Dy 2J Research Articles

Fatiguability and oxidative capacity of forelimb and hind limb muscles of dystrophic mice

Fatigue indices and succinic dehydrogenase (SDH) activities were determined in the extensor digitorum longus (EDL) and soleus muscles of the hind limb and the extensor carpi radialis longus of the forelimb in control and dystrophic mice aged 4 to 26 weeks. A good correlation was found between SDH activities and fatigue indices in muscles from normal mice. In the dystrophic (dy 2J) mice, however, this correlation was not present. The EDL muscles from 26-week-old dy 2J mice showed a much higher resistance to fatigue than age-matched controls but this was not accompanied by a significant change in SDH. The increased fatigue resistance in dy 2J EDL appeared between 8 and 12 weeks of age and was temporally correlated with the onset of fused bursts of spontaneous activity in the hind limb muscles. Nevertheless, there was no conclusive evidence for a link among this spontaneous activity, oxidative enzyme capacity, and fatigue resistance.

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 C57B1 6J dystrophic ( dy 2J dy 2J ) mice. This study reports a change in the metabolic profile of superficial gastrocnemius muscle fibers from dy 2J dy 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 dy 2J dy 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 dy 2J dy 2J fibers. One hypothesis suggests that the increase in oxidative capacity of the dy 2J dy 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 dy 2J dy 2J muscle. After phenytoin treatment the mean dy 2J dy 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 dy 2J dy 2J animals.

A comparative study of contractile responses in diaphragm muscles of normal and dystrophic ( [formula omitted]) 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 ( C57BL 6J dy 2J dy 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 m M and 146 m M 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 m M 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 m M 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.

Met-tRNA fMet binding in murine dystrophy

Dystrophic mice of the C57B1 dy 2J dy 2J strain and of the ReJ 129 dy dy strain and littermate controls were used to prepare met-tRNA fMet binding factors. The tissues were homogenized and fractions were obtained which contained ribosomes. The binding factors were assayed by the binding of [ 35S]methionyl-tRNA to control liver ribosomes. The binding, i.e. eukaryotic initiation factor 2 (elF 2) activity, was measured in brain, liver and muscle and in all of these tissues there was a significant decrease in the dystrophic mice. This decrease in initiation factor activity of hindleg muscle resembled, in the direction of the effect, the decrease in elongation factor activity of hindleg muscle resembled, in the of dy dy mice previously reported by our laboratory. Thus these two defects, taken together may help to explain the marked wasting of the muscles. The decrease in brain in both strains provides evidence for nervous tissue involvement in genetic dystrophy.

Enhanced natural killer (NK) cell activity and NK-sensitive thymic cells in murine muscular dystrophy

Studies have shown that there is an abnormality in the thymus of dystrophic mice with respect to age-dependent thymus weight changes and altered morphology (T. DeKretser and B. Livett, Nature (London), 263, 682, 1976). Recently, others have shown that natural killer (NK) cells can lyse cells of a large, immature, rapidly dividing cell subpopulation within the thymus of normal young (3 weeks of age) mice (M. Hansson, K. Karre, R. Kiessling, J. Roder, B. Anderson, and P. Hayry, J. Immunol., 123, 765, 1979). The NK susceptibility of dystrophic mouse thymocytes as targets was therefore studied. Spleen cells from normal (+/+) and dystrophic ( dy 2J dy 2J ) male C57BL/6J mice 8–10 weeks old were passed over nylon wool and the nonadherent cells were incubated with 51Cr-labeled YAC-1 lymphoma target cells or thymocytes in a 51Cr-release assay. Spleen cells from dystrophic mice killed twofold more YAC-1 target cells than did spleen cells from normal mice. Thymocytes from 3- to 4-week-old dystrophic mice were three to four times more susceptible to NK lysis by dystrophic mouse spleen cells as compared with normal mouse spleen cells. Spleen cells from dystrophic mice had the same NK activity against dystrophic and normal mouse thymocytes as targets. Normal mouse spleen cells killed three- to fourfold more dystrophic mouse thymocytes than that of normal mouse thymocytes as targets. Target cellbinding studies revealed that conjugate-forming cells from nylon nonadherent dystrophic mouse spleen cells were found to be two- to fourfold greater than for normal mouse spleen cells using YAC-1 tumor cells as targets. The number of lymphocytes bound per YAC-1 target cell ranged from 2 to 5 for dystrophic mouse spleen cells as compared with 1 to 2 for the normal control group. Using both normal and dystrophic mouse thymocytes as targets, the conjugate-forming cells from dystrophic mouse spleen cells were also found to be twofold greater than in the normal control group. Cold target inhibition studies revealed that the natural killing of dystrophic mouse thymocytes was due to a YAC-1-reactive NK cell. Effector cell depletion studies using monoclonal anti-Thy-1.2 plus complement treatment and plastic petri dish adherence also revealed that the natural killing of dystrophic mouse thymocytes was not due to either T lymphocytes or macrophages. Taken together, these results show an increase in NK-sensitive thymocyte targets in dystrophic mice, in combination with an increase in splenic NK activity.

Ultrastructural changes in muscle and motor end-plate of the dystrophic mouse

A comparative study of dystrophic mice ( C57BL 6J -dy 2J ) and normal littermates, 6 to 9 months old, has revealed numerous ultrastructural changes in the dystrophic soleus. Vacuoles, swollen mitochondria, vesicular aggregates, membranous bodies, Z-line degradation, localized hypercontraction, myofibrillar disorientation, and focal necrosis were common. We report here new observations. Discontinuous, dense bands (240 Å wide) were observed rarely between inner and outer membranes in some nuclei. Closely associated with infoldings of nuclear membrane were paracrystalline, vesicular structures, actin-like filaments, and autophagic vacuoles. With the degeneration of the nuclear membrane, actin-like filaments were observed within the nucleus. The primary synaptic clefts were widened and contained osmophilic-dense granules (diameter 0.2 to 0.7 μ). There was loss of secondary synaptic folds. Discontinuity of pre- and postsynaptic membranes could be observed, though rarely. Lysosome-like dense bodies were present among myofilaments. The myeloid bodies and autophagic vacuoles that were associated with nuclei, sarcoplasmic reticulum, and mitochondria gave a positive acid phosphatase reaction. Such degenerative changes were not observed in the normal soleus.

Changes in isometric contractile properties of fast-twitch and slow-twitch skeletal muscle of [formula omitted][formula omitted] dystrophic mice during postnatal development

Our primary aim was to determine if there exists a preferential involvement of the fast-twitch or slow-twitch skeletal muscle fibers in the dy 2J dy 2J strain of murine dystrophy. The changes in the contractile properties of the slow-twitch soleus (SOL) and the fast-twitch extensor digitorum longus (EDL) muscles of normal and dystrophic mice were studied at 4, 8, 12, and 32 weeks of age. Isometric twitch and tetanus tension were decreased in the 4- and 8-week-old dystrophic EDL compared with controls, this situation being reversed in the older animals. At 12 weeks, the dystrophic EDL generated 15% more tetanic tension than normal EDL and by 32 weeks no significant difference was seen between normal and dystrophic EDL twitch or tetanus tension. By 8 weeks, dystrophic EDL exhibited a prolonged time-to-peak twitch tension (TTP) and half-relaxation time ( 1 2 RT ) of the isometric twitch which continued to 32 weeks. For the dystrophic SOL, decreased twitch and tetanus tension was observed from 4 to 32 weeks. At 8 and 12 weeks, TTP and 1 2 RT of dystrophic SOL were prolonged. However, by 32 weeks there was no longer a significant difference seen in TTP or 1 2 RT between normal and dystrophic SOL. Our results appear to indicate that a loss of the primary control which is determining the fiber composition of the individual muscles is occurring as the dystrophic process advances.

Fiber type susceptibility in the dystrophic mouse

Slow-twitch soleus (SOL) and fast-twitch extensor digitorum longus (EDL) muscles from young normal and dystrophic ( dy 2J dy 2J ) mice were examined histochemically. Counts were made of total fiber content and of the proportions of various fiber types in whole cross sections. By 3 weeks of age the number of fibers in the SOL of dystrophic mice decreased by 16%. The proportions of both type I and type II ox fibers decreased approximately equal amounts. Even at this relatively early stage of the disease process, almost 20% of the fibers appeared abnormal and could not be clearly identified as belonging to a specific fiber type. Very few type I fibers with dystrophic characteristics were seen. However, this may simply reflect an alteration in staining characteristics which preceded structural changes in involved fibers. In 6- to 7-week-old EDL muscles the most marked change occurred in the proportion of type II glyc fibers. The percentage of these fibers in dy 2J mice was 8.4% compared with 57.3% in control animals. At this stage the total number of fibers in dy 2J EDL had decreased by 30%, although the number of type II ox fibers remained virtually constant. Various explanations for these findings are considered.

Slowing of fast-twitch muscle in the dystrophic mouse

Isometric twitch tension was measured in fast-twitch and slow-twitch muscles of normal and dystrophic ( C57Bl 6J dy 2J dy 2J ) mice in vivo. In dystrophic mice more than 6 months old the fast-twitch extensor digitorum longus (EDL) showed a prolongation of the time to peak tension as well as the time to relax to one-half peak tension ( 1 2 R T ) compared with age-matched controls. In younger dystrophic mice (4 to 6 weeks) the time to peak tension was prolonged but 1 2 R T not significantly so. This apparent “slowing” of dystrophic fast-twitch muscle was accompanied by a reduction in both cooling potentiation and post-tetanic potentiation toward values typical of slow-twitch muscle. Slow-twitch soleus muscle (SOL) of old mice was almost unaffected by the dystrophic process with regared to its contractile characteristics. However, there appeared to be a slight, but significant “speeding” of young dystrophic SOL compared with age-matched control muscles. This was apparent in reduced times to peak tension and half-relaxation as well as an enhanced cooling potentiation. We suggest that the altered contractile characteristics result from a change in some intrinsic property of the muscle fibers rather than from extrinsic factors such as the additional perimysial connective tissue seen in these muscles.

A maturational defect in passive membrane properties of dystrophic mouse muscle

Electrical properties, component ionic conductances, and histochemical characteristics of normal and dystrophic ( dy 2J dy 2J ) mouse extensor digitorum longus (EDL) and soleus (SOL) muscles were studied between 1 and 6 months of age. Normal EDL and SOL membrane electrical parameters were indistinguishable at 1 month. Between 1 and 3 months, membrane resistance ( R m) in normal EDL decreased progressively to a mature value one-half that found in SOL muscles. Measurements of component conductances indicated that this decrease in R m was due to a specific increase in membrane chloride conductance ( G C1) in EDL fibers. During the same time membrane capacitance increased slightly in both EDL and SOL. Dystrophic EDL failed to develop fully the changes in membrane resistance seen in controls, showing significant deviation at all time points beyond 1 month. An abnormal high-resistance subpopulation was evident in R m histograms of mature dystrophic EDL. Average membrane properties of dystrophic SOL appeared normal during the 6-month interval studied but significant changes in R m histograms were found. Histochemical analysis of normal and dystrophic EDL showed that both contained almost exclusively type II fibers (> 95%); normal and dystrophic SOL contained a nearly equal mixture of type I and type II fibers. Progressive degenerative changes were seen in both dystrophic muscles with age but were more severe in the SOL. The depressed average G C1 in the dystrophic EDL was due to a subpopulation of fibers (30% of total) having a high membrane resistance. Denervation was ruled out as a cause for this subpopulation by a lack of correlation between membrane resistance and resting potential. Histochemical evidence eliminated the possibility that this subpopulation was composed of slow-twitch fibers. Data are presented indicating that the subpopulation represents a group of dystrophic EDL fibers which fail to undergo normal maturation.

Studies in vivo of ribonucleic acid turnover in mice (strain C57 BL/6J) homozygous and heterozygous for the dystrophic gene, dy2J

In vivo incorporation of [5- 3H]uridine into RNA of 40-day-old mice, homozygous and heterozygous for the dy 2J dystrophic allele, has been examined. Increased incorporation is seen in leg skeletal muscle but no increase is liver tissue. Determination of DNA and RNA content of the muscle tissue examined showed that the increased incorporation was not due to muscle volume changes that are associated with wasting in dystrophic muscle. The results obtained with the dy 2J allele are discussed with respect to related investigation with the dy dystrophic allele.

Histochemical and structural analyses of the phenotypic expression of the dystrophic gene in the [formula omitted] and the [formula omitted] mice

Although both slow- and fast-twitch fibers of dystrophic genotype are affected by the disease hereditary muscular dystrophy, our histochemical evidence indicates a differential temporal involvement of different fiber types in the two strains of dystrophic mice investigated. Whereas in the 129 ReJ dy dy mice the disease is expressed early in white, fast-twitch glycolytic fibers, in the C57BL 6J dy 2J dy 2J mice the primary expression of the disease is noted in red, slow-twitch oxidative fibers. In the former, wasting progresses rapidly in leg muscles characterized by a large proportion of fast-twitch fibers; in the latter, there is a prolonged maintenance of the integrity of regions rich in these fibers. Of equal importance with the C57BL 6J dy 2J dy 2J mouse is the detection of small foci of destruction in regions rich in slow-twitch fibers as early as 2 to 3 weeks postnatally; wasting of these fibers ensues rapidly. Thus, in both strains, the fibers which are affected primarily show early alterations with a subsequent rapid progression of disease characteristics and the fibers which seem secondarily affected remain intact for prolonged periods. Our observations suggest an important role of the dy 2J dy 2J mouse as a “transitional” model to investigate a relationship between genotypically dystrophic muscle in which the phenotypic expression of the disease is present and genotypically dystrophic muscle spared phenotypic signs of the disease. The latter, characterized by multiple innervation, is never challenged to alter its metabolic or physiologic behavior during ontogeny.

Histochemical and morphological study of dystrophic ( [formula omitted]) and normal muscle

Morphological and histochemical techniques were utilized to study the soleus and gastrocnemius muscles of normal ( C57BL 6J +/+) and dystrophic ( C57BL 6J dy 2J dy 2J ) mice. This is the first such histochemical study of dy 2J dy 2J 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 dy 2J dy 2J 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 dy 2J dy 2J 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.

Comparison of γ-glutamyl transpeptidase activity in tissues of normal and dystrophic hamsters and mice

The activity of γ-glutamyl transpeptidase (γ-GT), a membrane-bound enzyme, was assayed by a sensitive fluorometric method in the brain, heart, kidney, liver, skeletal muscle and serum of normal and dystrophic hamsters and mice. Normal and dystrophic hamsters were of the golden brown and BIO 14.6 line respectively. Both normal and dystrophic mice were of the C57 BL/6J -dy 2J strain. Kidneys of both hamsters and mice contamed the highest enzyme activity followed by the brain, skeletal muscle, liver and heart. γ-GT activity was elevated in dystrophic skeletal muscles but decreased in the dystrophic kidneys and sera. In the brain, heart and liver of dystrophic animals from both species enzyme activity was similar to that in normals.

A study of functional denervation in fast and slow muscles of dystrophic mice of various ages

It has been claimed that some fibers in dystrophic mouse muscle are denervated and, furthermore, that there is no increase in motor unit size which would be expected to occur if reinnervation took place. If these observations are correct, it ought to be possible to demonstrate a mechanical correlate of the denervation phenomenon. The functional innervation ratio (ratio of maximum twitch tension in response to nerve stimulation × 100 to maximum twitch tension in response to direct muscle stimulation) has been determined for the extensor digitorum longus and soleus muscles of normal ( C57B1 6J ) and dystrophic ( dy 2J dy 2J ) mice of various ages. It is shown that although a fall in the functional innervation ratio is seen in both muscles, it occurs some time after the symptoms of the disease are apparent. This suggests that denervation may not be the primary event in murine muscular dystrophy. The fall in functional innervation ratio is apparently not due to an enhanced susceptibility of the dystrophic neuromuscular junction to anoxia, because dystrophic muscles showed the same response to a raised bath temperature as did normal muscles.

Some mechanical and electrical properties of distal hind limb muscles of genetically dystrophic mice (C57BL/6Jdy 2J/dy 2J)

Some mechanical and electrical properties of distal hind limb muscles of mice suffering from muscular dystrophy caused by the dy 2J allele have been defined. The dystrophic muscles were lighter and weaker than normal when subjected to either single or tetanic indirect stimulation. The twitch tetanus ratio was unchanged. The contraction time of the dystrophic muscles was prolonged, but time to half-relaxation was normal. All dystrophic muscle fibers generated an action potential when stimulated indirectly, and there was no myasthenialike response of the gross muscle EMG during high frequency stimulation. Resting membrane potentials were reduced in the dystrophic muscle fibers, and action potentials were slower and smaller in the dystrophic “fast-twitch” muscle fibers of the extensor digitorum longus muscle. Action potentials in the “slow twitch” muscle fibers of the soleus muscle were normal. Miniature end-plate potentials were present in all dystrophic muscle fibers examined. Many muscle fibers in both extensor digitorum longus and soleus muscles of dystrophic animals were sensitive to acetylcholine outside the end-plate region, and were also able to generate action potentials in the presence of 10 −6 m tetrodotoxin. Since no evidence of either functional or structural denervation was found, it is suggested that these are immature, regenerating muscle fibers. It is further suggested that the ability to regenerate in spite of the presence of the dystrophic gene is a major and possibly qualitative difference between murine dystrophy of the type dy 2J dy 2J , and dy dy in which regeneration of adult muscle is abortive. The implications of such a qualitative difference are discussed with reference to the current controversy concerning the etiology of the muscular dystrophies.

Ultrastructural alterations of the motor end plate in myotonic dystrophy of the mouse (dy2J dy2J).

The slow progression of weakness and atrophy of muscle and the prominence of myotonia suggested that the disease observed in the dy 2j strain of the Bar Harbor 129 mice is similar to the human form of myotonic dystrophy. Bright field microscopy and histochemical studies disclosed primary myopathic changes which were at first focal, becoming more diffuse as the disease progressed. The ultrastructural studies revealed two main types of alteration. The most obvious was the dystrophic process in muscle. Attention was focused on the second category of change, observed at every motor end plate, and consisting of a reduction in number and size and a distortion in shape of the terminal axoplasmic vesicles. In addition, at a number of end plates there was retraction of axolemma from the plasma membrane of the sareolemma. At still other end plates, processes of Schwann cytoplasm separated axolemma from plasmalemma. The fine structure of 46 motor end plates was altered while the structure of 12 motor end plates in the control muscles was normal. Considering the preservation of the post-synaptic architecture and the integrity of the axon with its myelin up to its termination, there is reason to believe that the described abnormality is primary at the motor end plate and not secondary to diseased nerve or muscle.

Muscular dystrophy in the mouse caused by an allele at the [formula omitted]-locus

We describe a progressive hereditary myopathy of mice caused by a second allele, dy 2J , at the dy -locus. Clinically and pathologically, the new syndrome is milder than the original muscular dystrophy, and both afflicted males and females have bred. Normal reproductive capacity of mutant mice will greatly facilitate studies on the pathogenesis of muscular dystrophy.