Dysferlin is a plasma membrane protein and is expressed early in human development

Recently, a single gene, DYSF, has been identified which is mutated in patients with limb-girdle muscular dystrophy type 2B (LGMD2B) and with Miyoshi myopathy (MM). This is of interest because these diseases have been considered as two distinct clinical conditions since different muscle groups are the initial targets. Dysferlin, the protein product of the gene, is a novel molecule without homology to any known mammalian protein. We have now raised a monoclonal antibody to dysferlin and report on the expression of this new protein: immunolabelling with the antibody (designated NCL-hamlet) demonstrated a polypeptide of approximately 230 kDa on western blots of skeletal muscle, with localization to the muscle fibre membrane by microscopy at both the light and electron microscopic level. A specific loss of dysferlin labelling was observed in patients with mutations in the LGMD2B/MM gene. Furthermore, patients with two different frameshifting mutations demonstrated very low levels of immunoreactive protein in a manner reminiscent of the dystrophin expressed in many Duchenne patients. Analysis of human fetal tissue showed that dysferlin was expressed at the earliest stages of development examined, at Carnegie stage 15 or 16 (embryonic age 5-6 weeks). Dysferlin is present, therefore, at a time when the limbs start to show regional differentiation. Lack of dysferlin at this critical time may contribute to the pattern of muscle involvement that develops later, with the onset of a muscular dystrophy primarily affecting proximal or distal muscles.     

Identical mutation in patients with limb girdle muscular dystrophy type 2B or Miyoshi myopathy suggests a role for modifier gene(s)

Limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM), a distal muscular dystrophy, are both caused by mutations in the recently cloned gene dysferlin, gene symbol DYSF. Two large pedigrees have been described which have both types of patient in the same families. Moreover, in both pedigrees LGMD2B and MM patients are homozygous for haplotypes of the critical region. This suggested that the same mutation in the same gene would lead to both LGMD2B or MM in these families and that additional factors were needed to explain the development of the different clinical phenotypes. In the present paper we show that in one of these families Pro791 of dysferlin is changed to an Arg residue. Both the LGMD2B and MM patients in this kindred are homozygous for this mutation, as are four additional patients from two previously unpublished families. Haplotype analyses suggest a common origin of the mutation in all the patients. On western blots of muscle, LGMD2B and MM patients show a similar abundance in dysferlin staining of 15 and 11%, respectively. Normal tissue sections show that dysferlin localizes to the sarcolemma while tissue sections from MM and LGMD patients show minimal staining which is indistinguishable between the two types. These findings emphasize the role for the dysferlin gene as being responsible for both LGMD2B and MM, but that the distinction between these two clinical phenotypes requires the identification of additional factor(s), such as modifier gene(s).     

Dysferlin and Myoferlin Regulate Transverse Tubule Formation and Glycerol Sensitivity

Dysferlin is a membrane-associated protein implicated in muscular dystrophy and vesicle movement and function in muscles. The precise role of dysferlin has been debated, partly because of the mild phenotype in dysferlin-null mice (Dysf). We bred Dysf mice to mice lacking myoferlin (MKO) to generate mice lacking both myoferlin and dysferlin (FER). FER animals displayed progressive muscle damage with myofiber necrosis, internalized nuclei, and, at older ages, chronic remodeling and increasing creatine kinase levels. These changes were most prominent in proximal limb and trunk muscles and were more severe than in Dysf mice. Consistently, FER animals had reduced ad libitum activity. Ultrastructural studies uncovered progressive dilation of the sarcoplasmic reticulum and ectopic and misaligned transverse tubules in FER skeletal muscle. FER muscle, and Dysf- and MKO-null muscle, exuded lipid, and serum glycerol levels were elevated in FER and Dysf mice. Glycerol injection into muscle is known to induce myopathy, and glycerol exposure promotes detachment of transverse tubules from the sarcoplasmic reticulum. Dysf, MKO, and FER muscles were highly susceptible to glycerol exposure in vitro, demonstrating a dysfunctional sarcotubule system, and in vivo glycerol exposure induced severe muscular dystrophy, especially in FER muscle. Together, these findings demonstrate the importance of dysferlin and myoferlin for transverse tubule function and in the genesis of muscular dystrophy.The muscular dystrophies are a heterogeneous group of genetic disorders characterized by progressive muscle loss and weakness. The mechanisms that underlie muscular dystrophy are diverse, including defective regeneration, plasma membrane instability, and defective membrane repair. Dysferlin (DYSF) has been implicated in all of these processes.^1,2 Autosomal recessive loss-of-function mutations in dysferlin cause three different forms of muscular dystrophy: limb-girdle muscular dystrophy type 2B, Miyoshi myopathy, and distal anterior compartment myopathy.^3–5 Mutations in dysferlin become clinically evident in the second to third decade or later, with muscle weakness. An early characteristic feature of dysferlin mutations is massively elevated serum creatine kinase levels. A spectrum of myopathic changes can be seen in muscle biopsy specimens from humans with dysferlin mutations, including dystrophic features, such as fibrofatty replacement and inflammatory infiltrates.Dysferlin is a 230-kDa membrane-inserted protein that contains at least six cytoplasmic C2 domains. C2 domains mediate protein-protein interactions and, in some cases, directly bind phospholipids and calcium. The C2 domains of dysferlin are highly related to those found in the membrane trafficking and fusion protein synaptotagmins.⁶ Dysferlin is highly expressed in adult skeletal muscle, whereas it is expressed at lower levels in muscle precursor cells, myoblasts.^1,7,8 On sarcolemma damage, dysferlin is found at the sites of membrane disruption and has been specifically implicated in resealing the sarcolemma.² Electron microscopy of skeletal muscle biopsy specimens from human dysferlin-mutant patients confirms discontinuity of the sarcolemma and reveals vesicles underneath the basal lamina, suggesting dysferlin plays an active role in vesicle fusion at the membrane lesion.⁹ Dysferlin also has been shown to interact with a variety of cytosolic and membrane-associated binding partners, including MG53, caveolin-3, AHNAK, and annexins A1 and A2.^10–13 Similar to dysferlin, MG53, caveolin-3, and the annexins have been implicated in membrane resealing, suggesting a large complex may act coordinately to seal the disrupted plasma membrane in a calcium-dependent manner.^13,14An increasing body of evidence suggests that dysferlin’s membrane-associated roles are not restricted to the sarcolemma. Dysferlin has been implicated in the development and maintenance of the transverse (T-) tubule, a muscle-specific membrane system essential for electromechanical coupling. The T-tubule is a membrane inversion of the sarcolemma that flanks the Z band of muscle, the anchor for sarcomeric proteins. Dysferlin associates with the T-tubule–like system in differentiated C2C12 myotubes,¹⁵ and dysferlin-null mouse muscle contains malformed T-tubules consistent with a role for dysferlin in the biogenesis and maintenance of the T-tubule system.¹⁶ In mature muscle damaged by stretch, dysferlin localizes to T-tubules, suggesting a reparative function for dysferlin at the T-tubule.¹⁷Dysferlin belongs to a family of proteins, the ferlins, that contains six family members. Myoferlin is a dysferlin homologue, which is 76% identical at the amino acid level.¹⁸ Such as dysferlin, myoferlin also contains at least six calcium-sensitive C2 domains, a carboxy-terminal transmembrane domain, an Fer domain, and a DysF domain.^17,19 Myoferlin is highly expressed in myoblasts and is markedly up-regulated in adult skeletal muscle on muscle damage.²⁰ Myoferlin, such as dysferlin, is required for normal myoblast fusion and muscle growth through regulating steps of vesicle trafficking and endocytic recycling.^1,20,21 Myoferlin, such as dysferlin, is required for the proper trafficking of and response to the insulin-like growth factor-1 receptor in muscle.²² Myoferlin interacts with endocytic recycling proteins EHD1 and EHD2, as well as AHNAK.^21,23,24 To date, no human forms of muscular dystrophy resulting from myoferlin mutations have been reported. However, mice lacking myoferlin show defects in muscle regeneration, establishing a role for myoferlin in muscle repair.²⁰We generated ferlin (FER) mice that carry both the dysferlin- and myoferlin-null loss of function mutations. We determined that FER mice have a more severe muscular dystrophy than dysferlin-null mice. In addition, FER muscle displays disorganization of the T-tubule system, dilated sarcoplasmic reticulum, and increased levels of serum glycerol. We revealed an enhanced sensitivity of Dysf, MKO, and especially FER myofibers to glycerol exposure, resulting in T-tubule vacuolation and disrupted membrane potential. Intramuscular glycerol injections into young FER muscle recapitulated the dystrophic phenotype characteristic of old FER muscle. Our data establish a role for both myoferlin and dysferlin in the biogenesis and remodeling of the sarcotubule system and suggest glycerol as a mediator of muscular dystrophy in dysferlin mutations.     

Calpain 3 is a modulator of the dysferlin protein complex in skeletal muscle

Muscular dystrophies comprise a genetically heterogeneous group of degenerative muscle disorders characterized by progressive muscle wasting and weakness. Two forms of limb-girdle muscular dystrophy, 2A and 2B, are caused by mutations in calpain 3 (CAPN3) and dysferlin (DYSF), respectively. While CAPN3 may be involved in sarcomere remodeling, DYSF is proposed to play a role in membrane repair. The coexistence of CAPN3 and AHNAK, a protein involved in subsarcolemmal cytoarchitecture and membrane repair, in the dysferlin protein complex and the presence of proteolytic cleavage fragments of AHNAK in skeletal muscle led us to investigate whether AHNAK can act as substrate for CAPN3. We here demonstrate that AHNAK is cleaved by CAPN3 and show that AHNAK is lost in cells expressing active CAPN3. Conversely, AHNAK accumulates when calpain 3 is defective in skeletal muscle of calpainopathy patients. Moreover, we demonstrate that AHNAK fragments cleaved by CAPN3 have lost their affinity for dysferlin. Thus, our findings suggest interconnectivity between both diseases by revealing a novel physiological role for CAPN3 in regulating the dysferlin protein complex.     

CD4+ cells,macrophages, MHC-I and C5b-9 involve the pathogenesis of dysferlinopathy

Objective: Dysferlin is a sarcolemmal protein that plays an important role in membrane repair by regulating vesicle fusion with the sarcolemma. Mutations in the dysferlin gene (DYSF) lead to multiple clinical phenotypes, including Miyoshi myopathy (MM), limb girdle muscular dystrophy type 2B (LGMD 2B), and distal myopathy with anterior tibial onset (DMAT). Patients with dysferlinopathy also show muscle inflammation, which often leads to a misdiagnosis as inflammatory myopathy. In this study, we examined and analyzed the dyferlinopathy-associated immunological features. Methods: Comparative immunohistochemical analysis of inflammatory cell infiltration, and muscle expression of MHC-I and C5b-9 was performed using muscle biopsy samples from 14 patients with dysferlinopathy, 7 patients with polymyositis, and 8 patients with either Duchenne muscular dystrophy or Becker muscular dystrophy (DMD/BMD). Results: Immunohistochemical analysis revealed positive staining for immune response-related CD4⁺ cells, macrophages, MHC-I and C5b-9 in dysferlinopathy, which is in a different mode of polymyositis and DMD/BMD. Conclusion: These results demonstrated the involvement of immune factors in the pathogenesis of dysferlinopathy.     

Novel sequence variants in dysferlin-deficient muscular dystrophy leading to mRNA decay and possible C2-domain misfolding

Mutations in the gene encoding dysferlin (DYSF) cause the allelic autosomal recessive disorders limb girdle muscular dystrophy 2B and Miyoshi myopathy. It encompasses 55 exons spanning 150 kb of genomic DNA. Dysferlin is involved in membrane repair in skeletal muscle. We identified three families with novel sequence variants in DYSF. All affected family members showed limb girdle weakness and had reduced or absent dysferlin protein on immunohistochemistry. All exons of DYSF were screened by genomic sequencing. Five novel variants in DYSF were found: two missense mutations (c.895G>A and c.4022T>C), one 5' donor splice-site variant (c.855+1delG), one nonsense mutation (c.1448C>A), and a variant in the 3'UTR of DYSF (c.*107T>A). All alterations were confirmed by restriction enzyme analysis and not found in 400 control alleles. Nonsense mediated RNA decay or changes in the three-dimensional protein structure resulting in intracellular dysferlin aggregates and finally the lack of dysferlin protein were identified as consequences of the novel DYSF variants.     

Heterogeneous characteristics of Korean patients with dysferlinopathy

Dysferlinopathy is caused by mutations in the DYSF gene. To characterize the clinical spectrum, we investigated the characteristics of 31 Korean dysferlinopathy patients confirmed by immunohistochemistry. The mean age of symptom onset was 22.23 ± 7.34 yr. The serum creatine kinase (CK) was highly increased (4- to 101-fold above normal). The pathological findings of muscle specimens showed nonspecific dystrophic features and frequent inflammatory cell infiltration. Muscle imaging studies showed fatty atrophic changes dominantly in the posterolateral muscles of the lower limb. The patients with dysferlinopathy were classified by initial muscle weakness: fifteen patients with Miyoshi myopathy phenotype (MM), thirteen patients with limb girdle muscular dystrophy 2B phenotype (LGMD2B), two patients with proximodistal phenotype, and one asymptomatic patient. There were no differences between LGMD2B and MM groups in terms of onset age, serum CK levels and pathological findings. Dysferlinopathy patients usually have young adult onset and high serum CK levels. However, heterogeneity of clinical presentations and pathologic findings upon routine staining makes it difficult to diagnose dysferlinopathy. These limitations make immunohistochemistry currently the most important method for the diagnosis of dysferlinopathy.     

Transgenic overexpression of dystroglycan does not inhibit muscular dystrophy in mdx mice

Recently, there have been a number of studies demonstrating that overexpression of molecules in skeletal muscle can inhibit or ameliorate aspects of muscular dystrophy in the mdx mouse, a model for Duchenne muscular dystrophy. Several such studies involve molecules that increase the expression of dystroglycan, an important component of the dystrophin-glycoprotein complex. To test whether dystroglycan itself inhibits muscular dystrophy in mdx mice, we created dystroglycan transgenic mdx mice (DG/mdx). The alpha and beta chains of dystroglycan were highly overexpressed along the sarcolemmal membrane in most DG/mdx muscles. Increased dystroglycan expression, however, did not correlate with increased expression of utrophin or sarcoglycans, but rather caused their decreased expression. In addition, the percentage of centrally located myofiber nuclei and the level of serum creatine kinase activity were not decreased in DG/mdx mice relative to mdx animals. Therefore, dystroglycan overexpression does not cause the concomitant overexpression of a utrophin-glycoprotein complex in mdx muscles and has no effect on the development of muscle pathology associated with muscular dystrophy.     

Dysferlin Deficiency and the Development of Cardiomyopathy in a Mouse Model of Limb-Girdle Muscular Dystrophy 2B

Limb-girdle muscular dystrophy 2B, Miyoshi myopathy, and distal myopathy of anterior tibialis are severely debilitating muscular dystrophies caused by genetically determined dysferlin deficiency. In these muscular dystrophies, it is the repair, not the structure, of the plasma membrane that is impaired. Though much is known about the effects of dysferlin deficiency in skeletal muscle, little is known about the role of dysferlin in maintenance of cardiomyocytes. Recent evidence suggests that dysferlin deficiency affects cardiac muscle, leading to cardiomyopathy when stressed. However, neither the morphological location of dysferlin in the cardiomyocyte nor the progression of the disease with age are known. In this study, we examined a mouse model of dysferlinopathy using light and electron microscopy as well as echocardiography and conscious electrocardiography. We determined that dysferlin is normally localized to the intercalated disk and sarcoplasm of the cardiomyocytes. In the absence of dysferlin, cardiomyocyte membrane damage occurs and is localized to the intercalated disk and sarcoplasm. This damage results in transient functional deficits at 10 months of age, but, unlike in skeletal muscle, the cell injury is sublethal and causes only mild cardiomyopathy even at advanced ages.Plasma membrane damage in mechanically active cells such as the myocyte is inevitable even under normal physiological conditions.^1,2 Since membranes are not self-sealing, effective and efficient repair mechanisms are necessary to maintain cell viability. Dysferlin plays a central role in this active repair mechanism in skeletal muscle. In the absence of dysferlin disruptions of the skeletal muscle plasma membrane are not repaired leading to cell death.³ Skeletal muscle can regenerate new cells from satellite cells but eventually even this response is exhausted, and lost myocytes are replaced by fat and fibrosis resulting in debilitating muscular dystrophy.Limb-girdle muscular dystrophy type 2 B (LGMD2B), Miyoshi myopathy, and distal myopathy of anterior tibialis are three clinically distinct forms of muscular dystrophy that are caused by mutations within the dysferlin (DYSF) gene resulting in severe to complete deficiency of dysferlin expression.^4,5 Clinically, these dysferlinopathies start in young adulthood with progressive muscle weakness and atrophy that advances to severe disability in older adulthood.Dysferlin is a 273 kDa membrane-spanning protein with multiple C2 domains that bind calcium, phospholipids, and proteins to then trigger signaling events, vesicle trafficking, and membrane fusion.^6,7 The name “dysferlin” reflects the homology with FER-1, the Caenorhabditis elegans spermatogenesis factor involved in the fusion of vesicles with the plasma membrane, as well as the dystrophic phenotype associated with its deficiency.⁵ Dysferlin is crucial to calcium dependent membrane repair in muscle cells.^3,8 In normal skeletal muscle, sarcolemma injuries lead to the accumulation of dysferlin-enriched membrane patches and resealing of the membrane in the presence of Ca²⁺.^3,9While the profound effect of dysferlin deficiency in skeletal muscle has been the subject of much investigation, the effect of dysferlin deficiency in cardiac muscle has largely been ignored. However, in 2004, Kuru et al¹⁰ reported on a 57-year-old Japanese woman with LGMD2B and dilated cardiomyopathy; more recently, Wenzel et al¹¹ described dilated cardiomyopathy in two out of seven patients with LGMD2B and other cardiac abnormalities in three of the others. These observations suggest that dysferlin deficiency can lead to cardiomyopathy as well as to muscular dystrophy. However, neither the morphological location of dysferlin in the cardiomyocyte nor the progression of the disease with age are known.Spontaneous mutations in the mouse are valuable resources in understanding human disease processes. Genetically defined mice develop dysferlinopathies closely resembling LGMD2B, Miyoshi myopathy, and distal myopathy of anterior tibialis.¹² In 2004, Ho et al¹² identified A/J mice as dysferlin deficient. A retrotransposon insertion in the dysferlin gene was found to result in a null allele, resulting in skeletal muscle dystrophy that shows histopathological and ultrastructural features that closely resemble the human dysferlinopathies of LGMD2B, Miyoshi myopathy, and distal myopathy of anterior tibialis.¹² The onset of dystrophic features in A/J mice begins in proximal limb muscles at 4 to 5 months of age and progresses to severe debilitating muscular dystrophy over several months. Ho et al¹² also found that human and murine dysferlin share very similar (approximately 90% identity) amino acid sequences. Cardiac muscle was not included in their study.Recently, Han et al,⁸ using sucrose gradient membrane fractionation on homogenates of wild-type C57BL/6J mouse heart muscle, showed that dysferlin is present in the cardiomyocyte plasma membrane and intracellular vesicle fractions. It was proposed that dysferlin is localized to the cardiomyocyte sarcolemma and some unidentified type of vesicles.⁸ Han et al⁸ in one study and Wenzel et al¹¹ in another study showed that the induction of significant cardiac stress lead to cardiac dysfunction in dysferlin-deficient mice, but to what extent dysferlin deficiency causes cardiomyopathy by aging alone in patients clinically affected with the debilitating effects of LGMD2B, Miyoshi myopathy, or distal myopathy of anterior tibialis is unknown.In this study, we used the A/J mouse model to study the effects of aging in mice affected by genetically determined dysferlin deficiency by using echocardiography and conscious electrocardiography to determine functional changes in vivo, followed postmortem by light and electron microscopy to determine associated morphological changes. We have determined that the normal primary location for dysferlin in the cardiomyocyte of control A/HeJ mice is the intercalated disk (ID), and to a lesser extent, to a distinctive transverse banding pattern within the sarcoplasm of the cardiomyocyte. We have also determined that in the dysferlin-deficient cardiomyocyte there is evidence of membrane damage at these locations. We also present data that show functional cardiac deficits were present in vivo at around 10 months of age then recovered by 12 months. Histopathology showed that under normal laboratory conditions dysferlin deficiency causes only a mild cardiomyopathy even at advanced ages, suggesting the possibility of dysferlin-independent membrane repair mechanisms in cardiac muscle that do not exist in skeletal muscle.     

Ahnak1 abnormally localizes in muscular dystrophies and contributes to muscle vesicle release

Ahnak1 is a giant, ubiquitously expressed, plasma membrane support protein whose function in skeletal muscle is largely unknown. Therefore, we investigated whether ahnak would be influenced by alterations of the sarcolemma exemplified by dysferlin mutations known to render the sarcolemma vulnerable or by mutations in calpain3, a protease known to cleave ahnak. Human muscle biopsy specimens obtained from patients with limb girdle muscular dystrophy (LGMD) caused by mutations in dysferlin (LGMD2B) and calpain3 (LGMD2A) were investigated for ahnak expression and localization. We found that ahnak1 has lost its sarcolemmal localization in LGMD2B but not in LGMD2A. Instead ahnak1 appeared in muscle connective tissue surrounding the extracellular site of the muscle fiber in both muscular dystrophies. The entire giant ahnak1 molecule was present outside the muscle fiber and did only partially colocalize with CD45-positive immune cell infiltration and the extracelluar matrix proteins fibronectin and collagenVI. Further, vesicles shedded in response to Ca(2+) by primary human myotubes were purified and their protein content was analysed. Ahnak1 was prominently present in these vesicles. Electron microscopy revealed a homogenous population of vesicles with a diameter of about 150?nm. This is the first study demonstrating vesicle release from human myotubes that may be one mechanism underlying abnormally localized ahnak1. Taken together, our results define ahnak1 in muscle connective tissue as a novel feature of two genetically distinct muscular dystrophies that might contribute to disease pathology.     

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.     

Dysferlin缺陷:肢带2B型肌营养不良与Miyoshi肌病的致病原因

目的　对临床怀疑为常染色体隐性遗传性肌营养不良一家系进行分析 ,以明确肌病类型并寻找其致病基因的分子缺陷。方法　用与 8种常染色体隐性遗传性肌营养不良基因连锁的短串联重复序列标记进行连锁分析 ,用与 5种肌营养不良相关的单克隆抗体作多重免疫印迹分析检测相应致病基因的编码产物 ;通过逆转录 - PCR扩增先证者致病基因的编码序列并测序 ,确定基因突变。结果　家系连锁分析显示在 DYSF基因附近的 D2 S337位点的优势对数记分值为 1.85 ,提示致病基因与 D2 S337连锁 ;免疫印迹分析提示患者DYSF基因的编码产物 dysferlin缺陷 ;测序证明先证者DYSF基因的c DNA第 6 4 2 9位发生纯合性del G突变。结论　综合研究结果和临床资料 ,这一家系中的先证者被诊断为 Miyoshi肌病 ,由DYSF基因纯合性缺失突变所导致。     

Clinical and pathological characteristics of four Korean patients with limb-girdle muscular dystrophy type 2B

Limb-girdle muscular dystrophy type 2B (LGMD2B), a subtype of autosomal recessive limb-girdle muscular dystrophy (ARLGMD), is characterized by a relatively late onset and slow progressive course. LGMD2B is known to be caused by the loss of the dysferlin protein at sarcolemma in muscle fibers. In this study, the clinical and pathological characteristics of Korean LGMD2B patients were investigated. Seventeen patients with ARLGMD underwent muscle biopsy and the histochemical examination was performed. For the immunocytochemistry, a set of antibodies against dystrophin, alpha, beta, gamma, delta-sarcoglycans, dysferlin, caveolin-3, and beta-dystroglycan was used. Four patients (24%) showed selective loss of immunoreactivity against dysferlin at the sarcolemma on the muscle specimens. Therefore, they were classified into the LGMD2B category. The age at the onset of disease ranged from 9 yr to 33 yr, and none of the patients was wheelchair bound at the neurological examination. The serum creatine kinase (CK) was high in all the patients (4010-5310 IU/L). The pathologic examination showed mild to moderate dystrophic features. These are the first Korean LGMD2B cases with a dysferlin deficiency confirmed by immunocytochemistry. The clinical, pathological, and immunocytochemical findings of the patients with LGMD2B in this study were in accordance with those of other previous reports.     

Shifts in macrophage phenotypes and macrophage competition for arginine metabolism affect the severity of muscle pathology in muscular dystrophy

Duchenne muscular dystrophy (DMD) is the most common, lethal, muscle-wasting disease of childhood. Previous investigations have shown that muscle macrophages may play an important role in promoting the pathology in the mdx mouse model of DMD. In the present study, we investigate the mechanism through which macrophages promote mdx dystrophy and assess whether the phenotype of the macrophages changes between the stage of peak muscle necrosis (4 weeks of age) and muscle regeneration (12 weeks). We find that 4-week-old mdx muscles contain a population of pro-inflammatory, classically activated M1 macrophages that lyse muscle in vitro by NO-mediated mechanisms. Genetic ablation of the iNOS gene in mdx mice also significantly reduces muscle membrane lysis in 4-week-old mdx mice in vivo. However, 4-week mdx muscles also contain a population of alternatively activated, M2a macrophages that express arginase. In vitro assays show that M2a macrophages reduce lysis of muscle cells by M1 macrophages through the competition of arginase in M2a cells with iNOS in M1 cells for their common, enzymatic substrate, arginine. During the transition from the acute peak of mdx pathology to the regenerative stage, expression of IL-4 and IL-10 increases, either of which can deactivate the M1 phenotype and promote activation of a CD163+, M2c phenotype that can increase tissue repair. Our findings further show that IL-10 stimulation of macrophages activates their ability to promote satellite cell proliferation. Deactivation of the M1 phenotype is also associated with a reduced expression of iNOS, IL-6, MCP-1 and IP-10. Thus, these results show that distinct subpopulations of macrophages can promote muscle injury or repair in muscular dystrophy, and that therapeutic interventions that affect the balance between M1 and M2 macrophage populations may influence the course of muscular dystrophy.     

Skeletal,cardiac and tongue muscle pathology,defective retinal transmission,and neuronal migration defects in the Large(myd) mouse defines a natural model for glycosylation-deficient muscle - eye - brain disorders

We have recently shown that a deletion in the Large gene, encoding a putative glycosyltransferase, is the molecular defect underlying the myodystrophy (previously myd; now Large(myd)) mouse. Here we show that the muscular dystrophy phenotype is not confined to skeletal muscle, but is also present in the heart and tongue. Immunohistochemistry indicates disruption of the dystrophin-associated glycoprotein complex (DGC) in skeletal and cardiac muscle. Quantitative western blotting shows a general increase in the expression of DGC proteins and of dysferlin and caveolin-3 in mutant skeletal muscle. In contrast, the expression of DGC proteins is reduced in cardiac muscle. Overlay assays show loss of laminin binding by alpha-dystroglycan in Large(myd) skeletal and cardiac muscle and in brain. We also show that the phenotype of Large(myd) mice is not restricted to muscular dystrophy, but also includes ophthalmic and central nervous system (CNS) defects. Electroretinograms of homozygous mutant mice show gross abnormalities of b-wave characteristics, indicative of a complex defect in retinal transmission. The laminar architecture of the cortices of the cerebrum and the cerebellum is disturbed, indicating defective neuronal migration. Thus, the phenotype of the Large(myd) mouse shows similarities to the heterogeneous group of human muscle eye brain diseases characterized by severe congenital muscular dystrophy, eye abnormalities and CNS neuronal migration defects. These diseases include Fukuyama-type muscular dystrophy and muscle-eye-brain disease, both of which are also due to mutations in predicted glycosylation enzymes. Therefore, the Large(myd) mouse represents an important animal model for studying the function of glycosylation in muscle, brain and retina.     

Dysferlin deficiency shows compensatory induction of Rab27A/Slp2a that may contribute to inflammatory onset

Mutations in the dysferlin gene cause limb girdle muscular dystrophy 2B (LGMD2B) and Miyoshi myopathy. Dysferlin-deficient cells show abnormalities in vesicular traffic and membrane repair although onset of symptoms is not commonly seen until the late teenage years and is often associated with subacute onset and marked muscle inflammation. To identify molecular networks specific to dysferlin-deficient muscle that might explain disease pathogenesis, muscle mRNA profiles from 10 mutation-positive LGMD2B/MM patients were compared with a disease control [LGMD2I; (n = 9)], and normal muscle samples (n = 11). Query of inflammatory pathways suggested LGMD2B-specific increases in co-stimulatory signaling between dendritic cells and T cells (CD86, CD28, and CTLA4), associated with localized expression of both versican and tenascin. LGMD2B muscle also showed an increase in vesicular trafficking pathway proteins not normally observed in muscle (synaptotagmin-like protein Slp2a/SYTL2 and the small GTPase Rab27A). We propose that Rab27A/Slp2a expression in LGMD2B muscle provides a compensatory vesicular trafficking pathway that is able to repair membrane damage in the absence of dysferlin. However, this same pathway may release endocytotic vesicle contents, resulting in an inflammatory microenvironment. As dysferlin deficiency has been shown to enhance phagocytosis by macrophages, together with our findings of abnormal myofiber endocytosis pathways and dendritic-T cell activation markers, these results suggest a model of immune and inflammatory network over-stimulation that may explain the subacute inflammatory presentation.     

Mutation finding in patients with dysferlin deficiency and role of the dysferlin interacting proteins annexin A1 and A2 in muscular dystrophies

Mutations in the DYSF gene underlie two main muscle diseases: Limb Girdle Muscular Dystrophy (LGMD) 2B and Miyoshi myopathy (MM). Dysferlin is involved in muscle membrane-repair and is thought to interact with other dysferlin molecules and annexins A1 and A2 at the sarcolemma. We performed genotype/phenotype correlations in a large cohort of dysferlinopathic patients and explored the possible role of annexins as modifier factors in LGMD-2B and MM. In particular, clinical examination, expression of sarcolemmal proteins and genetic analysis were performed on 27 dysferlinopathic subjects. Expression of A1 and A2 annexins was investigated in LGMD-2B/MM subjects and in patients with other muscle disorders. We identified 24 different DYSF mutations, 10 of them being novel. We observed no clear correlation between mutation type and clinical phenotype, but MM patients were found to display muscle symptoms significantly earlier in life than LGMD subjects. Remarkably, dysferlinopathic patients and subjects suffering from other muscular disorders expressed higher levels of both annexins compared to controls; a significant correlation was observed between annexin expression levels and clinical severity scores. Also, annexin amounts paralleled the degree of muscle histopathologic changes. In conclusion, our data indicate that the pathogenesis of different inherited and acquired muscle disorders involves annexin overexpression, probably because these proteins actively participate in the plasmalemma repair process. The positive correlation between annexin A1 and A2 and clinical severity, as well as muscle histopathology, suggests that their level may be a prognostic indicator of disease.