Muscle Nuclei Research Articles

Agrin regulates CLASP2-mediated capture of microtubules at the neuromuscular junction synaptic membrane

Agrin is the major factor mediating the neuronal regulation of postsynaptic structures at the vertebrate neuromuscular junction, but the details of how it orchestrates this unique three-dimensional structure remain unknown. Here, we show that agrin induces the formation of the dense network of microtubules in the subsynaptic cytoplasm and that this, in turn, regulates acetylcholine receptor insertion into the postsynaptic membrane. Agrin acted in part by locally activating phosphatidylinositol 3-kinase and inactivating GSK3β, which led to the local capturing of dynamic microtubules at agrin-induced acetylcholine receptor (AChR) clusters, mediated to a large extent by the microtubule plus-end tracking proteins CLASP2 and CLIP-170. Indeed, in the absence of CLASP2, microtubule plus ends at the subsynaptic muscle membrane, the density of synaptic AChRs, the size of AChR clusters, and the numbers of subsynaptic muscle nuclei with their selective gene expression programs were all reduced. Thus, the cascade linking agrin to CLASP2-mediated microtubule capturing at the synaptic membrane is essential for the maintenance of a normal neuromuscular phenotype.

Fractal Analysis and Biophysical Investigation of Muscular Tissue Damaged due to Low Temperature: A Pilot Study

Severe damage is produced in tissues by freezing and thawing. Until now, a great majority of the studies are performed qualitatively, lacking any quantitative approach. An important step is to choose the best option among different freezing methods. To approach the complex problem of damage produced in tissues by freezing, in this paper we present the classical mechanics approach and a quantitative study making use of a fractal methodology (evaluation of fractal dimension by box-counting method). A comparative fractal analysis between two different steps of freezing the human thoracic diaphragm muscle has been performed to quantify the voids and cracks produced by freezing (samples were placed in a cryostat chamber). Moreover, a standard Euclidean morphometry was performed to determine area and shape of the muscle nuclei after the two steps of freezing. Fractal dimension of the ice-tissue interface structures increased with decreasing temperature (p<0.0001), percentage of cell muscle decreased (p<0.01), while standard morphometry of the nuclei didnt show any modifications. Our results show the ability of the fractal approach to accurately quantify the damage produced by freezing and reveals that the lowest temperature produces the most damage.

A rabbit model for studying degeneration and regeneration properties of young striated muscle at different distraction rates

The present study evaluated the histological changes in the muscle tissue after limb lengthening in skeletally immature rabbits and assessed the effect of different lengthening rates on the regeneration and degeneration properties of striated muscle. Thirteen different lengthening protocols were applied on a total of 16 male domestic white rabbits divided into four groups. The histopathological changes were analysed by a semiquantitative method according to the scoring system of Lee et al. (1993). After evaluation of the five main degenerative parameters (muscle atrophy, internalisation of muscle nuclei, degeneration of the muscle fibre, perimysial and endomysial fibrosis, haematomas), it is evident that rabbits subjected to limb lengthening at a rate of 3.2 mm/day showed more degenerative changes than those limb-lengthened at 0.8 or 1.6 mm/day. Our study showed that the regenerative mechanisms were not endless. If the daily lengthening rate reached the 3.2 mm/day limit, the regenerating ability of the muscle decreased, and signs of degeneration increased significantly.

In ovotemperature manipulation differentially influences limb musculoskeletal development in two lines of chick embryos selected for divergent growth rates

Selective breeding has led to diverging phenotypic evolution in layer and broiler chickens through genomic and epigenetic modifications. Here we show that in ovo environmental manipulation differentially influences embryonic limb muscle phenotype in these two breeds. We demonstrate that raising incubation temperature from 37.5 to 38.5°C between embryonic days (ED) 4 and 7 increased motility and body mass in both layer and broiler embryos. In layers, this was accompanied by gastrocnemius muscle hypertrophy, increased fibre and nuclei numbers and a higher nuclei to fibre ratio (ED18), preceded by increased hindlimb Myf5 (ED5-8), Pax7 (ED5-10), BMP4 (ED6-9) and IGF-I (ED9-10, ED18) mRNAs. In broilers, the same temperature treatment led to reduced gastrocnemius cross-sectional area with fewer fibres and nuclei and an unchanged fibre to nuclei ratio (ED18). This was preceded by a delay in the peak of hindlimb Myf5 expression, increased Pax7 (ED5, ED7-10) and BMP4 (ED6-8) but reduced IGF-I (ED8-10) mRNAs. Rather than promoting myogenesis as in layer embryos, the temperature treatment promoted gastrocnemius intramuscular fat deposition in broilers (ED18) preceded by increased hindlimb PPARγ mRNA (ED7-10). The treatment increased tibia/tarsus bone length as well as femur cross-sectional area in both breeds, but femur length and bone to cartilage ratio in the femur and tibia/tarsus were only increased in treated layers (ED18). We conclude that in ovo temperature manipulation differentially affected the molecular regulation of hindlimb myogenic, adipogenic and growth factor expression in broiler and layer embryos, leading to differential changes in muscle phenotype. The underlying interactive mechanisms between genes and the environment need further investigation.

Examine the Eyes

In their article the authors report a common syndrome of therapy resistant, migraine-like abdominal pain in childhood, whose causes remain vastly unidentified and whose therapeutic success is mostly sought in a psychosomatic approach. The frequent involvement of the eyes in this syndrome is not mentioned, even though a direct circuit exists between the areas of the nuclei of the ocular muscles, the vestibular organs, the cerebellum, and the vegetative centers in the diencephalon, from which biohistorically the eyes developed. If the cooperation between the eyes is disrupted, diplopia results with substantial asthenopic symptoms, headache, and abdominal pain. Correcting visual impairment by correcting the visual defects such as hyperopia, myopia, astigmatism, especially the associated heterophoria (commonly known as “angle” visual defect), by using prismatic spectacles according to the measuring and correcting method after Haase in the Polatest may help. The symptoms mentioned above disappear pretty much immediately, as I have been able to observe in many hundreds of casesorganic findings, it is worth having the eyes examined by a specialist. Further information at www.ivbv.org

Troponin T Nuclear Localization and its Role in Aging Skeletal Muscle

Troponin T (TnT) is known to mediate the interaction between Tn complex and tropomyosin (Tm), which is essential for calcium-activated striated muscle contraction. This regulatory function takes place in the myoplasm, where TnT binds Tm. However, recent findings of Troponin I and Tm nuclear translocation in Drosophila and mammalian cells imply other roles for the Tn-Tm complex. We hypothesized that TnT plays a nonclassical role through nuclear translocation. By immunoblotting with two different antibodies that target the NH2- or COOH-terminal region, respectively, we show that fast skeletal muscle TnT3 is localized in the muscle nucleus in addition to the cytoplasm as either an intact or fragmented protein. By transient overexpressing DsRed fusion proteins of different TnT3 fragments in C2C12 skeletal muscle cells, full-length or the COOH-terminal fragment are predominantly localized in the nucleus, closely related to nucleolus. In contrast, TnT3 middle and NH2-terminal fragments are predominantly localized in the cytoplasm, suggesting divergent functions. RNA polymerase inhibition redistributed TnT3 and its COOH-terminus from the nucleolus, implying a role for TnT3 in rRNA processing. More importantly, the full-length TnT3 protein is less abundant in myonuclei from old (24 months) vs young (4 months) mice while the COOH-terminal fragment is more abundant in older mice. We also found that apoptosis markers increased in cells expressing the fluorescence-tagged full length or COOH-terminal (CT) TnT3 proteins, in contrast to those expressing the middle and NH2-terminal fragments. We propose that TnT3 plays an important role in aging muscle, by contributing to decreased myonuclear numbers, thus mediating age-related muscle damage and disease.

Biophysical Properties of a Novel Cationic Channel in the Outer and Inner Membranes of Nuclei from Adult Skeletal Muscle Fibers

In spite of some limited progress, the physiological role of cationic ion channels within the nuclear envelope remains largely unknown. Since biophysically distinct channels have been reported within the nuclear envelope of different cells, it is likely that nuclear membrane ion homeostasis is cell-type specific. Here we provide the first identification and characterization of the biophysical properties of the most prominently observed ion channel expressed within the inner and outer membrane of nuclei from adult skeletal muscle fibers. Excised inside-out single channel recordings were obtained from individual nuclei acutely isolated from Flexor Digitorum Brevis (FDB) fibers of wild-type mice. The outer membrane of nuclei was readily accessible following isolation. For measurements of channels from the inner membrane, nuclei were treated with 1 % (w/v) sodium citrate in order to remove the outer membrane. We found that the predominant ion channel expressed in both the inner and outer nuclear membrane was a cationic channel that conducts monovalent ions with slight preference for potassium over sodium ions (a PK/PNa ∼1.22). A 10,000-fold difference in the concentration of free Ca2+ between the pipette and bath solutions did not affect the channel reversal potential in symmetric KCl (∼0 mV), indicating that Ca2+ ions permeation is negligible. The maximum conductance of the channel in the outward direction was ∼162 pS. The mean open probability (PO) was ∼0.7 and voltage-independent between −50 mV to +50 mV. We suggest that this novel monovalent cationic channel within the inner and outer membrane of skeletal muscle nuclei provides a counter-current mechanism that minimizes voltage change across the nuclear membrane. This research was supported by NIH K01 award AR060831(to V.Y.) and NIH R01 grant AR44657 (to R.T.D).

Effect ofFerula hermonisroot extract on rat skeletal muscle adaptation to exercise

Ferula hermonis Boiss. is an aphrodisiac plant that grows in the Mediterranean region. It has been reported that treatment with acetonic extract from the root of this plant acutely increases serum testosterone in the rat. This study investigated the effects of F. hermonis extract alone or combined with exercise on rat skeletal muscle fibers. Adult male rats were divided into four groups: control-sedentary (CS) that had no treatment or exercise; ferula-sedentary (FS) that was orally treated with ferula extract at a dose of 60 mg/kg/rat every other day over a period of 20 d; control-exercised (CE) that was trained by swimming for 40 min every other day; and ferula-exercised (FE) that received ferula and performed exercise. At the end of experiments, the fiber diameter and number of muscle nuclei of tibialis anterior were measured by using immunofluorescent techniques and software analyses. The FE group showed significant increases in muscle weight, fiber size and nuclear number compared with the other groups. However, no significant changes in the aforementioned parameters were found among the CS, FS and CE groups. Ferula treatment and exercise were additive to each other. In conclusion, short-term exercise combined with administration of F. hermonis extract was more effective in enhancing the growth of skeletal muscle fibers than exercise alone.

Lysosomal action of intracrine angiotensin II. Focus on “Intracellular angiotensin II activates rat myometrium”

forty years ago, Robertson and Khairallah ([20][1]) demonstrated that tritiated angiotensin II (ANG II) rapidly localized in myocyte nuclei and mitochondria after intracardiac injection. This finding was confirmed and expanded upon over the subsequent decades with the result that the notion of

Endoplasmic reticulum stress response in P104L mutant caveolin-3 transgenic mice

Mutations in the caveolin-3 gene cause autosomal dominant limb-girdle muscular dystrophy 1C (LGMD1C). However, the precise molecular pathogenesis of caveolin-3-related muscular dystrophy remains uncertain. Here, we demonstrate the effect of gene dosage on the severity of the myopathic phenotype in P104L mutant caveolin-3 (mCav3(P104L)) transgenic mice, a model of LGMD1C. We analyzed the endoplasmic reticulum (ER) stress response in the transgenic mice and found upregulated transcription of the molecular chaperone, glucose-regulated protein (GRP78). Moreover, signaling downstream of GRP78 in the myofibers was activated toward apoptosis. However, terminal transferase dUTP nick end labeling assays detected a few apoptotic nuclei in transgenic mouse skeletal muscle, probably due to the transcriptional activation of Dad1, an anti-apoptotic factor in the ER. These findings suggest that the ER stress response caused by mCav3(P104L) plays a role in the pathogenesis of LGMD1C as a toxic gain of function effect.

IMPROVING METHODOLOGICAL STRATEGIES FOR SATELLITE CELLS COUNTING IN HUMAN MUSCLE DURING AGEING

Stereological methods, based on the optical disector principle and fluorescent staining, were developed for estimating frequency of satellite cells in skeletal muscles. The parameter NL(sc, fib) (number of satellite cells per fibre length) was compared with the parameter NN(sc, nucl) (the percentage of satellite cell nuclei in all muscle nuclei), most often published in the literature, by applying unbiased sampling and counting procedures and using a confocal microscope. The methods were tested in autopsy samples of four young vs. four old human vastus lateralis muscles. Both parameters NL(sc, fib) and NN(sc, nucl) declined during ageing. However, it appears that the two parameters cannot be substituted one by the other because the number of nuclei per fibre length tends to be increased during aging. Using the introduced methods, it is more straightforward to estimate NL(sc, fib) than NN(sc, nucl).

Aortic stiffness is associated with vascular calcification and remodeling in a chronic kidney disease rat model

Increased aortic pulse-wave velocity (PWV) reflects increased arterial stiffness and is a strong predictor of cardiovascular risk in chronic kidney disease (CKD). We examined functional and structural correlations among PWV, aortic calcification, and vascular remodeling in a rodent model of CKD, the Lewis polycystic kidney (LPK) rat. Hemodynamic parameters and beat-to-beat aortic PWV were recorded in urethane-anesthetized animals [12-wk-old hypertensive female LPK rats (n = 5)] before the onset of end-stage renal disease and their age- and sex-matched normotensive controls (Lewis, n = 6). Animals were euthanized, and the aorta was collected to measure calcium content by atomic absorption spectrophotometry. A separate cohort of animals (n = 5/group) were anesthetized with pentobarbitone sodium and pressure perfused with formalin, and the aorta was collected for histomorphometry, which allowed calculation of aortic wall thickness, medial cross-sectional area (MCSA), elastic modulus (EM), and wall stress (WS), size and density of smooth muscle nuclei, and relative content of lamellae, interlamellae elastin, and collagen. Mean arterial pressure (MAP) and PWV were significantly greater in the LPK compared with Lewis (72 and 33%, respectively) animals. The LPK group had 6.8-fold greater aortic calcification, 38% greater aortic MCSA, 56% greater EM/WS, 13% greater aortic wall thickness, 21% smaller smooth muscle cell area, and 20% less elastin density with no difference in collagen fiber density. These findings demonstrate vascular remodeling and increased calcification with a functional increase in PWV and therefore aortic stiffness in hypertensive LPK rats.

RNA processing is altered in skeletal muscle nuclei of patients affected by myotonic dystrophy

Myotonic dystrophies (DMs) are characterised by highly variable clinical manifestations consisting of muscle weakness and atrophy, and a wide spectrum of extramuscular manifestations. In both DM1 and DM2 forms, expanded nucleotide sequences cause the accumulation of mutant transcripts in the nucleus, thus deregulating the function of some RNA-binding proteins and providing a plausible explanation for the multifactorial phenotype of DM patients. However, at the skeletal muscle level, no mechanistic explanation for the muscle wasting has so far been proposed. We therefore performed a study in situ by immunoelectron microscopy on biceps brachii biopsies from DM1, DM2 and healthy subjects, providing the first ultrastructural evidence on the distribution of some nuclear ribonucleoprotein (RNP)-containing structures and molecular factors involved in pre-mRNA transcription and maturation in dystrophic myonuclei. Our results demonstrated an accumulation of splicing and cleavage factors in myonuclei of both DM1 and DM2 patients, suggesting an impairment of post-transcriptional pre-mRNA pathways. The transcription of the expanded sequences in DM myonuclei would therefore hamper functionality of the whole splicing machinery, slowing down the intranuclear molecular trafficking; this would reduce the capability of myonuclei to respond to anabolic stimuli thus contributing to muscle wasting.

P25 Double-blind, placebo-controlled, parallel group, phase III study comparing dichlorphenamide vs. placebo for the treatment of periodic paralysis (HYP HOP trial)

the AChR subunits alter the channel kinetics by prolonging the burst duration, thereby causing cationic overload and leading to endplate myopathy. In order to test new therapies and analyse the mechanisms of existing therapies for slow channel syndrome, we generated a transgenic mouse expressing the slow channel mutation L221F in the human e-subunit of the AChR. EGFP was inserted into the large cytoplasmic loop, located between the third and fourth transmembrane domains, to allow visualisation of the mutant e subunit (eL221F-EGFP). Transgene transcription was driven from the AChR b-subunit promoter to restrict expression to the post-synaptic nuclei in skeletal muscle. Transgenic mice were back-crossed with mice deficient in endogenous AChR, to generate mice that expressed only mutated e-subunit. The mice have no obvious defects at birth, thrive and survive for longer than 12 months but show fatiguability. Analysis of endplates from EDL, soleus and diaphragm muscles using fluorescently tagged a-bungarotoxin showed that eL221F-EGFP is correctly incorporated into endplate AChR. Electron microscopy showed a large variability in endplate morphology within the same muscle. Electrophysiological analysis demonstrated prolonged decay of the EPPs and MEPPs, thereby confirming slow channel characteristics of the eL221F-EGFP receptors. Our model accurately reflects the disease in humans and is currently being used to test the efficacy and mechanisms of novel and existing slow channel therapies.

Resistance Exercise Reduces Skeletal Muscle Cachexia and Improves Muscle Function in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic, systemic, autoimmune, inflammatory disease associated with cachexia (reduced muscle and increased fat). Although strength-training exercise has been used in persons with RA, it is not clear if it is effective for reducing cachexia. A 46-year-old woman was studied to determine: (i) if resistance exercise could reverse cachexia by improving muscle mass, fiber cross-sectional area, and muscle function; and (2) if elevated apoptotic signaling was involved in cachexia with RA and could be reduced by resistance training. A needle biopsy was obtained from the vastus lateralis muscle of the RA subject before and after 16 weeks of resistance training. Knee extensor strength increased by 13.6% and fatigue decreased by 2.8% Muscle mass increased by 2.1%. Average muscle fiber cross-sectional area increased by 49.7%, and muscle nuclei increased slightly after strength training from 0.08 to 0.12 nuclei/μm2. In addition, there was a slight decrease (1.6%) in the number of apoptotic muscle nuclei after resistance training. This case study suggests that resistance training may be a good tool for increasing the number of nuclei per fiber area, decreasing apoptotic nuclei, and inducing fiber hypertrophy in persons with RA, thereby slowing or reversing rheumatoid cachexia.

Soft tissue changes during distraction osteogenesis

This study aimed to investigate soft tissue changes in distraction osteogenesis depending on various distraction and consolidation periods. Fifteen male rabbits were divided into three groups and evaluated for five parameters. An external custom-made device was placed on the right side of the mandible. The postoperative latent period was 4 days. The groups were distracted for different periods (6 days, 3mm; 11 days, 5.5mm, 16 days, 8mm). For histomorphometric analyses, specimens were retrieved from both study and control sides of the mandible and were examined with light microscopy. The number of muscle fibres and nuclei on the study side increase proportionally with the distraction period. Regeneration in the first group continued in its natural pattern; in the second group, there seemed to be degeneration rather than regeneration; and in the third group there seemed to be a balance between regeneration and degeneration processes. In conclusion, when a massive amount of bone is to be obtained, instead of distracting the bone at once, it is thought to give better results if the total amount of distraction is divided into several time periods.

Thoracolumbar multisegmental motor responses in the upper and lower limbs in healthy subjects

Experimental design. This descriptive study presents muscular responses from both the upper and the lower extremities during T11-12 segmental stimulation. Neuro Lab of the Texas Woman's University (School of Physical Therapy, TX, USA). A total of 13 healthy subjects were electrically stimulated using surface electrodes. In trial 1, signals were recorded from the flexor hallucis brevis, soleus, vastus medialis and gluteus medius in the lower right extremity. In trial 2, responses were recorded from the abductor digiti minimi, abductor pollicis brevis (APB), flexor carpi radialis and biceps brachii in the right upper extremity. In trial 3, stimulation was carried out and signals were recorded for both the upper and the lower extremities simultaneously, using different muscle combinations. Five traces per muscle were averaged for each step of the testing. Amplitude and deflection latency were the measured parameters and were compared using descriptive statistics. Results showed signal amplitudes ranging from 85 to 821 μV in the upper extremity and from 582 to 3927 μV in the lower extremity, with the largest signal recorded in the soleus muscle and the APB. Response latency varies between 5.5 and 14 ms in the upper limbs and between 7.7 and 27 ms in the lower limbs and was comparable in bilateral recording. These muscular responses seem to be elicited from electrical stimulation of motor nuclei in lower limb muscles or from pathways to those nuclei in upper limb muscles, and could be useful in testing patients with spinal disorders.

SMAD signaling drives heart and muscle dysfunction in a Drosophila model of muscular dystrophy

Loss-of-function mutations in the genes encoding dystrophin and the associated membrane proteins, the sarcoglycans, produce muscular dystrophy and cardiomyopathy. The dystrophin complex provides stability to the plasma membrane of striated muscle during muscle contraction. Increased SMAD signaling due to activation of the transforming growth factor-β (TGFβ) pathway has been described in muscular dystrophy; however, it is not known whether this canonical TGFβ signaling is pathogenic in the muscle itself. Drosophila deleted for the γ/δ-sarcoglycan gene (Sgcd) develop progressive muscle and heart dysfunction and serve as a model for the human disorder. We used dad-lacZ flies to demonstrate the signature of TGFβ activation in response to exercise-induced injury in Sgcd null flies, finding that those muscle nuclei immediately adjacent to muscle injury demonstrate high-level TGFβ signaling. To determine the pathogenic nature of this signaling, we found that partial reduction of the co-SMAD Medea, homologous to SMAD4, or the r-SMAD, Smox, corrected both heart and muscle dysfunction in Sgcd mutants. Reduction in the r-SMAD, MAD, restored muscle function but interestingly not heart function in Sgcd mutants, consistent with a role for activin but not bone morphogenic protein signaling in cardiac dysfunction. Mammalian sarcoglycan null muscle was also found to exhibit exercise-induced SMAD signaling. These data demonstrate that hyperactivation of SMAD signaling occurs in response to repetitive injury in muscle and heart. Reduction of this pathway is sufficient to restore cardiac and muscle function and is therefore a target for therapeutic reduction.

Long Range Interactions Regulate Igf2 Gene Transcription during Skeletal Muscle Differentiation

The differentiation, maintenance, and repair of skeletal muscle is controlled by interactions between genetically determined transcriptional programs regulated by myogenic transcription factors and environmental cues activated by growth factors and hormones. Signaling through the insulin-like growth factor 1 (IGF1) receptor by locally produced IGF2 defines one such pathway that is critical for normal muscle growth and for regeneration after injury. IGF2 gene and protein expression are induced as early events in muscle differentiation, but the responsible molecular mechanisms are unknown. Here we characterize a distal DNA element within the imprinted mouse Igf2-H19 locus with properties of a muscle transcriptional enhancer. We find that this region undergoes a transition to open chromatin during differentiation, whereas adjacent chromatin remains closed, and that it interacts in differentiating muscle nuclei but not in mesenchymal precursor cells with the Igf2 gene found more than 100 kb away, suggesting that chromatin looping or sliding to bring the enhancer in proximity to Igf2 promoters is also an early event in muscle differentiation. Because this element directly stimulates the transcriptional activity of an Igf2 promoter-reporter gene in differentiating myoblasts, our results indicate that we have identified a bona fide distal transcriptional enhancer that supports Igf2 gene activation in skeletal muscle cells. Because this DNA element is conserved in the human IGF2-H19 locus, our results further suggest that its muscle enhancer function also is conserved among different mammalian species.

P143 NF-kappa B (NF-κB) and activator protein-1 (AP-1) DNA binding in the quadriceps of COPD patients

IntroductionAnimal work implicates NF-κB and AP-1 activation in muscle atrophy (Cai et al, 2004 and Costelli et al, 2005). Prior study of seven COPD patients with a low BMI and...