Freeze-fracture study of muscle plasmalemma in normal and dystrophic chickens

We compared the freeze-fracture morphology of the plasmalemma of the pectoralis major muscle taken from normal and dystrophic chickens at adult, embryonic, and early posthatching stages. Developmental changes were more conspicuous in surface caveolae than in intramembranous particles. The most striking differences between normal and dystrophic muscles were seen in the densities of the caveolae (17/μm² in the normal adult; 30/μm² in the dystrophic adult) and in their distribution (rectangular pattern in normals; random arrangement in dystrophics). These differences had already become significant at seven days posthatching and before the appearance of clinical symptoms. This is the earliest developmental stage at which morphologic abnormalities of plasmalemma have been reported for dystrophic muscle. Variations of surface topography and caveolar morphology, presumably representing secondary changes, were common in adult dystrophic muscle.     

Effect of low Ca2+ solution on muscle contraction of developing, preclinical dystrophic (dy2j) mice.

EDL muscles from normal and dystrophic (dy2j) mice aged 7 to 21 days of postnatal life were examined. Muscles were divided into 2 groups according to age, 7 to 14 days and 16 to 21 days postnatal, so as to assess age- and/or phenotype-related differences in the muscle response to low Ca2+ solution. Tension production was already much impaired in "predystrophic" muscles. At this stage, however, there was essentially no difference in twitch kinetics between normal and dystrophic muscles. Upon exposure to low Ca2+ solution, twitch responses of both normal and dystrophic muscles declined in a similar manner. In the youngest animals studied (7 to 14 days), the tetanic responses of both normal and dystrophic muscles to low Ca2+ solution were also similar. In animals 15 to 21 days old, however, the tetanic tension developed in low Ca2+ solution by dystrophic muscles, was significantly less than that of normal. Moreover, under these conditions (i.e., in low Ca2+ solution), and following tetanic stimulation, the membrane potential of dystrophic muscles in this age group was significantly more depolarized than that of normal muscles. Our results suggest that the ability of the cell to deal with extracellular Ca2+ is normal in predystrophic muscles up to 21 days of postnatal life. The results also clearly point to the fragility of the membrane in these muscles.     

Pro-opiomelanocortin neuropeptide receptors on developing and dystrophic muscle fibers

Autoradiography was used to investigate the presence of corticotropin and β-endorphin receptors on soleus, extensor digitorum longus, and diaphragm muscles of normal and dystrophic adult mice and developing mice. In both adult and developing mice, specific binding sites for both [¹²⁵I]ACTH and [¹²⁵I]β-endorphin were present in some fibers in all of the muscles examined. The specific binding sites appeared to be distributed over the length of the surface membrane in the fibers that expressed them. There were significantly higher proportions of fibers exhibiting the specific β-endorphin and the specific ACTH binding sites in the three muscle types in mice of 5 d of age compared to the muscles of the normal adult. There was also a higher proportion of fibers with the putative ACTH and β-endorphin receptors in the three muscle types in dystrophic mice.     

Developmental changes in succinate dehydrogenase activity in muscle fibers from normal and dystrophic mice

The question of whether or not the development of dystrophic muscles is similar to that of normal muscles, prior to the manifestations of the symptoms of the disease, is investigated here. The developmental change in the activity of succinate dehydrogenase was therefore measured in individual fibers of prospectively dystrophic muscles from 10- to 28-day-old mice (strain C57Bl/6J dy2j) and compared with that of muscles from normal mice of the same age. It was found that up to 10 days of age, muscle fibers from normal and prospective dystrophic animals had low succinate dehydrogenase activities, and were all more or less uniform. Thereafter in the normal muscle the overall activity of the enzyme increased and the fibers became more heterogeneous with age. By 21 days the extensor digitorum longus muscle resembled that of the adult. At that time, fibers from prospectively dystrophic muscles had lower succinate dehydrogenase activities and were more homogeneous. Thus fibers from prospectively dystrophic muscles fail to achieve their adult characteristics by 21 days. On the basis of these results, it is suggested that muscle maturation is retarded in dystrophic animals.     

Methylphenidate treatment increases Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase activity in the cerebrum of young and adult rats

Methylphenidate is a central nervous system stimulant used for the treatment of attention-deficit hyperactivity disorder. Na⁺, K⁺-ATPase is a membrane-bound enzyme necessary to maintain neuronal excitability. Considering that methylphenidate effects on central nervous system metabolism are poorly known and that Na⁺, K⁺-ATPase is essential to normal brain function, the purpose of this study was to evaluate the effect of this drug on Na⁺, K⁺-ATPase activity in the cerebrum of young and adult rats. For acute administration, a single injection of methylphenidate (1.0, 2.0, or 10.0 mg/Kg) or saline was given to rats on postnatal day 25 or postnatal day 60, in the young and adult groups, respectively. For chronic administration, methylphenidate (1.0, 2.0, or 10.0 mg/Kg) or saline injections were given to young rats starting at postnatal day 25 once daily for 28 days. In adult rats, the same regimen was performed starting at postnatal day 60. Our results showed that acute methylphenidate administration increased Na⁺, K⁺-ATPase activity in hippocampus, prefrontal cortex, and striatum of young and adult rats. In young rats, chronic administration of methylphenidate also enhanced Na⁺, K⁺-ATPase activity in hippocampus and prefrontal cortex, but not in striatum. When tested in adult rats, Na⁺, K⁺-ATPase activity was increased in all cerebral structures studied. The present findings suggest that increased Na⁺, K⁺-ATPase activity may be associated with neuronal excitability caused by methylphenidate.     

Response of a fast muscle from normal and dystrophic (dy2j) mice to a local decrease in extracellular Ca2+ induced at different stages of postnatal life

It has previously been reported that reducing Ca2+ entry into muscle fibres was beneficial to dystrophic muscles. In this study, we examined the effect, on the force output and contractile properties of the tibialis anterior muscle, of a local decrease in extracellular Ca2+, produced in normal and dystrophic mice at various stages of postnatal life by applying a small strip of silicon rubber containing a calcium chelator (BAPTA). Lowering extracellular Ca2+ in this way at an early stage of postnatal life (11-16 days) interferes with normal development in that, 3-5 weeks after the initial operation, the treated TA muscles from normal mice are weaker and their contractile speed is slower than that of their untreated counterparts. In contrast, the same procedure has a beneficial effect on dystrophic muscles in that they produce more force than untreated controls. Our results show that these changes are not related to changes in the total number of muscle fibres or fibre type proportions. These changes are temporary and by 8-12 weeks after the operation, the treated muscles are indistinguishable from controls. Finally, our results also indicate that skeletal muscles from older animals, both normal and dystrophic, become insensitive to this manipulation. These results provide the first evidence for a difference in the sensitivity of normal immature and normal adult skeletal muscles to their extracellular Ca2+ environment. They also suggest that in this context, dystrophic muscles might already differ from normal at a stage prior to the clinical expression of the symptoms of the disease.     

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.     

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.     

Muscle fiber growth and necrosis in dystrophic muscles: a comparative study between dy and mdx mice

Histopathological and morphometric studies of murine dystrophic muscles in the early postnatal period were performed. In dy mice, obtained by in vitro fertilization, scattered necrotic fibers were noted at 10 days of age followed by poorly compensated regeneration leading to muscle fiber loss, marked variation in fiber size, and fibrous and adipose tissue proliferation. In mdx mice, clusters of necrotic muscle fibers seen at 10-15 days were followed by well compensated regeneration with little fibrosis. There appeared to be no delay in muscle fiber growth and fiber type differentiation in both dy and mdx mice up to 10 days of age since there was no distinct difference in muscle fiber size, fiber type differentiation and the incidence of myosatellite cells between dystrophic muscles and those from age-matched control mice. Muscle fibers appeared to undergo necrosis only when they had reached a certain stage of maturation since at early stages of development or regeneration they rarely became necrotic. The difference in clinical symptoms between dy and mdx mice may result from differences in their regenerative response to necrosis.     

Isometric contractile properties of extensor digitorum longus muscle of the dystrophic hamster BIO 40.54

The contractile properties, including instantaneous stiffness, of normal and the genetically dystrophic BIO 40.54 hamster extensor digitorum longus muscle were compared at 60, 120, 170, 220, and 320 days of age. A general impairment in tension-generating ability of the dystrophic muscles was observed which correlated well with a corresponding change in the measured stiffness. A decrease in the mechanical half-relaxation time was observed at all age groups except 120 and 170 days of age. The data presented are discussed in terms of a defect in the Ca²⁺ regulatory functions and/or energy production and utilization in the dystrophic skeletal muscle cell.     

Microglial K+ channel expression in young adult and aged mice

The K⁺ channel expression pattern of microglia strongly depends on the cells' microenvironment and has been recognized as a sensitive marker of the cells' functional state. While numerous studies have been performed on microglia in vitro, our knowledge about microglial K⁺ channels and their regulation in vivo is limited. Here, we have investigated K⁺ currents of microglia in striatum, neocortex and entorhinal cortex of young adult and aged mice. Although almost all microglial cells exhibited inward rectifier K⁺ currents upon membrane hyperpolarization, their mean current density was significantly enhanced in aged mice compared with that determined in young adult mice. Some microglial cells additionally exhibited outward rectifier K⁺ currents in response to depolarizing voltage pulses. In aged mice, microglial outward rectifier K⁺ current density was significantly larger than in young adult mice due to the increased number of aged microglial cells expressing these channels. Aged dystrophic microglia exhibited outward rectifier K⁺ currents more frequently than aged ramified microglia. The majority of microglial cells expressed functional BK‐type, but not IK‐ or SK‐type, Ca²⁺‐activated K⁺ channels, while no differences were found in their expression levels between microglia of young adult and aged mice. Neither microglial K⁺ channel pattern nor K⁺ channel expression levels differed markedly between the three brain regions investigated. It is concluded that age‐related changes in microglial phenotype are accompanied by changes in the expression of microglial voltage‐activated, but not Ca²⁺‐activated, K⁺ channels. GLIA 2015;63:664–672     

Response of normal and dystrophic muscles to increased functional demand

Immature fast muscles appear to be unfavorably influenced by excessive activity and there is evidence suggesting that the maturation of muscles from animal models of muscular dystrophy and patients suffering from Duchenne dystrophy is impaired. We therefore examined the effects of chronic functional overload applied at different stages of postnatal life on a fast muscle in normal and dystrophic mice (C57B1/6J dy2j/dy2j). "Overload" of tibialis anterior muscle was produced by removal of its synergist extensor digitorum longus in one hind limb. Our results suggest that increased functional demand can be damaging to immature muscles and that in dystrophic animals, the inability to adjust to overload persists into adult life.     

CNS control of active sodium transport in muscle during progressive hypokalemia in the rat

The degree of plasma hypokalemia was graded with the duration of a potassium deficient diet. The intracellular Na⁺ and K⁺ contents ([Na]_i and [K]_i) of the innervated soleus muscle of hypokalemic rats were determined and plotted as a function of plasma K⁺ concentration following a potassium deficient diet during 1–8 weeks. The progression of plasma hypokalemia was highly correlated with both [Na]_i accumulation and [K]_i loss in the muscle. Denervation of muscles in the hypokalemic rat resulted in a rapid activation of the Na-pump in the denervated soleus muscle regardless of the degree of plasma hypokalemia. This pump activation in the denervated muscle was chronically maintained throughout the hypokalemia. Restoration of a normal, K⁺ containing, diet for 4 days in hypokalemic rats resulted in a complete recovery of normal Na⁺ and K⁺ contents in both the innervated muscle and plasma, while the [Na]_i and [K]_i in the denervated muscle was unaffected. The relationship between the CNS-induced inhibition of the muscle Na-pump and the plasma K⁺ levels in hypokalemic rats is discussed.     

A comparative study of contractile responses in diaphragm muscles of normal and dystrophic (C57BL6Jdy2Jdy2J) 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 ($\text{C57BL}\text{6J}\text{dy}{}^{\mathrm{2J}}\text{dy}{}^{\mathrm{2J}}$) 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.     

Effects of pH on membrane resistance in normal and dystrophic mouse skeletal muscle fibers

Membrane cable properties of skeletal muscle fibers of dystrophic mice (Rej-129) and their littermate controls were examined using a conventional two-microelectrode recording technique. Fibers from dystrophic mice had a decreased membrane resistivity (R_m) compared with those from normal mice (517 ± 27 vs 642 ± 34 Ω ? cm²), while the internal resistivities (R_i) did not differ significantly. The increase in membrane specific conductance was due to an increased Cl^? conductance (g_Cl) (2304 vs 1346 μS/cm² for normal fibers), although the K⁺ conductance (g_K) was actually decreased (234 vs 369 μS/cm² for normal fibers). With changes in pH, membrane conductances of normal and dystrophic skeletal muscle fibers varied differently, mainly due to differences in effects on the Cl^? conductance. This contrast may be due to altered regulation of internal pH in dystrophic muscle.     

Intracellular potassium activities in muscles of normal and dystrophic mice: An in vivo electrometric study

Intracellular potassium ion activity (aK_i⁺) was measured in vivo in gastrocnemius muscle fibers of normal and dystrophic ($\text{dy-2J}\text{dy-2J}$) $\text{C57B1}\text{6}\text{J}$ mice. Rapid measurements were made by means of a double-barrel ion selective microelectrode. The aK_i⁺ in muscles of dy-2J mice was about 8% lower than in muscles of normal mice. This reduction in aK_i⁺ was not sufficient to account for the low resting potential of the dy-2J fibers. The fibers measured were from the superficial region of the muscle, a region previously shown to be composed of fast-twitch fibers. This region shows only minor morphologic changes in young dy-2J mice. The reduction of aK_i⁺ in these fibers is consistent with the hypothesis that there is a general membrane defect associated with muscular dystrophy. It is also suggested, however, that the neurally derived pseudomyotonia associated with the dy-2J mutation and the concomitant increase in the oxidative capacity in the measured muscle fibers might also affect the concentration of intracellular K⁺.     

Spontaneous electrical activity in muscles of dystrophic (dy/dy) mice

Spontaneous electrical activity of tibialis anterior (TA) muscles was recorded using extracellular electrodes in dystrophic and phenotypically normal mice. Abnormal levels of spontaneous activity were recorded in the muscles of adult dystrophic mice. The activity was reduced by more than 75% after sciatic nerve section in anaesthetised dystrophic mice suggesting that most of the activity was neurally mediated. No abnormal activity was seen in the biceps brachii muscles of adult dystrophic mice. No abnormal spontaneous activity was seen in young (2-3-week-old) dystrophic mice. As degeneration of muscle fibres begins at about 7 days of age in dystrophic hindlimb muscles, the onset of the abnormal spontaneous EMG activity could not be considered causally related to muscle fibre degeneration.     

Endogenous modulators of brain Na, K-atpase at early postnatal stages of rat development

The presence of endogenous modulators (peaks I and II) of synaptosomal Na⁺, K⁺-ATPase activity from adult rat cerebral cortex was previously suggested. In this study, the presence of such modulators at different postnatal stages of rat development was examined and their effect was tested on Na⁺, K⁺-ATPase activity. Synaptosomal membrane Na⁺, K⁺-ATPase activity was enhanced 20–30% by peak I and inhibited 70–75% by peak II obtained from 4-, 10-, 20- and 35–40-day-old rats. A fraction purified from peak II by anionic exchange HPLC (termed II-E) highly inhibits enzyme activity and behaves as a ouabain-like factor. Inhibitory activity of a 4-day-old II-E fraction proved higher than the corresponding fraction obtained from adult rats. Since expression of cerebral Na⁺, K⁺-ATPase has been shown to increase 10-fold during development whereas peak II concentration was observed to remain constant, and given the higher potency of purified neonatal II-E fraction, the effect of the latter may be greater at early postnatal stages of development than during adult life. It is suggested that the II-E fraction, which contains an ouabain-like factor, may play a role in neuronal development.     

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.     

Observations on the efficiency of dystrophic muscle in vitro

A study of muscles of the dystrophic mouse has failed to substantiate earlier claims that these muscles were especially resistant to fatigue in vitro or that fast muscles are preferentially damaged. It has been found that the fast muscle selected for previous studies is very often unable to withstand isolation in an organ bath if it is working, and both the difficulty in removing the normal gastrocnemius muscle intact and the need to trim it surgically contribute independently toward its deterioration in vitro. The smaller dystrophic gastrocnemius muscle is less liable to excision damage, is able to satisfy its resting metabolic needs in nutrient solution, and requires no damaging dissection, but is nevertheless unable to recover normally from fatigue. Using EDL and soleus muscles which are small enough to withstand isolation in vitro, no differences are found between fatigue patterns of normal and dystrophic specimens. Responses to rest, KCl, and 2 mM caffeine are also quite similar, and the only distinguishing biomechanical characteristic we have found in dystrophic mouse muscle is a weaker contraction and a longer total twitch time.