Dy 2J Research Articles

Overexpression of LARGE suppresses muscle regeneration via down-regulation of insulin-like growth factor 1 and aggravates muscular dystrophy in mice.

Several types of muscular dystrophy are caused by defective linkage between α-dystroglycan (α-DG) and laminin. Among these, dystroglycanopathy, including Fukuyama-type congenital muscular dystrophy (FCMD), results from abnormal glycosylation of α-DG. Recent studies have shown that like-acetylglucosaminyltransferase (LARGE) strongly enhances the laminin-binding activity of α-DG. Therefore, restoration of the α-DG-laminin linkage by LARGE is considered one of the most promising possible therapies for muscular dystrophy. In this study, we generated transgenic mice that overexpress LARGE (LARGE Tg) and crossed them with dy(2J) mice and fukutin conditional knockout mice, a model for laminin α2-deficient congenital muscular dystrophy (MDC1A) and FCMD, respectively. Remarkably, in both the strains, the transgenic overexpression of LARGE resulted in an aggravation of muscular dystrophy. Using morphometric analyses, we found that the deterioration of muscle pathology was caused by suppression of muscle regeneration. Overexpression of LARGE in C2C12 cells further demonstrated defects in myotube formation. Interestingly, a decreased expression of insulin-like growth factor 1 (IGF-1) was identified in both LARGE Tg mice and LARGE-overexpressing C2C12 myotubes. Supplementing the C2C12 cells with IGF-1 restored the defective myotube formation. Taken together, our findings indicate that the overexpression of LARGE aggravates muscular dystrophy by suppressing the muscle regeneration and this adverse effect is mediated via reduced expression of IGF-1.

P.8.11 Glatiramer acetate improved motor conduction velocity in dy2J/dy2J mouse peripheral neuropathy

The dy 2J /dy 2J mouse model of laminin α 2-deficient congenital muscular dystrophy (MDC1A) has prominent peripheral neuropathy with slowed conduction. The conduction slowing may be due to reduced myelinated axon number, diameter, and atypical Schwann cell ensheathement/amyelination with aberrant basal lamina, nodal gap shortening, and decreased axon membrane excitability. Although no inflammatory mechanism was identified in dy 2J /dy 2J neuropathy, other genetically mediated neuropathies show low-grade inflammation and immune pathways play a pathological role. The immune modulating agent, Glatiramer acetate (GA) improves dy 2J /dy 2J mouse mobility and hind limb muscle strength, and shows significant beneficial effect in the neuropathy of experimental autoimmune neuritis (EAN). GA mechanisms of action from experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis studies are at various levels of the immune response (skewing T-cells from pro- to anti-inflammatory pathways) and generating neuroprotective activity (brain derived neurotrophic factor BDNF, anti-inflammatory cytokines). Because of GA’s beneficial effect in the dy 2J /dy 2J mouse model we evaluated it’s effect on dy 2J /dy 2J peripheral neuropathy motor conduction. Homozygous dy 2J /dy 2J and control mice were treated with GA or placebo for 12 weeks from 6 weeks of age. Outcome measures included sciatic-posterior tibial motor nerve conduction parameters (NCV, distal latency, and CMAP amplitude, area and dispersion) at 18 weeks. GA treated dy 2J /dy 2J mice showed significantly improved average sciatic-posterior tibial nerve motor conduction velocity 50.35 ± 2.9 m/s versus untreated dy 2J /dy 2J 34.49 ± 2.14 m/s ( p 2 J /dy 2J mouse model of congenital muscular dystrophy.

Losartan, a therapeutic candidate in congenital muscular dystrophy: Studies in the dy2J/dy2J Mouse

Lamininα2-deficient congenital muscular dystrophy type 1A (MDC1A) is a cureless disease associated with severe disability and shortened lifespan. Previous studies have shown reduced fibrosis and restored skeletal muscle remodeling following treatment with losartan, an angiotensin II type I receptor blocker. We therefore evaluated the effect of losartan treatment in the dy(2J) /dy(2J) mouse model of MDC1A. Homozygous dy(2J) /dy(2J) and control mice were treated with losartan or placebo for 12 weeks from 6 weeks of age. Outcome measures included hindlimb and forelimb muscle strength by Grip Strength Meter and quantitative muscle fibrosis parameters. Losartan's effects on transforming growth factor β (TGF-β) and mitogen-activated protein kinase (MAPK) signaling pathways were evaluated with Western blotting, immunofluorescence, and cytokine measurements. Losartan treatment was associated with significant impressive improvement in muscle strength and amelioration of fibrosis. Administration of losartan inhibited TGF-β signaling pathway, resulting in decreased serum TGF-β1 level and reduced downstream phosphorylated (P) Smad2/3 proteins. Moreover, losartan activated Smad7 protein, a key negative regulator of TGF-β signaling. In addition, losartan treatment inhibited the MAPK cascade as shown by decreased expression of P-p38 MAPK, P-c-jun-N-terminal kinase, and P-extracellular signal-regulated kinases 1 and 2 in the treated mice. Losartan, a commonly prescribed US Food and Drug Administration-approved medication for hypertension, demonstrated clinical improvement and amelioration of fibrosis in the dy(2J) /dy(2J) mouse model of MDC1A via TGF-β and MAPK signaling pathways. The results of this study support pursuing a clinical trial of losartan treatment in children with MDC1A.

The Ras Antagonist, Farnesylthiosalicylic Acid (FTS), Decreases Fibrosis and Improves Muscle Strength in dy2J/dy2J Mouse Model of Muscular Dystrophy

The Ras superfamily of guanosine-triphosphate (GTP)-binding proteins regulates a diverse spectrum of intracellular processes involved in inflammation and fibrosis. Farnesythiosalicylic acid (FTS) is a unique and potent Ras inhibitor which decreased inflammation and fibrosis in experimentally induced liver cirrhosis and ameliorated inflammatory processes in systemic lupus erythematosus, neuritis and nephritis animal models. FTS effect on Ras expression and activity, muscle strength and fibrosis was evaluated in the dy2J/dy2J mouse model of merosin deficient congenital muscular dystrophy. The dy2J/dy2J mice had significantly increased RAS expression and activity compared with the wild type mice. FTS treatment significantly decreased RAS expression and activity. In addition, phosphorylation of ERK, a Ras downstream protein, was significantly decreased following FTS treatment in the dy2J/dy2J mice. Clinically, FTS treated mice showed significant improvement in hind limb muscle strength measured by electronic grip strength meter. Significant reduction of fibrosis was demonstrated in the treated group by quantitative Sirius Red staining and lower muscle collagen content. FTS effect was associated with significantly inhibition of both MMP-2 and MMP-9 activities. We conclude that active RAS inhibition by FTS was associated with attenuated fibrosis and improved muscle strength in the dy2J/dy2J mouse model of congenital muscular dystrophy.

Fibrosis inhibition and muscle histopathology improvement in laminin‐α2‐deficient mice

In muscular dystrophies (MD) the loss of muscle and its ability to function are associated with fibrosis. We evaluated the efficacy of halofuginone in reducing fibrosis in the dy(2J)/dy(2J) mouse model of congenital MD. Mice were injected intraperitoneally with 5 microg of halofuginone 3 times a week for 5 or 15 weeks, starting at the age of 3 weeks. Halofuginone caused a reduction in collagen synthesis in hindlimb muscles. This was associated with reductions in the degenerated area, in cell proliferation, in the number of myofibers with central nuclei, with increased myofiber diameter, and with enhanced motor coordination and balance. Halofuginone caused a reduction in infiltrating fibroblasts that were located close to centrally nucleated myofibers. Our results suggest that halofuginone reduced the deleterious effects of fibrosis, thus improving muscle integrity. Halofuginone meets the criteria for a novel antifibrotic therapy for MD patients.

Animal Models for Genetic Neuromuscular Diseases

The neuromuscular disorders are a heterogeneous group of genetic diseases, caused by mutations in genes coding sarcolemmal, sarcomeric, and citosolic muscle proteins. Deficiencies or loss of function of these proteins leads to variable degree of progressive loss of motor ability. Several animal models, manifesting phenotypes observed in neuromuscular diseases, have been identified in nature or generated in laboratory. These models generally present physiological alterations observed in human patients and can be used as important tools for genetic, clinic, and histopathological studies. The mdx mouse is the most widely used animal model for Duchenne muscular dystrophy (DMD). Although it is a good genetic and biochemical model, presenting total deficiency of the protein dystrophin in the muscle, this mouse is not useful for clinical trials because of its very mild phenotype. The canine golden retriever MD model represents a more clinically similar model of DMD due to its larger size and significant muscle weakness. Autosomal recessive limb-girdle MD forms models include the SJL/J mice, which develop a spontaneous myopathy resulting from a mutation in the Dysferlin gene, being a model for LGMD2B. For the human sarcoglycanopahties (SG), the BIO14.6 hamster is the spontaneous animal model for delta-SG deficiency, whereas some canine models with deficiency of SG proteins have also been identified. More recently, using the homologous recombination technique in embryonic stem cell, several mouse models have been developed with null mutations in each one of the four SG genes. All sarcoglycan-null animals display a progressive muscular dystrophy of variable severity and share the property of a significant secondary reduction in the expression of the other members of the sarcoglycan subcomplex and other components of the Dystrophin-glycoprotein complex. Mouse models for congenital MD include the dy/dy (dystrophia-muscularis) mouse and the allelic mutant dy(2J)/dy(2J) mouse, both presenting significant reduction of alpha2-laminin in the muscle and a severe phenotype. The myodystrophy mouse (Large(myd)) harbors a mutation in the glycosyltransferase Large, which leads to altered glycosylation of alpha-DG, and also a severe phenotype. Other informative models for muscle proteins include the knockout mouse for myostatin, which demonstrated that this protein is a negative regulator of muscle growth. Additionally, the stress syndrome in pigs, caused by mutations in the porcine RYR1 gene, helped to localize the gene causing malignant hypertermia and Central Core myopathy in humans. The study of animal models for genetic diseases, in spite of the existence of differences in some phenotypes, can provide important clues to the understanding of the pathogenesis of these disorders and are also very valuable for testing strategies for therapeutic approaches.

Synaptic plasticity in the dy2J mouse model of laminin α2-deficient congenital muscular dystrophy

Laminin α2-deficient congenital muscular dystrophy is a debilitating disease affecting both muscle and neural tissue as a result of mutations in the LAMA2 gene. It presents at or soon after birth with muscle weakness and is further characterised by clinical central nervous system involvement. Laminin α2 is part of the extracellular matrix, linked to the cellular cystoskeleton via dystroglycan which is an integral part of the dystrophin–glycoprotein complex (DGC). We examined both short- and long-term synaptic plasticity in the C57BL6J/ dy 2J mouse, an animal model of laminin α2 deficient congenital muscular dystrophy. Using a cerebellar slice preparation, we show that the pre-synaptically mediated paired-pulse facilitation (PPF) was no different between dy 2J and littermate controls. Approximately half (7/12) the dy 2J Purkinje cells displayed a blunted LTD compared to littermate controls, and one third (4/12) of dy 2J Purkinje cells displayed LTP. This study demonstrates that a defective laminin α2 causes a disruption in long-term synaptic plasticity at the Purkinje cell-parallel fibre synapse.

Laminin α2 deficiency and muscular dystrophy; genotype-phenotype correlation in mutant mice

Deficiency of laminin α2 is the cause of one of the most severe muscular dystrophies in humans and other species. It is not yet clear how particular mutations in the laminin α2 chain gene affect protein expression, and how abnormal levels or structure of the protein affect disease. Animal models may be valuable for such genotype-phenotype analysis and for determining mechanism of disease as well as function of laminin. Here, we have analyzed protein expression in three lines of mice with mutations in the laminin α2 chain gene and in two lines of transgenic mice overexpressing the human laminin α2 chain gene in skeletal muscle. The dy 3K /dy 3K experimental mutant mice are completely deficient in laminin α2; the dy/dy spontaneous mutant mice have small amounts of apparently normal laminin; and the dy W /dy W mice express even smaller amounts of a truncated laminin α2, lacking domain VI. Interestingly, all mutants lack laminin α2 in peripheral nerve. We have demonstrated previously, that overexpression of the human laminin α2 in skeletal muscle in dy 2J /dy 2J and dy W /dy W mice under the control of a striated muscle-specific creatine kinase promoter substantially prevented the muscular dystrophy in these mice. However, dy W /dy W mice, expressing the human laminin α2 under the control of the striated muscle-specific portion of the desmin promoter, still developed muscular dystrophy. This failure to rescue is apparently because of insufficient production of laminin α2. This study provides additional evidence that the amount of laminin α2 is most critical for the prevention of muscular dystrophy. These data may thus be of significance for attempts to treat congenital muscular dystrophy in human patients.

Electrotransfer of naked DNA in the skeletal muscles of animal models of muscular dystrophies

The electrotransfer of naked DNA has recently been adapted to the transduction of skeletal muscle fibers. We investigated the short- and long-term efficacy of this methodology in wild-type animals and in mouse models of congenital muscular dystrophy (dy/dy, dy(2J)/dy(2J)), or Duchenne muscular dystrophy (mdx/mdx). Using a reporter construct, the short-term efficacy of fiber transduction reached 40% and was similar in wild-type, dy/dy and dy(2J)/dy(2J) animals, indicating that ongoing muscle fibrosis was not a major obstacle to the electrotransfer-mediated gene transfer. Although the complete rejection of transduced fibers was observed within 3 weeks in the absence of immunosuppression, the persistency was prolonged over 10 weeks when transient or continuous immunosuppressive regimens were used. Using therapeutic plasmids, we demonstrated that electrotransfer also allowed the transduction of large constructs encoding the laminin alpha2 chain in dy/dy mouse, or a chimeric dystrophin-EGFP protein in mdx/mdx mouse. The correct sarcolemmal localization of these structural proteins demonstrated the functional relevance of their expression in vivo, with a diffusion domain estimated to be 300 to 500 microm. However, degeneration-regeneration events hampered the long-term stability of transduced fibers. Given its efficacy for naked DNA transfer in these models of muscular dystrophies, and despite some limitations, gene electrotransfer methodology should be further explored as a potential avenue for treatment of muscular dystrophies.

Muscular transverse relaxation time measurement by magnetic resonance imaging at 4 Tesla in normal and dystrophic dy/dy and dy(2j)/dy(2j) mice.

Muscular transverse relaxation times values were measured in vivo in normal mice (strain C57BL6/J, n=14) and in murine models of human congenital muscular dystrophy (dy/dy, n=9; dy(2j)/dy(2j), n=8). A single-slice multi-echo sequence was used. Gastrocnemius/soleus complex, thigh and buttock muscles were studied. Muscular transverse relaxation times values were compared between different muscle groups in each type of animal and between animal groups. Differences were observed between normal and dy(2j)/dy(2j) mice from 3 to 12 weeks of age, and between normal and dy/dy mice at 6 weeks. In specific age ranges, the values of muscular transverse relaxation times in two dystrophic models are different from those in normal mice, and could thus be used as an index of modifications in dystrophic muscle to evaluate therapies.

Merosin and congenital muscular dystrophy

Merosin (also called as Laminin-2) is an isoform of laminin comprised of the alpha2, beta1 and gamma1 chains. In European populations, half of the patients with classical congenital muscular dystrophy have mutations of the LAMA2 gene (6q22-23) and present reduced or absence of laminin alpha2 chain. This form is generally referred to as merosin-deficient CMD. Merosin-deficient CMD is characterized by involvement of not only skeletal muscle but also central and peripheral nervous systems: Extensive brain white matter abnormalities are found by magnetic resonance imaging (MRI). However, most patients show no mental retardation. Recent case studies reported that some patients have several structural abnormalities such as abnormal cerebral cortical gyration, hypoplasia of cerebellum and pons, and dilation of ventricles. At present, functions of merosin related to muscle degeneration have not been fully elucidated. In addition, the mechanisms responsible for pathogenesis of diffuse brain white matter abnormalities remain to be determined. As mouse models for merosin-deficient CMD, three spontaneous mutants(dy, dy(2J), dy(PAS1)) and two mutants named dy(W) and dy(3K) by targeted gene disruption have been reported. These mice will help to elucidate the pathogenesis of merosin-deficient CMD and serve to develop therapy.

Identification of homozygous and heterozygous dy2J mice by PCR.

The dystrophia muscularis dy2J/dy2J mouse is an animal model for one form of human congenital muscular dystrophy. A point mutation in the gene coding for the laminin-2 alpha 2 chain leads to the expression of a truncated, partially functional protein. We developed a simple assay for the detection of the dy2J allele, which contains a new NdeI restriction site. Genomic DNA was prepared from animals of known status and amplified by PCR. The digestion of the PCR product with the restriction enzyme resulted in characteristic profiles. Then, this technique was used to identify heterozygous mice among unaffected animals of unknown status. Subsequently, the heterozygous genotype of these mice was confirmed by the birth of dystrophic offspring after mating. This technique allows the detection of the dy2J allele in heterozygous and homozygous animals at any age.

The laminin α2 expressed by dystrophic dy 2J mice is defective in its ability to form polymers

Mutations in LAMA2 cause severe congenital muscular dystrophy accompanied by nervous system defects [1]. Mice homozygous for the dy(2J) allele of LAMA2 express a laminin alpha2 subunit that has a deletion in the amino-terminal domain VI, providing an animal model for study of the molecular basis of congenital muscular dystrophy [2] [3]. Domain VI is predicted to be involved in laminin polymerization, along with amino-terminal domains from laminin beta and gamma chains [4]. In a solution-polymerization assay, we found that purified dy(2J) laminin assembled poorly and formed little polymer, in contrast to wild-type muscle laminin. Furthermore, dissolution of the collagen IV network caused dy(2J) laminin to be released into solution, indicating that laminin polymers within the skeletal muscle basement membrane were defective. In addition to loss of polymerization, dy(2J) laminin had a reduced affinity for heparin. Finally, recombinant laminin engineered with the dy(2J) deletion was more sensitive to proteolysis and was readily cleaved near the junction of domains V and VI. Thus, the dy(2J) deletion selectively disrupts polymer formation, reduces affinity for heparin, and destabilizes domain VI. These are the first specific functional defects to be identified in a muscular dystrophy laminin, and it is likely that these defects contribute to the abnormalities seen in dy(2J)/dy(2J) muscle and nerve.

Response of a fast muscle from normal and dystrophic (dy 2j) mice to a local decrease in extracellular Ca 2+ induced at different stages of postnatal life

It has previously been reported that reducing Ca 2+ 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 Ca 2+, 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 Ca 2+ 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 Ca 2+ 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.

Comparison of fiber size and phenotypic gene expression in muscles of dystrophic C57BL/6J DY2J/DY2J mice

Considerable evidence has shown a correlation between fiber size and degree of necrosis in dystrophic muscles of hamsters, X-linked muscular dystrophy (MDX) mice and humans. It has been proposed that small-caliber fibers have an immunity to the phenotypic expression of the dystrophic gene(s). The results from the present study show a discordance between fiber size and necrosis in dystrophic muscles of C57BL/6J dy 2J dy 2J mice. Extensor carpi radialis longus and brevis muscles (ECRL and ECRB respectively) were compared in normal and dystrophic 2-week, 4-week and 12-month animals by measuring the mean cross-sectional area of type II fibers, determination of relative proportions of types IIA and IIB fibers and calculation of percentage of fibers exhibiting centronucleation in an entire cross-section of muscle (stained for haematoxylin and eosin or ATPase). The ECRL and ECRB muscles were found to have identical sizes of fiber at each of the 3 ages studied and similar proportions of fiber types, yet the former muscle developed and retained significantly more necrosis (manifest as centronucleation) than the latter.

Effect of denervation on adenine nucleotides in skeletal muscle from normal and dystrophic mice

The effect of denervation on the adenine nucleotide content of fast-and slow-twitch skeletal muscle of the C57BL mouse was studied by high-performance liquiud chromatography. From the adenine nucleotide content the energy charge, a measure of high-energy phosphate available to the cell, was calculated. The energy charge of the extensor digitorum longus muscle was significantly higher than that of the same muscle from dystrophic mice ( C57BL 6J dy 2j dy 2j ) and on denervation decreased to the values found in the innervated muscle from dystrophic animals. Denervation of the muscle in dystrophic mice did not change the energy charge of that muscle. The energy charge of the soleus muscle from both normal and dystrophic mice was similar and did not change on denervation. It is proposed that in the dystrophic process a functional denervation of skeletal muscle occurs which preferentially affects fast-twitch muscle, leading to a reduction in the energy charge.

Age-related changes in oxidative capacity of the gastrocnemius muscle in normal and dystrophic ( [formula omitted]) mice

The hind limb muscles of dy 2J dy 2J mice appear more oxidative than normal hind limb muscles when assayed histochemically. In some cases, biochemical assay of oxidative capacity does not match histochemical data, and in some dy 2J mice, particularly old animals, there appears to be a decline in biochemically assayed oxidative enzymes. The current study cyto-biochemically assayed succinic dehdrogenase activity of the gastrocnemius muscle in dy 2J and normal mice. The assays were conducted over a maturation time course of 1 to 6 months of age. Additionally, the gastrocnemius muscle was divided into two distinct regions, a superficial region, containing largely glycolytic fast-twitch fibers (SGM), and a deeper region of mixed, largely oxidative fibers (DGM) in normal animals. Assay of the whole muscle and the two regions of each individual muscle showed that the dy 2J muscle increased in SDH activity with maturation and was significantly greater than the change observed in normal muscle during the same period. The increase in the whole muscle SDH activity was accounted for increases noted in the SGM. The dy 2J DGM, which showed marked morphologic degeneration, had neither an increase nor a decrease in measured SDH activity. Michaelis-Menton analysis of the enzymatic assay indicated that the V max, and not the K m , of the dystrophic muscle enzyme system was higher than normal, suggesting a change in quantity of enzyme present, and not altered function of the system. The observed increases occurred for 4 months of maturation and then began a decline, which was noted at 6 months. Coupled with the time course of gastrocnemius muscle degeneration (the glycolytic region was slow—very little by 4 months; the mixed was fast—highly degenerated by 1 to 2 months), the results of this study suggest that oxidative capacity in the dy 2J gastrocnemius muscle increased with maturation until it became obscured by inherant muscle wasting in a particular dy 2J muscle region. By comparison, analysis of the large glycolytic triceps muscle, a forelimb muscle which does not receive pseudomyotonia, indicated that this muscle did not increase in SDH activity in dystrophic animals, which showed the abnormal increase in the activity of the hind limb gastrocnemius. Thus the relationship of pseudomyotonia to increasing oxidative activity in the hind limb muscles of dystrophic animals was evident.

Intracellular sodium-activity at rest and after tetanic stimulation in muscles of normal and dystrophic ( [formula omitted]) [formula omitted] mice

Intrafiber Na +-activity, ( a Na) i, was measured in vivo in single muscle fibers in the gastrocnemius and soleus muscles of normal and dystrophic ( dy 2J dy 2J ) C57BL 6J mice by means of double-barrel Na +-selective microelectrodes. Values of ( a Na) i in normal gastrocnemius and soleus muscles were 9.8 ± 1.0 m M ( x ± SE; N = 15 ) and 13.7 ± 1 m M ( N = 19), respectively. Values of ( a Na) i in dystrophic gastrocnemius and soleus muscles were 18.7 ± 1.3 m M ( N = 10) and 26.2 ± 3.3 m M ( N = 15), respectively. The soleus muscle of both normal and dystrophic mice was “Na + loaded” by an intermittent tetanus delivered via the sciatic nerve. Muscles from both normal and dystrophic mice could extrude the Na + load at similar rates. The results suggest that in the dystrophic mice the elevated ( a Na) i is not due to inability of the Na + pump to respond to a Na + load; rather, it may be due to an alteration in the affinity of the pump for Na +, resulting in a different “set point” for ( a Na) i.

Natural killer cell activity in murine muscular dystrophy III. NK-sensitive myoblast cells and lack of NK activity in beige/dystrophic hybrid mice

The NK-susceptibility of dystrophic mouse myoblast cells was investigated. Spleen cells from 8- to 10-week-old normal (+/+) and dystrophic (dy 2J/dy 2J) male C57BL/6J mice were fractionated on Percoll density gradients and the cells at each density interface were incubated with either 51Cr-labeled YAC-1 or myoblast cells in a 6 hr 51Cr-release assay. Myoblast target cells were obtained from either heterozygous (+/dy 2J) or homozygous (dy 2J/dy 2J) muscle cultures or a transformed tetraploid myoblast line (M14D2). The data indicate that the interface between the 50 and 60% (1.060–1.075 g/ml) Percoll density fractions of spleen cells from either normal or dystrophic mice contains the largest proportion of asialo GM-1 positive and NK-1 positive cells displaying NK activity. Myoblast cells from either heterozygous (phenotypically normal) or homozygous dystrophic mice were not significantly different in susceptibility to NK-mediated lysis by Percoll enriched normal or dystrophic mouse NK cells. However, dystrophic mouse spleen cells had the highest NK activity against both myoblast targets as compared with normal mouse spleen cells. The transformed myoblast cell line, M14D2, was significantly less susceptible to NK-mediated lysis by dystrophic mouse spleen cells when compared with the freshly cultured myoblast target cells. Target cell binding studies revealed that conjugate forming cells from the 50% Percoll density interface of dystrophic mouse spleen cells were approximately twofold greater than that of normal mouse spleen cells against either heterozygous or homozygous dystrophic mouse myoblast targets. Cold target inhibition studies revealed that the natural killing of dystrophic mouse myoblast cells was due to a YAC-1 reactive NK cell. Breeding experiments between C57BL/ 6J homozygous “beige” (bg J/bg J) mutant mice and dystrophic (dy 2J/dy 2J) mice produced beige/ dystrophic hybrid mice which displayed clinical symptoms of the dystrophy process by 3 to 4 weeks of age. Spleen cells from these hybrid mice showed no significant differences in NK activity against YAC-1 target cells when compared with homozygous beige mice. Taken together, these results demonstrate the first reported evidence that murine myoblasts are susceptible to NK-mediated lysis. In addition, the data indicate that although dystrophic mouse NK cells recognize myoblast cells as targets, the NK cell studies with the beige/dystrophic hybrid mice do not indicate a direct in vivo role for NK cells in the dystrophy process.

Number and size of motoneurons in a forelimb motor nucleus of normal and dystrophic ( [formula omitted]) mice

The method of retrograde axonal transport of horseradish peroxidase (HRP) was used to identify the motoneurons that innervate the distal forelimb muscles via the ulnar nerve in normal and dystrophic ( C56BL 6J dy 2j dy 2j ) mice. In both normal and dystrophic mice this motor nucleus was located in spinal segments C6 through T1. No clear division, on the basis of size, into alpha and gamma motoneuron populations was apparent. The motoneurons of dystrophic mice were fewer in number (26.5 vs. 35) but larger in cross-sectional area (780 vs. 674 μm 2) than those of age-matched control mice. These results are quantitatively similar to those reported for the hind limb soleus muscle of dystrophic mice and suggest that the motoneuronal changes are a reflection of the dystrophic process rather than the associated spontaneous action potential generation seen in the dystrophic hind limb muscles.