Myosin isoforms in anuran skeletal muscle: their influence on contractile properties and in vivo muscle function

Functional studies on isolated single anuran skeletal muscle cells represent classic experiments from which much of our understanding of muscle contraction mechanisms have been derived. Because of their superb mechanical stability when isolated, single anuran fibers provide a uniquely powerful model system that can be exploited to understand the relationship between myosin heavy chain (MHC) and myosin light chain (MLC) composition and muscle fiber function. In this review, we summarize historic and recent studies of MHC and MLC expression patterns in the fiber types of anuran species. We extend the traditional classification scheme, using data from recent reports in which frog MHCs have been cloned, to reveal the molecular basis of frog muscle fiber types. The influence of MHC and MLC isoforms on contractile kinetics of single intact fibers is reviewed. In addition, we discuss more subtle questions such as variability of myosin coexpression along a single cell, and its potential influence on contractile function. The frog jump is used as a model system to elucidate principles of muscular system design, including the role of MHC isoforms on in vivo muscle function. Sequence information is used from cloned frog MHCs to understand the role of specific regions of the myosin motor domain in regulating contractile function and the evolutionary origins of fast and slow amphibian MHCs. Finally, we offer promising future possibilities that combine molecular methods (such as recombinant gene transfer) with single cell contractile measurements to address questions regarding myosin structure/function and gene regulation.     

Ice crystal growth in skeletal muscle fibres

Ice crystal growth was studied in rapidly frozen skeletal muscle fibres which were treated with cryo-protective additives (glycerol, DMSO, sucrose) or which were untreated. Freeze cleaving and etching was the basic method, with conventional plastic embedding and cryo-ultramicrotomy as complementary techniques. Extensive crystal growth occurred during freezing in all unprotected fibres. Just below the fibre surface the crystals were numerous but small, while deeper in the fibre they were fewer but larger. The deeper within the specimen a fibre was located, the larger, in general, was the crystal size. The crystal volume density was about 55%, irrespective of crystal size. Ice recrystallization was practically absent at the temperature normally used in cryo-sectioning (–70°C). Anti-freeze treatment eliminated crystal growth. If the anti-freeze agents were used in non-toxic concentrations, however, their effect on crystal growth was very limited. ‘Dry’-cut, freeze-dried ultra-thin cryosections of protected and unprotected fibres confirmed these observations, while sections obtained by ‘wet’ cryo-cutting showed no apparent signs of crystal growth. In plastic sections of frozen and thawed fibres a previous occurrence of crystals was only slightly indicated. In interpreting the ultrastructure in ‘wet’-cut cryo-sections of unprotected frozen mucle fibres, the distorting effects of ice crystals through mechanical compression and alterations in sectioning conditions, must be taken into consideration. Crystal growth also strongly limits the possibilities of using ‘dry’-cut sections of untreated frozen tissue for analytical electron microscopy; only the most superficial parts of the fibres seem to be suitable for microanalysis.     

Estimating length density and quantifying anisotropy in skeletal muscle capillaries

The accurate estimation of stereological parameters defined on anisotropic structures is a long-standing problem. In this paper we seek to estimate the capillary length density J_v in skeletal muscle tissue. A well-known model for directional anisotropy in space, namely the ‘spherical normal’ or ‘Fisher axial distribution’ model, is found to fit the relevant data satisfactorily. Based on this model, a short-cut estimation method is proposed and illustrated with a numerical example. This method essentially consists in taking the ratio of mean capillary profile counts, as obtained from transversal and longitudinal sections of the muscle tissue, and making use of a table or a graph given in the paper to estimate J_v. The conditions under which the methods are applicable and practicable are discussed in detail. Apart from an accurate estimation of J_v, an important feature of our method is the possibility of quantifying the degree of anisotropy by a coefficient K (called the concentration parameter of the Fisher axial distribution), which enjoys both a biological significance and a sound statistical basis.     

Trunk and lower extremity muscle activity in seated weight-moving tasks: Three-dimensional analyses of intersubject and intertask differences

A three-dimensional musculoskeletal model was used to predict the trunk and lower extremity muscle activity required to stabilize the body while performing dynamic tasks in the seated posture. We studied seven subjects performing four tasks consisting of cyclic or single-directional movements of a hand-held weight in the sagittal plane. Five different optimization schemes involving the minimization of muscle forces, muscle stresses, or joint force components were used to predict the 64 muscle force-time histories. A quantitative method was used to compare prediction schemes by correlating predicted muscle forces with measured myoelectric data and ranking the efficacy of each scheme for different tasks and subjects. The results showed that (1) the trunk and lower extremity muscles play an important role in stabilizing the seated posture in these tasks. (2) the most successful muscle force prediction scheme was not the same for all seven subjects performing a given task, or for a given subject performing all four tasks, and (3) these linear optimization techniques successfully predicted activity in up to 10 of the 15 muscles whose myoelectric signals were actually monitored.     

Effects of veratrine on skeletal muscle mitochondria: ultrastructural, cytochemical, and morphometrical studies

The alkaloid veratrine is a lipid-soluble neurotoxin, which target voltage-gated Na+ channels for their primary action. Recently, we showed that this alkaloid may cause myonecrosis and evidences suggest mitochondria as one of its cell targets. Herein, we investigate the effects caused by variable concentration of veratrine (250 and 550 microg/mL) on mitochondrial oxygen consumption, respiratory chain enzymes activities, and ultrastructure, combining electron microscopy with cytochemical and biochemical approaches. The results showed different sort of ultrastructural changes, both in isolated and intramuscular mitochondria. Veratrine decreased mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH-d), succinic dehydrogenase (SDH), and cytochrome oxidase (COX) activities, significantly and dose-dependently inhibited the state 3 respiration rate, respiratory control ratio (RCR), and ADP/O on isolated rat skeletal muscle mitochondria, whereas state 4 was unaffected. A tendency of increase in mitochondria diameter was seen with 250 microg/mL veratrine. We conclude that the alkaloid would probably act on mitochondrial membrane phospholipid configuration, which would explain the changes observed.     

Parametric study of a Hill-type hyperelastic skeletal muscle model

Hill's one-dimensional three-element model has often been used for formulating a three-dimensional skeletal muscle constitutive model, which generally involves several material parameters. However, only few of these parameters have physical meanings and can be experimentally determined. In this paper, a parametric study of a Hill-type hyperelastic skeletal muscle model is performed. First, the Hill-type hyperelastic skeletal muscle model is formulated, containing 13 material parameters. The values or value ranges of these parameters are discussed. The muscle model is then used to predict the behaviour of New Zealand white rabbit hind leg muscle tibialis anterior and a sensitivity study of several parameters is performed. Results show that some parameters in the muscle model can be experimentally determined, some parameters have their own value ranges and the muscle model can predict the experimental data by tuning the parameters within their value ranges. The results from the sensitivity study can help understand how some parameters influence the total muscle stress.     

Hypothalamic regulatory peptides and their receptors: Cytochemical studies of their role in regulation at the adenohypophyseal level

Hypothalamic regulatory peptides bind to specific receptors on target cells in the pituitary and control secretion. They in turn can be regulated at the pituitary level by steroid and peptide modulators. Affinity cytochemical techniques are important tools for the identification of specific target binding sites for these regulatory peptides. This presentation reviews the work in which potent, biotinylated ligands of gonadotropin releasing hormone (bio-GnRH), corticotropin releasing hormone (bio-CRH), and arginine vasopressin (bio-AVP) were applied to study the target cell responses. Bio-GnRH, bio-CRH, and bio-AVP bind to membrane receptors on specific anterior pituitary cells. Dual labeling for either gonadotropin or adrenocorticotropin (ACTH) antigens further identified the target cells. After 1–3 minutes, the label was in patches or capped on the surface. After 3 minutes, it was internalized in small vesicles and sent to receptosomes and vacuoles in the Golgi complex. Eventually the biotinylated peptides, or a metabolite, was found in the lysosomes (multivesicular bodies) and a subpopulation of secretory granules. The route and rate of uptake was similar to that described for the classical receptor-mediated endocytosis process. In contrast, intermediate lobe corticotropes internalized the bio-CRH in less than 1 minute. The route through the Golgi complex appeared to be bypassed. Instead the labeled peptide was in vesicles, on the membranes of scattered vacuoles, and in multivesicular bodies. Modulation of ligand binding by steroids showed that changes in receptor numbers correlated with changes in the number of cells that bound the ligand. In male rats, dihydrotestosterone reduced the percentage of GnRH-bound cells by 50%. Most of the reduction appeared in cells that stored luteinizing hormone (LH) antigens. In diestrous female rats, estradiol increased the percentage of bio-GnRH-bound cells. However, the steroid decreased the percentage of GnRH-bound cells in cells from proestrous rats. Glucocorticoids decreased the percentage of CRH-bound corticotropes in as little as 10 minutes. Potentiation of secretion by these ligands was correlated with increases in the percentage of ligand-bound cells. AVP pretreatment of corticotropes increased the percentage of cells that bound bio-CRH. It also increased the rate of receptor-mediated endocytosis of CRH and changed the route so that the Golgi complex was bypassed. This effect could be mimicked by activation of its second messengers (calcium and protein kinase C). Similarly, CRH pretreatment increased the percentage of corticotropes that bound AVP. Thyrotropin releasing hormone (TRH) pretreatment also increased the percentage of thyrotropes that bound AVP. Finally, calcium or sodium channel blockers altered CRH binding so that fewer cells were labeled. This binding by CRH was not dependent on extracellular calcium and tests with a calcium channel agonist showed that it was related to activation of calcium channels. To summarize, these affinity cytochemical studies have identified specific target cells in the pituitary for GnRH, CRH, and AVP. They have also identified heterogeneity in the population. They have demonstrated new information about the direct modulatory effects of steroids, ion channels, and neuropeptides on neuropeptide binding by subpopulations of these target cells.     

Calibration for quantitative X-ray microanalysis of skeletal muscle cells in culture.

Skeletal muscle in culture is used to demonstrate that the intracellular concentration of elements in tissue culture cells, especially of diffusible ions such as sodium, chlorine, potassium, and calcium can be measured by X-ray microanalysis in the scanning electron microscope. Quantitative analysis is possible by comparison with X-ray spectra of cells incubated in electrolyte solutions of known concentration. The minimum detectable concentration is approximately 2 mM.     

基于CATIA的单齿辊破碎机齿辊三维模型建立及有限元分析

运用CATIAV5,对单齿辊破碎机齿辊进行建模并进行有限元应力分析以检验其强度,为单齿辊破碎机的优化设计做初步准备.     

Imaging and elasticity measurements of the sarcolemma of fully differentiated skeletal muscle fibres

This study aimed to describe the three-dimensional structure and the elastic properties of the sarcolemma of adult, fully differentiated, skeletal muscle fibres combining Atomic Force Microscopy (AFM) and optical microscopy. Single fibres were enzymatically dissociated from Flexor Digitorum Brevis of adult mice and were maintained in culture up to 3 weeks. On the sixth day after dissociation, the upper surface of intact fibres, either alive in solution or fixed and kept in solution or fixed and exposed in air, was analysed with AFM. The most prominent features in AFM images were periodic transversal foldings with an interval that corresponded to the sarcomere length. More detailed analysis of the topography profile showed that the depth in the folding decreased with increasing sarcomere length and that the crests of the foldings corresponded to the Z-lines. Minor periodic structures could be detected in the valleys between the major foldings. AFM images also showed deep depressions on the sarcolemma likely corresponding to openings of T tubules and caveolae. Two-dimensional elasticity maps were obtained using AFM as an indenter and showed that the crests of the transversal foldings correspond to higher stiffness regions. This study provides the first complete three-dimensional topography and mechanical characterization of intact, living skeletal muscle fibres and might form the basis for further investigations aimed to compare healthy and dystrophic muscles.     

Similar features in Z bands of both skeletal and cardiac muscle revealed by image enhancement

We have shown previously that the small square (ss) and basket weave (bw) states of the Z band lattice in cardiac and skeletal muscle are related to the contractile state of the muscle. We have used two-dimensional image processing techniques on digitized electron micrographs to enhance the structural features of each projected lattice form in cardiac and skeletal muscle. Four different processing techniques were employed to assess the effect of enhancement artifacts on the resulting Z band images. We observed only slight differences between enhanced images of a particular Z band form produced by the four different techniques. Every enhanced image showed an approximate four-fold symmetry independent of muscle type or Z band lattice form. Each enhanced image showed four cross-connecting Z-filaments which appeared to connect each axial filament to the four nearest axial filaments. In bw images from both cardiac and skeletal muscle, axial filaments had a greater apparent diameter and a greater interaxial filament spacing than in the ss images. In both muscle types, the cross-connecting Z-filaments appeared to overlap half-way between axial filaments in the ss images while the bw images showed no such overlap. These structural features are consistent with a dynamic Z band lattice that participates in muscle contraction.     

Regulation and functional significance of utrophin expression at the mammalian neuromuscular synapse

Duchenne muscular dystrophy (DMD) is caused by the absence of full-length dystrophin molecules in skeletal muscle fibers. In normal muscle, dystrophin is found along the length of the sarcolemma where it links the intracellular actin cytoskeleton to the extracellular matrix, via the dystrophin-associated protein (DAP) complex. Several years ago, an autosomal homologue to dystrophin, termed utrophin, was identified and shown to be expressed in a variety of tissues, including skeletal muscle. However, in contrast to the localization of dystrophin in extrajunctional regions of muscle fibers, utrophin preferentially accumulates at the postsynaptic membrane of the neuromuscular junction in both normal and DMD adult muscle fibers. Since it has recently been suggested that the upregulation of utrophin might functionally compensate for the lack of dystrophin in DMD, considerable interest is now directed toward the elucidation of the various regulatory mechanisms presiding over expression of utrophin in normal and dystrophic skeletal muscle fibers. In this review, we discuss some of the most recent data relevant to our understanding of the impact of myogenic differentiation and innervation on the expression and localization of utrophin in skeletal muscle fibers.     

Correcting the axial shrinkage of skeletal muscle thick sections visualized by confocal microscopy

Confocal microscopy is a suitable method for measurements and visualization of skeletal muscle fibres and the neighbouring capillaries. When using 3D images of thick sections the tissue deformation effects should be avoided. We studied the deformation in thick sections of the rat skeletal muscle from complete stacks of images captured with confocal microscope. We measured the apparent thickness of the stacks and compared it to the slice thickness deduced from calibrated microtome settings. The ratio of both values yielded the axial scaling factor for every image stack. Careful sample preparation and treatment of the tissue cryosections with cold Ringer solution minimize the tissue deformation. We conclude that rescaling by the inverse of the axial scaling factor of the stack of optical slices in the direction of the microscope optical axis satisfactorily corrects the axial deformation of skeletal muscle samples.     

Nanoinhomogeneities in glasses and their role in optical memory phenomena and charge transfer processes

To explain the basic features of the response of disordered materials (glasses) to irradiation by light, a new approach based on the assumption that glasses have the so-called “medium-range” order (inhomogeneities with the characteristic scale of ∼1 nm) is proposed and justified. The structure of inhomogeneities depends on the bond type: glasses with covalent directed bonds (strong glasses) at nanometer scales are similar to their crystalline ancestors. Glasses with non-directed bonds (ion and van der Waals bonds) do not form crystalline nanoinhomogeneities, though the existence of a certain medium-range order is typical for them. Using the concept of an inhomogeneous structure of glasses at nanometer scales, it is possible to explain the physical properties demonstrated by these materials: structural factor, specific features of the density of vibrational states in the terahertz range, mean free paths at frequencies below the boson peak frequency, optical memory phenomenon, and specific features of charge transfer. The proposed approach is an alternative for the widely used approach that involves construction of specific defects understood as violations of coordination of individual atoms, as quasi-molecular complexes, or as two-level states with no particular information about them. This approach reflects the specific features of the vitreous state, is universal, and is not based on a particular chemical nature of the material.     

Mass customization: manufacturing issues and taxonomic analyses

As the field of mass customization (MC) attains the status of a mature discipline, two significant research deficits stand out. First, a through metareview of the entire body of MC research that looks at the application value and rigorousness of research is overdue. Second, manufacturing issues, especially those pertaining to quality and the supply chain have been largely ignored. This issue is dedicated to both of these important areas of research. The conclusion with regards to the status of the MC field is that it is currently vibrant, with growing research volume and applications. The manufacturing issues dealt with in this issue are strategically important, dealing with quality and customization issues. The work on quality is the first of its kind: it seeks to generate a defect-tracking matrix consistent with product configurations, enabling agile identification of defects in a mass customization environment. The use of discrete event simulation to deal with the dynamically evolving customized demand so as to minimize cost and schedule disruption is innovative, timely, and profound.

Some limitations of the use of tannic acid as a marker of damaged skeletal muscle fibres

Mouse biceps brachii muscles containing single transverse incisions were treated with low molecular weight tannic acid. In the absence of fibre degeneration, intracellular spread of tannic acid was confined to a short distance along the fibres from the damaged portions of the cell membranes. Beyond this, damaged and undamaged fibres could not be distinguished from one another. Tannic acid is not a reliable marker of localized trauma to skeletal muscle fibres.     

Coordinated expression of myosin heavy chains, metabolic enzymes, and morphological features of porcine skeletal muscle fiber types

Combined methodologies of electrophoresis, immunoblots, immunohistochemistry, histochemistry, and photometric image analysis were applied to characterize porcine skeletal muscle fibers according to their myosin heavy chain (MyHC) composition, and to determine on a fiber-to-fiber basis the correlation between contractile [MyHC (s), myofibrillar ATPase (mATPase), and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) isoforms], metabolic [succinate dehydrogenase (SDH), and glycerol-3-phosphate dehydrogenase (GPDH) activities, glycogen, and phospholamban (PLB) contents], and morphological [cross-sectional area (CSA), capillary, and nuclear densities] features of individual myofibers. An accurate delineation of MyHC-based fiber types was obtained with the immunohistochemical method developed. This protocol showed a high sensitivity and objectivity to delineate hybrid fibers with overwhelming dominance of one MyHC isoform. The phenotypic differences in contractile, metabolic, and morphological properties seen between fiber types were related with MyHC content. Slow fibers had the lowest mATPase activity (related to shortening velocity), the highest SDH activity (oxidative capacity), the lowest GPDH activity (glycolytic metabolism), and glycogen content, the smallest CSA, the greatest capillary, and nuclear densities, and expressed slow SERCA isoform and PLB, but not the fast SERCA isoform. The reverse pattern was true for pure IIB fibers, whereas type IIA and IIX fibers had intermediate properties. Hybrid fibers had mean values intermediate in-between their respective pure phenotypes. Discrimination of myofibers according to their MyHC content was possible on the basis of their contractile and non-contractile profiles. These intrafiber interrelationships suggest that myofibers of control pigs exhibit a high degree of co-ordination in their physiological, biochemical, and anatomical features. This study may well be a useful baseline for future work on the pig meat industry and also offers new prospects for muscle fiber typing in porcine experimental studies.     

Laminins: structure and genetic regulation

The laminins form a large family of modular proteins found in basement membranes, but also elsewhere. They function as structural components and are essential for morphogenesis, but in addition interact with cell surface receptors such as integrins and alpha-dystroglycan. By virtue of their receptor interactions, they initiate intracellular signalling events that regulate cellular organization and differentiation. The many interactions of laminins are mediated by binding sites, often contributed by single domains, which may differ between different forms of laminin. In the present article, we describe how the diversity of laminins and the genetic regulation of the expression of different laminin forms lead to the formation of extracellular matrices with variable laminin composition and thereby different biological properties.