Potassium and caffeine contractures in limb muscles of normal and dystrophic (C57 BL/6J dy/dy) mice

The strength of contractures, produced by 15 to 146 mM [K]₀ (as l-glutamate), was measured in isolated small bundles of muscle fibers from the fast-twitch extensor digitorum longus and from the slow-twitch soleus of normal and dystrophic (C57 BL/6J dy^2J/dy^2J) mice. The analysis of the relation between the maximal amplitude of the contracture vs the membrane potential and the time constant of relaxation of the K-contractures has shown that dystrophy induced an attenuation of the differences between fast- and slow-twitch muscles. The repriming of K-contractures was more affected by changes in [Ca]₀ in normal soleus than in normal extensor digitorum longus and this difference was unaffected by dystrophy. For both types of muscles, the ability of caffeine to produce contractures was reduced in dystrophic muscle and this modification was not related to a change in the fiber typing.     

A comparative study of contractile responses in diaphragm muscles of normal and dystrophic ( ) mice

Potassium and caffeine contractures of isolated small bundles (100 to 200 μm diameter) of muscle fibers isolated from the diaphragm of normal and dystrophic ( ) mice were compared. In diaphragms of pathologic mice (3 to 5 months old) the resting potential, the characteristics of the twitch, and some histological examinations were typical of dystrophic muscles. The slopes of the relationships between the steady membrane potential and log [K]₀ were similar for the two types of cells. In 110 mM and 146 mM K there were no significant differences in the time course of the contractures and reduction in [Ca]₀ decreased the time to peak and the time constant of relaxation to the same extent; the relative efficiency of [Mg]₀ compared with [Ca]₀ was equivalent. Repriming of K contractures at different external calcium concentrations indicated that the normal diaphragm did not have any special advantage. The exposure of isolated strips to a solution containing caffeine resulted in a similar increase of the strength of the regularly evoked twitch responses. However, the contractures elicited by 1.25 to 20 mM caffeine showed a subsensitivity of the dystrophic diaphragm (K_mDys = 9.3 K_mN) and the rate of relaxation was significantly slower than in normal muscle (in 20 mM caffeine, 50% decay time for normal muscle was 25.2 ± 7.6 s and for dystrophic muscle 54.8 ± 11.2 s. THese results suggest an absence of major alterations in the mechanism of excitation-contraction coupling associated with dystrophy, except for a change in the specific element of the sarcoplasmic reticulum where caffeine acts.     

Chronic phenytoin administration alters the metabolic profile of superficial gastrocnemius muscle fibers in dystrophic mice

Phenytoin is known to reduce neural overactivity (pseudomyotonia) affecting the hind limb musculature in $\text{C57B1}\text{6J}$ dystrophic ($\text{dy}{}^{\mathrm{2J}}\text{dy}{}^{\mathrm{2J}}$) mice. This study reports a change in the metabolic profile of superficial gastrocnemius muscle fibers from $\text{dy}{}^{\mathrm{2J}}\text{dy}{}^{\mathrm{2J}}$ animals after chronic phenytoin treatment. The superficial gastrocnemius muscle region from normal mice is composed of 98% fast-twitch glycolytic muscle fibers. In dystrophic mice these fibers (FG) show increased oxidative capacity without evidence of morphologic degeneration during the first few months ex utero. Many of these fibers also store abnormally large amounts of glycogen as determined by periodic acid-Schiff histochemistry. After 104 days of phenytoin treatment, the $\text{dy}{}^{\mathrm{2J}}\text{dy}{}^{\mathrm{2J}}$ FG muscle fibers showed a reduction in abnormally high oxidative capacity as monitored by succinic dehydrogenase activity; there was also a reduction of glycogen storage in a number of $\text{dy}{}^{\mathrm{2J}}\text{dy}{}^{\mathrm{2J}}$ fibers. One hypothesis suggests that the increase in oxidative capacity of the $\text{dy}{}^{\mathrm{2J}}\text{dy}{}^{\mathrm{2J}}$ superficial gastrocnemius muscle fibers is the expected result of overstimulation by the pseudomyotonia. Our experiments indicated that the abnormal metabolic profile observed in those fibers can be altered simply by a reduction in pseudomyotonia. These results mimic those seen after short-term denervation of the same $\text{dy}{}^{\mathrm{2J}}\text{dy}{}^{\mathrm{2J}}$ muscle. After phenytoin treatment the mean $\text{dy}{}^{\mathrm{2J}}\text{dy}{}^{\mathrm{2J}}$ superficial gastrocnemius muscle fiber cross-sectional area was significantly increased compared with untreated animals. Cursory examination of the degenerated deep region of this same muscle suggested that similar changes did not occur after drug treatment. This suggests that the pseudomyotonia was partially different from the factor(s) causing early degeneration of the oxidative muscle fibers in the $\text{dy}{}^{\mathrm{2J}}\text{dy}{}^{\mathrm{2J}}$ animals.     

Restoration of functional continuity in dystrophic murine muscle after crushing

The recovery of functional continuity after crush injury was measured by a simple electrophsiological technique in semitendinosus muscles of normal and dystrophic mice of the $\text{C57BL}\text{6J}\text{(dy}{}^{\mathrm{2J}}\text{dy}{}^{\mathrm{2J}}\text{)}$ strain. In contrast to virtually complete restitution in normal muscles, only one-third of fibers regained continuity in dystrophic muscles. The study also confirmed the lower resting membrane potentials of dystrophic fibers and the presence of “functional” denervation in some of them.     

The number and size of motoneurons in the soleus motor nucleus of the normal and dystrophic (C57BL/6J dy2J/dy2J) mouse

The method of retrograde axonal transport of horseradish peroxidase (HRP) was used to identify the motoneurons that innervate the soleus muscle in normal and dystrophic (C57BL/6J dy^2J/dy^2J) mice. In both normal and dystrophic animals the soleus motor nucleus was located in spinal segments L3 and L4, in a medial position in the lateral division of lamina IX. Measurements of the motoneuron soma area of HRP-labeled cells in normal and dystrophic animals showed that motoneuron size was bimodally distributed in both cases but that mean soma areas of motoneurons of both the large and small cell component from the dystrophic animals were larger. The number of alpha motoneurons was reduced in the dystrophic animals compared with the normal animals, the mean number of alpha motoneurons being 20 in the case of dystrophic animals and 25 in the case of normal animals.     

Rate and extent of functional reinnervation in fast-twitch and slow-twitch muscles of the dystrophic mouse (C57Bl6Jdy2jdy2j)

The extent of functional reinnervation of fast-twitch extensor digitorum longus muscle in dystrophic and normal mice was determined at various times after nerve transection. Functional reinnervation was assessed by measuring the twitch tension evoked by stimulation of the nerve central to the site of transection. In control mice aged 4 to 6 weeks at the time of denervation, complete reinnervation was observed after 6 weeks. In dystrophic mice of the same age reinnervation was clearly impaired. The ratio of functional innervation of the operated leg to that of the contralateral unoperated leg was only 0.62 after 6 weeks. In older dystrophic mice (4 to 6 months at the time of nerve transection) the reinnervation ratio was even lower, 0.43 after 12 weeks. Reinnervation of slow-twitch soleus muscle was assessed 8 weeks after denervation and was also found to be reduced in the older dystrophic animals. The extent of reinnervation was reflected in the measured values of muscle weight, twitch tension per unit wet weight, and twitch time course. The impairment of reinnervation of dystrophic muscle is consistent with, but not proof of, a neurogenic defect in murine muscular dystrophy.     

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.     

Electrophysiology of the neuromuscular junction of the laminin-2 (merosin) deficient C57 BL/6J dy/dy dystrophic mouse

The C57 BL/6J dy^2J/dy^2J dystrophic mouse expresses an abnormal truncated form of the α2 subunit of the protein laminin-2 (or merosin), which is unable to form a stable link between the extracellular matrix and the dystrophin-associated proteins, resulting in muscular dystrophy. Morphological abnormalities of the peripheral nervous system and neuromuscular junction have also been reported. The electrophysiological properties of the neuromuscular junctions of diaphragm, extensor digitorum longus (EDL), and soleus from C57 BL/6J dy^2J/dy^2J mice and controls are described. No evidence for the presence of denervated fibres were found. Mean MEPP amplitudes were significantly increased in EDL and soleus but reduced in the diaphragm from affected mice. Mean MEPP frequencies were raised in all the dy^2J/dy^2J muscles studied. dy^2J/dy^2J muscles were paralysed by low concentrations of μ-conotoxin suggesting that embryonic (tetrodotoxin and μ-conotoxin resistant) sodium channels are not widespread on dy^2J/dy^2J muscle as has previously been reported. EPP latencies were significantly prolonged in the diaphragm and EDL but not soleus from dy^2J/dy^2J mice. Quantal contents were higher in all dy^2J/dy^2J muscles. In the dy^2J/dy^2J diaphragm failures in neurotransmission occurred and a faster rate of rundown of EPPs was apparent. Some changes appear from a direct effect of dystrophy, whilst increased MEPP frequency and quantal content, and failures in neurotransmission indicate neuronal abnormalities.     

Potassium and caffeine contractures in limb muscles of normal and dystrophic (C57 BL/6J dy2J/dy2J) mice

The strength of contractures, produced by 15 to 146 mM [K]0 (as L-glutamate), was measured in isolated small bundles of muscle fibers from the fast-twitch extensor digitorum longus and from the slow-twitch soleus of normal and dystrophic (C57 BL/6J dy2J/dy2J) mice. The analysis of the relation between the maximal amplitude of the contracture vs the membrane potential and the time constant of relaxation of the K-contractures has shown that dystrophy induced an attenuation of the differences between fast- and slow-twitch muscles. The repriming of K-contractures was more affected by changes in [Ca]0 in normal soleus than in normal extensor digitorum longus and this difference was unaffected by dystrophy. For both types of muscles, the ability of caffeine to produce contractures was reduced in dystrophic muscle and this modification was not related to a change in the fiber typing.     

The plasmin-catalyzed hydrolysis of Nα-CBZ-L-lysine p-nitrophenyl ester

Several details of the hydrolysis of N^α-CBZ-L-lysine $\text{p}$-nitrophenyl ester catalyzed by human plasmin are described. The reaction proceeds $\text{via}$ a Michaelis-Menten type mechanism with k_cat = 40/sec, K_m = 2.0 × 10^?5 M and $\text{k}{}_{\mathrm{cat}}\text{K}{}_{\mathrm{m}}\text{= 2.0 × 10}{}^6\text{/M sec}$ at pH 6.0. When the initial substrate concentration was 1.04 × 10^?4 M, no substrate and/or product activation and/or inhibition could be detected. The reversible inhibitor 6-amino-hexanoic acid partially inhibited the activation of plasminogen by streptokinase and had a small effect upon the hydrolysis of CLN by plasmin. The activity toward the synthetic ester was also found to be proportional with the fibrinolytic activity, as determined by a conventional fibrin tube assay. In analogy with thrombin and trypsin, these data suggest that the plasmin-catalyzed hydrolysis of carboxylic acid derivatives proceeds $\text{via}$ an acyl-enzyme intermediate.     

Mitochondrial calcium content and oxidative phosphorylation in heart and skeletal muscle of dystrophic mice

Mitochondrial calcium overloading was investigated in the genetically dystrophic mouse (strains $\text{129}\text{ReJ}\text{dy}\text{dy}$) as a possible contributing factor to the development of muscle fiber necrosis. Mitochondrial calcium concentrations were significantly elevated in both skeletal muscle and heart organelles. Because mitochondria were isolated in the presence of ruthenium red this finding was not the result of an artefact of isolation. State 3 respiration rates and concomitantly the respiratory control ratios were slightly decreased in skeletal muscle, but not in heart mitochondria. This abnormality could result from calcium overloading in a small fraction of the mitochondria. Fractionation of skeletal muscle mitochondria on sucrose gradients gave two distinct populations of dystrophic organelles, one with high calcium, whereas normal skeletal muscle mitochondria and heart organelles showed only one broad band on the gradient. The results support the idea that both skeletal muscle and heart are affected in dystrophic mice, strain $\text{129}\text{ReJ}\text{dy}\text{dy}$ and also that in the dystrophic mouse the process of cell necrosis is associated with cellular calcium overloading.     

Beneficial effects of transplanting normal limb-bud mesenchyme into dystrophic mouse muscles

A new technique is being developed to remedy muscle weakness of hereditary myopathies. Mesenchymal cells dissected from limb-buds of day-12 normal mouse embryos were transplanted into the right solei of 20-day-old normal or dystrophic C56BL/6J-dy^2J mice. Host and donors were immunocompatible. Unoperated left solei served as controls. Sham control solei receiving similar surgical treatment but no mesenchyme transplant did not differ from contralateral, unoperated solei. Six to seven months postoperatively the test solei (8 normal and 15 dystrophic) exhibited greater cross-sectional area, total fiber number, and twitch and tetanus tensions than their contralateral controls. Test dystrophic solei contained more normal-appearing and less abnormal-appearing fibers than their controls. Their mean fiber resting potential was intermediate between those of normal and dystrophic controls. There is no difference in twitch time course between test and control solei. The results indicate that such transplantation improves the structure and function of the dystrophic muscles.     

Surface density of T tubules in normal and dystrophic mouse muscle

Although, as shown in previous studies, fast-twitch fibers from the superficial region of the dystrophic gastrocnemius of the mouse (C57BL/6J dy^2J/dy^2J) show some abnormality, including abnormal end-plates and elevated mitochondrial volume percentages, the transverse tubular system in these fibers appears normal. Measurement of the surface density of the system reveals no significant difference between normal and dystrophic fast-twitch fibers, unlike the situation in dystrophic fast-twitch chicken muscle fibers.     

The number and size of motoneurons in the soleus motor nucleus of the normal and dystrophic (C57BL/6J dy/dy) mouse

The method of retrograde axonal transport of horseradish peroxidase (HRP) was used to identify the motoneurons that innervate the soleus muscle in normal and dystrophic (C57BL/6J dy^2J/dy^2J) mice. In both normal and dystrophic animals the soleus motor nucleus was located in spinal segments L3 and L4, in a medial position in the lateral division of lamina IX. Measurements of the motoneuron soma area of HRP-labeled cells in normal and dystrophic animals showed that motoneuron size was bimodally distributed in both cases but that mean soma areas of motoneurons of both the large and small cell component from the dystrophic animals were larger. The number of alpha motoneurons was reduced in the dystrophic animals compared with the normal animals, the mean number of alpha motoneurons being 20 in the case of dystrophic animals and 25 in the case of normal animals.     

Improvement of motor conduction velocity in hereditary neuropathy of LAMA2-CMD dy2J/dy2J mouse model by glatiramer acetate

ObjectiveGlatiramer acetate (GA), an agent modulating the immune system, has been shown to cause significantly improved mobility and hind limb muscle strength in the dy^2J/dy^2J mouse model for LAMA2-congenital muscular dystrophy (LAMA2-CMD). In view of these findings and the prominent peripheral nervous system involvement in this laminin-α2 disorder we evaluated GA’s effect on dy^2J/dy^2J motor nerve conduction electrophysiologically.MethodsLeft sciatic-tibial motor nerve conduction studies were performed on wild type (WT) mice (n = 10), control dy^2J/dy^2J mice (n = 11), and GA treated dy^2J/dy^2J mice (n = 10) at 18 weeks of age.ResultsControl dy^2J/dy^2J mice average velocities (34.49 ± 2.15 m/s) were significantly slower than WT (62.57 ± 2.23 m/s; p < 0.0005), confirming the clinical observation of hindlimb paresis in dy^2J/dy^2J mice attributed to peripheral neuropathy. GA treated dy^2J/dy^2J mice showed significantly improved average sciatic-tibial motor nerve conduction velocity versus control dy^2J/dy^2J (50.35 ± 2.9 m/s; p < 0.0005).ConclusionIn this study we show for the first time improvement in motor nerve conduction velocity of LAMA2-CMD dy^2J/dy^2J mouse model’s hereditary peripheral neuropathy following GA treatment.SignificanceThis study suggests a possible therapeutic effect of glatiramer acetate on hereditary peripheral neuropathy in this laminin-α2 disorder.     

Study of neurotrophism in normal/dystrophic parabiotic mice

The trophic influences of nerve and muscle on one another were studied in normal and dystrophic littermates of C57BL/6J dy^2J mice parabiosed at 20 to 23 days after birth. Each parabiont had a soleus muscle cross-reinnervated by a tibial nerve of its partner. Ultrastructural abnormalities of muscle and endplate were quantified and compared 6 to 7 months postoperatively. The dystrophic nerve degenerated despite reinnervation to a normal muscle. The normal muscle did not prevent the dystrophic nerve from degenerating, and the dystrophic nerve induced degenerative changes in the reinnervated normal muscle. Normal nerve did not retard the genetically programmed degeneration of the dystrophic muscle. The dystrophic muscle, however, did not appear to cause normal nerve terminals to degenerate. We conclude that both nerve and muscle cells in dystrophic mice express characteristics of muscular dystrophy. Muscle fibers of a few motor units further suffer from abnormal neurotrophic influence because of the degeneration of the motor neurons. Myotrophic influence on nerve was not observed.