Intracellular calcium in canine muscle biopsies

Intracellular staining for calcium was studied in muscle biopsies from 15 dogs by the alizarin red S (ARS) stain. Rare positive fibres were present in normal muscle and in denervation atrophy. The percentage of positive fibres was slightly increased in polymyositis, dermatomyositis and canine temporal/masseter myositis and markedly increased in progressive muscular dystrophy. Calcium-positive fibres were usually so-called large-dark (hypercontracted) fibres or necrotic fibres, although there was occasional staining of normal and atrophied fibres. These results indicate the probable involvement of calcium in muscle injury in canine inflammatory myopathies and in canine muscular dystrophy. In addition, use of the ARS stain appears to be useful for detecting the earliest lesions of acute muscle fibre injury.     

Expression of transforming growth factor-beta 1 and its relation to endomysial fibrosis in progressive muscular dystrophy.

Progressive muscular dystrophy is characterized by muscle fiber necrosis, regeneration, and endomysial fibrosis. Although absence of dystrophin has been known as the cause of muscle fiber degeneration, pathogenesis of interstitial fibrosis is still unknown. Transforming growth factor-beta 1 (TGF-beta 1) induces accumulation of extracellular matrix in various diseases, such as liver cirrhosis and interstitial pneumonitis. To investigate its function on the pathogenesis of progressive muscular dystrophy, it was necessary to determine the degree of TGF-beta 1 expression and the site of TGF-beta 1 immunoreactivity. In Duchenne muscular dystrophy and most of Becker muscular dystrophy, high TGF-beta 1 immunoreactivity expressed on muscle fibers and extracellular space. In other myopathies with endomysial fibrosis, however, TGF-beta 1 was seldom observed. We also examined the immunoreactivity of the latent TGF-beta binding protein, which is bound to the TGF-beta precursors. In all Duchenne muscular dystrophy and half of Becker muscular dystrophy cases, high latent TGF-beta 1 binding protein immunoreactivity was seen, but in other myopathies its immunoreactivity was seldom seen on muscle fibers or extracellular space. Therefore TGF-beta 1 may play an important role in synthesis and accumulation of extracellular matrix in progressive muscular dystrophy.     

Platelet-derived growth factor and its receptors are related to the progression of human muscular dystrophy: an immunohistochemical study

This study has examined the immunological localization of platelet-derived growth factor (PDGF)-A, PDGF-B, and PDGF receptor (PDGFR) alpha and beta to clarify their role in the progression of muscular dystrophy. Biopsied frozen muscles from patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and congenital muscular dystrophy (CMD) were analysed immunohistochemically using antibodies raised against PDGF-A, PDGF-B, and PDGFR alpha and beta. Muscles from two dystrophic mouse models (dy and mdx mice) were also immunostained with antibodies raised against PDGFR alpha and beta. In normal human control muscle, neuromuscular junctions and vessels were positively stained with antibodies against PDGF-A, PDGF-B, PDGFR alpha and PDGFR beta. In human dystrophic muscles, PDGF-A, PDGF-B, PDGFR alpha and PDGFR beta were strongly immunolocalized in regenerating muscle fibres and infiltrating macrophages. PDGFR alpha was also immunolocalized to the muscle fibre sarcolemma and necrotic fibres. The most significant finding in this study was a remarkable overexpression of PDGFR beta and, to a lesser extent, PDGFR alpha in the endomysium of DMD and CMD muscles. PDGFR was also overexpressed in the interstitium of muscles from dystrophic mice, particularly dy mice. Double immunolabelling revealed that activated interstitial fibroblasts were clearly positive for PDGFR alpha and beta. However, DMD and CMD muscles with advanced fibrosis showed very poor reactivity against PDGF and PDGFR. Those findings were confirmed by immunoblotting with PDGFR beta. These findings indicate that PDGF and its receptors are significantly involved in the active stage of tissue destruction and are associated with the initiation or promotion of muscle fibrosis. They also have roles in muscle fibre regeneration and signalling at neuromuscular junctions in both normal and diseased muscle.     

HISTOMETRICAL and HISTOLOGICAL CHANGES OF THE SKELETAL MUSCLE IN NEUROMUSCULAR DISORDERS – AN AUTOPSY STUDY

Systemic hlstometrical and histological examinations of major skeletal muscles were performed by using autopsy cases with simple atrophy, neurogenic muscular atrophy, Duchenne type progressive muscular dystrophy, myositis of myasthenia gravis, and autopsy control cases. In hlstometrical studies, the shortest diameters of muscle fibers were measured and arranged in histograms. Volume ratio of stroma to muscle was measured by point-counting method.
Histometrical studies revealed the following results: (1) averages of muscle fiber diameters in controls showed the largest value In the muscles of the upper and lower extremities, and the smallest value in the lingual muscle; (2) in simple atrophy, neurogenic muscular atrophy, progressive muscular dystrophy and myositis, a decrease in muscle fiber diameters was more prominent in the muscles of the lower extremities than those of the upper extremities; (3) patterns of histograms of muscle fiber diameters were classified into six types, and in simple atrophy, almost one-half of muscles examined belonged to type 3 histogram, which had the mode situated at a relatively small diameter and a not so high kurtosls; (4) volume ratios of stroma to muscle Increased most in both muscular dystrophy and long-standing neurogenic muscular atrophy, moderately in myositis, and mildly in simple atrophy; and (5) hlstometrical changes In myasthenia gravis were minimal.     

Ocular myopathies

Ocular myopathies are manifested by primary and progressive involvement of extraocular muscles. In most cases of involvement of extra-ocular muscles a biopsy from somatic muscles studied by histochemistry and electron microscopy permits to make the diagnosis of the underlying condition. The two main clinico-pathological types of ocular myopathies are the oculocraniosomatic syndrome (Kearns-Sayre syndrome) and oculopharyngeal muscular dystrophy. The oculocraniosomatic syndrome is a multisystemic disorder and its histopathological hallmark is the presence of ragged-red muscle fibres which contain aggregates of abnormal mitochondria, often with paracrystalline inclusions. In the oculopharyngeal muscular dystrophy are observed muscle fibres with rimmed vacuoles and intranuclear tubular filamentous inclusions about 8.5 nm in external diameter. The rimmed vacuoles may occur in other muscle diseases but the intranuclear inclusions appear to be specific for oculopharyngeal muscular dystrophy. Their nature is unknown.     

Lamin A N-terminal phosphorylation is associated with myoblast activation: impairment in Emery-Dreifuss muscular dystrophy

Background: Skeletal muscle disorders associated with mutations of lamin A/C gene include autosomal Emery–Dreifuss muscular dystrophy and limb girdle muscular dystrophy 1B. The pathogenic mechanism underlying these diseases is unknown. Recent data suggest an impairment of signalling mechanisms as a possible cause of muscle malfunction. A molecular complex in muscle cells formed by lamin A/C, emerin, and nuclear actin has been identified. The stability of this protein complex appears to be related to phosphorylation mechanisms.

Objective: To analyse lamin A/C phosphorylation in control and laminopathic muscle cells.

Methods: Lamin A/C N-terminal phosphorylation was determined in cultured mouse myoblasts using a specific antibody. Insulin treatment of serum starved myoblast cultures was carried out to evaluate involvement of insulin signalling in the phosphorylation pathway. Screening of four Emery–Dreifuss and one limb girdle muscular dystrophy 1B cases was undertaken to investigate lamin A/C phosphorylation in both cultured myoblasts and mature muscle fibres.

Results: Phosphorylation of lamin A was observed during myoblast differentiation or proliferation, along with reduced lamin A/C phosphorylation in quiescent myoblasts. Lamin A N-terminus phosphorylation was induced by an insulin stimulus, which conversely did not affect lamin C phosphorylation. Lamin A/C was also hyperphosphorylated in mature muscle, mostly in regenerating fibres. Lamin A/C phosphorylation was strikingly reduced in laminopathic myoblasts and muscle fibres, while it was preserved in interstitial fibroblasts.

Conclusions: Altered lamin A/C interplay with a muscle specific phosphorylation partner might be involved in the pathogenic mechanism of Emery–Dreifuss muscular dystrophy and limb girdle muscular dystrophy 1B.

     

Function Induced Modifications of Gene Expression: an Alternative Approach to Gene Therapy of Duchenne Muscular Dystrophy

In Duchenne muscular dystrophy a large gene that codes for dystrophin is altered. The possibility that the defective gene/protein could be at least in part substituted by other molecules that the diseased muscle is able to produce and that have a function similar to that of dystrophin is being discussed. Muscle fibres have a tremendous adaptive potential, and the expression of several protein isoforms can be induced by either stretch or long-term change of activity. The exploitation of this ability of muscle cells to express new genes, which would code for proteins that will not be alien to the individual, for treatment of Duchenne muscular dystrophy is being considered. The argument for this approach is strengthened by results that in patients with Duchenne muscular dystrophy the progress of the disease can be slowed with changes of muscle activity.     

生长相关蛋白及其mRNA在大鼠损伤坐骨神经中的表达

为了研究大鼠周围神经损伤后远侧端组织中生长相关蛋白 (GAP-4 3 )及其 m RNA的表达 ,采用正常 SD大鼠坐骨神经横断损伤的模型 ,于术后不同时间取坐骨神经远侧端组织 ,以 Western Blot和 RT-PCR法检测 GAP-4 3及其 m RNA的表达。RT-PCR法检测结果显示 ,正常大鼠坐骨神经组织中 GAP-4 3 m RNA表达量较低 ,而损伤坐骨神经的远侧段 GAP-4 3 m RNA的表达量明显增加 ,于损伤第 2周时达高峰。用抗 GAP-4 3抗体进行 Western Blot检测 ,结果表明 ,正常坐骨神经 43 k Da处出现弱阳性反应的蛋白区带 ,而损伤后坐骨神经远侧段 43 k Da处阳性反应条带着色明显加深 ,损伤后第 3周时阳性反应着色最强。本研究结果提示 ,GAP-4 3及其 m RNA在大鼠损伤坐骨神经远侧段组织中表达增强 ,其 m RNA表达上调高峰在神经损伤后的第 2周 ;而其蛋白合成增加最为明显的时间是在神经损伤后的第 3周。     

Absence of alpha 7 integrin in dystrophin-deficient mice causes a myopathy similar to Duchenne muscular dystrophy

Both the dystrophin-glycoprotein complex and alpha7beta1 integrin have critical roles in the maintenance of muscle integrity via the provision of mechanical links between muscle fibres and the basement membrane. Absence of either dystrophin or alpha7 integrin results in a muscular dystrophy. To clarify the role of alpha7 integrin and dystrophin in muscle development and function, we generated integrin alpha7/dystrophin double-mutant knockout (DKO) mice. Surprisingly, DKO mice survived post-natally and were indistinguishable from wild-type, integrin alpha7-deficient and mdx mice at birth, but died within 24-28 days. Histological analysis revealed a severe muscular dystrophy in DKO mice with endomysial fibrosis and ectopic calcification. Weight loss was correlated with the loss of muscle fibres, indicating that progressive muscle wasting in the double mutant was most likely due to inadequate muscle regeneration. The data further support that premature death of DKO mice is due to cardiac and/or respiratory failure. The integrin alpha7/dystrophin-deficient mouse model, therefore, resembles the pathological changes seen in Duchenne muscular dystrophy and suggests that the different clinical severity of dystrophin deficiency in human and mouse may be due to a fine-tuned difference in expression of dystrophin and integrin alpha7 in both species. Together, these findings indicate an essential role for integrin alpha7 in the maintenance of dystrophin-deficient muscles.     

Amyloidose bei Muskeldystrophie

Mutations in the gene encoding dysferlin (DYSF) cause limb-girdle muscular dystrophy 2B (LGMD2B) and Miyoshi myopathy (MM). We were able to examine eight patients suspected of LGMD2B clinically, histochemically. The genotype was determined in every case. We found sarcolemmal and interstitial amyloid deposits in four muscle sections. All of the mutations associated with amyloid were located in the N-terminal region of dysferlin, and dysferlin clearly proved to be a component of the amyloid deposits. Dysferlin-deficient muscular dystrophy is the first muscular dystrophy in which amyloidosis is involved. This fact must be considered in the process of developing therapeutic strategies. The influence of the amyloid deposits on the pathogenesis of the disease and the possible involvement of other organs in the progressive course are as yet unclear.     

Becker muscular dystrophy (BMD) and Klinefelter''s syndrome: a possible cause of variable expression of BMD within a pedigree.

We describe a man with Becker muscular dystrophy whose weakness was minimal in contrast to that of his more severely affected nephews. This man had a Klinefelter karyotype (47,XXY) and his mild symptoms may be attributed to him being heterozygous for the muscular dystrophy gene. This is the first report of a person with both Klinefelter's syndrome and Becker muscular dystrophy. This combination may be one explanation for the variable expression of X linked muscular dystrophy noted in some pedigrees.     

生长相关蛋白在损伤坐骨神经中的表达

目的：研究大鼠周围神经损伤与再生过程中生长相关蛋白的表达。方法：取正常SD大鼠坐骨神经和离断损伤后不同时间近侧端和远侧端神经组织，采用Anti-GAP－43抗体以Western blot方法检测GAP－43的表达变化。结果：NC膜上43kDa位置出现阳性反应条带，在正常坐骨神经中反应微弱，损伤后明显加强，随损伤时间延长，神经近侧端的反应无明显改变，神经元侧端的反应逐渐加强，以第3周为最强，之后逐渐减弱，结论：GAP－43在正常坐骨神经中低表达，损伤后近侧端与远侧端表达均增加，远侧端的表达增加以第3周为高峰。     

脑缺血再灌注后脑梗塞周围区生长相关蛋白-GAP43表达的变化及外源性神经生长因子的影响

目的研究大鼠脑缺血再灌注后生长相关蛋白-43(GAP-43)表达的变化规律及外源性神经生长因子(NGF)的影响.方法成年健康雌性Wistar大鼠72只,随机分为假手术组、自然恢复组、人工脑脊液组和NGF治疗组.采用线栓法建立大脑中动脉缺血再灌注(MCAO)动物模型,应用免疫组织化学方法观察脑缺血再灌注后GAP-43的表达.结果(1)脑缺血再灌注6 h后,缺血周围区GAP-43表达逐渐增高,第7天达高峰,以后逐渐降低,第21天仍有表达.(2)应用外源性NGF后,GAP-43表达较对照组有所增高,但无显著性差异(P＞0.05).结论提示中枢神经系统损伤后,神经元具有再生和修复的可塑性,外源性NGF对GAP-43的调节有待于进-步研究.     

Myopathology of non-infectious inflammatory myopathies - the current status

Besides the classical inflammatory myopathies (IM), dermatomyositis (DM), polymyositis, and inclusion body myositis, the much larger spectrum of IM includes focal and nodular myositis, granulomatous myositis, macrophagic myofasciitis, graft vs. host myositis, eosinophilic myositis, and other immune-associated conditions, some of them only recently described. In addition, paraneoplastic, statin-induced and critical illness myopathies have been considered immune-associated IM. Infectious, i.e., bacterial, viral, and parasitic IM are much less frequent in the northern hemisphere. In IM, muscle biopsy is an essential diagnostic procedure to initiate therapy. The myopathological spectrum encompasses disease-specific histopathological features, such as perifascicular atrophy in DM, non-necrotizing granulomas in sarcoid myopathy, autophagic vacuoles with tubulofilamentous inclusions in inclusion body myositis, rarely electron microscopic criteria, such as undulating tubules in endothelial cells of DM specimens, and, foremost, immunohistochemical findings. These latter features concern inflammatory infiltrates, the muscle parenchyma, the interstitial compartment, and the vasculature with varying involvement of each component in the different IM. Differences in immunohistochemical parameters among the IM, such as major histocompatibility complexes I and II, cytokines, cell adhesion molecules, different types of inflammatory cells, metalloproteinases, and complement factors procure a large gamut of data, the individual patterns of which characterize the myopathology of individual IM.     

Mdm muscular dystrophy: interactions with calpain 3 and a novel functional role for titin's N2A domain

Human tibial muscular dystrophy and limb-girdle muscular dystrophy 2J are caused by mutations in the giant sarcomeric protein titin (TTN) adjacent to a binding site for the muscle-specific protease calpain 3 (CAPN3). Muscular dystrophy with myositis (mdm) is a recessive mouse mutation with severe and progressive muscular degeneration caused by a deletion in the N2A domain of titin (TTN-N2ADelta83), disrupting a putative binding site for CAPN3. To determine whether the muscular dystrophy in mutant mdm mice is caused by misregulation of CAPN3 activity, genetic crosses with CAPN3 overexpressing transgenic (C3Tg) and CAPN3 knockout (C3KO) mice were generated. Here, we report that overexpression of CAPN3 exacerbates the mdm disease, leading to a shorter life span and more severe muscular dystrophy. However, in a direct genetic test of CAPN3's role as a mediator of mdm pathology, C3KO;mdm double mutant mice showed no change in the progression or severity of disease indicating that aberrant CAPN3 activity is not a primary mechanism in this disease. To determine whether we could detect a functional deficit in titin in a non-disease state, we examined the treadmill locomotion of heterozygous +/mdm mice and detected a significant increase in stride time with a concomitant increase in stance time. Interestingly, these altered gait parameters were completely corrected by CAPN3 overexpression in transgenic C3Tg;+/mdm mice, supporting a CAPN3-dependent role for the N2A domain of TTN in the dynamics of muscle contraction.     

The action potential-evoked sarcoplasmic reticulum calcium release is impaired in mdx mouse muscle fibres

The mdx mouse, a model of the human disease Duchenne muscular dystrophy, has skeletal muscle fibres which display incompletely understood impaired contractile function. We explored the possibility that action potential-evoked Ca²⁺ release is altered in mdx fibres. Action potential-evoked Ca²⁺-dependent fluorescence transients were recorded, using both low and high affinity Ca²⁺ indicators, from enzymatically isolated fibres obtained from extensor digitorum longus (EDL) and flexor digitorum brevis (FDB) muscles of normal and mdx mice. Fibres were immobilized using either intracellular EGTA or N -benzyl- p -toluene sulphonamide, an inhibitor of the myosin II ATPase. We found that the amplitude of the action potential-evoked Ca²⁺ transients was significantly decreased in mdx mice with no measured difference in that of the surface action potential. In addition, Ca²⁺ transients recorded from mdx fibres in the absence of EGTA also displayed a marked prolongation of the slow decay phase. Model simulations of the action potential-evoked transients in the presence of high EGTA concentrations suggest that the reduction in the evoked sarcoplasmic reticulum Ca²⁺ release flux is responsible for the decrease in the peak of the Ca²⁺ transient in mdx fibres. Since the myoplasmic Ca²⁺ concentration is a critical regulator of muscle contraction, these results may help to explain the weakness observed in skeletal muscle fibres from mdx mice and, possibly, Duchenne muscular dystrophy patients.     

Fibre-related nitric oxide synthase (NOS) in Duchenne muscular dystrophy

Nitric oxide (NO) mediates fundamental physiological actions on skeletal muscle. The loss of NO synthase (NOS) from the sarcolemma was assumed to be associated with development of Duchenne muscular dystrophy (DMD). We have, however, recently reported that, in contrast to the commonly accepted view, NOS expression in DMD myofibres is up-regulated. This poses the question of the fibre type-specific NOS expression in DMD muscles and how the NOS expression is related to the regeneration or degeneration status. To address this issue, we examined localization of NOS isoforms I, II and III in skeletal muscles of DMD patients employing immunohistochemical labelling with tyramide signal amplification complemented with enzyme histochemistry. We found that NOS immunolabelling as well as metabolic enzyme activity in DMD muscles were heterogeneously distributed along the fibre length of DMD muscle fibres revealing regenerating and degenerate (hypercontracted) fibres as well as normal segments. Like in normal muscles, positive NOS immunoreactivity was found to be associated with fast-oxidative glycolytic (FOG) phenotype. The regeneration status of NOS-positive segments was deduced from the presence of neonatal and developmental myosin heavy chains. High NOS expression in regenerating DMD muscle fibres can be well reconciled with reports about the protective role of endogenous NO in inflammatory diseases and in muscle repair.     

The prion protein in human neuromuscular diseases

The basis of human prion diseases affecting the nervous system is accumulation of a disease-associated conformer (PrPSc) of the normal cellular prion protein (PrPC). Earlier studies demonstrated increased expression of PrPC in inclusion body myositis (IBM), dermato-, and polymyositis, as well as neurogenic muscle atrophy. To define the spectrum and reliability of PrPC immunoreactivity, its expression was examined systematically in a series of pathologically characterized muscular disorders by means of immunohistochemistry, confocal laser microscopy, and immunogold electron microscopy. Anti-PrPC immunolabelling of rimmed vacuoles was observed in IBM, inclusions of myofibrillary myopathy, targets, regenerating, and atrophic fibres, mononuclear cells, in addition to ragged red fibres in mitochondrial myopathies, and focal sarcolemmal immunostaining in non-diseased controls. Quantitative analysis demonstrated that, in neurogenic muscle lesions, anti-PrPC staining detects a significantly broader spectrum of fibres than anti-vimentin or anti-NCAM. In dystrophic muscle, PrPC expression was mainly restricted to regenerating fibres. In IBM, PrPC expression was not confined to rimmed vacuoles or vacuolated fibres and only a small percentage (7.1%) of rimmed vacuoles were PrPC positive. Ultrastructurally, PrPC was observed in the cytoplasm of lymphocytes, in the myofibrillar network of targets, and in rimmed vacuoles. Knowledge of disease circumstances with altered expression of PrPC is important in the setting of a potentially increased chance for extraneural PrPC-PrPSc conversion. In addition, our observations suggest that PrPC may have a general stress-response effect in various neuromuscular disorders.