Distribution of the ten known laminin chains in the pathways and targets of developing sensory axons

Laminins are heterotrimers of α, β and γ chains. At present, five α, three β, and two γ chains have been described. The best characterized laminin (laminin 1 = α1, β1, γ1) promotes neurite outgrowth from virtually all classes of developing neurons, implying that laminins may serve as axon guidance molecules in vivo. Moreover, different laminin trimers exert distinct effects on subsets of laminin-1-responsive cells, suggesting that isoform diversity may underlie some axonal choices in vivo. As a first step toward evaluating these hypotheses, we have documented the expression patterns of all 10 known laminin chains in the peripheral nervous system and spinal cord of the murine embryo. The α2, α4, β1, and γ1 chains are expressed in peripheral axonal pathways by embryonic day (E) 11.5, when sensory and motor axonal outgrowth is underway. Thus, laminins (but not laminin 1) may promote peripheral axonal outgrowth. By E 13.5, laminin chains are differentially expressed in the limb-bud, with prominent expression of α2 and α4 in muscle and of α3 and α5 in skin. This pattern raises the possibility that laminin isoform diversity contributes to the ability of cutaneous and muscle sensory axons to distinguish their targets. Later in development, some chains (e.g., α2, α4, and β1) are downregulated in peripheral nerve while others (e.g., γ1), continue to be expressed by Schwann cells into adulthood. In contrast to peripheral nerves and ganglia, laminin chains are expressed at low levels, if at all, in the developing spinal cord gray matter. J. Comp. Neurol. 378:547–561, 1997. © 1997 Wiley-Liss, Inc.     

Desmin and vimentin in regenerating muscles.

Desmin is a normal constituent of skeletal muscle fibers; vimentin is contained in myoblasts and connective tissue cells. The intracellular localization of both intermediate filament proteins in regenerating rat muscles was investigated by immunohisto- and immunocytochemistry. Necrosis was induced by hot Ringer solution. Desmin and vimentin were diffusely distributed in myoblasts and young myotubes. Both proteins became arranged in a sarcomeric fashion between the Z-lines when the sarcomeres got into register. Desmin reactivity persisted, but vimentin disappeared after about 2 weeks. Traces were found for up to 4 weeks. This observation is in contrast to findings in fetal and cultured muscles in which several authors did not find expression of vimentin after myoblast fusion. The presence of vimentin may well be a useful marker for regenerated muscle fibers in muscle biopsies from patients with neuromuscular disorders.     

Reinnervation of dystrophic muscles

OBJECTIVE: To analyse the regenerating capability of the peripheral nerve fibers and the capability of the muscle fibers to accept the regenerating and new nerve sprouts in myotonic dystrophy (MD). MATERIAL AND METHODS: One male, aged 58 years, diagnosed of MD at the age of 30 years, suffered neuralgic amyotrophy in the right shoulder girdle 4 weeks before admission. Needle EMG and nerve conduction studies were performed on admission and 6, 12, and 18 months later. RESULTS: On admission there were atrophy and absence of voluntary contraction in deltoids, supra- and infraspinatus muscles. EMG showed abundant fibrillations, positive sharp waves and myotonic bursts in these muscles without voluntary activity, consistent with axonal neuropathy of both axillary and suprascapular nerves. The follow-up showed signs of reinnervation 6 months later and slight loss of long duration and high amplitude MUPs at 18 months of evolution, with good clinical recovery. This is compatible with chronic neurogenic atrophy, presumably as an expression of type grouping. CONCLUSIONS: The reinnervation capability of the nerve fibers and the capability of muscle fibers membrane to accept regenerating and new nerve sprout remain in MD. Myotonic bursts persist after total denervation.     

Distribution of acetylcholinesterase activity in normal,dystrophic, and denervated muscles of the chicken

One distinctive property of denervated and genetically dystrophic muscles of chickens is high acetylcholinesterase activity found in the fibers. The distribution of AChE activity in single fibers from these muscles was studied by using fresh frozen serial sections, a specific histochemical stain, and photodensitometry. The results confirm the findings of a previous report (22) that high AChE activity occurs only around the neuromuscular junction region in fibers from biceps muscles of 6-week-old dystrophic chickens. In contrast, both normal and dystrophic biceps muscle of 6-week old chickens that had been denervated for 5 days exhibited high AChE activity throughout the length of the fibers. These results suggest that changes in AChE activity due to denervation are superimposed on the activity already present in dystrophy. The data support the idea that inherited muscular dystrophy in the chicken involves a specific defect in the regulation of AChE and perhaps other molecules around the neuromuscular junction.     

Morphologic changes in extraocular muscles of the dystrophic hamster.

Cardiomyopathic hamsters (UM-X7.1 strain) have demonstrable myopathy involving both skeletal and cardiac muscle. In addition, they have multiple ocular abnormalities. In this study, extraocular muscles were examined by light and electron microscopy. Changes observed within affected muscle fibers were variable and included coagulation necrosis, lysis of myofibrils, mitochondrial changes, and infiltration by phagocytic cells. Regenerative changes included duplication of myoblast nuclei, proliferation of sarcoplasmic reticulum, and myofibrillogenesis. The lesions are presumably myogenic in origin. The cardiomyopathic hamster may be useful as an animal model for certain types of ocular myopathy in man.     

Antidystrophin stains triadic junctions in regenerating rat muscles.

Dystrophin has biochemically been found in the sarcolemma and in junctional t-tubules, but immunocytochemistry shows reactivity at the sarcolemma only. In the present study, normal and regenerating soleus muscles of rat were perfused for 10 minutes with 2% formaldehyde; isolated fibers were stained with polyclonal antidystrophins and HRP and embedded in epoxy. Staining of triadic junctions in normal fibers was ambiguous but, in regenerated fibers, 4 weeks after injury it was distinct. Immature myotubes 3 days after injury showed reactivity at the sarcolemma and at various internal membranes. The nonselective staining of internal membranes may be due to secondary binding of the reaction product, and supports the view that dystrophin is cytoplasmic before it becomes restricted to the sarcolemma and t-tubules.     

Peripheral mechanisms of fatigue in muscles of normal and dystrophic mice.

We evaluated the contribution of different processes to fatigue of normal and dystrophic mouse muscles using an in vitro electromyography chamber. Fatigue was induced by repetitive nerve stimulation at 30 Hz for 0.5 s, every 2.5 s until tension decreased by about 50%. We monitored the compound nerve action potential (AP), compound muscle AP, and isometric tension responses to nerve stimulation, and compound muscle AP and tension responses to direct muscle stimulation. In normal mice, about 50% reduction in nerve-evoked tension occurred by 2.4 min in extensor digitorum longus (EDL), 4.8 min in diaphragm, and 9 min in soleus. Analysis of the responses revealed that the fatigue was caused by failure of more than one process in all muscles, and failure of nerve conduction did not contribute to fatigue in any muscle. Failure of neuromuscular transmission, muscle membrane excitation, and excitation-contraction (E-C) coupling and contractility accounted for 55, 45, and 0%, respectively, of the fatigue in EDL, for 21, 74, and 5% of the fatigue in diaphragm, and for 2, 54, and 44% of the fatigue in soleus. In dystrophic mice, while about 50% reduction in nerve-evoked tension occurred by 8.1 min in EDL and 5.6 min in diaphragm, only 29% reduction in tension occurred by 80 min in soleus. Failure of neuromuscular transmission, muscle membrane excitation, E-C coupling and contractility accounted for 22, 63 and 15% of the fatigue in EDL, for 21, 79, and 0% of the fatigue in diaphragm, and for 15, 59, and 26% of the fatigue in soleus. The proportion of slow-twitch oxidative fibers was more than normal in dystrophic EDL, but the same as normal in dystrophic diaphragm and soleus. The slower onset of fatigue was attributable to lesser failure of neuromuscular transmission in dystrophic EDL, and to lesser failure of E-C coupling and contractility in dystrophic soleus.     

Histochemical changes in fast and slow muscles of nutritionally dystrophic rabbits.

Rabbits were rendered dystrophic by feeding them a diet deficient in vitamin E and their fast-twitch EDL and slow-twitch SOL muscles were examined histochemically. The soleus muscle of control rabbits consisted largely of type I fibres with occasional areas of scattered type II fibres. In the nutritionally dystrophic rabbits type II fibres were consistently found homogeneously distributed throughout the entire muscle and in increased proportion. A very similar pattern was observed in the solei of rabbits following sciatic nerve section. The normal EDL contained three fibre types (I, IIoxidative and IIglycolytic). Vitamin E deficiency appeared to be associated with a shift towards an increase in the proportion of IIglycolytic fibres at the expense of IIoxidative. In denervation as well as vitamin E deficiency the type I fibres of the EDL appeared to be spared. A small number of the E-deficient rabbits exhibited degenerative changes in their sciatic and sural nerves. When animals were both denervated and E-deprived the resulting muscle changes were very much more severe than in the case of either challenge in isolation. We suggest that although some of the signs of vitamin E deficiency resemble those of a neural defect there is, in addition, a direct myopathic effect.     

Sarcomere length in normal and dystrophic chick muscles

An electron microscope study of 2- and 8-week-old normal and dystrophic chickens compared sarcomere lengths in relaxed and passively extended Patagialis (PAT) muscles. Sarcomeres were measured in dystrophic muscles only in fibers which exhibited no morphological signs of degeneration. Sarcomere lengths were not different from each other in normal muscles of 2- and 8-week-old chickens. Passive extension of the normal wing increased mean sarcomere length by 44%. Sarcomere lengths in unstretched dystrophic PAT muscles were 22 and 25% longer than unstretched normal sarcomeres at 2 and 8 weeks of age. Passive extension of the wing further increased sarcomere length of 2-week-old dystrophic muscles to the length of stretched sarcomeres in 2-week-old normal muscles. In 8-week-old dystrophic chickens, the wings could be passively extended to only 134 degrees, rather than the normal range of 180 degrees. In this case, passive extension of the wings did not further increase the length of sarcomeres. Increased sarcomere lengths in dystrophic muscles may indicate that dystrophic muscle fibers are being subjected to greater degrees of passive tension than normal muscle fibers during early stages of growth. Passive tension is known to promote fiber hypertrophy, nuclear proliferation, and increased oxidative metabolism in normal muscle. These responses to passive tension are also characteristic of prenecrotic stages of muscular dystrophy in chickens.     

Increased laminin a expression in regenerating myofibers neuromuscular disorders

Laminin is a basement membrane (BM) glycoprotein composed of three of five subunits, the A, M, B1, B2, and the S chain. Four forms of laminin, A-B1-B2, A-S-B2, M-B1-B2, and M-S-B2, have been identified. Laminin is implicated in various biological processes such as cell adhesion and differentiation. We studied immunohistochemically the expression of the four laminin subunits A, M, B1, B2 as well as of neural cell adhesion molecule (N-CAM, CD56), a marker of regenerating myofibers, in various neuromuscular disorders. In normal muscle, the predominant subunits of myofiber laminin were M, B1, and B2. The A chain was only faintly expressed in myofiber BM. In inflammatory myopathies and dystrophinopathies myofiber laminin A expression was greatly increased. An average of 80% and 63% of laminin A–positive myofibers in inflammatory myopathies and dystrophinopathies, respectively, were additionally CD56 positive. Laminin A and CD56 expression in denervating diseases and mitochondrial myopathies were negligible. Expression of M, B1, and B2 subunits did not seem to be altered in the diseased conditions examined above. The data suggest that laminin A is upregulated in inflammatory myopathies and dystrophinopathies and, most markedly in regenerating myofibers. © 1995 John Wiley & Sons, Inc.     

Effects of laminin on functional reinnervation of target organs by regenerating axons.

Reinnervation of sweat glands, skeletal muscle and skin was studied in the mouse paw after section of the sciatic nerve and repair by entubulation with collagen conduits, with and without a coating of laminin. After operation, sweat glands activated by pilocarpine reappeared at 32 days and increased in number to 67% of control counts. Muscle action potentials (during sciatic nerve stimulation) were recorded from the interosseus muscle at 39 days. The potential amplitudes increased to 30% of controls. The first withdrawal response from pin prick to the paw was observed at about 34 days, the response score increasing to 66% of control. There were no significant differences at any test interval between the control group and the group with laminin added.     

Response of dystrophic muscles to reduced load

We have previously reported that, in dystrophic mice, functional overload has a damaging effect on the tibialis anterior (TA) muscle. In the present study, we have examined the effect of a load reduction on the TA and extensor digitorum longus (EDL) muscles. Our results show that reducing the passive load to which these muscles are subjected in dystrophic mice by resecting the Achilles tendon has a beneficial effect. The force output of the "released" EDL muscle improved, while the time course of contraction and relaxation of the "released" TA muscle became faster. Also in this muscle, resistance to fatigue became significantly greater. Low frequency electrical stimulation of the "released" muscles via implanted electrodes had little effect on their force output. It led, however, to a relative speeding of their time course of contraction and relaxation and to a further increase in their resistance to fatigue. Taken together, our results suggest that the beneficial effect of low frequency electrical stimulation on the force output of weak dystrophic muscles, described in the preceding paper, might be conditioned by the load to which these muscles are subjected.     

Nonequivalence of surgical and natural denervation in dystrophic mouse muscles

The electrical “cable” properties of fibers in the soleus muscle of normal and dystrophic mice were measured in vitro at 37C with intracellular microelectrodes. The motor nerve of the dystrophic muscle was stimulated to determine whether or not the fibers chosen for measurement were functionally innervated. Normal and dystrophic muscles which had been surgically denervated were also studied. Specific membrane resistance was higher in normal than in dystrophic muscle fibers; the respective mean values were 1516 and 636 ohm cm². Dystrophic fibers which were not functionally innervated had significantly higher resistance than those which were innervated, but in neither case did values approach the normal. In both normal and dystrophic muscles, previous sectioning of the motor nerve led to an increase in muscle fiber input resistance. Surgically denervated dystrophic fibers had considerably higher resistance than measured in fibers which had become functionally denervated during the course of the disease. The results suggest that denervation per se cannot account for the differences between normal and dystrophic muscle fibers.     

Energetics of isometric contraction in dystrophic fast and slow muscles

The amount of phosphoryl creatine (PC) hydrolysed during a ten-second isometric contraction was measured in the biceps brachii (fast) and soleus (slow) muscles of adult normal and dystrophic mice (Re 129 strain) following inhibition of glycolysis and oxidative phosphorylation. The dystrophic muscles were found to have a lower isometric economy (tension-time integral per μmol PC) than the normal muscles. This was particularly so in the case of the fast biceps brachii muscle which is affected by dystrophy to a greater extent than the slow soleus muscle. The isometric economy of the dystrophic muscles was lower even when the results were based on a total creatine rather than on a weight basis. This suggests there may be some defect in the contractile proteins of dystrophic muscle. The normal soleus muscle was found to be approximately three times more economical in maintaining tension than the normal biceps brachii muscle. This indicates the adaptation of slow muscles such as the soleus are used for maintenance of posture.     

Nerve growth factor expression in human dystrophic muscles

Nerve growth factor (NGF) is a neurotrophin that is expressed during muscle development and is also capable of favoring muscle regeneration in experimental studies. The presence of NGF in muscular dystrophies, such as Duchenne and Becker muscular dystrophies, has never been fully explored. By means of immunohistochemistry, we show that regenerating muscle fibers from such patients consistently express NGF, as do myofibroblasts and mast cells. By contrast, rest fibers from dystrophic patients, as well as muscle fibers from healthy, control patients and even regenerative muscle fibers in polymyositis do not show NGF immunoreactivity. The paracrine effect of NGF on muscle regeneration, as well as its chemoattractant capacities for mast cells, may contribute to explaining why regenerating fibers most frequently occur in clusters and why mast cells are more numerous in dystrophic muscles. Moreover, being a mediator of wound healing and tissue fibrosis, NGF may contribute to long-term muscle regeneration impairment by tissue fibrosis in the muscular dystrophies.     

Structural changes in myosin in dystrophic, denervated and regenerating muscle in mice

The technique of sodium dodecyl sulphate-polyacrylamide gel electrophoresis was used to resolve the large and small polypeptide chains of mouse myosin and to determine their molecular weights. In myosin preparations from dystrophic, denervated and regenerating muscle a decrease in amount of the large sub-unit (molecular weight 200,000) was accompanied by an increase in smaller protein chains. There was evidence that some of the smaller chains in dystrophic myosin were due to proteolytic activity. These smaller chains repeatedly co-precipitated with myosin at low ionic strength and were not removed by gel filtration through Sephadex G-200, indicating that they were tightly bound to the myosin molecule. Normal myosin contained three low molecular weight proteins (26,500, 19,000 and 16,500). The 19,000 molecular weight protein was not detectable in myosin preparations from denervated muscle, regenerating muscle and normal muscle treated with extraction medium previously used to extract dystrophic muscle, but Ca²⁺-activated ATPase was unaffected. In dystrophic myosin both the 19,000 and 16,500 molecular weight proteins were undetectable and the level of Ca²⁺-ATPase was reduced.     

Comparison of force and stiffness in normal and dystrophic mouse muscles

Isometric force and stiffness of fast- and slow-twitch muscles of affected and normal mice of the 129/ReJ dy/dy strain were studied at rest and during active contraction at a variety of lengths. Dystrophic muscles developed less force in response to stimulation, but the resting stiffness was not reduced as much, particularly at long muscle lengths. This is consistent with the replacement of muscle fibers by connective tissue that is considerably less elastic. When second and third stimuli are superimposed on the rising phase of a twitch in a normal muscle, a less-than-linear summation of force and stiffness generation (early depression) is followed by a more-than-linear summation (later facilitation). Dystrophic muscles showed a smaller early depression and a greater later facilitation of force and active muscle stiffness. Many of these phenomena can be predicted from a simple model of Ca2+ release and binding to troponin.     

Calmodulin content and Ca-activated protease activity in dystrophic hamster muscles

As part of a study on the implication of elevated Ca²⁺ levels in the myofibrillar degeneration seen in dystrophic muscle, the content of calmodulin and the activity of Ca²⁺ -activated neutral protease (CANP) have been measured in normal and dystrophic (UM-X7.1) hamsters. Calmodulin levels, expressed as micrograms ± SEM per gram wet weight were highest in brain (385 ± 24.7), followed by tongue (93.88 ± 3.93), heart (42.13 ± 2.93), and skeletal muscle (31.69 ± 1.42). No significant increases in calmodulin were observed in the dystrophic tissues thus suggesting that the Ca²⁺ accumulations observed in dystrophic muscles are unrelated to changes in calmodulin levels. Because of the complexity of regulation of CANP, a time-dependent study was done using extracts of skeletal, heart, and tongue muscles. Marginal increases in dystrophic CANP were seen in skeletal muscle at all times studied and in the heart and tongue at initial time points only. The data are discussed in terms of rising levels of Ca²⁺ in muscles of the UM-X7.1 hamster being sufficient to increase CANP activity (without increasing content) to where it causes Z-line dissolution.