Calcium-mediated myopathy at neuromuscular junctions of normal and dystrophic muscle

Previous studies showed that a myopathy which can be produced in vertebrate muscle by inactivating esterases is mimicked by exposure to carbamylcholine and requires agonist-receptor interaction and extracellular calcium. A most consistent aspect of the myopathy is dissolution of Z-disks in myofibrils near the postsynaptic membrane. Using mouse extensor digitorum longus (EDL) muscles in vitro, we found that leupeptin partially protects the Z-disks from dissolution. Chloroquine had much less, if any, effect. These data are compatible with the suggestion that prolonged agonist action at the neuromuscular junction results in the activation of the calcium-activated protease known to destroy the Z-disk protein. Because dystrophic mouse muscles reportedly have increased activities of calcium-activated proteases, we compared the response of normal and dystrophic EDL muscle. These muscles showed no significant difference after 3 h in Krebs baths, but when carbachol was added, there was a significantly greater amount of Z-disk damage in dystrophic muscles than in muscles from wild types (129 ReJ) or from albino mice. As in normal muscle, the agonist-induced myopathy in dystrophic muscle is both calcium- and protease dependent.     

Repeated stimulation of the dystrophic mouse neuromuscular junctions

Morphometric changes produced by repeated stimulation of the neuromuscular junctions (NMJs) have been studied by electron microscopy in 769 profiles of normal, heterozygous dystrophic, and dystrophic mice. The study indicates that in animals not stimulated but anesthetized for 40 min, there is no significant difference in the profile area between the normal, the heterozygous dystrophic, and dystrophic mice. There is also no significant difference between these three groups in the number of clear vesicles and of coated vesicles per micron2 of terminal. Following stimulation, the profile area of NMJs is not significantly increased in the normal and in the heterozygous dystrophic mice. However, the profile area is increased by 50% in the dystrophic animals. This is perhaps due to changes in the shape of the nerve terminal in stimulated dystrophic animals. However, the number of the clear vesicles per micron2 is significantly reduced to a similar extent in the three groups. These results indicate that the recycling of vesicular membrane is normal in dystrophic animals.     

Fragmentation analysis of normal and dystrophic avian muscle

A difference between normal and dystrophic avian muscle was demonstrated by comparing the patterns of fragmentation of muscle during homogenization. Fragmentation was monitored by morphological methods and by viscometry. This method of fragmentation analysis depends on the principle that the viscosity of a suspension is an exponential function of the partial volume fraction occupied by the suspended particles; the more a tissue is fragmented into smaller pieces, the greater the viscosity of the resulting homogenate. Increasing the duration of homogenization of either fresh muscle in buffer or glycerinated muscle in relaxing solution gradually increased the viscosity of the homogenate of normal muscle, whereas the viscosity of the homogenate of dystrophic muscle remained approximately constant. This difference in viscosity indicated that dystrophic muscle was sheared into larger pieces which were resistant to further fragmentation. Electron microscopic examination showed that the homogenate of dystrophic muscle contained rows of sarcomeres, whereas normal muscle was sheared into amorphous masses of myofilaments.     

Electrophysiologic differences between normal and dystrophic avian muscle

In the avian model of muscular dystrophy, electrophysiologic studies have shown alterations in the action potential characteristics of dystrophic muscle in vitro, supporting the notion that a membrane defect exists in avian dystrophy. As neurogenic and vascular etiologies have also been proposed, we examined the characteristics of action potentials recorded in a novel in vivo preparation of the extensor digitorum communis muscle in 8-week-old normal and dystrophic chickens. To facilitate intracellular recording, dantrolene sodium was used to attenuate the muscle twitch. Results showed that although the resting membrane potential, action potential amplitude and the action potential maximum rate of rise were similar in normal and dystrophic cells, the action potential duration at half the maximum amplitude was increased in dystrophic cells. This observation has not been previously reported for dystrophic avian muscle and suggests that a defect in the sarcolemmal potassium conductance is an early change in dystrophic avian muscle.     

Molecular phenotyping of the mouse ky mutant reveals UCP1 upregulation at the neuromuscular junctions of dystrophic soleus muscle

The ky mutant mouse displays a muscular dystrophy that affects almost exclusively slow type muscles in which persistent muscle regeneration, neuromuscular junction instability and an absence of the hypertrophic response are prominent features. In order to gain insights into the pathogenesis of this muscular dystrophy we have undertaken RNA profiling of the extensor digitorum longus, a fast unaffected muscle, and the highly pathological soleus slow muscle, followed by further expression studies to validate the results. In dystrophic soleus, there is a coordinated change in the expression level of genes encoding energy transducing mitochondrial proteins and an increase in the expression of stretch response genes. Upregulation of uncoupling proteins 1 and 2 is a unique molecular signature of the ky muscular dystrophy and was further characterised at the protein level. Our results show a spatial and temporal association between disorganisation of acetylcholine receptor clusters and upregulation of uncoupling protein 1. There is also evidence of a breakdown of neuromuscular junction muscle-specific kinase-dependent signalling in adult mutant soleus. Sarcolemma-associated proteins implicated in muscular dystrophies revealed no differences on microarrays and were confirmed as normally distributed by immunofluorescence. Altogether, the data presented suggest that the ky muscular dystrophy develops by a distinctive pathogenic mechanism.     

Single-fiber electromyography for studying the effect of betamethasone on normal neuromuscular junctions in man

The effects of steroids on normal end-plate in vivo were evaluated in man by using single-fiber electromyography (SFEMG). SFEMG was performed on voluntarily activated extensor digitorium communis muscle under basal conditions and 1 week later an intramuscular injection of 4 mg betamethasone per day was administered for 7 days. Ten patients with low back pain were examined. Steroid therapy did not induce any statistically significant variation either of the mean jitter value or of the percentage of recordings with abnormally high jitter. The results of this study do not give evidence of a direct effect of steroids on normal neuromuscular junction.     

Inactivity-induced remodeling of neuromuscular junctions in rat diaphragmatic muscle

We hypothesized that inactivity-induced remodeling of neuromuscular junctions (NMJs) depends on fiber type and the match between muscle fiber and motoneuron (MN) activities. Two inactivity models were studied in rat diaphragmatic muscle: spinal hemisection at C2 (SH), where both diaphragmatic muscle fibers and phrenic MNs were inactive, and tetrodotoxin (TTX) nerve blockade, where only muscle fibers were inactive. After 2 weeks of inactivity, there was increased number of pre- and postsynaptic branches (fragmentation) of NMJs at type IIx/b fibers in both models. In addition, planar NMJ areas at type IIx/b fibers in the SH model were enlarged. In contrast, NMJs at type I and IIa fibers were unaffected in both SH and TTX models. Functionally, neuromuscular transmission in diaphragmatic muscle fibers improved in the SH model, but worsened in the TTX model, compared to controls. These results suggest that NMJ remodeling depends on the level of MN activity. The relative preservation of NMJs at type I and IIa fibers suggests a potential for recovery from diaphragmatic paralysis in the clinical setting, at least for respiratory behaviors.     

Abnormal neuromuscular junctions in the lateral rectus muscle of wobbler mice

We have studied the lateral rectus muscles and neuromuscular junctions (NMJs) of abducens motoneurons in wobbler (wr/wr) mutant mice from 26 to 58 days of age. The muscles of wr/wr weighed about 70% of the weight of littermate controls and were composed of fiber types comparable to those of controls, as assayed by succinate dehydrogenase activity. The most obvious difference between wr/wr and control NMJs was a reduction in the length of the postjunctional membrane of wr/wr mice. The mutant muscle endplate membrane was only about 70% (6.58 micron) the length of control muscle regions (9.44 micron). There were no obvious differences at the light microscopic level in the distribution of acetylcholine (ACh) receptors at junctional regions or staining of acetylcholinesterase, as assayed with alpha-bungarotoxin binding or enzyme histochemistry. Indirect immunocytochemical studies using antibodies directed against the subunits of the ACh receptor failed to indicate an abnormal presence of immature receptors clustered at the NMJs of wr/wr mice. Our findings suggest that the formation or maintenance of normal postjunctional folds and the differentiation of receptors at the junctions are under independent control during development. Furthermore, the wobbler mutation may affect muscle cell differentiation as well as neuronal differentiation. This mutant mouse should prove a useful model for study of postjunctional fold formation and function.     

The role of collagen crosslinking in the increased stiffness of avian dystrophic muscle

The resting tension and stiffness in the range of sarcomere lengths 2.4-3.6 microns were studied in highly inbred normal and dystrophic chicken pectoral muscle bundles, and the results were compared with the collagen content and the extent of crosslinkage of the collagen. All parameters increased in the order normal homozygote (003/003) less than heterozygote (003/433) less than dystrophic homozygote (433/433) chickens, with the data from the heterozygotes being halfway between the two homozygotes, thus exhibiting a semi-dominant inheritance pattern. In separate experiments, lathyrism was induced by treating normal (412/412) and dystrophic (413/413) chickens with alpha-acetoaminonitrile, an inhibitor of lysyl oxydase, the enzyme responsible for the initiation of collagen crosslinkage formation. These experiments showed that the tension and stiffness in response to passive stretch did not change with lathyrism in normal muscles, whereas the tension and stiffness decreased significantly with lathyrism in dystrophic muscles. The collagen content did not change with lathryrism in both normal and dystrophic muscles. These results indicate that the increased content of collagen crosslinkages is the basis for the increased resting tension and stiffness in the dystrophic muscles of the chicken, and that the effects can be reversed by treatment with an inhibitor of collagen crosslinkage formation.     

The location of neuromuscular junctions on regenerating adult mouse muscle in culture

The location of neuromuscular junctions which form in vitro between regenerating adult mouse muscle fibres and sections of embryonic mouse spinal cord was examined. The position of the original motor end-plates on the explanted muscle fibres was determined by using either rhodamine-labelled α-bungarotoxin (RαBT) binding to the acetylcholine receptors, or by stains to demonstrate acetylcholinesterase (AChE) also located at the end-plate. In this culture system, the explanted muscle fibres degenerate and regenerate to form new myotubes which develop cross-striations and contractions. The location of the newly-formed neuromuscular junctions in these mature cultures was then demonstrated using RαBT-binding to acetylcholine receptors, silver impregnation and cholinesterase techniques. Less than half the new neuromuscular junctions were at the original end-plate areas indicating that, at least in this system, junctions can form at sites other than those of the original end-plate.     

A new stain for quantitative measurement of sprouting at neuromuscular junctions

A new combined stain for the simultaneous demonstration of motor nerve terminals and cholinesterase at neuromuscular junctions is described. It employs bromoindoxyl acetate dye-staining for cholinesterase and silver-gold impregnation for nerve terminals. The clarity and reliability of the stain permit quantitative measurements of neuromuscular junctions in order to evaluate nerve terminal sprouting as well as other pathological changes. The method is rapid, reproducible, and simple, and it is well suited for the processing of large numbers of frozen sections.     

Dystrophin and a dystrophin-related protein in intrafusal muscle fibers,and neuromuscular and myotendinous junctions

Summary To determine whether or not and how dystrophin exists in neuromuscular junctions (NMJs) and myotendinous junctions (MTJs), we studied the mid-belly and peripheral portions of control and mdx muscles, immunohistochemically and immunoelectrophoretically, using six kinds of polyclonal antibodies, and an antibody against a dystrophin-related protein (DRP). In controls these regions and the polar region of intrafusal muscle fibers showed a rather clearer immunohistochemical dystrophin reaction than those of extrafusal muscle fibers with all antibodies used. In the muscles of mdx mice NMJs only showed a positive dystrophin reaction with the c-terminal antibody, that is, no reaction with the other five antibodies, and MTJs in mdx showed a positive reaction with the c-terminal antibody and a faint to negative reaction with the other five antibodies. In biopsied human muscles NMJs and MTJs also showed a clear reaction with all ten antibodies, i.e., six polyclonal and four monoclonal ones. Although an immunohistochemical DRP reaction was clearly seen at NMJs, only a faint or no reaction was seen on MTJs and on intrafusal muscle fibers in both mouse and human materials. Western blot analysis of control mouse muscle for dystrophin showed a clearer band for the peripheral portion, which contains many MTJs, than for the mid-belly portion. These data suggest that dystrophin really exists on MTJs, and that dystrophin and DRP exist on NMJs in mouse and human muscles.     

Skeletal muscle lysosomes: Comparison of lysosomes from normal and dystrophic avian pectoralis muscle as a function of age

The properties of skeletal muscle lysosomes from normal and dystrophic chickens were studied to assess their involvement in the dystrophic process. A method is described for isolation of a three-to-sevenfold purified lysosome fraction with 29–33% yield. Lysosomal enzymes in crude homogenates and isolated lysosome-enriched fractions from dystrophic muscle exhibit decreased latency for N-acetyl β-D -glucosaminidase, acid phosphatase, and cathepsin D. However, no differences in the fragility of lysosomes in isolated lysosome-enriched fractions from normal and dystrophic muscle were observed using shear, sonication and detergent stress. Lower percent recovery, enrichment factor and percent latency of acid phosphatase compared to N-acetyl-β-D -glucosaminidase and cathepsin D were observed from both normal and dystrophic muscle. These results are consistent with the presence of a significant amount of nonlysosomal acid phosphatase activity in skeletal muscle.     

Increased resistance of the collagen in avian dystrophic muscle to collagenolytic attack: evidence for increased crosslinking

Fibrous collagen from normal (line 412) and dystrophic (line 413) chicken pectoral muscles was examined for susceptibility to bacterial collagenase by a pH-stat titration method to assess the kinetics of peptide bond hydrolysis. These experiments showed that there was a profound difference in the kinetics of hydrolysis using fibrous collagen purified from normal and dystrophic muscle. Although normal collagen was readily hydrolyzed by the collagenase, dystrophic collagen was not hydrolyzed in the presence of 200 microM Ca and was hydrolyzed slowly in the presence of 5 mM Ca. At this high Ca concentration, the Km for collagenase was six times higher in dystrophic collagen than in normal, whereas the maximum reaction rate (Vmax) remained the same. After CNBr digestion, peptides from both sources were readily hydrolyzed by collagenase in the presence of 200 microM Ca, and there was no noticeable difference in the reaction kinetics. The above results were not modified whether the chicken was sacrificed on day 4 or day 98 post-hatching, and the insusceptibility to collagenase preceded the clinical manifestations of dystrophy. The structure of both fibrous and CNBr-treated collagen from normal and dystrophic muscle was compared. One and two-dimensional gel electrophoreses showed identical peptide maps. Gel filtration of CNBr peptides showed in the excluded volume the presence of twice as much high-molecular-weight (presumably crosslinked) peptides from dystrophic collagen compared with normal. We conclude that the intermolecular crosslinks are more extensively formed in dystrophic collagen and that this increased crosslinking results in the increased resistance of the dystrophic collagen to collagenase.     

Congenital neuromuscular disease with uniform type-1 fibers, presenting early stage dystrophic muscle pathology

We report two male siblings presenting with severe hypotonia, generalized muscle atrophy, multiple joint contractures and respiratory failure. The serum creatine kinase levels were within normal limits, 75 IU/l in the younger boy and 123 IU/l in the older one. Muscle biopsies at the age of 28 days in the younger boy and 48 days in the older one revealed dystrophic pathology with increased interstitial fibrous tissue, scattered basophilic fibers and an increased number of undeveloped type-2C fibers. Although the elder brother died from respiratory failure at 4 months of age, the younger child has been sustained with mechanical ventilation, and has been exhibiting non-progressive muscle symptoms. Upon re-biopsy of the younger sibling at the age of 3 years, neither basophilic regenerating fibers nor degenerating fibers were found. All muscle fibers were found to be extremely atrophic and behaved mostly like type-1 fibers, displaying the features of congenital neuromuscular disease with uniform type-1 fibers. Since early biopsies in congenital myopathies reveal numerous undifferentiated immature muscle fibers, it is difficult to make a definite diagnosis, unless we recognize disease-specific cytoplastic abnormalities of nemaline body formation and abnormalities of core structure.     

A diencephalic slice preparation and chamber for studying neuronal thermosensitivity

An in vitro slice preparation and electrophysiological recording chamber for studying neuronal thermosensitivity throughout the hypothalamus are described. A series of eight, 300 microns thick, horizontal tissue slices encompassing most of the hypothalamus are prepared from male Sprague-Dawley rats. Horizontal slice maps showing the major nuclei and fiber tracts are provided. Horizontal tissue slices contain many hypothalamic nuclei as well as the medial forebrain bundle, a large fiber tract interconnecting these nuclei. Three water-perfused thermodes directly beneath the tissue slices are used to produce discrete thermal stimulations of rostral, middle, and caudal nuclear regions. Fine thermocouples monitor slice temperature over each thermode. Limiting microelectrode explorations to regions directly over a thermode eliminates the problems of temperature gradients and permits more accurate manipulation of temperature at the recording site. While this preparation is ideal for characterizing hypothalamic neuronal thermosensitivity, it is also appropriate for electrophysiological studies of other hypothalamic functional systems.     

Comparison of the development of isometric contractile properties of embryonic avian normal and dystrophic skeletal muscle

Embryonic posterior latissimus dorsi (PLD) muscles were isolated at 24-h intervals between days 14 and 20 in ovo from a line of normal chickens (412) and a line afflicted with hereditary muscular dystrophy (413), and their isometric contractile properties were compared. The results demonstrated differences in the isometric contractile responses between normal and dystrophic embryonic PLD muscles. The normalized twitch and tetanic tensions were significantly less for the dystrophic muscle immediately before hatching. Some kinetics of the isometric responses were also different between normal and dystrophic muscles. At embryonic day 16 the times to one-half peak twitch tension, to peak twitch tension, and to one-half peak tetanic tension were significantly longer for the dystrophic muscles. The maximum rate of tetanic force development at days 14, 16, and 18 was lower in the dystrophic muscles. At embryonic day 18 the twitch relaxation of the dystrophic muscle was significantly slower. The results indicated that as early as the final week in ovo, the dystrophic PLD produced less tension and, in some respects, was slower than the normal muscle. Moreover, the differences in the kinetics of the responses were transient, i.e., differences in the kinetics that were observed at day 16 in ovo were not seen closer to hatching.     

Stiffness and contractile properties of avian normal and dystrophic muscle bundles as measured by sinusoidal length perturbations

Both tension and stiffness as a function of muscle length were measured under relaxing conditions on isolated small bundles of chemically skinned myofibers from normal and dystrophic chicken pectoral muscles. It was shown that the dystrophic muscle was stiffer than normal muscle and developed more tension for the same amount of stretch. A fraction of stiffness was not removed by extraction with either 0.6 M KI or with 5 M guanidine HCl mixed with 1% mercaptoethanol. The stiffness of dystrophic muscle was also unaffected by treatment with bacterial collagenase under conditions that destroyed the stiffness of tendon. Nyquist plots of normal and dystrophic muscles during calcium-activated isometric contraction were very similar and were characteristic of fast-twitch muscle, as evidenced by three clear exponential processes. The normal appearance of the Nyquist plot of dystrophic muscle demonstrates that cross-bridge function is not altered, and the characteristic slowing of contraction and relaxation is not a consequence of a fast-to-slow transformation of muscle types. The increased stiffness of dystrophic muscle may be a very fundamental change in the biomechanics of dystrophy. We postulate that the stiffness is mediated by an altered form of collagen, which is collagenase-resistant by virtue of excessive crosslinking.