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.     

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.     

Organ culture studies of coupled fetal cord and adult muscle from normal and dystrophic mice

Organotypic cultures of adult mouse muscle, both normal (+/+) and dystrophic ($\text{dy}\text{dy}$), coupled with embryonic spinal cord from both +/+ and $\text{dy}\text{dy}$ mice were studied. Regeneration in muscle cultures without cord was rare. Regardless of the type of embryonic cord (+/+ or $\text{dy}\text{dy}$) coupled with muscle, normal muscle consistently regenerated, exhibiting cross-striations and spontaneous contractions; dystrophic muscle produced myoblastic outgrowth followed by abortive cell fusion, and atypical degenerating myotubes. Dystrophic muscle regeneration as scored by cellular outgrowth and myotube formation was precocious compared to that of normal. The large majority of dystrophic regenerated myotubes were flat and unstriated, and exhibited no spontaneous contractions in contrast to the rounded, striated, spontaneously contracting tubes of normal muscle. It is concluded that cultured adult myogenic cells which are genetically dystrophic are capable only of abortive regeneration, which is not “cured” by the presence of normal fetal neural tissue. The sequence of regeneration of adult normal muscle in vitro is not altered when muscle is cultured in the presence of genetically dystrophic fetal spinal cord. These results are compared with other in vitro studies of dystrophic muscle development.     

Normal and dystrophic human muscle in vitro: an argument against denervation

Normal and dystrophic human intercostal muscle has been studied under controlled conditions in vitro. The diseased muscles were weaker and slower to contract and relax than controls but the dystrophic fibers were neither resistant to fatigue nor unusually sensitive to caffeine or nitrate ion. It was not possible to explain the findings on the basis of denervation, and we suggest that both the altered muscle properties and the presynaptic defects recorded by other observers may arise from the same disturbance in trophic function of the neuron.     

A developmental change in the content of parvalbumin in normal and dystrophic mouse (mdx) muscle

A highly sensitive enzyme immunoassay for mouse parvalbumin was developed in this study. The amount of parvalbumin was determined by a sandwich enzyme immunoassay method using anti-parvalbumin IgG-coated polystyrene balls and an anti-parvalbumin Fab'-horseradish peroxidase conjugate. Parvalbumin could not be detected in normal and dystrophic skeletal muscles of newborn mice. In normal mice, it appeared in the first postnatal week and increased linearly thereafter until the 12th week in fast twitch muscle. Rapid increase in parvalbumin was seen during 3rd and 8th week. On the other hand, parvalbumin detected in the first postnatal week increased gradually, but did not yet reach the adult level at the 16th postnatal week in slow twitch muscle. In mdx mice, fast twitch muscles such as the gastrocnemius and tibialis anterior were found to contain significantly decreased amounts of parvalbumin, compared with those in control mice. In fast twitch muscle parvalbumin in mdx mice could not be detected in the newborn, increased until 4th week and thereafter did not increase as that in normal mice. In slow twitch muscle the postnatal increase in parvalbumin content was not different from that in control mice. These results suggest that the decrease in the content of parvalbumin in dystrophic muscle may contribute to the elevation of the level of sarcoplasmic free Ca2+ and the activated Ca2(+)-dependent proteolysis.     

Similar in vitro fatigue patterns of normal and BIO 40.54 dystrophic hamster extensor digitorum longus muscle.

Fatigue patterns of normal and BIO 40.54 dystrophic hamster extensor digitorum longus (EDL) muscles were studied in vitro (22°C) at 60, 120, 170, and 320 days of age. The diseased muscle showed similar rates of tension decline compared to their normal counterparts when stimulated intermittently (a twitch or tetanus every 90 s) for 3 h or in rapid succession (1-s tetanus every 5 s) until tetanic tension was decreased 50%. Electron microscopic observations revealed a subsarcolemmal accumulation of enlarged mitochondria in dystrophic muscle compared to normal EDL. These results suggest that the availability of adenosine triphosphate for cross-bridge formation may not be impaired in dystrophic hamster muscles.     

Histochemical and autoradiographic study of injured and uninjured dystrophic ( ) and normal muscle

Morphological and histochemical techniques were utilized to study the soleus and gastrocnemius muscles of normal ( +/+) and dystrophic ( ) mice. This is the first such histochemical study of muscle. The use of the succinic dehydrogenase and acid-stable and alkali-stable adenosine triphosphatase reactions revealed that the dystrophic soleus muscles exhibited decreased numbers of intermediate type fibers, variable numbers of red type fibers, and increased numbers of “abnormal” type fibers compared to normal soleus muscles. The dystrophic intermediate fibers were hypertrophied, the red fibers tended to remain unchanged, and the abnormal fibers were atrophied in comparison to the normal muscle fibers. The gastrocnemius, unlike the soleus, was preferentially more severely “dystrophic” in one region (red) of the muscle than another (white). The altered histochemical staining of the superficial white region of the gastrocnemius resulted in a sharp decrease (13% to 16%) in the number of white fibers in a region almost 100% white in the normal animal. There is a possibility that indirect effects (i.e., altered functional demands) of the disease are responsible for the observed histochemical and morphological changes in the dystrophic muscle fibers.     

The chicken dystrophic model: Does hypersensitivity to glucocorticoids cause atrophy?

The characteristics of the glucocorticoid receptor population in skeletal muscle and liver from line 412 (normal) and line 413 (dystrophic) chickens were examined. The results demonstrate that in cytosol of pectoralis major muscle from line 412 animals there is a precipitous decrease in the number of receptor sites that occurs between 7 and 14 days ex ovo, followed by a slow decline in the number of sites as the animal matures. The results obtained from the examination of pectoralis major muscle from line 413 are considerably different. Although there is a decrease in the number of sites in the cytosol of pectoralis major muscle from line 413 animals it is delayed by at least 2 weeks. In addition, the number of sites in these muscles is still at least twice that observed in the muscle from normal line 413 animals. Even at 60 days ex ovo the number of receptors in pectoralis major muscle from line 413 animals is more than twice that observed in the comparable muscle from line 412 animals. In addition, the gastrocnemius muscle from older animals (90 days ex ovo) was examined. By comparing this red-fiber muscle from line 412 and line 413 animals, we found a significantly higher number of sites in the gastrocnemius muscle from dystrophic animals. No difference was observed in the glucocorticoid receptor population in the livers of normal and dystrophic animals.     

The isometric responses of fast and slow twitch muscles from normal and genetically dystrophic hamsters

There has been a need for some time to examine the effects of the dystrophic process upon the mechanical properties of the limb muscles of the dystrophic hamster. This paper reports the findings of such a study by using an in vivo technique to record the isometric twitch characteristics of the extensor digitorum longus and soleus muscles. Normal and dystrophic animals have been examined at various ages as it has been reported that the disease is progressive. Normal muscle characteristics fall in line with those already published for small mammals. Dystrophic muscles show little difference from normal muscles with respect to their twitch characteristics. Only at the 60 day age point was the time to half relaxation of dystrophic muscles longer than its normal counterpart. Differences were found between normal and dystrophic muscle weights and between the ratio of muscle weight to body weight. It is proposed that these result probably from increased hydration of the muscles caused secondarily to the cardiomyopathy.     

Increased binding of alpha-bungarotoxin in dystrophic mouse muscle.

The content of ¹²⁵I-labeled α-bungarotoxin in muscles of normal and dystrophic mice was measured after in vivo administration of a lethal dose of the toxin. Both the uptake of toxin in vivo and the amount retained after washing in vitro were higher in most of the dystrophic hind limb muscles studied than in corresponding controls. In addition, there was an increase of toxin-binding sites which progressed with time after surgical denervation of normal muscles but the corresponding increase in dystrophic muscles was considerably less. However, autoradiography of dystrophic muscles indicates that the toxin is localized at end plate regions and that extrajunctional receptors still appear after surgical denervation.     

Axoplasmic flow of protein in the sciatic nerve of mice with experimentally induced myopathy.

An experimental myopathy was induced in hind-limb muscles of the mouse by ligating the common iliac artery and subsequently administering serotonin. A marked muscle weakness developed after the serotonin injection, and histological examination showed large areas of fiber degeneration interspersed with regenerating fibers. Axoplasmic flow of ¹⁴C-labeled proteins along the sciatic nerve was measured in these mice and compared with the flow patterns found previously in dystrophic mice. In the dystrophic mouse fast flow of protein was increased but no corresponding increase was found in mice with experimental myopathy. This result is consistent with the proposal that changes in flow patterns in the dystrophic mice are primarily of neural origin.     

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.     

Lipid changes in Duchenne muscular dystrophy

Thin layer chromatographic analysis of lipid extracts of rectus abdominis and gastrocnemius muscles from controls and patients with severe sex-linked Duchenne muscular dystrophy shows the dystrophic tissue to contain more sphingomyelin, less lecithin plus choline plasmalogen, and more total cholesterol than normal. Comparison of normal, dystrophic, and immature muscle suggests that these observations can be interpreted as showing a similarity between dystrophic and immature muscle and in this respect human Duchenne dystrophy resembles hereditary muscular dystrophy in the mouse. Although sphingomyelin was present in apparently normal amount in muscle biopsies from patients with various other neuromuscular disorders, it was raised in two cases showing evidence of peripheral neuropathy.     

Muscle regeneration and mitochondrial calmitine increase in the dystrophic dy/dy mouse after intramuscular chlorpromazine injection

We studied the effect of chlorpromazine injection on the gastrocnemius muscles of C57BL/6J dy/dy dystrophic mice. Changes in mitochondrial calmitine concentrations and differences in microscopy studies, fibre typing and morphometry were compared in gastrocnemius muscles of dystrophic and control mice before and 2 and 21 days after injection. In both cases, calmitine reduction associated with muscle degeneration was observed 2 days after drug injection. Calmitine then increased, reaching a level at day 21 nearly identical to that of controls before injection. This increase was associated with muscle regeneration. These results clearly indicate that dystrophic mouse muscle can regenerate calmitine after drug-induced damage.     

Increase in oxidative capacity of muscle fibers in dystrophic mice and correlation with overactivity in these fibers

The abnormally dark color of the gastrocnemius muscle in C57B16J dy-2J dystrophic mice is due, at least in part, to the increase in oxidative capacity of the fast-twitch fibers found in the crown of the diseased muscle. These fibers show an increased succinic dehydrogenase activity compared to normal crown fibers, and contain a higher mitochondrial volume percent compared to normal fibers using quantitative electron microscopy. They also show an abnormal amount of spontaneous activity in the form of a peripherally derived pseudomyotonia. Increased fiber activity and artifical stimulation of muscle fibers are known to cause an increase in oxidative capacity of mammalian skeletal muscle fibers. It is clear from the quantitative microscopy data, that although these fibers show an increase in mitochondrial volume and a concomitant increase in Z-disk thickness, they do not resemble the underlying oxidative fibers in either parameter. Furthermore, their ATPase activities show fast-twitch characteristics. The fibers in the crown of the dystrophic muscle do not result from fast-twitch glycolytic fiber degeneration leaving only the underlying oxidative fibers as occurs in the mouse dy mutation. The hypothesis is presented that the overactivity endured by these fibers may be the cause of their increased oxidative capacity.     

AN ELECTRON MICROSCOPIC STUDY OF SATELLITE CELLS AND REGENERATION IN DYSTROPHIC MOUSE MUSCLE

Triceps and gastrocnemius muscles from dystrophic (129 Rej dy/dy) and normal mice were examined by electron microscopy at different stages in development for evidence of regeneration. Mitotic satellite cells were present only in dystrophic muscle. Myoblasts containing myofilaments, and multinucleate myotubes were observed within foci of regeneration. Approximately 50% of the myotubes showed features indicative of degeneration or abnormal development. These features included the presence of membrane whorls, and myofibrillar and sarcolemma breakdown. Quantitative studies suggest that the number of satellite cells is increased in dystrophic muscle. It is concluded that there are sufficient satellite cells in dystrophic mouse muscle to allow regeneration, and they are able to proliferate and form well differentiated myotubes. However, subsequent development of the myotubes can be ineffective, or 'abortive', reducing the regenerative capacity to the muscle.     

Functional properties of muscles transplanted between normal and dystrophic mice

Tibialis anterior muscles were transplanted between 12-week-old normal and dystrophic mice with intact or polydimethyl silicone-capped peroneal nerve. After 150 days the transplants were removed and their isometric twitch contraction properties were studied in vitro at 20 C. Intact normal and dystrophic muscles of equivalent age were used as controls. Dystrophic muscles developed lower twitch and tetanus tension than normal muscles and showed prolonged half relaxation time. The contraction time and twitch/tetanus ratio of both types of muscle were similar. Of all transplantations performed, only those in normal mice with intact nerve responded upon stimulation. Both normal and dystrophic transplants in normal hosts showed similar isometric properties. Although intact dystrophic muscles and viable dystrophic transplants in normal hosts were similar in weight, the transplants developed about three to four times more tension. In addition, dystrophic transplants showed relaxation times similar to normal muscles. It is suggested that the dystrophic lesion in mice may have a neural origin.     

The effect of age on the nucleic acid content of slow- and fast-twitch muscle in normal and dystrophic mice and their litter mates

RNA, DNA, and NCP content were measured in fast- and slow-twitch skeletal muscle of normal and dystrophic mice (HDM) and their littermates at ages 4 through 29 weeks.In normal and litter mate mice RNA and DNA content were far greater in the soleus than in the gastrocnemius while the RNA/DNA ratio and NCP content were greater in the gastrocnemius. In dystrophic mice, however, the differences between nucleic acid content of the 2 muscles were far less, apparently due to a proportionately higher content in the dystrophic gastrocnemius. Due to a proportionately lower ratio in the gastrocnemius, dystrophic RNA/DNA ratios for the 2 muscles were essentially the same.Age had a marked effect on the nucleic acid content of both muscles in all 3 mice types but to varying degrees. In the soleus, RNA and DNA content rapidly decreased until 9 to 10 weeks of age followed by a gradual decline. Soleus RNA/DNA ratios showed little change with age except in the HDM mice in which there was a significant overall decline. In the gastrocnemius, RNA content followed the same pattern but with a smaller decline in the younger ages. Age had no affect on DNA content in the normal gastrocnemius, but there was a significant decline in the HDM gastrocnemius. RNA/DNA gastrocnemius ratios showed marked fluctuations in both normal and dystrophic mice but did not appear to be affected by age.Depending upon age and muscle type, dystrophic RNA content was significantly higher in both muscle types from 5 to 9 weeks of age, and then decreased below normal levels. DNA content of both dystrophic soleus and gastrocnemius was also elevated in the young mice. In the soleus, DNA also decreased below normal values during middle age but then increased again in the middle and older age mice. The RNA/DNA ratio of the dystrophic gastrocnemius was decreased in most age groups. The ratio for the soleus, however, had 3 phases; a reduced ratio in the young mice, a higher ratio in the middle-aged mice, and a reduced ratio in the oldest mice.     

Fatigability of normal and dystrophic chicken muscle in vivo

Changes in the indirectly elicited isometric twitch tension, tetanic tension, twitch-to-tetanus ratio, and post-tetanic potentiation in response to fatigue were examined in the posterior latissimus dorsi muscles of normal and genetically dystrophic New Hampshire chickens. Contractile parameters were studied during 3-h fatigue stimulations and during continuous infusion of potassium chloride (0.24 m equiv/min) initiated at the conclusion of the fatigue period. Both twitch and tetanic tension of dystrophic muscles showed a relative resistance to fatigue; no significant changes in either twitch-to-retanus ratio or post-tetanic potentiation occurred during the fatigue period. In contrast, twitch and tetanic tension of normal muscles decreased more rapidly and to a greater extent in response to fatigue. The twitch-to-tetanus ratio decreased and post-tetanic potentiation increased such that after 30 min they were not significantly different from values seen in dystrophic muscles. Potassium chloride infusion produced a significant recovery (two- to ninefold improvement) of the fatigued twitch response to dystrophic muscle but did not have a significant effect on fatigued normal muscles. A comparison of directly and indirectly elicited twitch contractions indicated that part of the decrement of contractile response in dystrophic muscle was due to synaptic failure at the neuromuscular junction and that potassium chloride infusion resulted in restoration of neuromuscular transmission. It is suggested that the difference in fatigue pattern observed between normal and dystrophic muscle was a function of an altered distribution in the physiological types of motor units present in the diseased muscle.