont- and Cont+. Supplementation of Lys, Met, and Lys+Met improved G:F ( < 0.05) compared to Cont- during the ZH feeding period (d 111 to 134) as well as the entire feeding period ( < 0.05). Zilpaterol hydrochloride increased carcass ADG ( < 0.05) when compared to non-ZH-fed steers. Methionine and Lys+Met treatments had heavier HCW ( < 0.02) than that of Cont-. Yield grade was decreased ( < 0.04) for Cont+ steers compared to steers treated with Lys, Lys+Met, and Cont-. Tenderness was reduced ( < 0.05) with ZH regardless of AA supplementation. Lysine, Met, Lys+Met, and Cont+ had less tender steaks ( < 0.05) throughout all aging groups compared to Cont-. Steaks from Lys-treated steers were less tender ( < 0.05) than those of Cont+ during the 7- and 14-d aging periods. Nuclei density was the greatest with Cont- cattle compared to all other treatments suggesting a dilution effect of the nuclei in the larger muscle fibers with ZH feeding. Supplementation of Met in conjunction with ZH feeding increased ADG and HCW although this may lead to decreased tenderness even after aging for 28 d. These findings indicated that steers fed ZH may require additional AA absorbed from the small intestine to maximize performance.

615. Characterizing the Endogenous MTM1 Promoter

X-linked myotubular myopathy (XLMTM) is caused by a deficiency in the phosphatase myotubularin 1 (MTM1), and affects 1 in 50,000 males. Dysregulation of MTM1 expression and function directly affects the myocyte morphology as noted by centrally located nuclei of skeletal muscle fibers. Clinically, patients present with severe hypotonia, leading to respiratory failure by one year of age and those who survive have a high risk of developing hepatis peliosis in early adolescence. Currently patients with XLMTM receive palliative care, as there is no FDA approved treatment. Initial studies have already demonstrated great promise using adeno-associated (AAV)-based gene therapy strategies in treating MTM1 deficiency. However, toxicity in cardiac muscle after delivery of AAV-DESPro-Mtm1 has been observed in mice, possibly related to the level of Mtm1 expression. Here, we characterized a region of the endogenous MIM1 promoter (MTMPro) using a Luciferase Reporter Assay in vitro and in vivo, in order to regulate the expression of MTM1 associated with toxicity.A 1.3kb region of DNA upstream of the human MIM1 gene was analyzed and confirmed to contain promoter elements. The DNA sequence was examined using TFSEARCH and results indicate the presence of both ubiquitous and muscle-specific transcription factorbinding sites. MTMPro was cloned in front of a luciferase reporter gene and its activity was first verified in HEK293 cells, murine myoblasts, and HepG2 cells, in which we found expression of the two promoters to be cell type specific. C57BL/6 mice were administered by an intravenous injection of rAAV2/9 vectors containing DESPro-Luc and MTMPro-Luc. Bioluminescent scanning and tissue analysis was completed one month post-injection. Preliminary results indicate that MTMPro expresses at a higher level than DESPro in most tissues. The luciferase gene was then swapped out for a codon optimized human MIM1 transgene. These vectors were first administered locally to the Tibialis Anterior of wt mice. Myotubularin 1 was observed via western blot and shown to be functionally active from the rAAV2/9-DESPro-hMIM1 samples. Levels of hMTM1 were below detection in the rAAV2/9-MTMPro-hMTM1 samples. The cause of the discrepancy between the reporter and therapeutic vectors will be further investigated. We believe that systemically delivering a vector containing either the MTMPro or a lower dose of DESPro to drive MTM1 expression in an AAV9 vector will result in therapeutic levels of gene expression without causing cellular toxicity in a mouse model of XLMTM.

Expression of the myostatin gene in the adductor muscle of the Pacific lion-paw scallop Nodipecten subnodosus in association with growth and environmental conditions.

The cDNA sequence of the myostatin gene in the Pacific lion-paw Nodipecten subnodosus (Ns-mstn) was characterized, and the temporal expression during grow-out was analyzed for the first time in a scallop. Ns-mstn encodes a 459-amino-acid protein in which two propeptide proteolytic sites were identified, the previously recognized (RSKR) and a second one at position 266-269 aa (RRKR). The alternative furin cleavage site could be related with post-translational processing, or it could be a tissue-specific mechanism for signaling activity. The Ns-mstn transcript was located by in situ hybridization in sarcomeres and around the nucleus of muscle fibers. The temporal expression analysis by qPCR in the adductor muscle showed that Ns-mstn expression was significantly different (P < 0.05) between months during the grow-out period, increasing largely during the summer months when both biomass and muscle weight did not increase or even decreased; muscle fiber size and number were found to decrease significantly. Exogenous and endogenous factors such as high temperature and low food availability, as well as gametogenesis and reproduction, can be associated with the growth pattern and Ns-mstn expression changes. Our results indicate that MSTN is involved in adductor muscle growth regulation in N. subnodosus as it occurs in vertebrate skeletal muscle although Ns-mstn expression in non-muscle organs/tissues suggests additional functions.

Exercise Intolerance and Myoglobinuria Associated with a Novel Maternally Inherited MT-ND1 Mutation.

The most common clinical phenotype caused by a mtDNA mutation in complex I of the mitochondrial respiratory chain is Leber hereditary optic neuropathy. We report a family with a novel maternally inherited homoplasmic mtDNA m.4087A>G mutation in the ND1 gene (MT-ND1) associated with isolated myopathy, recurrent episodes of myoglobinuria, and rhabdomyolysis. DNA from blood in seven family members and muscle from four family members were PCR amplified and sequenced directly and assessed for the m.4087A>G variation in MT-ND1. Mitochondrial enzyme activity in all muscle biopsies was measured. PCR and direct sequencing of the MT-ND1 genes from blood showed that all seven family members were homoplasmic for the m.4087A>G mutation (NC_012920.1:c.781A>G). The mutation predicts a threonine to alanine substitution at position 261 (p.T261A). The same mutation was found in muscle of all four family members available for muscle biopsy, and biochemical analyses revealed an isolated complex I defect in muscle of all family members (range 22-52% of normal). Muscle morphology showed severe myopathic changes with internal nuclei in multiple fibers of all family members. Monosymptomatic myopathy with recurrent myoglobinuria is a rare phenotype of mitochondrial myopathies. We report this phenotype in a family affected by a novel homoplasmic mutation in MT-ND1. It is the first time such a phenotype has been associated with complex I gene mutations and a homoplasmic mutation of mtDNA.

Lack of the serum- and glucocorticoid-inducible kinase SGK1 improves muscle force characteristics and attenuates fibrosis in dystrophic mdx mouse muscle.

Duchenne muscular dystrophy (DMD) is a human genetic disease characterized by fibrosis and severe muscle weakness. Currently, there is no effective treatment available to prevent progressive fibrosis in skeletal muscles. The serum- and glucocorticoid-inducible kinase SGK1 regulates a variety of physiological functions and participates in fibrosis stimulation. Here, we investigated whether SGK1 influences structure, function and/or fibrosis of the muscles from the mdx mouse, an animal model for DMD. As expected, mdx muscles showed the typical pathological features of muscular dystrophy including fiber size variations, central nuclei of muscle fibers, fibrosis in the diaphragm, and force reduction by 30-50%. Muscles from sgk1 (-/-) mice were histologically overall intact and specific force was only slightly reduced compared to wild-type muscles. Surprisingly, soleus and diaphragm muscles of mdx/sgk1 (-/-) mice displayed forces close to wild-type levels. Most muscle fibers of the double mutants contained central nuclei, but fibrosis was not observed in any of the tested limb and diaphragm muscles. We conclude that the sole lack of SGK1 in mouse muscle does not lead to pronounced changes in muscle structure and function. However, dystrophin-deficient mdx muscle seems to benefit from SGK1 deficiency. SGK1 appears to be an important enzyme in the process of fibrotic remodeling and subsequent weakness of dystrophin-deficient mouse muscle.

An image processing approach to analyze morphological features of microscopic images of muscle fibers

We present an image processing approach to automatically analyze duo-channel microscopic images of muscular fiber nuclei and cytoplasm. Nuclei and cytoplasm play a critical role in determining the health and functioning of muscular fibers as changes of nuclei and cytoplasm manifest in many diseases such as muscular dystrophy and hypertrophy. Quantitative evaluation of muscle fiber nuclei and cytoplasm thus is of great importance to researchers in musculoskeletal studies. The proposed computational approach consists of steps of image processing to segment and delineate cytoplasm and identify nuclei in two-channel images. Morphological operations like skeletonization is applied to extract the length of cytoplasm for quantification. We tested the approach on real images and found that it can achieve high accuracy, objectivity, and robustness.

Isolation, culture, and transplantation of muscle satellite cells.

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors. However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors. However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

Quantitative Analysis of Tendon Structural Integrity in Chickens using Fourier Transform-Second Harmonic Generation Imaging

Degenerative changes in tendons are common in human and land-dwelling vertebrates of all species, and are associated with both age and use. Tendon rupture and inflammation are common causes of lameness in broilers and breeders at multiple ages and has become a very important issue in the poultry industry attempts to analyze tendon health have relied primarily on visual assessment. The ability to conduct meaningful experiments on causes and interventions would be much improved by a direct assay of tendon structural integrity. The objective of this study is to test whether we can use Fourier transform-second harmonic generation (FT-SHG) imaging technique to quantify the differences in collagen fiber organization and nuclei morphology between normal and injured chicken tendons. Tendon injury/degeneration was induced by rearing chickens in wire-floor pens and feeding them an oxidized fat diet. Injured tendons had greater collagen fiber degradation/degeneration; more randomly organized collagen fibers, higher cellularity and more circular-shaped nuclei than normal tendons, indicating loss of collagen tertiary structure and lateral compression in injured tendons. Therefore, analysis of collagen fiber organization and nuclear morphology using FT-SHG imaging is a potential diagnostic tool to evaluate tendon structural health and to test the effects of nutrients and intervention strategies on the structural development of tendons in chickens.

Myotonia in DNM2-related centronuclear myopathy

Centronuclear myopathy (CNM) is a rare hereditary myopathy characterized by centrally located muscle fiber nuclei. Mutations in the dynamin 2 (DNM2) gene are estimated to account for about 50% of CNM cases. Electromyographic recordings in CNM may show myopathic motor unit potentials without spontaneous activity at rest. Myotonic discharges, a distinctive electrical activity caused by membrane hyperexcitability, are characteristic of certain neuromuscular disorders. Such activity has been reported in only one CNM case without a known genetic cause. We sequenced the DNM2 gene and the genes associated with myotonia (CLCN1, SCN4A, DMPK and ZNF9) in a sporadic adult patient with CNM and myotonic discharges. Sequencing the entire coding region and exon-intron boundaries revealed a heterozygous c.1106g-a substitution in exon 8, resulting in a R369Q change in the DNM2. Sequencing the CLCN1, SCN4A, DMPK and ZNF9 genes ruled out mutations in these genes. This is the first report of DNM2-related CNM presenting with myotonia. The diagnosis of CNM should be considered in patients with myotonic discharges of an unknown cause.

P.4.8 X-linked benign form of myotubular myopathy (XLMTM) with necklace fibres: A case report

X-linked myotubular myopathy (XLMTM) is usually a severe condition caused by mutations in myotubularin gen MTM1, (Xq28), a lipid inositide phosphatase acting in remodeling of membranes. Most of the affected patients present a severe neonatal form with hypotonia, weakness, facial diplegia, difficulties in sucking in addition to other symptoms dying within the first months. The biopsy features are consistent with the presence of central nuclei in most fibres. However, a more benign phenotype with a distinctive hallmark has recently been described. Herein, we present a four year old boy affected by this benign form. Patient was born by urgent caesarean section due to foetal bradycardia. He weighted 3.030 g. Presented axial hypotonic, scarce movements and respiratory distress, needing CPAP for few days. No tube feeding was required. Motor development was delayed. He had always difficulties in getting up from the floor. At 4 years he showed, facial weakness, weak cry, axial and head flexor muscles weakess. Cough was ineffective but no hypercapnia was detected. CK was normal. EMG was myopathic. Muscle biopsy showed two population of fibres. Most of the smaller one, revealed a “necklace” appearance. Genetic studies confirmed c.605T > C in exon 8 leading to p.I202S mutation in MTM1 gene. Necklace fibres constitute a hallmark for suspect diagnosis of this benign phenotype of XLMTM associated with mutation in the MTM1 gene. X-linked myotubular myopathy (XLMTM) is usually a severe condition caused by mutations in myotubularin gen MTM1, (Xq28), a lipid inositide phosphatase acting in remodeling of membranes. Most of the affected patients present a severe neonatal form with hypotonia, weakness, facial diplegia, difficulties in sucking in addition to other symptoms dying within the first months. The biopsy features are consistent with the presence of central nuclei in most fibres. However, a more benign phenotype with a distinctive hallmark has recently been described. Herein, we present a four year old boy affected by this benign form. Patient was born by urgent caesarean section due to foetal bradycardia. He weighted 3.030 g. Presented axial hypotonic, scarce movements and respiratory distress, needing CPAP for few days. No tube feeding was required. Motor development was delayed. He had always difficulties in getting up from the floor. At 4 years he showed, facial weakness, weak cry, axial and head flexor muscles weakess. Cough was ineffective but no hypercapnia was detected. CK was normal. EMG was myopathic. Muscle biopsy showed two population of fibres. Most of the smaller one, revealed a “necklace” appearance. Genetic studies confirmed c.605T > C in exon 8 leading to p.I202S mutation in MTM1 gene. Necklace fibres constitute a hallmark for suspect diagnosis of this benign phenotype of XLMTM associated with mutation in the MTM1 gene.