Muscular dystrophy in adult and aged anti-NGF transgenic mice resembles an inclusion body myopathy

The role of nerve growth factor (NGF) and its receptors in the physiology of skeletal muscles has not been extensively studied in animal models. We describe the production of transgenic lines of mice expressing a neutralizing antibody against NGF (alphaD11) and the morphological and histochemical analysis of skeletal muscles from adult and aged anti-NGF mice. This study reveals that the chronic deprivation of NGF results in a decreased size of myofibers of dorsal and hindlimb muscles in adult but not in postnatal day (P)2 mice. In myofibers from adult anti-NGF mice, the presence of central nuclei, vacuolization of the cytoplasm, and inflammatory cell infiltration was observed. The immunohistochemical analysis of these muscular fibers revealed an upregulation of p75 expression, a decrease in adenosine triphosphatase (ATP)ase activity, and a subsarcolemmal Congo Red-positive staining. Immunostaining with an antibody against amyloid precursor protein showed an increased labeling of the cytoplasm of myofibers from adult and aged anti-NGF mice. These features are reminiscent of human myopathies, such as inclusion body myositis. We conclude that NGF deficits might be relevant for a class of human myopathies.     

Effects of fetal spinal cord tissue transplants and cycling exercise on the soleus muscle in spinalized rats.

Studies were carried out to determine if an intraspinal transplant (Trpl) of fetal spinal cord tissue or hind limb exercise (Ex) affected the changes in myosin heavy chain (MyHC) composition or myofiber size that occur following a complete transection (Tx) of the lower thoracic spinal cord of the adult rat. In one group of animals, transplants were made acutely, whereas in a second group, daily cycling exercise was initiated 5 days after injury, with animals in both groups being sacrificed 90 days after injury. The soleus muscle is normally composed of myofibers expressing either type I (90%) or type IIa (10%) MyHC. Following a spinal transection, expression of type I MyHC isoform decreased (18% of myofibers), type IIa MyHC expression increased (65% of myofibers), and the majority of myofibers (80%) expressed type IIx MyHC. Most myofibers coexpressed multiple MyHC isoforms. Compared with Tx only, with Ex or with Trpl, there was a decrease in the number of myofibers expressing type I or IIa isoforms but little change in expression of IIx MyHC. Myofibers expressing the IIb isoform appeared in several transplant recipients but not after exercise. Transection resulted in atrophy of type I myofibers to approximately 50% of normal size, whereas myofibers were significantly larger after exercise (74% of control) and in Trpl recipients (77% of control). Type IIa myofibers also were significantly larger in Trpl recipients compared with the Tx only group. Overall, the mean myofiber size was significantly greater after exercise and in Trpl recipients compared with myofibers in Tx only animals. Thus, although neither strategy shifted the MyHC profile towards the control, both interventions influenced the extent of atrophy observed after spinalization. These data suggest that palliative strategies can be developed to modulate some of the changes in hind limb muscles that occur following a spinal cord injury.     

Continuous myofiber remodeling in uninjured extraocular myofibers: myonuclear turnover and evidence for apoptosis

Unlike normal mature limb skeletal muscles, in which satellite cells are quiescent unless the muscle is injured, satellite cells in mammalian adult extraocular muscles (EOM) are chronically activated. This is evidenced by hepatocyte growth factor, the myogenic regulatory factor, Pax-7, and the cell-cycle marker, Ki-67, localized to the satellite cell position using serial sections and the positional markers laminin and dystrophin. Bromodeoxyuridine (brdU) labeling combined with dystrophin immunostaining showed brdU-positive myonuclei, presumably the result of fusion of activated satellite cells into existing myofibers. One new myonucleus was added to every 1000 myofibers in cross-section using a 12-hour brdU-labeling paradigm. The EOM thus appear to retain a stable nuclear population by an opposing process of apoptosis that results in myonuclear removal as visualized by terminal deoxynucleotidyltransferase-mediated nick end labeling (TUNEL). Activated caspase-3 was present in localized cytoplasmic domains extending from 10 to 210 microm within individual myofibers, suggesting segmental cytoplasmic reorganization. Understanding the cellular mechanisms that maintain this process of continuous myonuclear addition and removal in normal adult EOM may suggest new hypotheses to explain the preferential involvement or sparing of these muscles in skeletal muscle disease.     

Continuous myonuclear addition to single extraocular myofibers in uninjured adult rabbits

Extraocular muscles (EOM) are unique among mammalian skeletal muscles in that they normally express molecules associated with muscle development and regeneration. In this study we show that satellite cells of EOM, unlike those of other skeletal muscles, continually divide in the normal, uninjured adult. Adult EOM contained activated satellite cells positive for the myogenic regulatory factor MyoD. EOM satellite cells did not require a prolonged activation period prior to onset of cell division and differentiation in vitro. EOM satellite cells incorporated bromodeoxyuridine (brdU), a marker for cell division, and with longer postlabeling survival, brdU-labeled nuclei populated EOM myofibers. This was not seen with leg muscle. These findings suggest the possibility that continual division of satellite cells and fusion of their daughter myocytes with existing adult EOM myofibers contribute to the unique sparing or susceptibility of EOM to certain muscle diseases.     

Expression of cell surface and cytoskeleton developmentally regulated proteins in adult centronuclear myopathies.

In order to evaluate the developmental status of myofibers in 3 cases of adult centronuclear myopathies (CNM) with type I predominance, we searched for the expression of (a) developmentally regulated cytoskeleton proteins (myosin heavy chains (MHC), vimentin, desmin), and (b) cell surface molecules (neural cell adhesion molecules isoforms, NCAM). Desmin intermediate filaments were overexpressed in some fibers with centrally located nuclei and radially organized. Muscle fibers do not express vimentin. These findings were not observed in muscle biopsies from disease controls with numerous central nuclei. Few myofibers (less than 5%) expressed developmental MHC together with either embryonic NCAM or adult NCAM and rare fibers only expressed adult NCAM. Most of the remaining fibers neither expressed NCAM nor developmental MHC but were slow MHC positive. These features do not favor the hypothesis of a general arrest of muscle fiber maturation in adult CNM. It is more likely that fibers undergo a very slow developmental process with a long delay of innervation as shown by some fibers with NCAM expression. Nevertheless, innervation appears to be successful, as suggested by the large number of NCAM negative fibers. Moreover, the abnormal myofiber distribution could be related to this functional disturbance of innervation.     

Neural cell adhesion molecule in normal, denervated, and myopathic human muscle

The neural cell adhesion molecule (N-CAM) is a cell-surface glycoprotein that may mediate some intercellular adhesive interactions in the nervous system. In adult rat muscle, N-CAM is concentrated near neuromuscular junctions and on satellite cells, but is nearly undetectable in nonsynaptic portions of myofibers. However, N-CAM is abundant throughout myofibers in denervated and regenerating muscles. Using affinity-purified antibodies to N-CAM, we were able to demonstrate a similar distribution and regulation of N-CAM in human muscle. Myofiber N-CAM was not detectable immunohistochemically in any of 10 normal biopsies or in 4 biopsies that were abnormal but showed no evidence of fiber denervation or regeneration. N-CAM was present, however, at end plates, nerves, and satellite cells in normal human muscle. In contrast, myofiber N-CAM was detected in 16 of 16 patients with histological evidence of denervation and in 10 of 10 patients who had myopathy with degenerating/regenerating myofibers. In addition, 2 of 2 histologically nondiagnostic biopsies from patients with amyotrophic lateral sclerosis contained N-CAM-positive myofibers. Immunoblot analysis also detected N-CAM in denervated and myopathic, but not normal, human muscle. These results suggest that N-CAM may play a role in muscle reinnervation or regeneration and that N-CAM immunohistochemistry may complement conventional techniques in the diagnosis of neuromuscular disease.     

Human fetal muscle-specific antigen is restricted to regenerating myofibers in diseased adult muscle

A monoclonal antibody, 5.1H11, directed against human fetal muscle and myogenic cells in tissue culture, was used for immunofluorescence analysis of frozen sections of muscle biopsies from 24 patients with different diseases of muscle. The staining pattern was highly specific; detectable levels of the 5.1H11 antigen were restricted to regenerating myofibers as assessed by comparison with serial sections stained with hematoxylineosin. There was no appreciable staining of intrafusal, normal adult extra fusal, denervated, degenerating, or necrotic muscle fibers. Thus, the 5.1H11 antibody allows unambiguous identification of regenerating myofibers in biopsy specimens.     

Overactivity of exercise-sensitive cation channels and their impaired modulation by IGF-1 in mdx native muscle fibers: beneficial effect of pentoxifylline

Cell-attached patch-clamp recordings on native striated myofibers from adult dystrophic mdx mice revealed a higher occurrence and open probability compared to non-dystrophic wild-type myofibers of a 30 pS voltage-insensitive Ca2+-permeable channel, inhibited by Gd3+, streptomycin and ruthenium red. Myofibers from in vivo exercised animals had higher channel occurrence and/or open probability. Insulin-like growth factor 1 (3.3 nM) induced and/or enhanced channel activity, via PI3 kinase, in wild-type but not in mdx myofibers. Interestingly, in both genotypes the current was silenced by db-cAMP or pentoxifylline, a phosphodiesterase inhibitor. The channel activity/occurrence in pentoxifylline-treated exercised mdx (50 mg/kg/day i.p. for 4-8 weeks) overlapped that of exercised wild-type mice. Thus, a growth factor-sensitive current, likely due to a TRP channel, is activated in vivo by exercise in native striated fibers; its deregulation in the absence of dystrophin may contribute to Ca2+ homeostasis alteration. The possibility to pharmacologically counteract abnormal channel activity discloses important therapeutic application.     

The contribution of myofibers and fibrocytes to palmitate oxidation in skeletal muscle

Myofibers and fibrocytes were separated from a cell suspension of rat m. flexor digitorum brevis. The procedure was successful only when myofibers were intentionally damaged prior to separation. Density gradient centrifugation yielded a fraction containing 82% of fibrocytes and 20% of myofibers and a second fraction with the complementary 18% and 80% of fibrocytes and myofibers, respectively. Since the damaged myofibers had a negligible palmitate oxidation activity, the relative contribution of fibrocytes and myofibers to palmitate oxidation in the parent cell suspension with intact myofibers could be established, and amounted to 13% and 87%, respectively.     

Plasticity of skeletal muscle differentiation generating stem cell-like phenotype: possible application to cell therapy for muscular diseases]

Drug therapy, gene therapy, and cell therapy may be effective to degenerative muscular diseases caused by genetic mutations including muscular dystrophy. Stem cells including embryonic stem (ES) cells and adult stem cells (tissue stem cells) are generally believed to be applicable to cell therapy. However, both types of cells have several problems to be solved for the cell therapy. We have shown that the expression of SV40 large T antigen in terminally differentiated mouse C2 skeletal muscle myotubes induce mitosis and dedifferentiation. This finding has exploded the concept that terminally differentiated cells never proliferate. It remains to be determined, however, whether mature myofibers in vivo are capable of dedifferentiation and proliferation. Here we present the data showing that mouse myofibers dedifferentiate and proliferate to form many mononucleated cells. Eventually, these dedifferentiated cells redifferentiate and regenerate myofibers. The isolated dedifferentiated cells show stem cell-like phenotype. These cells are expected to be applied to the cell therapy for degenerative muscular diseases.     

Necrosis,sarcolemmal damage and apoptotic events in myofibers rejected by CD8+ lymphocytes: Observations in nonhuman primates

To detect the mechanisms of death in allogeneic myofibers rejected by the immune system, myoblasts were allotransplanted in muscles of macaques immunosuppressed with tacrolimus. Immunosuppression was stopped 1 month later to induce a massive rejection of allogeneic myofibers. Grafted sites were biopsied at 2-week intervals and analyzed by histology. The loss of allogeneic myofibers was rapid and concomitant with an intense infiltration of CD8+ lymphocytes. Several necrotic myofibers were observed in the lymphocyte accumulations by intracellular complement immunodetection. Dystrophin and spectrin immunodetection showed sarcolemmal damage in myofibers surrounded and invaded by CD8+ lymphocytes. Active caspase-3 was immunodetected in some myofibers surrounded by CD8+ lymphocytes. This is the first evidence that the collapse of myofibers attacked by T lymphocytes occurs by necrosis possibly due to damage of the sarcolemma. Caspase 3 is activated at least in some myofibers, but there was no evidence of a complete classical process of apoptosis.     

Full-length dystrophin cDNA transfer into skeletal muscle of adult mdx mice by electroporation

We showed that a LacZ expression plasmid (pCAG-lacZ) injection followed by electroporation increased the expression of the LacZ gene in the skeletal muscles of adult mdx mice up to ninefold higher as compared with simple intramuscular DNA injection. When full-length mouse dystrophin plasmid (pCAG-dys) and pCAG-lacZ were co-transfected by electroporation, 56% of dystrophin-positive fibers were stained for beta-galactosidase activity suggesting most of these myofibers are not revertants but transfected ones. Our data indicate that electroporation in vivo could introduce large full-length dystrophin cDNA into skeletal muscle of adult mdx mice.     

Myofiber differentiation in normal and hypotrophied canine pectineal muscles

The myofiber type composition of pectineal muscles was determined in 21 mixed-breed pups at 1 through 79 days of age and in 22 8-week-old-pups (4 mixed-breed, 5 greyhound and 5 German shepherd pups with normal muscles, and 8 German shepherd pups with myofiber hypotrophy). Sections were stained for the histochemical demonstration of myofibrillar adenosine triphosphatase by incubation in media at pH 9.8 without preincubation and following preincubation in acid media at pH 4.5 and at pH 4.3. At day 1 most myofibers were type 2C (alkaline and acid stable) while the remainder were type 1A myofibers (alkaline labile; acid stable) which had differentiated prenatally. Subsequently, the number of type 2C myofibers decreased while the number of type 1A and type 2A myofibers (alkaline stable; acid labile at pH 4.5 and pH 4.3) increased. Type 1B myofibers (partially alkaline stable; acid stable) were first observed at day 12 and were variably present through day 79. Type 1B myofibers appeared to be a transition form of type 2C to type 1A differentiation. Type 2B myofibers (alkaline stable; acid stable at pH 4.5; acid labile at pH 4.3) were not observed. Hypotrophied muscles contained more type 2C and fewer type 2A myofibers and all myofiber types were smaller than normal. These results suggest that the pathogenesis of this disorder involves both myofiber growth and differentiation, particularly differentiation of type 2A myofibers.     

Maturation, dystrophic changes and the continuous production of fibers in skeletal muscle regenerating in the absence of nerve

To investigate how much skeletal muscle depends on influences coming from peripheral nerve, we studied muscle regeneration induced by bupivacaine in the permanently denervated soleus muscle of adult rats. Using light and electron microscopy, we studied the extent of maturation attained by regenerated myofibers and their ability to regenerate again after repeated bupivacaine injury. Morphometric analyses showed that within the first two weeks after injury, the regenerated denervated fibers increased in diameter, matured and then became atrophic. By electron microscopy the morphological characteristics of mature fibers are evident, but final differentiation into adult fiber types is not attained. This is in keeping with previously reported biochemical results. A new phase of massive regeneration occurred when bupivacaine treatment was repeated, thus demonstrating that regenerated myofibers devoid of innervation keep their intrinsic ability to regenerate after injury. Spontaneous though scanty regeneration also occurred in denervated regenerated muscle at late stages, when atrophy and dystrophy of fibers became prominent. Concomitantly the relative number of myosatellite cells remained high. These morphological observations further support the hypothesis that fiber regeneration participates in the maintenance of chronically denervated muscles.     

The effect of X-irradiation on skeletal muscle regeneration in the adult rat

Muscle regeneration was induced by transplanting the extensor digitorum longus (EDL) muscle in adult rats to examine the effect of X-irradiation on muscle regeneration. The EDL muscles were removed, irradiated with X-rays to administer 650 R, 2,000 R or 10,000 R, and transplanted into the original animal and location. Muscles from non-irradiated control group and each irradiated group were analyzed morphologically at 4, 7, 14 and 30 days post-transplantation. The regeneration pattern in the non-irradiated and 650-R irradiated muscles was similar. A majority of myofibers underwent degeneration followed by regeneration from the precursor myosatellite cells. The myosatellite cells proliferated, differentiated into myoblasts and then fused to form myotubes and myofibers. Muscles exposed to 2,000 R underwent initial degeneration and myosatellite cell activation, however, considerably fewer myotubes regenerated in these muscles. In muscles exposed to 10,000 R, again myofiber degeneration and myosatellite cell activation was evident, but these cells remained undifferentiated and did not fuse to form myotubes. These results show a dose-dependent inhibition in muscle regeneration due to irradiation.     

Selective type II muscle fiber hypertrophy in severe infantile spinal muscular atrophy

The diagnostic muscle biopsy finding in severe infantile spinal muscular atrophy (Werdnig-Hoffmann disease, SMA type 1) is considered to be large-group atrophy with isolated clusters of hypertrophic type I myofibers. We present a unique case of severe infantile spinal muscular atrophy with selective hypertrophy of type II myofibers. A male infant presented at age 2 months with breathing difficulties and by age 4 months was hypotonic and weak. Electromyography revealed denervation in all extremity muscles, and nerve conduction velocities were normal but with small compound muscle action potentials. Quadriceps muscle biopsy revealed many hypertrophied type II myofibers (myofibers with a mean least diameter of 25.4 microns). In contrast, the largest type I myofibers were 20 microns in least diameter (mean diameter, 14.9 microns), and there was a normal-size population of type II fibers (mean diameter, 15.7 microns). In addition, sheets of atrophic type I and type II fibers averaged 2.0 microns in least diameter. Sural nerve biopsy was normal. Breathing difficulties progressed, with death ensuing at age 5 1/2 months. Autopsy revealed atrophy of ventral spinal roots with normal dorsal roots. There was loss of anterior horn cells, while remnant neurons were reduced in size. No other pathologic changes were identified. This case indicates that in severe infantile spinal muscular atrophy, relative sparing of the motor units with type II myofibers may occur.     

Immunoreactivity of antibodies raised against synthetic peptide fragments predicted from mid portions of dystrophin cDNA

We synthesized 3 peptide fragments predicted by residues 2354-2368 (peptide I), 2310-2324 (peptide II) and 2255-2269 (peptide III) on the mid-portion of the human dystrophin cDNA map where the most frequent intragenic deletions occurred in Duchenne muscular dystrophy. Rabbit antibodies against these peptides were raised and cryosections of 47 biopsied muscles were studied immunohistochemically. The 47 biopsied muscles included the quadriceps femoris muscles of 8 Duchenne muscular dystrophy patients, 8 child and 5 adult normal controls, 1 facioscapulohumeral dystrophy, 2 limb girdle dystrophy, 3 myotonic dystrophy, 3 polymyositis, 1 mitochondrial myopathy, 1 nemaline myopathy, 3 amyotrophic lateral sclerosis and the extensor digitorum longus muscles of 6 mdx mice (C57BL/10ScSn-mdx) and 6 normal control mice (C57BL/10ScSn). The peptide I antiserum continuously stained the myofiber surface membranes in 8 child and 5 adult normal control muscles, and in 14 other muscles from various neuromuscular diseases, but failed to stain the surface membranes in normal control mice. The surface membranes of 8 Duchenne muscles were not stained by the peptide I antiserum except for a few myofibers. Although the ELISA titers of peptide I, II and III antibodies were high, immunostaining by peptide II antiserum showed no reaction in the myofibers of any of the biopsied muscles, and immunostaining by peptide III antiserum revealed faint reactions on the myofiber surface membranes of all biopsied muscles, including the mdx control mouse muscles except for the Duchenne and mdx myofibers.     

不同血清型的重组腺相关病毒载体对成熟肌纤维转导的研究

目的探讨不同血清型对成熟肌纤维的转导效率.方法我们用不同的血清型AAV（rAAV-1,rAAV-2,rAAV-3和rAAV-5）表达LacZ和GDNF基因研究在小鼠骨骼肌转导效率,转基因表达用组化方法或ELISA来评定. 结果在3周龄的小鼠中,LacZ组化染色显示rAAV5在慢和快的肌纤维中都有效的转导,而rAAV2和rAAV3优先在慢纤维中转导.在8周龄的小鼠（成年鼠）中,rAAV3和rAAV5在慢纤维和快纤维中都有效的转导.用rAAV3-LacZ或rAAV5-LacZ优先转导的慢纤维与rAAV2相比没有显著性差异.用8周龄的小鼠肌肉注射不同血清型的rAAV-GDNF后,结果显示rAAVl-GDNF表达最高;rAAV2、rAAV3和rAAV5在骨骼肌的表达也有效.结论在肌肉基因转导中,rAAV3和rAAV5介导的载体都是有效的,能克服rAAV2所受到的限制.     

Slow-type C-protein in dystrophic chicken fast pectoralis muscle

C-protein isoform expression in hereditary dystrophic chicken skeletal muscle was compared with that in normal chicken muscle during postnatal development by immunocytochemical and immunoblot methods. In the pectoralis muscle (PM) of both normal and dystrophic chicken, slow- and fast-type C-proteins were coexpressed in the vast majority of myofibers at neonatal age, but the slow C-protein disappeared, leaving continued expression of only the fast-type C-protein as muscle development progressed up to 2 weeks posthatch. In the dystrophic chicken PM, however, myofibers containing slow-type C-protein reappeared about 1 month posthatch and increased in number with the progression of muscular dystrophy. We conclude that C-protein isoform expression in dystrophic myofibers resembles that in neonatal myofibers and that the expression of slow-type C-protein can be seen as a marker for chicken muscular dystrophy.     

Membrane skeleton of innervated and denervated fast- and slow-twitch muscle

We used confocal microscopy and immunoblotting to study membrane skeletal proteins of fast-twitch (extensor digitorum longus) and slow-twitch (soleus) muscles of the adult rat. In the extensor digitorum longus (EDL), beta-spectrin concentrates in costameres, whereas dystrophin is enriched at costameres but is also present in intercostameric regions. In the soleus, beta-spectrin and dystrophin underlie much of the sarcolemma, and intercostameric regions are difficult to detect. The EDL sarcolemma reorganizes following denervation to resemble soleus sarcolemma, but denervation does not significantly affect the latter. Consistent with these observations, soleus contains similar amounts of dystrophin but more beta-spectrin than EDL. Denervation increases beta-spectrin levels only in the EDL and dystrophin levels in both muscles. Denervation does not affect beta-fodrin, a beta-spectrin homolog expressed in embryonic myofibers. Thus, neuromuscular activity controls sarcolemmal organization and the levels of beta-spectrin and dystrophin, but not postnatal downregulation of beta-fodrin. The differences in organization of the sarcolemma may underlie the differential susceptibility of fast and slow myofibers to dystrophinopathies.