The X-linked muscle-wasting disease Duchenne muscular dystrophy is caused by mutations in the gene encoding dystrophin. There is currently no effective treatment for the disease; however, the complex molecular pathology of this disorder is now being unravelled. Dystrophin is located at the muscle sarcolemma in a membrane-spanning protein complex that connects the cytoskeleton to the basal lamina. Mutations in many components of the dystrophin protein complex cause other forms of autosomally inherited muscular dystrophy, indicating the importance of this complex in normal muscle function. Although the precise function of dystrophin is unknown, the lack of protein causes membrane destabilization and the activation of multiple pathophysiological processes, many of which converge on alterations in intracellular calcium handling. Dystrophin is also the prototype of a family of dystrophin-related proteins, many of which are found in muscle. This family includes utrophin and α-dystrobrevin, which are involved in the maintenance of the neuromuscular junction architecture and in muscle homeostasis. New insights into the pathophysiology of dystrophic muscle, the identification of compensating proteins, and the discovery of new binding partners are paving the way for novel therapeutic strategies to treat this fatal muscle disease. This review discusses the role of the dystrophin complex and protein family in muscle and describes the physiological processes that are affected in Duchenne muscular dystrophy.

Function and genetics of dystrophin and dystrophin-related proteins in muscle

The repair process of damaged tissue involves the coordinated activities of several cell types in response to local and systemic signals. Following acute tissue injury, infiltrating inflammatory cells and resident stem cells orchestrate their activities to restore tissue homeostasis. However, during chronic tissue damage, such as in muscular dystrophies, the inflammatory-cell infiltration and fibroblast activation persists, while the reparative capacity of stem cells (satellite cells) is attenuated. Abnormal dystrophic muscle repair and its end stage, fibrosis, represent the final common pathway of virtually all chronic neurodegenerative muscular diseases. As our understanding of the pathogenesis of muscle fibrosis has progressed, it has become evident that the muscle provides a useful model for the regulation of tissue repair by the local microenvironment, showing interplay among muscle-specific stem cells, inflammatory cells, fibroblasts and extracellular matrix components of the mammalian wound-healing response. This article reviews the emerging findings of the mechanisms that underlie normal versus aberrant muscle-tissue repair.

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Aberrant repair and fibrosis development in skeletal muscle

Chelating agents were introduced into endodontics as an aid for the preparation of narrow and calcified root canals in 1957 by Nygaard-Ostby. A liquid solution of ethylenediaminetetraacetic acid (EDTA) was thought to chemically soften the root canal dentine and dissolve the smear layer, as well as to increase dentine permeability. Although the efficacy of EDTA preparations in softening root dentine has been debated, chelator preparations have regained popularity recently. Almost all manufacturers of nickel-titanium instruments recommend their use as a lubricant during rotary root canal preparation. Additionally, a final irrigation of the root canal with 15-17% EDTA solutions to dissolve the smear layer is recommended in many textbooks. This paper reviews the relevant literature on chelating agents, presents an overview of the chemical and pharmacological properties of EDTA preparations and makes recommendations for their clinical use.

Chelating agents in root canal treatment: mode of action and indications for their use.

Mutations in dystrophin cause Duchenne muscular dystrophy (DMD), but absent dystrophin does not invariably cause necrosis in all muscles, life stages and species. Using DNA microarray, we established a molecular signature of dystrophinopathy in the mdx mouse, with evidence that secondary mechanisms are key contributors to pathogenesis. We used variability controls, adequate replicates and stringent analytic tools, including significance analysis of microarrays to estimate and manage false positive rates. In leg muscle, we identified 242 differentially expressed genes, >75% of which have not been previously reported as altered in human or animal dystrophies. Data provide evidence for coordinated activity of numerous components of a chronic inflammatory response, including cytokine and chemokine signaling, leukocyte adhesion and diapedesis, invasive cell type-specific markers, and complement system activation. Selective chemokine upregulation was confirmed by RT-PCR and immunoblot, and may be a key determinant of the nature of the inflammatory response in dystrophic muscle. Up-regulation of secreted phosphoprotein 1 (minopontin, osteopontin) mRNA and protein in dystrophic muscle identified a novel linkage between inflammatory cells and repair processes. Extracellular matrix genes were up-regulated in mdx to levels similar to those in DMD. Since, unlike DMD, mdx exhibits little fibrosis, data suggest that collagen regulation at post-transcriptional stages mediates extensive fibrosis in DMD. Taken together, these data identify a relatively neglected aspect of DMD, suggest new treatment avenues, and highlight the value of genome-wide profiling in study of complex disease processes.

A chronic inflammatory response dominates the skeletal muscle molecular signature in dystrophin-deficient mdx mice

The effects of botulinum toxin-A was compared on both extrafusal and intrafusal muscle fibers in the biceps femoris of Wistar rats. Four days after injection no action potentials were elicited with stimulation single-fiber electromyography on the injected side. Fourteen days after injection, jitter became measurable and these values were increased on the injected side. Extrafusal muscle fibers began to atrophy on the 4th day and this continued to the 14th day postinjection. Atrophy was also evident and progressive in intrafusal muscle fibers. Increased terminal innervation ratios, end-plate spread of cholinesterase, and increased density of very small myelinated fibers in large intramuscular nerves were observed 14 days postinjection. Both extrafusal and intrafusal fibers are cholinergically innervated, and both were progressively affected by botulinum toxin, perhaps varying in degree only. In addition to partial denervation, Botulinum toxin effects in dystonia may also be related to modified spindle afferent discharge.

Extrafusal and intrafusal muscle effects in experimental botulinum toxin-A injection

The permeability of the normal muscle spindle capsule to the entrance of an exogenous protein tracer was assessed by the use of horseradish peroxidase (HRP). Adult mice were injected intravenously with a solution of HRP (MW 40,000). After varying intervals, ranging from two to 240 minutes, animals were perfused aortically with fixative, and anterior tibialis muscles were removed, processed, and examined. The results were evaluated by electron microscopic cytochemical techniques, and attention was directed to the movement of tracer and its relationship to the outer and inner capsule of the muscle spindle.



HRP was first demonstrated in cytoplasmic vesicles crossing the continuous capillary endothelium supplying the muscle spindle, and was then detected in the interstitial space surrounding the outer capsule. In polar regions, HRP had entered the periaxial space by two minutes, and it was seen in cytoplasmic vesicles of the attenuated outer capsule. Later, HRP abutted the sarcolemma of intrafusal fibers, and by 12.5 minutes there was evidence of tracer in T-tubules of these muscle cells. Whereas at no time was tracer observed traversing any of the numerous intercellular junctions of the capsule, it appeared that polar regions were leaky and open-ended at their distal portions.



The sensory equatorial zone was considerably less permeable to the entrance of tracer. Whereas HRP was visible initially in cytoplasmic vesicles of the subjacent capillary endothelium, it was not until 12.5 minutes that tracer could be demonstrated within some vesicles of equatorial outer capsule cells. At later times, a small amount of HRP was observed in the equatorial periaxial space, where it was phagocytosed and finally sequestered by cells of the inner capsule as membrane-bound lysosomal deposits. Consequently, at no time was tracer incorporated into either intrafusal fibers or their sensory nerve terminals.



Thus, in contrast to polar regions, the muscle spindle capsule in the equatorial zone appears to be effective in preventing the indiscriminate penetration of HRP from the bloodstream. This suggests dissimilar paths of tracer movement from the microvasculature into sensory and non-sensory regions of the muscle spindle.

Ultrastructural study of a blood-muscle spindle barrier after systemic administration of horseradish peroxidase.

Background: Mast cells are indigenous connective tissue cells that function in the process of inflammation and edema. Their numbers were studied in a quantitative morphological study of the soleus muscles from 32-week-old and 56-week-old normal and genetically dystro- phic dy 2J and mdx mice to determine the incidence of mast cells in muscle to increasing age and to normal and myopathic conditions. Methods: Soleus muscles from normal C57Bl/J and from dystrophic C57Bl/SnJ (dy 2J /dy 2J ) and C57BL/10ScSn mdx mice were processed for examination by light and electron microscopy. Quantitation of mast cells was performed on semi-thick sections and expressed as an average of cells per millimeter squared of muscle tissue. Results: Mast cells were observed in the connective tissue interstitium that normally separates skeletal muscle into fascicles. Their cytoplasmic granules stained metachromatically with toluidine blue and often ob- scured the single, centrally placed nucleus. They occurred singly or in small groups and were most frequently seen adjacent to neurovascular elements within the muscle, and in many cases were closely associated with the outer capsular regions of muscle spindles. Between the 32- and 56-week-old groups in each strain, an age-related increase in mast cell numbers was observed. In the dystrophic conditions, the dy 2J and mdx skeletal muscles exhibited a two- to four-fold increase in mast cells when compared to normals in both age groups. Extensive connective tissue proliferation and sites of necrotic and regenerating muscle were common features in both myopathies. Conclusions: Results of this study indicate that a significantly higher number of mast cells which exist in dy 2J and mdx murine skeletal muscles may be related to the high amount of connective tissue infiltration and extensive muscle fiber remodelling in these conditions. Moreover, the close proximity of mast cells to muscle spindles and nerve fascicles suggests that these cells may play a role in modulating their activities. Anat. Rec. 247:341-349, 1997. r 1997 Wiley-Liss, Inc.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/%28SICI%291097-0185%28199703%29247%3A3%3C341%3A%3AAID-AR5%3E3.0.CO%3B2-X

Quantitative Morphology of Mast Cells in Skeletal Muscle of Normal and Genetically Dystrophic Mice

The ultrastructure of inner capsule cells of the vertebrate muscle spindle was studied by transmission electron microscopy and compared with that of homologous cells in the tendon organ. Aside from variations in their complexity and pattern of organization, cells of the inner capsule in these two sensory receptors exhibited marked similarities in fine structure. The virtual absence of basal lamina in the region of the nucleated soma as well as on the branching cytoplasmic extensions of these cells was noted. In the inner capsule of both end organs, three kinds of intercellular specialization were encountered. Cell processes were typically linked together at multiple sites by intermediate junctions. In addition, focal points of membrane fusion between two or more cellular profiles were identified as tight junctions. In more extensive regions of plasma membrane overlap, gap junctions were also discerned. It seems probable that these sites along the inner capsule represent areas of mechanical and electrical linkage, enabling contiguous cells to function as a synchronous unit. Tight junctions may also provide the inner capsular sheath with specific permeability-barrier characteristics. Elements of the Golgi complex and associated presecretory vesicles and cytoplasmic granules were prominent. Their presence implicates these cells in the elaboration of the paracellular connective-tissue matrix occupying the intracapsular spaces of both receptors. The close resemblance of these cells to endoneurial fibroblasts of peripheral nerve and to hyalocytes of the vitreous body is emphasized. It is likely that, regardless of species examined, cells of the inner capsule in both receptors play an overall protective role in the formation, maintenance and regulation of their luminal paracellular contents.

Comparative ultrastructure of the inner capsule of the muscle spindle and the tendon organ

Muscle spindles in the soleus of 1-year-old dystrophic mice of the C57BL/6J dy2J/dy2J strain were studied by microscopic and morphometric methods, and comparisons were made with those in age-matched normal tissue. Transverse epon sections were cut through various regions of an individual receptor, and subsequent 90 degrees reorientation enabled longitudinal examination of the same spindle. In dystrophy, alterations were detected in the outer capsule and consisted of a significant increase in its overall thickness in equatorial regions. Perineurial proliferation accompanied histiocyte and collagen infiltration. Within the equator, intrafusal fibers and sensory terminals appeared unaffected by dystrophy. Alterations in the intrafusal fibers were restricted to polar zones where the mean diameters of chain and bag fibers were significantly reduced. Polar chain fibers exhibited a greater degree of atrophy in dystrophy with a 40% diminution in size. Ultrastructural changes in intrafusal fiber polar regions were less pronounced compared to the surrounding dystrophic muscle. Mitochondrial alterations in affected intrafusal fibers included intramatrix inclusions and glycogen deposition. Vacuolization of the sarcoplasmic reticulum and subsarcolemmal tubular aggregates were also observed in polar regions of dystrophic chain fibers. Regional variation in spindle involvement in advanced murine dystrophy provides evidence that the equatorial contents of this receptor are sequestered from the deleterious effects of the disease. Capsular thickening in the equator may be an adaptive response, preventing the intrafusal fibers from undergoing the moderate change and atrophy observed at their polar ends.

Alterations in muscle spindle morphology in advanced stages of murine muscular dystrophy

Muscle spindles in 2 synergistic avian skeletal muscles, the anterior (ALD) and posterior (PLD) latissimus dorsi, were studied by light and electron microscopy to determine whether morphological or quantitative differences existed between these sensory receptors. Differences were found in the density, distribution and location of muscle spindles in the 2 muscles. They also differed with respect to the morphology of their capsules and intracapsular components. The slow ALD possessed muscle spindles which were evenly distributed throughout the muscle, whereas in the fast PLD they were mainly concentrated around the single nerve entry point into the muscle. The muscle spindle index (number of spindles per gram wet muscle weight) in the ALD was more than double that of its fast-twitch PLD counterpart (130.5±2.0 vs 55.4±2.0 respectively, n=6). The number of intrafusal fibres per spindle ranged from 1 to 8 in the ALD and 2 to 9 in the PLD, and their diameters varied from 5.0 to 16.0 μm and 4.5 to 18.5 μm, respectively. Large diameter intrafusal fibres were more frequently encountered in spindles of the PLD. Unique to the ALD was the presence of monofibre muscle spindles (12.7% of total spindles observed in ALD) which contained a solitary intrafusal fibre. In muscle spindles of both the ALD and PLD, sensory nerve endings terminated in a spiral fashion on the intrafusal fibres in their equatorial regions. Motor innervation was restricted to either juxtaequatorial or polar regions of the intrafusal fibres. Outer capsule components were extensive in polar and juxtaequatorial regions of ALD spindles, whereas inner capsule cells of PLD spindles were more numerous in juxtaequatorial and equatorial regions. Overall, muscle spindles of the PLD exhibited greater complexity with respect to the number of intrafusal fibres per spindle, range of intrafusal fibre diameters and development of their inner capsules. It is postulated that the differences in muscle spindle density and structure observed in this study reflect the function of the muscles in which they reside.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1469-7580.1999.19430381.x

Pierre R. Dow

Papers

Ultrastructural study of a blood-muscle spindle barrier after systemic administration of horseradish peroxidase.

Quantitative Morphology of Mast Cells in Skeletal Muscle of Normal and Genetically Dystrophic Mice

Comparative ultrastructure of the inner capsule of the muscle spindle and the tendon organ

Alterations in muscle spindle morphology in advanced stages of murine muscular dystrophy

Structure, distribution and innervation of muscle spindles in avian fast and slow skeletal muscle