An electron-microscopic study of the T-system in progressive muscular dystrophy (Duchenne) using lanthanum

doi:10.1016/0022-510X(80)90124-0

Journal of the Neurological Sciences

Volume 44, Issues 2–3, January 1980, Pages 161-168

https://doi.org/10.1016/0022-510X(80)90124-0 Get rights and content

Abstract

Muscle biopsies in 4 cases of Duchenne muscular dystrophy and two normal controls were examined by electron microscopy using lanthanum as a tracer for assessment of the T-system.

Lanthanum was localized in the extracellular spaces, the basement membranes, intercellular spaces between muscle fibers and satellite cells, subsarcolemmal vesicles (caveola), motor end-plate, and the T-system in the controls. The T-system was sometimes connected with the caveolae, and was located at right angles to the myofibrils in the controls.

In Duchenne muscular dystrophy, the T-system showed irregularities of position, tangle formations and dilatation of the diameters when observed using lanthanum as a tracer, even in muscle fibers which under light- and electron-microscopic examinations did not show any changes. Structure of the triad was destroyed and lanthanum was localized in the sarcoplasmic reticulum (SR).

The changes of the T-system and triad seemed to play an important role in the high level of creatine phosphokinase (CPK) in the serum of Duchenne muscular dystrophy patients who do not show any clinical symptoms. Because of the destruction of the triad, CPK will easily flow out to the exterior of muscle fibers via the T-system.

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Introducing a mammalian nerve-muscle preparation ideal for physiology and microscopy, the transverse auricular muscle in the ear of the mouse
2020, Neuroscience
Citation Excerpt :
The first is ruthenium red, a colloidal substance that stains intensely with osmium during the normal workup of muscles for EM (Blanquet, 1976a, 1976b; Dierichs, 1979) and thus dramatically displays T-tubule distribution (Goldstein, 1969; Baldwin, 1970, 1981; Zampighi et al., 1975; Forbes and Sperelakis, 1979). The second compound used for this purpose (a colloid whose molecular weight is totally unknown and is not likely to be uniform, but which is intrinsically electronic-dense) is lanthanum hydroxide (Zacks and Saito, 1970; Fahimi and Cotran, 1971; Oguchi and Tsukagoshi, 1980; Oguchi et al., 1982; Sonoda et al., 1993; Dauber et al., 2000), originally used to stain the extracellular spaces in brain (Doggenweiler and Frenk, 1965; Brightman and Reese, 1969; Gobel, 1971; Sotelo and Llinas, 1972), and most famous for having percolated into electrical synapses and demonstrating the fundamental structure of the “gap junction” (the 'gap' in fact being the space accessible to the La +++ colloid, (Revel and Karnovsky, 1967; Goodenough and Revel, 1970; McNutt and Weinstein, 1970; Peracchia, 1973). This colloid can do a magnificent job of staining muscle T-tubules, but does not easily penetrate most muscles' connective-tissue capsules, so has not been used in recent decades; however, we have found that it penetrates readily into mouse ear muscles (not shown).
A new mammalian neuromuscular preparation is introduced for physiology and microscopy of all sorts: the intrinsic muscle of the mouse ear. The great utility of this preparation is demonstrated by illustrating how it has permitted us to develop a wholly new technique for staining muscle T-tubules, the critical conductive-elements in muscle. This involves sequential immersion in dilute solutions of osmium and ferrocyanide, then tannic acid, and then uranyl acetate, all of which totally blackens the T-tubules but leaves the muscle pale, thereby revealing that the T-tubules in mouse ear-muscles become severely distorted in several pathological conditions. These include certain mouse-models of muscular dystrophy (specifically, dysferlin-mutations), certain mutations of muscle cytoskeletal proteins (specifically, beta-tubulin mutations), and also in denervation-fibrillation, as observed in mouse ears maintained with in vitro tissue-culture conditions. These observations permit us to generate the hypothesis that T-tubules are the “Achilles' heel” in several adult-onset muscular dystrophies, due to their unique susceptibility to damage via muscle lattice-dislocations. These new observations strongly encourage further in-depth studies of ear-muscle architecture, in the many available mouse-models of various devastating human muscle-diseases. Finally, we demonstrate that the delicate and defined physical characteristics of this 'new' mammalian muscle are ideal for ultrastructural study, and thereby facilitate the imaging of synaptic vesicle membrane recycling in mammalian neuromuscular junctions, a topic that is critical to myasthenia gravis and related diseases, but which has, until now, completely eluded electron microscopic analysis. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.
Impairment of caveolae formation and T-system disorganization in human muscular dystrophy with caveolin-3 deficiency
2002, American Journal of Pathology
Citation Excerpt :
In addition, some of the large vacuolated structures showed continuity with the plasma membrane, possibly representing abnormal caveolae-like membrane invaginations or disrupted T-tubule openings. Furthermore, some of the vacuolated structures were also associated with lanthanum positive honeycomb structures, a known indicator of abnormal proliferation of the T-tubule system (Figure 4D).17,18 The described abnormalities were seen in all LGMD-1C muscle samples.
Caveolin-3, a muscle specific caveolin-related protein, is the principal structural protein of caveolar membranes. We have recently identified an autosomal dominant form of limb girdle muscular dystrophy (LGMD-1C) that is due to caveolin-3 deficiency and caveolin-3 gene mutations. Here, we studied by electron microscopy, including freeze-fracture and lanthanum staining, the distribution of caveolae and the organization of the T-tubule system in caveolin-3 deficient human muscle fibers. We found a severe impairment of caveolae formation at the muscle cell surface, demonstrating that caveolin-3 is essential for the formation and organization of caveolae in muscle fibers. In addition, we also detected a striking disorganization of the T-system openings at the sub-sarcolemmal level in LGMD-1C muscle fibers. These observations provide new perspectives in our understanding of the role of caveolin-3 in muscle and of the pathogenesis of muscle weakness in caveolin-3 deficient muscle.
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Behavior of sarcotubular system formation in experimentally induced regeneration of muscle fibers
1984, Journal of the Neurological Sciences
To examine the behavior of transverse (T)-tubule formation in experimentally-induced regenerating fibers, a local anesthetic, bupivacaine hydrochloride, was injected directly into the rat soleus muscle to cause myonecrosis. The regenerating fibers following necrosis were then examined by electron microscopy using lanthanum nitrate which clearly demonstrated the sarcotubular system. In the early stage of regeneration within 7 days after muscle necrosis, the T-tubules seemed to be composed of markedly proliferated subsarcolemmal caveolae with occasional honeycomb structure formation. Around 10 days, the T-tubules in regenerating fibers were tortuously and irregularly arranged with focal dilatation in diameter, and extended longitudinally along the axis of well organized myofibrils. As the regenerating fibers matured, the sarcotubular system, irregular in course and in shape, gradually became organized into a regular transverse position against the myofibrils, along with a marked decrease in longitudinally arranged tubular components. These morphological findings of the early T-tubule formation seen in the present study were similar to those found in early myogenesis, and in biopsied muscles from cases of polymyositis and progressive muscular dystrophy.
Ultrastructural changes in muscle and motor end-plate of the dystrophic mouse
1983, Experimental Neurology
A comparative study of dystrophic mice ( $C57BL 6J -dy^{2J}$ ) and normal littermates, 6 to 9 months old, has revealed numerous ultrastructural changes in the dystrophic soleus. Vacuoles, swollen mitochondria, vesicular aggregates, membranous bodies, Z-line degradation, localized hypercontraction, myofibrillar disorientation, and focal necrosis were common. We report here new observations. Discontinuous, dense bands (240 Å wide) were observed rarely between inner and outer membranes in some nuclei. Closely associated with infoldings of nuclear membrane were paracrystalline, vesicular structures, actin-like filaments, and autophagic vacuoles. With the degeneration of the nuclear membrane, actin-like filaments were observed within the nucleus. The primary synaptic clefts were widened and contained osmophilic-dense granules (diameter 0.2 to 0.7 μ). There was loss of secondary synaptic folds. Discontinuity of pre- and postsynaptic membranes could be observed, though rarely. Lysosome-like dense bodies were present among myofilaments. The myeloid bodies and autophagic vacuoles that were associated with nuclei, sarcoplasmic reticulum, and mitochondria gave a positive acid phosphatase reaction. Such degenerative changes were not observed in the normal soleus.
Plasma acetylcholinesterase in duchenne muscular dystrophy
1983, Experimental Neurology
Muscle acetylcholinesterase (AChE) is unregulated in animal and human muscular dystrophies and its activity is elevated in plasma of dystrophic chickens, probably due to a leakage from affected muscles. It is possible to measure AChE activity in human plasma in spite of high butyrylcholinesterase activity if acetyl-β-methylcholine is used as the substrate and butyrylcholinesterase is inhibited by iso-OMPA. It has been found that, unlike in chickens, the plasma AChE activity in human newborns is not higher than that in adults. The AChE activity in plasma of children afflicted by Duchenne muscular dystrophy does not differ from that found in plasma of normal boys of the same age. In this respect Duchenne muscular dystrophy differs from chicken muscular dystrophy as well as from a neurogenic muscle disease (amyotrophic lateral sclerosis) in man.

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An electron-microscopic study of the T-system in progressive muscular dystrophy (Duchenne) using lanthanum

Abstract

Clin. Chim. Acta

Progr. Biophys. Mol. Biol.

Electron microscope study of sectioned breast muscle of domestic fowl

Amer. J. Anat.

Study of sarcolemmal integrity in myopathic muscle

Neurology (Minneap.)

Comparative study of the T-system in various neuromuscular diseases

Neurology (Minneap.)

Anastomosis of transverse tubules with terminal cisternae in polymyositis

Neurology, (Minneap.)

Entry of a dye into the sarcotubular system of muscle

Nature (Lond.)

Permeability studies in heat-induced injury of skeletal muscle using lanthanum as fine structural tracer

Amer. J. Path.

Sarcolemmal invaginations and the T-system in fish skeletal muscle

Nature (Lond.)