Localization of sarcoglycan, neuronal nitric oxide synthase, beta-dystroglycan, and dystrophin molecules in normal skeletal myofiber: triple immunogold labeling electron microscopy.

In order to investigate the mode of existence of the sarcoglycan complex, neuronal nitric oxide synthase (nNOS), beta-dystroglycan, and dystrophin in the normal skeletal myofiber, we examined the ultrastructural localization and mutual spatial relationship of nNOS, beta-dystroglycan, dystrophin, and the individual components of the sarcoglycan complex by using triple immunogold labeling electron microscopy. Each molecule of alpha-, beta-, gamma- and delta-sarcoglycans is located intracellularly or extracellularly near the muscle plasma membrane mostly in accordance with the sarcoglycan antigenic sites against which the antibodies were generated. The association of different two and/or three sarcoglycan molecules out of alpha-, beta-, gamma- and delta-sarcoglycan molecules was frequently observed. Each molecule of nNOS, beta-dystroglycan, and dystrophin was ultrastructurally noted along the cell surface of normal skeletal myofibers. Moreover, the close relation of a sarcoglycan molecule with beta-dystroglycan and dystrophin, and the association of nNOS with dystrophin were also confirmed ultrastructurally. Thus, this study demonstrated that the constituting molecules of the sarcoglycan complex, nNOS, beta-dystroglycan, and dystrophin existed in the form of a cluster at the normal muscle plasma membrane. The association of nNOS with dystrophin and its associated glycoproteins may form a macromolecular signaling complex at the muscle plasma membrane.     

Developments in gene therapy for muscular dystrophy

Gene therapy for muscular dystrophy (MD) presents significant challenges, including the large amount of muscle tissue in the body, the large size of many genes defective in different muscular dystrophies, and the possibility of a host immune response against the therapeutic gene. Overcoming these challenges requires the development and delivery of suitable gene transfer vectors. Encouraging progress has been made in modifying adenovirus (Ad) vectors to reduce immune response and increase capacity. Recently developed gutted Ad vectors can deliver full-length dystrophin cDNA expression vectors to muscle tissue. Using muscle-specific promoters to drive dystrophin expression, a strong immune response has not been observed in mdx mice. Adeno-associated virus (AAV) vectors can deliver small genes to muscle without provocation of a significant immune response, which should allow long-term expression of several MD genes. AAV vectors have also been used to deliver sarcoglycan genes to entire muscle groups. These advances and others reviewed here suggest that barriers to gene therapy for MD are surmountable.     

Role of nitric oxide in the pathogenesis of muscular dystrophies: a "two hit" hypothesis of the cause of muscle necrosis.

Although the genetic and biochemical bases of many of the muscular dystrophies have been elucidated, the pathophysiological mechanisms leading to muscle cell death and degeneration remain elusive. Among the most well studied of the dystrophies are those due to defects in proteins that make up the dystrophin-glycoprotein complex (DGC). There has been much interest in the role of nitric oxide (NO(*)) in the pathogenesis of these diseases because the enzyme that synthesizes NO(*), nitric oxide synthase (NOS), is associated with the DGC. Recent studies of dystrophies related to DGC defects suggest that one mechanism of cellular injury is functional ischemia related to alterations in cellular NOS and disruption of a normal protective action of NO(*). This protective action is the prevention of local ischemia during contraction-induced increases in sympathetic vasoconstriction. However, the loss of this protection, alone, does not explain the subsequent muscle cell death and degeneration since mice lacking neuronal NOS (the predominant isoform expressed in muscle) do not develop a muscular dystrophy. Thus, there must be additional biochemical changes conferred upon the cells by these DGC defects, and these changes are discussed in terms of a proposed "two hit" hypothesis of the pathogenetic mechanisms that underlie the muscular dystrophies. According to this hypothesis, pathogenic defects in the DGC have at least two biochemical consequences: a reduction in NO(*)-mediated protection against ischemia, and an increase in cellular susceptibility to metabolic stress. Either one alone may be insufficient to lead to muscle cell death. However, in combination, the biochemical consequences are sufficient to cause muscle degeneration. The role of oxidative stress as a final common pathophysiologic pathway is discussed in terms of data showing that oxidative injury precedes pathologic changes and that muscle cells with defects in the DGC have an increased susceptibility to oxidant challenges. Accordingly, this "two hit" hypothesis may explain many of the complex spatial and temporal variations in disease expression that characterize the muscular dystrophies, such as grouped necrosis, a pre-necrotic phase of the disease, and selective muscle involvement.     

Dystrophin and utrophin: genetic analyses of their role in skeletal muscle

Since the identification of dystrophin as the causitive factor in Duchenne muscular dystrophy, there has been substantial progress in understanding the functions and interactions of this protein. Dystrophin has been shown to interact with a group of peripheral- and trans-membrane proteins known as the dystrophin-associated protein complex (DAPC) and mutations in some of the members of this complex have been shown to account for other forms of muscular dystrophy. This review summarizes the experiments using transgenic and knockout mouse models that have defined the roles of dystrophin, and the dystrophin-related protein utrophin at the skeletal muscle membrane and at the neuromuscular junction. These studies are presented in the context of other known interactions at the muscle membrane. Studies of the dystrophin-deficient mdx mouse have lead to a greater understanding of the human disease. Knockouts and transgenics of utrophin have shown this protein to be sufficient to functionally compensate for dystrophin. Dystrophin transgenic mice combined with the mdx mouse have been used to study the function of specific domains of the dystrophin protein. Together these animal models have led to a delineation of protein functions and localization patterns that will be useful for the generation of potential therapies for DMD.     

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.     

Can scanning near‐field optical microscopy be compared with confocal laser scanning microscopy? A preliminary study on α‐sarcoglycan and β1D‐integrin in human skeletal muscle

The dystrophin–glycoprotein complex and the vinculin–talin–integrin system constitute, together a protein machinery, called costameres. The dystrophin–glycoprotein complex contains, among other proteins, also dystrophin and the sarcoglycans subcomplex, proteins playing a key role in the pathogenesis of many muscular dystrophies and linking the cytoplasmic myofibrillar contractile elements to the signal transducing molecules of the extracellular matrix, also providing structural support to the sarcolemma. The vinculin–talin–integrin system connects some components of the extracellular matrix with intermediate filaments of desmin, forming transverse bridges between Z and M lines. In our previous reports we always studied these systems by confocal laser scanning microscopy (CLSM). In this paper we report on the first applications of optical near‐field fluorescence microscopy to the spatial localization of α‐sarcoglycan and β1D‐integrin in human skeletal muscle fibres in order to better compare and test the images obtained with conventional CLSM and with scanning near‐field optical microscopy (SNOM). In addition, the analysis of the surface morphology, and the comparison with the fluorescence map is put forward and analyzed for the first time on human muscle fibres. In aperture‐SNOM the sample is excited through the nanometre‐scale aperture produced at the apex of an optical fibre after tapering and subsequent metal coating. The acquisition of the topography map, simultaneously to the optical signal, by SNOM, permits to exactly overlap the fluorescence images obtained from the two consecutive scans needed for the double localization. Besides, the differences between the topography and the optical spatial patterns permit to assess the absence of artefacts in the fluorescence maps. Although the SNOM represented a good method of analysis, this technique remains a complementary method to the CLSM and it can be accepted in order to confirm the hypothesis advanced by CLSM.     

Immunocytochemical analysis of human muscular dystrophy

Immunocytochemistry is an essential tool for the assessment of muscle biopsies from patients with muscular dystrophy, especially the recessive forms. Antibodies can detect primary defects when there is an alteration in expression, in particular in Xp21 muscular dystrophies, Emery-Dreifuss muscular dystrophy, the limb-girdle dystrophies caused by abnormal expression of the sarcoglycans, and in the form of congenital muscular dystrophy linked to the gene for laminin alpha2. Absence of a protein is easily observed and reduction in expression can be assessed provided adequate controls and baselines are established. Assessment of secondary defects can also be of diagnostic value; they widen the understanding of pathology changes, and are helping in the development of therapeutic strategies.     

Plasma membrane cytoskeleton of muscle: a fine structural analysis

The discovery of dystrophin and its definition as the causative molecule in Duchenne Muscular Dystrophy has led to a renewed interest in the molecular structure of the muscle fiber plasma membrane and its association with the extracellular basal lamina. The original identification of dystrophin gave credence to the possibility that the plasma membrane of the muscle fiber may be highly organized and involved in maintaining appropriate homeostasis in this actively contracting cellular system. In this review, we examine the currently known members of the muscle fiber plasma membrane cytoskeleton and the interactions that occur between the different members of this complex using histological, electron microscopic, and confocal methods. From our studies and others cited in this review, it is clear that the dystrophin cytoskeletal complex is not completely understood and component molecules continue to be discovered. Perhaps equally importantly, currently defined molecules (such as alpha-actinin or neuronal nitric oxide synthase) are being recognized as being specifically associated with the complex. What is striking from all of the studies, to date, is that while we are able to identify members of the dystrophin cytoskeletal complex and while we are able to associate mutations of individual molecules with disease(s), we are still unable to truly define the roles of each of the molecules in maintaining the normal physiology of the muscle fiber.     

Eye muscle sparing by the muscular dystrophies: lessons to be learned?

The devastating consequences of the various muscular dystrophies are even more obvious when a muscle or muscle group is spared. The study of the exceptional cell or tissue responses may prove to be of considerable value in the analysis of disease mechanisms. The small muscles responsible for eye movements, the extraocular muscles, have functional and morphological characteristics that set them aside from other skeletal muscles. Notably, these muscles are clinically unaffected in Duchenne/Becker, limb-girdle, and congenital muscular dystrophies, pathologies due to a broken mechanical or signaling linkage between the cytoskeleton and the extracellular matrix. Uncovering the strategies used by the extraocular muscles to "naturally" protect themselves in these diseases should contribute to knowledge of both pathogenesis and treatment. We propose that careful investigation of the cellular determinants of extraocular muscle-specific properties may provide insights into how these muscles avoid or adapt to the cascade of events leading to myofiber degeneration in the muscular dystrophies.     

Adenovirus mediated gene transfer to skeletal muscle

Transfer of therapeutic genes into muscle tissue has promise for the treatment of a variety of muscular dystrophies. Various vectors have been used to deliver genes to skeletal muscle but their application has faced several major limitations including: (1) the lack of transgene persistence caused by the immune rejection of transduced myofibers and/or vector toxicity, and (2) the maturation dependence of viral transduction. While the immunorejection and/or cytotoxic problems are being overcome with the development of new vectors, maturation-dependent viral transduction is still a major hurdle in gene transfer to skeletal muscle. Poor adenoviral transduction in mature myofibers has been attributed to: (1) the extracellular matrix of mature myofibers may form a physical barrier and prevent the passage of large viral particles; (2) viral receptors are down-regulated with muscle maturation; and (3) loss of myoblasts with muscle maturation, which serve as intermediaries in the viral transduction. In this review, we will focus on recent developments in overcoming those hurdles of gene therapy in skeletal muscle, especially to adenovirus (Ad), including: (1) new mutant vectors lacking all viral genes to decrease immunogenicity, and hence, improve persistence of transgene expression in muscle in vivo; (2) using tissue specific promoters to evade immunorejection; (3) permeabilization of the extracellular matrix; (4) modifying the viral receptors in mature myofibers; and (5) myoblast or muscle stem cell mediated ex vivo gene transfer.     

Role of nitric oxide and nitric oxide synthases in experimental models of denervation and reinnervation

Nitric oxide (NO) is a short-living free molecule synthesized by three different isoforms of nitric oxide synthases (NOS)—neuronal NOS, endothelial NOS, and inducible NOS—associated with neuromuscular transmission, muscle contractility, mitochondrial respiration, and carbohydrate metabolism in skeletal muscle. Neuronal NOS is constitutively expressed at the muscle fiber sarcolemma linked to the dystrophin-glycoprotein complex and concentrated at the neuromuscular endplate. There is increasing evidence that altered expression of neuronal NOS plays a role in muscle fiber damage in neuromuscular diseases such as dystrophinopathies and denervating disorders. Although there have been some previous conflicting results on the neuronal NOS expression pattern in denervated muscle fibers, it is now well established that denervation is associated with a down-regulation and disappearance of sarcolemmal neuronal NOS at synaptic/extrasynaptic or both sites. As NO has been shown to induce collapse and growth arrest on neuronal growth cones, down-regulation of sarcolemmal neuronal NOS may contribute to axonal regeneration and attraction to muscle fibers aiming at the formation of new motor endplates providing reinnervation and reconstitution of NOS expression. As NO serves as a retrograde messenger, it may trigger structural downstream events responsible for neuromuscular synaptogenesis and preventing polyneural innervation. Nevertheless, decreased NO production in denervation reduces the cytoprotective scavenger function of NO for superoxide anions promoting oxidative stress that is likely to be involved in muscle fiber damage and death. However, the multifaced role of NOS and NO under physiological and pathological conditions remains poorly understood on the basis of the current knowledge. Microsc. Res. Tech. 55:181–186, 2001. © 2001 Wiley-Liss, Inc.     

Inactivation of myosin heavy chain genes in the mouse: diverse and unexpected phenotypes

Myosin heavy chain (MyHC) is a critical component of the cellular contractile apparatus. The mammalian genome contains two nonmuscle, two smooth muscle, and eight striated muscle isoforms of MyHC. Within each class of genes, there is extremely high sequence homology among different MyHC isoforms, raising the question of whether these isoforms are functionally redundant or whether they perform unique roles in cell function. Recently, strains of mice null for four different MyHC isoforms have been generated. Mice null for the nonmuscle II-B isoform experience significant prenatal lethality and surviving animals have several cardiac abnormalities [Tullio et al. (1997) Proc Natl Acad Sci USA 94:12407-12412]. Mice homozygous null for alpha cardiac MyHC are embryonic lethal, while heterozygous mice are viable but also have numerous cardiac defects [Jones et al. (1996) J Clin Invest 98:1906-1917]. Mice null for IIb or IId adult skeletal MyHC are viable but have skeletal muscle abnormalities compared to wild type mice, despite compensation of a neighboring MyHC gene [Acakpo-Satchivi et al. (1997) J Cell Biol 139:1219-1229]. Both IIb and IId null mice show significant decreases in body mass. Mean muscle mass is also significantly decreased in both null strains but the extent and the pattern of affected muscles differs between the two strains. Both strains show evidence of skeletal muscle pathology but again the pattern and extent differ between the two strains. Finally, both adult skeletal strains demonstrate distinct impairments in contractile function when compared to wild type. Together these observations support the hypothesis that the different isoforms of MyHC are functionally unique and cannot substitute for one another.     

Myotonic dystrophy protein kinase of the cardiac muscle: evaluation using an immunochemical approach

Myotonic dystrophy (DM) is an inherited multisystem disorder characterized by the presence of a high polymorphic expansion of trinucleotide (CTG) repeat in the 3' untranslated region of the DM protein kinase (DMPK) gene. However, the role of myotonic dystrophy protein kinase (DMPK) has yet to be elucidated. Studies aimed to discover possible physiological targets of DMPK indicated several subcellular localization sites, such as neuromuscular junctions, myotendinous junctions, and terminal cisternae of the sarcoplasmic reticulum in the skeletal muscle and intercalated discs in the cardiac muscle. Here, we extend our previous observations on the localization of DMPK at gap junction (GJ) level in the heart, taking advantage of the polyclonal peptide-specific anti-DMPK antibodies raised against two different domains of the protein. DMPK was detected by immunofluorescence at the intercalated disc level by both antibodies. Double immunofluorescence staining experiments performed with each anti-DMPK and anti-connexin43 showed colocalization of the two antigens. Immunoblot analysis of partially purified GJs showed co-sedimentation of DMPK and connexin43. We conclude that GJs are a genuine localization site of DMPK. Given the known regulation exerted by protein kinases on assembly, trafficking, gating, and disassembly of connexins, such a localization may be relevant to the functional role of connexins. DM is the most common muscular dystrophy in adults, and is known by the cardiac involvement that is a common feature in DM patients. Localization of DMPK at GJ in relation to DM is also briefly discussed.     

Perisynaptic Schwann cells of the vertebrate motor endplate bear modified cilia

Perisynaptic Schwann cells (PSCs), descendants of the myelinating Schwann cells, cover the axon terminal of the vertebrate motor endplate of the skeletal muscle fiber. PSCs are assumed to support the function of the axon terminal. This function suggests a net material transport in the direction of the axon terminal. Morphologically it is to be expected that these cells have a cytoskeleton aligned to the axon terminal. Investigations clarifying this statement have not yet been undertaken. From previous investigations we know, however, that the PSCs have a microtubule-organizing center, which is a part of this cytoskeleton. The centrioles of the organizing center may also participate in the formation of a modified cilium structure whose function is unknown. In the present investigation, characteristic ultrastructural features of the modified cilium structure and its relationship to the Golgi apparatus and the axon terminal are presented. A function for the modified cilium structure is discussed.     

Variations of gait parameters in Duchenne muscular dystrophy

The gait parameters (speed, stride length and cadence) of nine boys with Duchenne muscular dystrophy were compared with those of 21 normal boys in the same age range. Differences found were due to the altered ability to control their dynamic state and, to a lesser extent, physical limitations of joint range. This simple method of quantifying gait is proposed as a way in which progression or response to treatment in muscular dystrophy might be monitored. The information obtained may alert the clinician to the fact that the progressive muscle weakness and joint contractures have begun to cause compensatory mechanisms during walking to fail. It is also useful to obtain information on gait in clinical treatment trials as there are very few reliable methods for testing function in muscular dystrophy.     

The role of 5′-adenosine monophosphate-activated protein kinase (AMPK) in skeletal muscle atrophy

As a key coordinator of metabolism, AMP-activated protein kinase (AMPK) is vitally involved in skeletal muscle maintenance. AMPK exerts its cellular effects through its function as a serine/threonine protein kinase by regulating many downstream targets and plays important roles in the development and growth of skeletal muscle. AMPK is activated by phosphorylation and exerts its function as a kinase in many processes, including synthesis and degradation of proteins, mitochondrial biogenesis, glucose uptake, and fatty acid and cholesterol metabolism. Skeletal muscle atrophy is a result of various diseases or disorders and is characterized by a decrease in muscle mass. The pathogenesis and therapeutic strategies of skeletal muscle atrophy are still under investigation. In this review, we discuss the role of AMPK in skeletal muscle metabolism and atrophy. We also discuss targeting AMPK for skeletal muscle treatment, including exercise, AMPK activators including 5-amino-4-imidazolecarboxamide ribonucleoside and metformin, and low-level lasers. These studies show the important roles of AMPK in regulating muscle metabolism and function; thus, the treatment of skeletal muscle atrophy needs to take into account the roles of AMPK.     

How to estimate the reliability of a weld joint under conditions of cyclic loading according to the results of a nondestructive check

The problem of how to determine the reliability of thin-walled structures as a function of the number of loading cycles and degree of inspection of the weld joints with blind cracklike holes, whose depth and length are independent random values, is solved. The reliability is characterized by the probability of whether the defect sizes belong to the admitted region, whose boundary is determined by the set of the initial sizes of the defects, which rises past the limit state of the predetermined number of loading cycles.     

Morphological and ultrastructural evaluation of the golden retriever muscular dystrophy trachea,lungs, and diaphragm muscle

Duchenne muscular dystrophy (DMD) is a genetic disease, characterized by atrophy and muscle weakness. The respiratory failure is a common cause of early death in patients with DMD. Golden retriever muscular dystrophy (GRMD) is a canine model which has been extensively used for many advances in therapeutics applications. As the patients with DMD, the GRMD frequently died from cardiac and respiratory failure. Observing the respiratory failure in DMD is one of the major causes of mortality we aimed to describe the morphological and ultrastructural data of trachea, lungs (conductive and respiratory portion of the system), and diaphragm muscle using histological and ultrastructural analysis. The diaphragm muscle showed discontinuous fibers architecture, with different diameter; a robust perimysium inflammatory infiltrate and some muscle cells displayed central nuclei. GRMD trachea and lungs presented collagen fibers and in addition, the GRMD lungs showed higher of levels collagen fibers that could limit the alveolar ducts and alveoli distension. Therefore, the most features observed were the collagen areas and fibrosis. We suggested in this study that the collagen remodeling in the trachea, lungs, and diaphragm muscle may increase fibrosis and affect the trachea, lungs, and diaphragm muscle function that can be a major cause of respiratory failure that occur in patients with DMD. Microsc. Res. Tech. 77:857–861, 2014. © 2014 Wiley Periodicals, Inc.     

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.     

Expression of Leu-19 (CD56, N-CAM) and nitric oxide synthase (NOS) I in denervated and reinnervated human skeletal muscle.

Neural cell adhesion molecule (N-CAM, Leu-19, CD 56) expression appears during muscle fiber regeneration and after denervation. Sarcolemma-associated nitric oxide synthase (NOS) I, however, disappears from denervated myofibers. The dynamics of expression of both proteins were studied in 5 cases of acute/subacute denervation, 28 cases of chronic denervation with and without collateral reinnervation, 5 cases of the intermediate type spinal muscular atrophy (SMA 2), and in 2 normal biopsies. NOS I and its NADPH diaphorase (NADPHd) activity disappeared from the sarcolemma region shortly after denervation, and before the appearance of denervation atrophy. N-CAM was found diffusely distributed in the sarcoplasm at the most severe phase of denervation atrophy in the majority of highly atrophic fibers. During reinnervation, NOS I expression remained absent and in part of the cases the target/targetoid phenomenon appeared. In parallel with the increase in volume of the reinnervated muscle fibers, the intensity of N-CAM immunoreactivity decreased progressively. After full restitution of muscle fiber caliber, the target/targetoid phenomenon and N-CAM immunostaining disappeared completely, and, finally, NOS I reappeared in the sarcolemma region. The sarcolemmal expression of dystrophin and dystrophin-associated proteins was unchanged during denervation. NOS I was completely absent in children with SMA 2, since the protein does not appear before 5 years of age in skeletal muscle, while N-CAM was very intensely expressed in the sarcoplasm of highly atrophic denervated muscle fibers. In conclusion, this study suggests that innervation is an important factor for selective gene expression and positioning of NOS I and N-CAM in skeletal muscle and gives practical information for the assessment of the phase and developmental stage of the denervation and reinnervation process.