Appearance of acetylcholinesterase and creatine kinase in plasma of normal chickens after denervation.

Evidence that acetylcholinesterase (AChE) activity is released from normal chick embryonic muscle fibers and from muscles of chickens with inherited muscular dystrophy suggested that denervated chick muscles, which have AChE properties similar to dystrophic muscles, would also release AChE. Bilateral denervation of the breast and wing muscles of normal chickens was followed by the appearance of AChE activity, distinguished from plasma cholinesterase by differential substrate hydrolysis, inhibitor sensitivity, and electrophoretic migration. Plasma creatine kinase (CK) activity was also elevated after denervation.     

Incidence of acetylcholinesterase in the sarcoplasm of human and chicken muscles

Fifty-nine biopsies of human muscle, 53 of them abnormal, 6 normal, were studied for the histochemical localization of acetylcholinesterase (AChE) using frozen sections and light microscopy. In addition to AChE which was found at the myoneural and myotendon junction, specific staining was found around the periphery of many fibers from normal and abnormal muscles. Moreover, AChE activity was found to be high in the sarcoplasm of more than 10% of the fibers from 28 biopsies of abnormal muscle including cases of hemiplegia, spinal cord injury, denervation and neuropathy, infantile spinal muscle atrophy, Duchenne, limb-girdle and facioscapulohumeral dystrophies, Schwartz-Jampel syndrome and a myasthenic syndrome. Of the muscles from experimental animals examined, only the Rhesus monkey exhibited AChE around the periphery of the fibers, and only the dystrophic chicken and not the dystrophic mouse or hamster, showed extensive sarcoplasmic AChE. Histograms of muscle fiber diameters indicated that AChE in the sarcoplasm was associated with fibers of all sizes, depending on the nature of the disorder examined. Fibers containing AChE were smaller than unstained fibers in dystrophic chicken muscle. The results suggest that in the human, sarcoplasmic AChE is reversibly repressed during muscle maturation and that its mode of regulation by motor neurons is similar to that found in the chicken.     

Terminal Schwann cell structure is altered in diaphragm of mdx mice

The diaphragm muscle of the mdx mouse is a model system of Duchenne muscular dystrophy, since it completely lacks dystrophin and shows severe fiber necrosis and loss of specific muscle force by 4-6 weeks of age. Changes in neuromuscular junction structure also become apparent around 4 weeks including postsynaptic acetylcholine receptor declustering, loss of postsynaptic junctional folds, abnormally complex presynaptic nerve terminals, and muscle fiber denervation. Normally, terminal Schwann cells (TSCs) cap both nerve terminals and acetylcholine receptors at the neuromuscular junction, and play a crucial role in regeneration of motor axons following muscle denervation by guiding axons to grow from innervated junctions to nearby denervated junctions. However, their role in restoring innervation in dystrophic muscle is unknown. We now show that TSCs fail to cap fully the neuromuscular junction in dystrophic muscle; TSCs extend processes, but the organization of these extensions is abnormal. TSC processes of dystrophic muscle do not form bridges from denervated fibers to nearby innervated endplates, but appear to be directed away from these endplates. Adequate signaling for TSC reactivity is present, since significant muscle fiber denervation and acetylcholine receptor declustering are present. Thus, significant structural denervation is present in the diaphragm of mdx mice and the ability of TSCs to form bridges between adjacent endplates to guide reinnervation of muscle fibers is impaired, possibly attenuating the ability of dystrophic muscle to recover from denervation and ultimately leading to muscle weakness.     

Passive stretch of adult chicken muscle produces a myopathy remarkably similar to hereditary muscular dystrophy

The wings of 10 chickens between 1 and 5 years of age were passively extended. An increase in plasma creatine phosphokinase activity was observed in 30 min, continued to rise for 24 h, and then declined, suggesting mechanically induced damage to muscle fibers. Wing muscles were removed and examined histologically at various times after stretch. Patagialis muscles, but not biceps brachii, showed the development of muscle fiber pathology. The patagialis muscle is less active than the biceps brachii and is stretched to a greater degree by wing extension. Susceptibility of muscles to development of pathology appeared to be correlated with the age of the chickens. Pathology was remarkably similar to that observed in young chickens with hereditary muscular dystrophy. Necrotic fibers exhibiting segmental necrosis, abnormal shapes, enlargement, splitting, vacuolation, and phagocytosis were evident. Of particular interest was the appearance of abnormal clusters of acetylcholinesterase activity along the sarcolemma. These sites were shown to appear on fibers of 2-week-old dystrophic chicks prior to necrosis and increase in plasma creatine phosphokinase activity. It is suggested that aging of inactive muscles may promote adhesions between muscle fibers rendering them susceptible to damage when stretched and that necrosis of dystrophic fibers may be initiated by a similar mechanism. Such could occur if the genetic defect resulted in interfiber adhesions. Support for this hypothesis by other reports in the literature is discussed.     

Distribution of extrajunctional acetylcholinesterase in muscle of normal and dystrophic chickens.

A specific histochemical staining of serial cross sections of frozen muscle samples for AChE activity was performed to investigate the distribution of sarcoplasmic AChE activity and its relationship to the motor end plate in individual muscle fibers of 1-, 2-, and 6-week-old normal and dystrophic chickens. A photographic cytophotometric technique was used to determine AChE activity. There were no differences between normal and dystrophic muscle fibers in the distribution or level of AChE activity at both 1 and 2 weeks of age. By 6 weeks, AChE activity had spread to either side of the motor end plates for approximately five times the distance found in normal fibers. In addition, the level of AChE activity had almost tripled in dystrophic fibers in comparison to normal fibers. These findings suggest that dystrophic chicken muscle develops similarly to normal muscle with respect to AChE localization and level of activity for at least 2 weeks following hatching, and then AChE spreads along the muscle fiber from the motor end plate. The data are consistent with the idea that there is a myogenic defect in the maturation of AChE regulation associated with the motor end plate of dystrophic chickens.     

Plasma acetylcholinesterase in Duchenne muscular dystrophy

Muscle acetylcholinesterase (AChE) in 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-beta-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.     

Tissue acetylcholinesterase in plasma of chick embryos and dystrophic chickens

The plasma cholinesterases of chickens (line 304) with inherited muscular dystrophy and 2 normal lines were examined from 12 days of incubation to 14 weeks after hatching. Acetylcholinesterase (AChE) and non-specific cholinesterase (BChE) activities were distinguished by spectrophotometric, electrophoretic and titrimetric analyses using acetylcholine, butyrylcholine, and their thioester analogs acetylthiocholine and butyrylthiocholine as substrates and 284C51 and iso-OMPA as selective inhibitors. Plasma from normal and dystrophic embryos and from dystrophic chicks had high AChE activity. For example, by 12 weeks of age, 37% of plasma acetylthiocholine hydrolysis was inhibited by the anti-AChE agent 284C51. The results support the view that embryo plasma contains AChE and BChE forms. After hatching the AChE forms decrease greatly in normal plasma and a shift in BChE forms occurs. Later, the AChE forms return in dystrophic line plasma. AChE activity in plasma was circumstantially associated with multiple molecular forms of AChE in the sarcoplasm of embryo and dystrophic muscles and it is likely that these muscles are sources of the plasma AChE activity.The results also confirmed that acetyl-β-methylcholine, unlike the situation which exists in mammals, is hydrolyzed by both AChE and BChE forms in the chicken, and cannot be used to distinguish these cholinesterases.     

Multiple forms of acetylcholinesterase in nutritional and inherited muscular dystrophy of the chicken

Isozymes of acetylcholinesterase (AChE) are found in the cytoplasm of chick embryo muscle. They are maintained in muscles from birds with inherited muscular dystrophy and return with denervation but not tenotomy of normal muscle. The present study examined AChE activity in pectoral and lateral adductor muscles of birds with nutritional muscular dystrophy brought on by diets deficient in vitamin E, selenium, and sulfur amino acids, and containing excess arginine. Cytochemical and acrylamide gel electrophoretic studies showed that cytoplasmic AChE activity and embryo AChE forms appeared in pectoral muscle from vitamin E-deficient birds after 6 weeks of age. The cytoplasmic activity and embryo isozymes did not appear in muscles from birds grown with a vitamin E supplement. Cytoplasmic AChE activity was localized in focal regions of intact muscle fibers. The lateral adductor muscle of the leg was little affected. The results suggest that vitamin E deficiency interrupts neuromuscular interactions that suppress embryo AChE isozyme in “white” muscle of the chicken.     

Reinnervation is followed by necrosis in previously denervated skeletal muscles of dystrophic hamsters

Hind leg muscles of dystrophic hamsters were continually denervated by multiple crushes of the sciatic nerve to as long as 93 days of age. In these muscles, the prevalence of centronucleated fibers which is a cumulative index of prior necrosis, remained very low. In control dystrophic muscles the prevalence of centronucleated fibers increased steadily to approximately 80% where it leveled off. By omitting further crushes in other groups of animals, previously denervated muscles became adequately reinnervated. In the reinnervated muscles the prevalence of centronucleated fibers steadily increased throughout the necrotic phase of dystrophy at a rate that was comparable to corresponding stages of the natural history of the disease. These experiments indicated that continued denervation was effective in negating skeletal muscle fiber necrosis throughout the necrotic phase and that the electromechanical activity of muscle fibers which allows muscle fiber necrosis was not a time-locked factor.     

Glycogen cycle enzymes and cyclic nucleotides in avian muscular dystrophy

Fast-frozen pectoralis muscle samples were taken from normal chickens (lines 200 and 412) and chickens having hereditary muscular dystrophy (line 304). The glycogen phosphorylase activity ratio (activity without AMP/activity with AMP) was significantly greater in dystrophic muscles (0.306 +/- 0.046) than it was in normal muscles (0.090 +/- 0.023). Glucagon treatment did not cause any changes in phosphorylase activity ratios. Isoproterenol treatment of both normal and dystrophic muscles raised the phosphorylase activity ratio of normal muscles to 0.446 +/- 0.054, which was not significantly different from that of the dystrophic muscles. The dystrophic muscles had significantly less glycogen than normal muscles (23.3 +/- 2.8 compared with 36.8 +/- 2.8 mumoles glucosyl units/g of muscle). There was no relationship of muscular dystrophy to total phosphorylase activity (measured in the presence of 1 mM AMP) and to glycogen synthase activities measured without and with glucose 6-phosphate. Normal muscles had 28% less cAMP and 49% less cGMP than dystrophic muscles, but these differences were eliminated by treatment of the chickens with glucagon.     

Membrane cable properties of normal and dystrophic chicken muscle fibers

Values of fiber radius obtained by square pulse analysis and histological measurements indicated that the innervated dystrophic fibers which had been examined with microelectrodes were hypertrophic. The specific membrane resistance of dystrophic fibers was also greater than normal. In addition, experimentally induced compensatory hypertrophy of innervated nondystrophic (normal) fibers of the posterior latissimus dorsi led to alterations in several membrane characteristics which resulted in values resembling those of innervated dystrophic fibers. Twenty-one days after denervation, the values for the cable properties of normal and dystrophic fibers were increased, yet similar values were attained for the space constant, specific membrane resistance, and membrane capacitance. In both normal and dystrophic muscles which were denervated for 21 days the fiber radius decreased 40%. To study the mechanism underlying the increase of the specific membrane resistance after denervation, the resting membrane conductance was selectively altered. In solutions of low pH (5.0) where chloride conductance was presumably reduced, the space constant, time constant and specific membrane resistance of innervated normal and dystrophic fibers were increased and approached values obtained from 21-day denervated muscles. In contrast, solutions of low pH had no marked effects on 21-day denervated normal and dystrophic muscles. It is suggested that the increased values for these cable properties from denervated normal and dystrophic posterior latissimus dorsi muscles may be partially due to reduced potassium and chloride conductances. Furthermore, the presence of hypertorphic fibers may be a significant morphological adaptation in dystrophic muscles.     

Simultaneous cytochemical demonstration of muscle fiber types and acetylcholinesterase in muscle fibers of dystrophic chickens.

A cytochemical procedure is described which allows the simultaneous observation of different types of muscle fibers and their motor end plates. The procedure utilizes an assay for myofibrillar adenosine 5′-triphosphatase (ATPase) activity followed by an assay for acetylcholinesterase (AChE) activity. This combined assay eliminates the necessity for using serial sections for observation of these two parameters. This combined assay increases the apparent AChE activity such that sites of AChE activity are revealed which are not visualized when using the AChE assay alone. In muscles from chicks with hereditary muscular dystrophy, it is shown that initially dystrophic fibers contain nuclei which react strongly for AChE activity. Subsequently many fibers exhibit an intense reaction for AChE activity over a major portion of their cell surface. AChE activity is also found along the splits of fragmenting fibers and on the periphery of necrotic vacuoles.     

Pathology of skeletal muscle and intramuscular nerves in infantile neuroaxonal dystrophy

Summary Biopsies of the biceps muscle and sural nerve were taken from a girl aged 2 years with infantile neuroaxonal dystrophy (INAD).In addition to the typical axonal spheroid bodies in a number of the i. m. nerve fibers, the neuromuscular junctions (NMJs) and motor nerve endings also contained axonal swellings. The sural nerve, except for three dystrophic fibers, was almost completely normal.A teased nerve preparation showed four additional abnormal fibers with focal axonal enlargement similar to those in giant axonal neuropathy (GAN).These results suggest that a biceps muscle biopsy may be more useful than a sural nerve biopsy for the diagnosis of INAD, because the muscle contains abnormal peripheral nerves and NMJs in high frequency.     

Abnormal distribution of skeletal muscle acetylcholinesterase molecular forms in dystrophic mice

Acetylcholinesterase (AChE) activities and molecular forms, as separated by density gradient centrifugation, were studied in dystrophic and clinically normal mouse muscle. Dystrophic hemidiaphragms exhibited normal AChE activity, but there was little or no 10 S enzyme, a form that constitutes 27% of control tissue AChE. The 10 S-AChE abnormality was similarly present in dystrophic extensor digitorum longus (EDL) muscle, but this muscle exhibited significantly reduced AChE activity. The EDL muscles also had reduced 16 S-AChE but normal 4 S enzyme activity. Chronic denervation of EDL muscles resulted in proportionally similar reductions of weight, total AChE, and 16 S enzyme in dystrophic and control muscles. We conclude that murine dystrophy involves some alterations that resemble denervation, but that there are major qualitative and quantitative differences in AChE that cannot be explained by a denervation-like 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.     

Muscle activity pattern regulates postnatal development of acetylcholinesterase molecular forms in normal mice and mice with motor endplate disease

We have studied the relative contributions of muscle activity and nerve-supplied materials to the regulation of AChE molecular forms during postnatal development of muscles in normal mice and in mice with motor endplate disease (med mice). Onset of this hereditary disease causes a progressive failure of evoked release of ACh from the motor neuron, which prevents contraction in muscles such as biceps and soleus. In these innervated but inactive muscles, one can examine the consequences of inactivity on the distribution of AChE forms. In normal mouse biceps the distribution of AChE forms, as shown by sucrose-gradient analysis, change substantially after birth; the most dramatic alteration is an increase in G4 AChE from 15 to 45% of total AChE during the third postnatal week. AChE profiles in normal or med biceps are indistinguishable until 10-12 d after birth, but the changes in distribution of AChE forms does not occur in med biceps nor in normal biceps denervated 2 weeks after birth. In contrast, the distributions of AChE forms in a predominantly slow muscle, the soleus, are similar in med and normal mice both early (10 d) and late (20 d) in the course of the disease, and the distributions are affected little by denervation. The profiles of AChE forms seen in normal soleus at all times studied resembled those seen in newborn biceps or biceps inactivated by denervation or the med disease. We conclude that neither innervation, age-dependent changes intrinsic to muscle, nor muscle activity is sufficient to induce the changes we seen in AChE forms in biceps. These results support the hypothesis that neonatal, inactive, or tonically active muscles produce an intrinsic pattern of AChE molecular forms, and that a phasic pattern of activity induces a postnatal redistribution of the AChE molecular forms expressed by the muscle.     

Baclofen,procainamide, verapamil,and prenylamine in hereditary muscular dystrophy of the chicken

Two “myotonia antagonists” (baclofen and procainamide) and two “calcium antagonists” (verapamil and prenylamine) were evaluated for their effects on hereditary muscular dystrophy of the chicken. Righting ability of dystrophic chicks was improved only by baclofen and procainamide. Plasma creatine kinase activity of dystrophic chicks was reduced 45% after chronic administration of baclofen, but was still nearly nine-fold greater than activity in plasma of normal chicks. Baclofen and verapamil both reduced acetylcholinesterase activity in pectoralis major muscles of dystrophic chicks by about 40%, but these values were still significantly greater than those measured in muscles of normal chicks. The data provide further support for the concept that impaired righting ability of young dystrophic chickens is associated with the presence of myotonia in affected muscles, but do not rule out the possibility that certain of the biochemical features of the avian dystrophy may involve calcium.     

Changes in genotypic expression, development, and the effects of chronic penicillamine treatment on the electrical properties of the posterior latissimus dorsi muscle in two lines of normal and dystrophic chickens.

When line 304 dystrophic chickens were outcrossed, a new line of genetically dystrophic chickens designated 413 was produced together with a genetically related normal animal (line 412). The expected alteration in membrane resistance in the surface fibers of the posterior latissimus dorsi (PLD) muscle was eliminated in this new line of dystrophic chickens. Although the input resistance of PLD fibers from both lines 200 and 304 chickens declined simultaneously from 1 to 2 Mω at 5 weeks ex ovo to 0.3 Mω at 17 to 33 weeks ex ovo, specific membrane resistance, membrane space and time constants, and fiber radius of line 304-dystrophic fibers were significantly greater than in line 200-normal fibers at various times ex ovo. Only the membrane time constant and capacitance of PLD fibers of line 413-dystrophic chickens were greater than in line 412-normal at 8 weeks ex ovo. Other differences in electrical properties were also seen. Only in line 413-dystrophic chickens were the resting membrane potentials and action potential properties significantly different from line 412. Outcrossing of line 304 chickens [has thus] revealed that various electrical properties of muscle such as membrane resistance are under genetic control but did not influence the development of dystrophy. On the other hand, alterations in action potential activity persists after outcrossing and appear to have a greater role in the symptomatology of muscular dystrophy. A therapeutic approach to the disease using oral d-penicillamine treatment was initiated 9 days ex ovo in normal (line 412) and dystrophic (line 413) chickens of either sex and continued 7 to 10 weeks. An improvement in the righting ability of treated vs. untreated dystrophic birds was first noted at 6 weeks ex ovo, and by 8 weeks, treated dystrophic chickens were always able to right themselves whereas untreated dystrophic chickens failed 75% of the time. The body weights and rates of growth in untreated normal and dystrophic chickens were similar but that of treated birds were 35 to 40% lower. The drug had no effect on the resting membrane potential of PLD surface fibers in treated chickens. The beneficial effect of the drug was initially correlated with the ability of the PLD muscle to respond with a twitch to single indirect stimulation and to the absence of a decrementing response in muscle to repetitive nerve stimulation. Penicillamine only decreased the action potential threshold in dystrophic chickens. Penicillamine treatment reduced the depression of the probability of release of transmitter in dystrophic muscle. The higher threshold for excitation and greater resting membrane potential in the newer line of dystrophic chickens may account in part for the inability of these chickens to right themselves when placed on their backs. It is suggested that the beneficial effect of penicillamine is related to an alteration of synaptic function and lowering of the threshold for excitation in muscle.     

Expression of mouse agrin in normal,denervated and dystrophic muscle

Agrin is a heparan sulfate proteoglycan that is required for the development of postsynaptic specializations at the neuromuscular junction. An alternatively spliced isoform of agrin that lacks this activity is found in basement membranes of several tissues including embryonic muscle. Overexpression of a miniaturized form of this agrin isoform ameliorates the severe muscle dystrophy of laminin alpha2-deficient mice, a mouse model for merosin-deficient congenital muscle dystrophy. Several lines of evidence indicate that this amelioration is based on the high-affinity binding of the mini-agrin to the laminins and to alpha-dystroglycan. Here, we used antibodies raised against mouse agrin to evaluate protein expression in adult muscle of normal and dystrophic mice. We find that expression of agrin in non-synaptic region varies greatly between different muscles in wild-type mice and that its levels are altered in dystrophic muscle.     

Regenerative capability of skeletal muscle in chicken muscular dystrophy

To examine the morphological sequence of regenerating fibers after myonecrosis in dystrophic muscles, 0.5 ml of 0.5% bupivacaine hydrochloride (BPVC) (Marcaine) solution, a local anesthetic with a cytotoxic effect on the muscle fibers, was injected directly into the dystrophic (line 413) and nondystrophic (line 412) posterior latissimus dorsi (PLD) muscles of young and adult chickens. Although the dystrophic muscles after BPVC injection showed a rapid recovery with a similar tempo to that of nondystrophic ones, they showed different morphological behavior in the early phase of regeneration, including marked variability in the size of fibers and in the intracytoplasmic enzyme activities of nicotinamide adenine dinucleotide, reduced-tetrazolium reductase (NADHTR), acetylcholinesterase (AChE), and nonspecific esterase (NSE).