Nuclei Of Skeletal Muscle Fibers Research Articles

Skeletal Muscle Nuclei in Mice are not Post-mitotic.

The skeletal muscle research field generally accepts that nuclei in skeletal muscle fibers (ie, myonuclei) are post-mitotic and unable to proliferate. Because our deuterium oxide (D2O) labeling studies showed DNA synthesis in skeletal muscle tissue, we hypothesized that resident myonuclei can replicate in vivo. To test this hypothesis, we used a mouse model that temporally labeled myonuclei with GFP followed by D2O labeling during normal cage activity, functional overload, and with satellite cell ablation. During normal cage activity, we observed deuterium enrichment into myonuclear DNA in 7 out of 7 plantaris (PLA), 6 out of 6 tibialis anterior (TA), 5 out of 7 gastrocnemius (GAST), and 7 out of 7 quadriceps (QUAD). The average fractional synthesis rates (FSR) of DNA in myonuclei were: 0.0202±0.0093 in PLA, 0.0239±0.0040 in TA, 0.0076±0. 0058 in GAST, and 0.0138±0.0039 in QUAD, while there was no replication in myonuclei from EDL. These FSR values were largely reproduced in the overload and satellite cell ablation conditions, although there were higher synthesis rates in the overloaded PLA muscle. We further provided evidence that myonuclear replication is through endoreplication, which results in polyploidy. These novel findings contradict the dogma that skeletal muscle nuclei are post-mitotic and open potential avenues to harness the intrinsic replicative ability of myonuclei for muscle maintenance and growth.

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.

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.

Modeling Oculopharyngeal Muscular Dystrophy in Myotube Cultures Reveals Reduced Accumulation of Soluble Mutant PABPN1 Protein

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease caused by an alanine tract expansion mutation in poly(A) binding protein nuclear 1 (expPABPN1). To model OPMD in a myogenic and physiological context, we generated mouse myoblast cell clones stably expressing either human wild type (WT) or expPABPN1 at low levels. Transgene expression is induced on myotube differentiation and results in formation of insoluble nuclear PABPN1 aggregates that are similar to those observed in patients with OPMD. Quantitative analysis of PABPN1 in myotube cultures revealed that expPABPN1 accumulation and aggregation is greater than that of the WT protein. We found that aggregation of expPABPN1 is more affected than WT PABPN1 by inhibition of proteasome activity. Consistent with this, in myotube cultures expressing expPABPN1, deregulation of the proteasome was identified as the most significantly perturbed pathway. Differences in the accumulation of soluble WT and expPABPN1 were consistent with differences in ubiquitination and rate of protein turnover. This study demonstrates, for the first time to our knowledge, that, in myotubes, the ratio of soluble/insoluble expPABPN1 is significantly lower compared with that of the WT protein. We suggest that this difference can contribute to muscle weakness in OPMD.

Molecular and phenotypic characterization of a mouse model of oculopharyngeal muscular dystrophy reveals severe muscular atrophy restricted to fast glycolytic fibres

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by ptosis, dysphagia and proximal limb weakness. Autosomal-dominant OPMD is caused by a short (GCG)(8-13) expansions within the first exon of the poly(A)-binding protein nuclear 1 gene (PABPN1), leading to an expanded polyalanine tract in the mutated protein. Expanded PABPN1 forms insoluble aggregates in the nuclei of skeletal muscle fibres. In order to gain insight into the different physiological processes affected in OPMD muscles, we have used a transgenic mouse model of OPMD (A17.1) and performed transcriptomic studies combined with a detailed phenotypic characterization of this model at three time points. The transcriptomic analysis revealed a massive gene deregulation in the A17.1 mice, among which we identified a significant deregulation of pathways associated with muscle atrophy. Using a mathematical model for progression, we have identified that one-third of the progressive genes were also associated with muscle atrophy. Functional and histological analysis of the skeletal muscle of this mouse model confirmed a severe and progressive muscular atrophy associated with a reduction in muscle strength. Moreover, muscle atrophy in the A17.1 mice was restricted to fast glycolytic fibres, containing a large number of intranuclear inclusions (INIs). The soleus muscle and, in particular, oxidative fibres were spared, even though they contained INIs albeit to a lesser degree. These results demonstrate a fibre-type specificity of muscle atrophy in this OPMD model. This study improves our understanding of the biological pathways modified in OPMD to identify potential biomarkers and new therapeutic targets.

G.P.7.13 Gene expression profiling in a skeletal muscle cell model of oculopharyngeal muscular dystrophy reveals an extracellular matrix defect

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset, usually autosomal dominant myopathy. OPMD is caused by a small expansion of an alanine (Ala) tract at the N-terminus of the nuclear poly(A)-binding protein (PABPN1) and is clinically characterised by progressive ptosis, dysphagia and limb muscle weakness. The pathological hallmark is the accumulation of unique tubulofilamentous inclusions (INI) within the nuclei of skeletal muscle fibers. These inclusions contain PABPN1 and also sequester ubiquitin, proteasome subunits and heat shock proteins as well as poly(A) RNA.

Blood vessels and desmin control the positioning of nuclei in skeletal muscle fibers

Skeletal muscle fibers contain hundreds to thousands of nuclei which lie immediately under the plasmalemma and are spaced out along the fiber, except for a small cluster of specialized nuclei at the neuromuscular junction. How the nuclei attain their positions along the fiber is not understood. Here we show that the nuclei are preferentially localized near blood vessels (BV), particularly in slow-twitch, oxidative fibers. Thus, in rat soleus muscle fibers, 81% of the nuclei appear next to BV. Lack of desmin markedly perturbs the distribution of nuclei along the fibers but does not prevent their close association with BV. Consistent with a role for desmin in the spacing of nuclei, we show that denervation affects the organization of desmin filaments as well as the distribution of nuclei. During chronic stimulation of denervated muscles, new BV form, along which muscle nuclei align themselves. We conclude that the positioning of nuclei along muscle fibers is plastic and that BV and desmin intermediate filaments each play a distinct role in the control of this positioning.

Oculopharyngeal muscular dystrophy: Potential therapies for an aggregate-associated disorder

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset, autosomal dominant disease caused by the abnormal expansion of a polyalanine tract within the coding region of poly(A) binding protein nuclear 1 (PABPN1). The resultant mutant PABPN1 forms aggregates within the nuclei of skeletal muscle fibres. The mechanism by which the polyalanine expansion mutation in PABN1 causes disease is unclear. However, the mutation is thought to confer a toxic gain-of-function on the protein. Despite controversy over the role of aggregates, it has been consistently shown that agents that reduce aggregate load in cell models of OPMD also reduce levels of cell death. Recently generated animal models of OPMD will help elucidate the mechanism of disease and allow the trial of potential therapeutics. Indeed, administration of known anti-aggregation drugs attenuated muscle weakness in an OPMD mouse model. This suggests that anti-aggregation therapies may be beneficial in OPMD.

Trehalose reduces aggregate formation and delays pathology in a transgenic mouse model of oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease that presents in the fifth or sixth decade with dysphagia, ptosis and proximal limb weakness. OPMD is caused by the abnormal expansion of a polyalanine tract within the coding region of polyA binding protein nuclear 1 (PABPN1). The resultant mutant PABPN1 forms aggregates within the nuclei of skeletal muscle fibres. We have previously described a transgenic mouse model of OPMD that recapitulates the human disease and develops progressive muscle weakness accompanied by the formation of aggregates in skeletal muscle nuclei. The chemical chaperone trehalose has been used effectively to alleviate symptoms in a mouse model of Huntington's disease and is thought to elicit its effect by binding and stabilizing partially folded polyglutamine proteins and inhibiting the formation of aggregates. Here, we show that trehalose reduces aggregate formation and toxicity of mutant PABPN1 in cell models. Furthermore, oral administration of trehalose attenuated muscle weakness, reduced aggregate formation and decreased the number of TUNEL-labelled nuclei in skeletal muscle in an OPMD transgenic mouse model. Thus, anti-aggregation therapy may prove effective in the treatment of human OPMD.

Doxycycline attenuates and delays toxicity of the oculopharyngeal muscular dystrophy mutation in transgenic mice

The muscular dystrophies are a heterogeneous group of disorders for which there are currently no cures. Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant late-onset, progressive disease that generally presents in the fifth or sixth decade with dysphagia, ptosis and proximal limb weakness. OPMD is caused by the abnormal expansion of a (GCG)n trinucleotide repeat in the coding region of the poly-(A) binding protein nuclear 1 (PABPN1) gene. In unaffected individuals, (GCG)6 codes for the first six alanines in a homopolymeric stretch of ten alanines. In most individuals with OPMD this (GCG)6 repeat is expanded to (GCG)8-13, leading to a stretch of 12-17 alanines in mutant PABPN1. PABPN1 with an expanded polyalanine tract forms aggregates consisting of tubular filaments within the nuclei of skeletal muscle fibers. We have developed a transgenic mouse model of OPMD that manifests progressive muscle weakness accompanied by intranuclear aggregates and TUNEL-stained nuclei in skeletal muscle fibers. The onset and severity of these abnormalities were substantially delayed and attenuated by doxycycline treatment, which may exert its therapeutic effect by reducing aggregates and by distinct antiapoptotic properties. Doxycycline may represent a safe and feasible therapeutic for this disease.

Neuregulin-2 is synthesized by motor neurons and terminal Schwann cells and activates acetylcholine receptor transcription in muscle cells expressing ErbB4

Acetylcholine receptor ( AChR) genes are transcribed selectively in synaptic nuclei of skeletal muscle fibers, leading to accumulation of the mRNAs encoding AChR subunits at synaptic sites. The signals that regulate synapse-specific transcription remain elusive, though Neuregulin-1 is considered a favored candidate. Here, we show that motor neurons and terminal Schwann cells express neuregulin-2, a neuregulin-1-related gene. In skeletal muscle, Neuregulin-2 protein is concentrated at synaptic sites, where it accumulates adjacent to terminal Schwann cells. Neuregulin-2 stimulates AChR transcription in cultured myotubes expressing ErbB4, as well as ErbB3 and ErbB2, but not in myotubes expressing only ErbB3 and ErbB2. Thus, Neuregulin-2 is a candidate for a signal that regulates synaptic differentiation.

Oestrogen receptor beta is expressed in adult human skeletal muscle both at the mRNA and protein level.

There are two known oestrogen receptors (ER), oestrogen receptor alpha (ERalpha) and the recently cloned oestrogen receptor beta (ERbeta). ERalpha mRNA has been detected in mouse, rat, bovine and human skeletal muscle. ERbeta mRNA has been detected in bovine skeletal muscle. To our knowledge, no study has investigated the expression of oestrogen receptor beta in human skeletal muscle. Therefore, the primary aim of the present investigation was to study ERbeta mRNA and protein expression in human skeletal muscle. In addition the ERalpha expression was also studied. Muscle biopsies were taken from vastus lateralis in six healthy adults (three women and three men). mRNA expression was detected with real-time PCR (TaqMan) and protein localization by immunohistochemistry. A clear expression of ERalpha and ERbeta mRNA was seen in skeletal muscle in all subjects. The ERalpha mRNA expression was 180 fold higher compared with that of ERbeta mRNA. Immunohistochemistry demonstrated positive staining for ERbeta, but not for ERalpha, with localization to the nuclei of skeletal muscle fibres. On average, 70% of all nuclei were ERbeta-positive. The present study shows for the first time ERbeta mRNA and protein expression in human skeletal muscle tissue in both males and females.

The lipid phosphatase myotubularin is essential for skeletal muscle maintenance but not for myogenesis in mice.

Myotubularin is a ubiquitously expressed phosphatase that acts on phosphatidylinositol 3-monophosphate [PI(3)P], a lipid implicated in intracellular vesicle trafficking and autophagy. It is encoded by the MTM1 gene, which is mutated in X-linked myotubular myopathy (XLMTM), a muscular disorder characterized by generalized hypotonia and muscle weakness at birth leading to early death of most affected males. The disease was proposed to result from an arrest in myogenesis, as the skeletal muscle from patients contains hypotrophic fibers with centrally located nuclei that resemble fetal myotubes. To understand the physiopathological mechanism of XLMTM, we have generated mice lacking myotubularin by homologous recombination. These mice are viable, but their lifespan is severely reduced. They develop a generalized and progressive myopathy starting at around 4 weeks of age, with amyotrophy and accumulation of central nuclei in skeletal muscle fibers leading to death at 6-14 weeks. Contrary to expectations, we show that muscle differentiation in knockout mice occurs normally. We provide evidence that fibers with centralized myonuclei originate mainly from a structural maintenance defect affecting myotubularin-deficient muscle rather than a regenerative process. In addition, we demonstrate, through a conditional gene-targeting approach, that skeletal muscle is the primary target of murine XLMTM pathology. These mutant mice represent animal models for the human disease and will be a valuable tool for understanding the physiological role of myotubularin.

Muscle cells become necrotic rather than apoptotic during reperfusion of ischaemic skeletal muscle.

While necrosis is known as a major mechanism for the loss of viability of skeletal muscle following ischaemia and reperfusion, much less is known of the role of apoptosis. In this study rat hind limbs were subjected to 2 h of tourniquet ischaemia, then reperfused for either 0, 0.25, 0.5, 1, 3, 8, 16 or 24 h (n = 6 per group). Mean viability of muscle, assessed by tetrazolium dye reduction, after 2 h ischaemia and 24 h reperfusion was 17%. Histological examination revealed disrupted, necrotic muscle fibres from 30 min to 24 h reperfusion. Apoptotic nuclei were identified by haematoxylin staining and TUNEL, terminal deoxynucleotidyl transferase mediated dUTP nick end labelling. No TUNEL-positive cells were observed at the end of the ischaemic period, but a small number of TUNEL-positive endothelial and smooth muscle cells were found at 30 min reperfusion, with a progressive increase in their number up to 24 h reperfusion. Apoptotic neutrophils were detected after 8-24 h reperfusion. At no stage was apoptosis seen in the nuclei of skeletal muscle fibres. It appears that apoptosis plays no role in the death of muscle fibres after ischaemia-reperfusion injury to skeletal muscle.

Electron-microscopic investigation of satellite cell formation in skeletal muscle during physical exertion

The content of condensed chromatin is increased in nuclei of skeletal muscle fibers of rats after repeated physical exertion continued until exhaustion. Some muscle fiber nuclei, together with part of the sarcoplasm, were separated from the muscle fibers. Satellite cells formed from the separated parts of the muscle fibers.

INTRANUCLEAR SYNTHESIS OF POLYGLUCOSE PARTICLES UNDER HISTOCHEMICAL CONDITIONS IN THE NORMAL RAT

Polyglucose particles artificially synthesized under histochemical conditions from glucose 1-phosphate by phosphorylase and branching glycosyltransferase in the nuclei of skeletal muscle fibers, satellite cells and endothelial cells of the blood capillary of the normal rat were studied at the electronmicroscopic level.Newly formed polyglucose particles appeared in the karyolymph and interchromatin area of nuclei as a large macromolecular structure of spheroidal branching bodies. The size of intranuclear polyglucose particles ranging from 400 Å to 1600 Å in diameter was slightly larger than the cytoplasmic particles.There was no evidence that newly formed particles in the nuclei had a relation to the chromatin or the nuclear membrane.

Studies on isolated human skeletal muscle fibers: Including a proposed pattern of nuclear distribution and a concept of nuclear territories

Studies of human skeletal muscle fibers from tissue specimens isolated by supersonic cavitation, employing a Willens Polytron, show that the number of nuclei per millimeter, the surface area of fiber per nucleus, and the cytoplasmic volume per nucleus increase with fiber diameter, for fibers from the same muscle, from muscle to muscle, and from patient to patient. Skeletal muscle fibers from patients with Pompe's disease, muscular dystrophy (both autosomal and sex linked recessive juvenile forms), Riley-Day central autonomic dysfunction, and cardiomyopathy with skeletal muscle weakness show increased N/mm. and decreased A/N and V/N values, whereas fibers from patients with the Down syndrome show low N/mm. and high A/N and V/N values. Possibly abnormal fiber values were also observed in familial cerebellar atrophy with mental retardation, in 18 trisomy, Jansky-Bielschowsky disease, and ataxia-telangiectasia. Skeletal muscle fibers from autosomal recessive (“limb girdle”) muscular dystrophy show segmental, and those from Duchenne's dystrophy diffuse, hypernucleation. In Pompe's disease, acid mucopolysaccharide occurs in discrete “packages” in skeletal muscle, muscles of respiration showing less acid mucopolysaccharide than others. Evidence that the nuclei of skeletal muscle fibers are arranged in a pattern based on a hexagonal array, such that the fibers are covered by nuclear territories of relatively uniform size and shape, is presented, models of such architecture are presented, and possible biochemical implications of nuclear territories are discussed. Evidence that division of fiber nuclei is amitotic is presented. The concept that nuclear rows or chains in skeletal muscle fibers are nuclear clones is proposed, and possible biochemical implications of this arrangement are also discussed.