Functional Deficits in nNOSμ-Deficient Skeletal Muscle: Myopathy in nNOS Knockout Mice

Skeletal muscle nNOSμ (neuronal nitric oxide synthase mu) localizes to the sarcolemma through interaction with the dystrophin-associated glycoprotein (DAG) complex, where it synthesizes nitric oxide (NO). Disruption of the DAG complex occurs in dystrophinopathies and sarcoglycanopathies, two genetically distinct classes of muscular dystrophy characterized by progressive loss of muscle mass, muscle weakness and increased fatigability. DAG complex instability leads to mislocalization and downregulation of nNOSμ; but this is thought to play a minor role in disease pathogenesis. This view persists without knowledge of the role of nNOS in skeletal muscle contractile function in vivo and has influenced gene therapy approaches to dystrophinopathy, the majority of which do not restore sarcolemmal nNOSμ. We address this knowledge gap by evaluating skeletal muscle function in nNOS knockout (KN1) mice using an in situ approach, in which the muscle is maintained in its normal physiological environment. nNOS-deficiency caused reductions in skeletal muscle bulk and maximum tetanic force production in male mice only. Furthermore, nNOS-deficient muscles from both male and female mice exhibited increased susceptibility to contraction-induced fatigue. These data suggest that aberrant nNOSμ signaling can negatively impact three important clinical features of dystrophinopathies and sarcoglycanopathies: maintenance of muscle bulk, force generation and fatigability. Our study suggests that restoration of sarcolemmal nNOSμ expression in dystrophic muscles may be more important than previously appreciated and that it should be a feature of any fully effective gene therapy-based intervention

Differentiation of activated satellite cells in denervated muscle following single fusions in situ and in cell culture

Satellite cells represent a cellular source of regeneration in adult skeletal muscle. It remains unclear why a large pool of stem myoblasts in denervated muscle does not compensate for the loss of muscle mass during post-denervation atrophy. In this study, we present evidence that satellite cells in long-term denervated rat muscle are able to activate synthesis of contractile proteins after single fusions in situ. This process of early differentiation leads to formation of abnormally diminutive myotubes. The localization of such dwarf myotubes beneath the intact basal lamina on the surface of differentiated muscle fibers shows that they form by fusion of neighboring satellites or by the progeny of a single satellite cell following one or two mitotic divisions. We demonstrated single fusions of myoblasts using electron microscopy, immunocytochemical labeling and high resolution confocal digital imaging. Sequestration of nascent myotubes by the rapidly forming basal laminae creates a barrier that limits further fusions. The recruitment of satellite cells in the formation of new muscle fibers results in a progressive decrease in their local densities, spatial separation and ultimate exhaustion of the myogenic cell pool. To determine whether the accumulation of aberrant dwarf myotubes is explained by the intrinsic decline of myogenic properties of satellite cells, or depends on their spatial separation and the environment in the tissue, we studied the fusion of myoblasts isolated from normal and denervated muscle in cell culture. The experiments with a culture system demonstrated that the capacity of myoblasts to synthesize contractile proteins without serial fusions depended on cell density and the availability of partners for fusion. Satellite cells isolated from denervated muscle and plated at fusion-permissive densities progressed through the myogenic program and actively formed myotubes, which shows that their myogenic potential is not considerably impaired. The results of this study suggest that under conditions of denervation, progressive spatial separation and confinement of many satellite cells within the endomysial tubes of atrophic muscle fibers and progressive interstitial fibrosis are the important factors that prevent their normal differentiation. Our findings also provide an explanation of why denervated muscle partially and temporarily is able to restore its functional capacity following injury and regeneration: the release of satellite cells from their sublaminal location provides the necessary space for a more active regenerative process.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47397/1/418_2005_Article_12.pd

Generator with modulated magnetic flux for wind turbines

In the paper, the concept of a permanent magnet synchronous generator (PMSG) with uniquely designed stator windings for wind turbines is presented. Two 3-phase windings in the stator are used, one of which is connected in the star, while the other in the delta configuration. Six-pulse rectifiers, mutually coupled by the pulse transformer, whose primary winding is supplied by the so-called “current modulator”, are placed at the outputs of both windings. The modulator output current should meet all the necessary and strict requirements. Both rectifiers operate on a common DC circuit. These solutions provide the sinusoidal magnetomotive force in the stator of the PMSG and the quasi-sinusoidal (taking into account the non-linearity of the magnetic circuit) magnetic flux. In light of the generator principle, it has been called the “PMSG with modulated the magnetic flux”. The slightly higher complexity in the structure of the generator, as compared to the normal three-phase construction, is compensated by the exceptional simplicity of the power electronics section of the system, which allows high efficiency to be reached. The current modulator (as well as the pulse transformer) is a power electronics converter with a relatively low output power as compared to the overall output power of the system. In comparison to other known solutions, the expected cost of the system should be lower. It is also expected that a high degree of reliability in terms of its operation will be achieved, and consequently, that the the ongoing costs of its maintenance will be reduced. In the paper, concept, theoretical basis of operation, and results of the studies of the simulation models of the generator, including the basic power electronics section, are presented

Effect of inspiratory threshold loading on ventilatory kinetics during constant-load exercise.

Humoral factors play an important role in the control of exercise hyperpnea. The role of neuromechanical ventilatory factors, however, is still being investigated. We tested the hypothesis that the afferents of the thoracopulmonary system, and consequently of the neuromechanical ventilatory loop, have an influence on the kinetics of oxygen consumption (VO2), carbon dioxide output (VCO2), and ventilation (VE) during moderate intensity exercise. We did this by comparing the ventilatory time constants (tau) of exercise with and without an inspiratory load. Fourteen healthy, trained men (age 22.6 +/- 3.2 yr) performed a continuous incremental cycle exercise test to determine maximal oxygen uptake (VO2max = 55.2 +/- 5.8 ml x min(-1) x kg(-1)). On another day, after unloaded warm-up they performed randomized constant-load tests at 40% of their VO2max for 8 min, one with and the other without an inspiratory threshold load of 15 cmH2O. Ventilatory variables were obtained breath by breath. Phase 2 ventilatory kinetics (VO2, VCO2, and VE) could be described in all cases by a monoexponential function. The bootstrap method revealed small coefficients of variation for the model parameters, indicating an accurate determination for all parameters. Paired Student's t-tests showed that the addition of the inspiratory resistance significantly increased the tau during phase 2 of VO2 (43.1 +/- 8.6 vs. 60.9 +/- 14.1 s; P < 0.001), VCO2 (60.3 +/- 17.6 vs. 84.5 +/- 18.1 s; P < 0.001) and VE (59.4 +/- 16.1 vs. 85.9 +/- 17.1 s; P < 0.001). The average rise in tau was 41.3% for VO2, 40.1% for VCO2, and 44.6% for VE. The tau changes indicated that neuromechanical ventilatory factors play a role in the ventilatory response to moderate exercise

Two-dimensional polymorphism and melting in a monolayer of nitric oxide adsorbed on graphite

Three two-dimensional (2D) solids γ, δ and β have been observed at low temperature by neutron scattering in the first monolayer of nitric oxide adsorbed on graphite. The structure of two of these solids, y and β, has been determined. In both cases, nitric oxide molecules are dimerized, but in the β phase N2O2 molecules stand up whereas in the y solid they lie down on the substrate. The β and y configurations are analogous to the packing of two planes of the nitric oxide bulk crystal namely the (001) plane for the β phase and the (101) plane for the y solid. The intermediate δ structure is not resolved but a possible unit cell is given. Increasing the temperature results in a melting of the 2D solids. A particular emphasis is made on the β-liquid transition which is shown to be first order. Other first order transitions are also observed like the β solid ⇄ δ solid, liquid ⇄ gas and y solid ⇄ gas transformations. All these results combined with previous adsorption isotherm measurements are put together in a proposed 2D phase diagram displaying three triple points, one critical temperature and one tricritical point.Trois solides bidimensionnels (2D) y, δ, β, sont observés à basse température par diffusion de neutrons dans une monocouche d'oxyde nitrique adsorbé sur du graphite. La structure de deux de ces solides, y et β, est déterminée. Dans les deux cas, les molécules d'oxyde nitrique sont dimérisées, mais dans la phase β les molécules N2O2 sont debout, tandis que dans le solide y elles sont couchées sur le substrat. Les configurations β et y sont analogues à l'empilement de deux plans de l'oxyde nitrique massique ; il s'agit du plan (001) pour la phase β et du plan (101) pour le solide y. La structure δ intermédiaire n'est pas résolue mais une maille est proposée. Si on augmente la température, les solides 2D fondent. On s'attache particulièrement à l'étude de la transition β-liquide qui s'avère être du premier ordre. D'autres transitions du premier ordre sont aussi observées comme les transformations β ⇄ δ, liquide ⇄ gaz et y ⇄ gaz. Tous ces résultats, ainsi que des mesures d'isothermes d'adsorption obtenues précédemment, sont rassemblés dans un diagramme de phase 2D qui présente trois points triples, une température critique et un point tricritique

Hypernitrosylated ryanodine receptor calcium release channels are leaky in dystrophic muscle

Duchenne muscular dystrophy is characterized by progressive muscle weakness and early death resulting from dystrophin deficiency. Loss of dystrophin results in disruption of a large dystrophin glycoprotein complex, leading to pathological calcium (Ca2+)-dependent signals that damage muscle cells. We have identified a structural and functional defect in the ryanodine receptor (RyR1), a sarcoplasmic reticulum Ca2+ release channel, in the mdx mouse model of muscular dystrophy that contributes to altered Ca2+ homeostasis in dystrophic muscles. RyR1 isolated from mdx skeletal muscle showed an age-dependent increase in S-nitrosylation coincident with dystrophic changes in the muscle. RyR1 S-nitrosylation depleted the channel complex of FKBP12 (also known as calstabin-1, for calcium channel stabilizing binding protein), resulting in 'leaky' channels. Preventing calstabin-1 depletion from RyR1 with S107, a compound that binds the RyR1 channel and enhances the binding affinity of calstabin-1 to the nitrosylated channel, inhibited sarcoplasmic reticulum Ca2+ leak, reduced biochemical and histological evidence of muscle damage, improved muscle function and increased exercise performance in mdx mice. On the basis of these findings, we propose that sarcoplasmic reticulum Ca2+ leak via RyR1 due to S-nitrosylation of the channel and calstabin-1 depletion contributes to muscle weakness in muscular dystrophy, and that preventing the RyR1-mediated sarcoplasmic reticulum Ca2+ leak may provide a new therapeutic approach

Exploration fonctionnelle respiratoire : explorer l’enfant

International audienceIn paediatrics, the pulmonary function test (PFT) is most often performed to support the diagnosis or in follow-up of asthma patients. Whatever the pathology responsible for respiratory symptoms and/or functional impairment, repeated PFTs make it possible to establish a prognosis (pulmonary function trajectories…) and to orient preventive interventions. PFT can be performed routinely from the age of three years, provided that the following requirements are met: suitable techniques and equipment, staff trained to apply the techniques and to receive young children, reference values for each technique indicating the limits of normal values and of between-test significant variation. From the age of three, children can be subjected to tidal breathing measurement of: resistance of the respiratory system (oscillometry, Rrs; airflow interruption, Rint) or of airways specific resistance (sRaw) and functional residual capacity (by applying a dilution technique). With maturity, the child will become capable of mobilizing his or her slow vital capacity to measure total lung capacity (TLC), once again by applying a dilution technique, then later by breathing against a closed shutter (plethysmography TLC and Raw). Finally, the child will be able to carry out forced expiration (forced spirometry) along with all of the other PFTs. It is important to take into account the paediatric adaptations specified in the international recommendations regarding the performance, reproducibility and quality of PFTs targeting this population.L’exploration fonctionnelle respiratoire (EFR) de l’enfant est le plus souvent réalisée pour étayer le diagnostic ou participer au suivi d’un asthme. Quelle que soit la pathologie respiratoire motivant l’EFR, sa répétition permet d’établir un pronostic (trajectoires) et de résistance guider les actions de prévention. Une EFR est réalisable en routine dès l’âge de 3 ans sous réserve de disposer : de techniques et matériels de mesure adaptés, d’un personnel formé à ces techniques et à l’accueil des jeunes enfants, de normes pour chaque technique avec les limites de la normale et celles de la variation significative. Dès cet âge, l’enfant peut réaliser en volume courant des mesures : de résistance du système respiratoire (par oscillométrie, Rrs, ou interruption du débit, Rint) ou spécifique des voies aériennes (sRaw), du volume de repos par une technique de dilution. Avec la maturité deviendront possible la mobilisation de la capacité vitale lente et donc de capacité pulmonaire totale (CPT) en dilution, puis des respirations contre une valve fermée (CPT en pléthysmographie, Raw), et enfin l’expiration forcée (spirométrie forcée) et toutes les autres mesures de fonction respiratoire. Il faut appliquer les aménagements pédiatriques des recommandations internationales concernant la réalisation, la reproductibilité et la qualité des mesures d’EFR