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.     

Myoferlin, a candidate gene and potential modifier of muscular dystrophy

Dysferlin, the gene product of the limb girdle muscular dystrophy (LGMD) 2B locus, encodes a membrane-associated protein with homology to Caenorhabditis elegans fer-1. Humans with mutations in dysferlin ( DYSF ) develop muscle weakness that affects both proximal and distal muscles. Strikingly, the phenotype in LGMD 2B patients is highly variable, but the type of mutation in DYSF cannot explain this phenotypic variability. Through electronic database searching, we identified a protein highly homologous to dysferlin that we have named myoferlin. Myoferlin mRNA was highly expressed in cardiac muscle and to a lesser degree in skeletal muscle. However, antibodies raised to myoferlin showed abundant expression of myoferlin in both cardiac and skeletal muscle. Within the cell, myoferlin was associated with the plasma membrane but, unlike dysferlin, myoferlin was also associated with the nuclear membrane. Ferlin family members contain C2 domains, and these domains play a role in calcium-mediated membrane fusion events. To investigate this, we studied the expression of myoferlin in the mdx mouse, which lacks dystrophin and whose muscles undergo repeated rounds of degeneration and regeneration. We found upregulation of myoferlin at the membrane in mdx skeletal muscle. Thus, myoferlin ( MYOF ) is a candidate gene for muscular dystrophy and cardiomyopathy, or possibly a modifier of the muscular dystrophy phenotype.     

Muscle‐specific calpain‐3 is phosphorylated in its unique insertion region for enrichment in a myofibril fraction

CAPN3 (also called p94/calpain‐3) is a skeletal muscle‐specific calpain, an intracellular cysteine protease. Loss of CAPN3 protease activity and/or structural functions cause limb‐girdle muscular dystrophy type 2A (LGMD2A). However, the precise mechanism of action of CAPN3 in skeletal muscles in vivo remains largely elusive. By studying the protein modifications that regulate CAPN3 activity, we found that CAPN3 was phosphorylated. By performing mutagenesis and mass spectrometry analyses, we identified two Ser residues at positions 629 and 636 in human CAPN3 that are phosphorylated and showed that S629 is a major phosphorylation site. Intriguingly, rapid and exhaustive autolysis of CAPN3 was slightly attenuated by the substitution of S629. In skeletal muscles, phosphorylated CAPN3 was enriched in the myofibril fraction. These results imply that phosphorylated CAPN3 is a myofibril structural component and/or participates in myofibril‐based signaling pathways, rather than functions as a protease. We evaluated the relationship between phosphorylated CAPN3 and the pathology of LGMD2A. The level of phosphorylated CAPN3 was greatly reduced in LGMD2A muscles. Our findings suggest that phosphorylated CAPN3 is involved in the pathology of LGMD2A through defects in myofibril integrity and/or signaling pathways. This is the first report that phosphorylation of CAPN3 may be involved in its physiological function.     

Protein studies in dysferlinopathy patients using llama-derived antibody fragments selected by phage display

Mutations in dysferlin, a member of the fer1-like protein family that plays a role in membrane integrity and repair, can give rise to a spectrum of neuromuscular disorders with phenotypic variability including limb-girdle muscular dystrophy 2B, Myoshi myopathy and distal anterior compartment myopathy. To improve the tools available for understanding the pathogenesis of the dysferlinopathies, we have established a large source of highly specific antibody reagents against dysferlin by selection of heavy-chain antibody fragments originating from a nonimmune llama-derived phage-display library. By utilizing different truncated forms of recombinant dysferlin for selection and diverse selection methodologies, antibody fragments with specificity for two different dysferlin domains could be identified. The selected llama antibody fragments are functional in Western blotting, immunofluorescence microscopy and immunoprecipitation applications. Using these antibody fragments, we found that calpain 3, which shows a secondary reduction in the dysferlinopathies, interacts with dysferlin.     

Aberrant dysferlin trafficking in cells lacking caveolin or expressing dystrophy mutants of caveolin-3

Mutations in the dysferlin (DYSF) and caveolin-3 (CAV3) genes are associated with muscle disease. Dysferlin is mislocalized, by an unknown mechanism, in muscle from patients with mutations in caveolin-3 (Cav-3). To examine the link between Cav-3 mutations and dysferlin mistargeting, we studied their localization at high resolution in muscle fibers, in a model muscle cell line, and upon heterologous expression of dysferlin in muscle cell lines and in wild-type or caveolin-null fibroblasts. Dysferlin shows only partial overlap with Cav-3 on the surface of isolated muscle fibers but co-localizes with Cav-3 in developing transverse (T)-tubules in muscle cell lines. Heterologously expressed dystrophy-associated mutant Cav3R26Q accumulates in the Golgi complex of muscle cell lines or fibroblasts. Cav3R26Q and other Golgi-associated mutants of both Cav-3 (Cav3P104L) and Cav-1 (Cav1P132L) caused a dramatic redistribution of dysferlin to the Golgi complex. Heterologously expressed epitope-tagged dysferlin associates with the plasma membrane in primary fibroblasts and muscle cells. Transport to the cell surface is impaired in the absence of Cav-1 or Cav-3 showing that caveolins are essential for dysferlin association with the PM. These results suggest a functional role for caveolins in a novel post-Golgi trafficking pathway followed by dysferlin.     

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.     

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.     

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.     

人CAPN3基因在骨骼肌和白细胞中存在不同的剪切

目的 比较CAPN3基因在外周血白细胞和骨骼肌组织中的剪切变异,探讨用外周血白细胞CAPN3 mRNA进行基因诊断的可行性.方法 抽提正常人外周血和骨骼肌组织中总RNA,通过逆转录-聚合酶链反应和DNA测序确定CAPN3基因的cDNA序列,比较外周血白细胞和骨骼肌组织中CAPN3cDNA序列.结果 由骨骼肌组织抽提的RNA进行逆转录合成cDNA为CAPN3基因全长cDNA,包含全部24个外显子;而由外周血白细胞抽提的RNA进行逆转录合成cDNA为CAPN3基因非全长cDNA,包含23个外显子,缺失了第15外显子.结论 人的CAPN3基因在骨骼肌和外周血自细胞中存在着不同的剪切方式,若用外周血抽提RNA进行CAPN3基因的编码序列分析时会漏检第15外显子的突变.这提示从cDNA水平分析CAPN3基因突变时应采用患者肌肉组织,而非外周血白细胞.     

Dysferlin mutations in LGMD2B, Miyoshi myopathy, and atypical dysferlinopathies

DYSF encoding dysferlin is mutated in Miyoshi myopathy and Limb-Girdle Muscular Dystrophy type 2B, the two main phenotypes recognized in dysferlinopathies. Dysferlin deficiency in muscle is the most relevant feature for the diagnosis of dysferlinopathy and prompts the search for mutations in DYSF. DYSF, located on chromosome 2p13, contains 55 coding exons and spans 150 kb of genomic DNA. We performed a genomic analysis of the DYSF coding sequence in 34 unrelated patients from various ethnic origins. All patients showed an absence or drastic decrease of dysferlin expression in muscle. A primary screening of DYSF using SSCP or dHPLC of PCR products of each of 55 exons of the gene was followed by sequencing whenever a sequence variation was detected. All together, 54 sequence variations were identified in DYSF, 50 of which predicting either a truncated protein or one amino-acid substitution and most of them (34 out of 54) being novel. In 23 patients, we identified two pathogenic mutations, while only one was identified in 11 patients. These mutations were widely spread in the coding sequence of the gene without any mutational "hotspot."     

UMD-DYSF, a novel locus specific database for the compilation and interactive analysis of mutations in the dysferlin gene

Mutations in the dysferlin gene (DYSF) lead to a complete or partial absence of the dysferlin protein in skeletal muscles and are at the origin of dysferlinopathies, a heterogeneous group of rare autosomal recessive inherited neuromuscular disorders. As a step towards a better understanding of the DYSF mutational spectrum, and towards possible inclusion of patients in future therapeutic clinical trials, we set up the Universal Mutation Database for Dysferlin (UMD‐DYSF), a Locus‐Specific Database developed with the UMD® software. The main objective of UMD‐DYSF is to provide an updated compilation of mutational data and relevant interactive tools for the analysis of DYSF sequence variants, for diagnostic and research purposes. In particular, specific algorithms can facilitate the interpretation of newly identified intronic, missense‐ or isosemantic‐exonic sequence variants, a problem encountered recurrently during genetic diagnosis in dysferlinopathies. UMD‐DYSF v1.0 is freely accessible at www.umd.be/DYSF/. It contains a total of 742 mutational entries corresponding to 266 different disease‐causing mutations identified in 558 patients worldwide diagnosed with dysferlinopathy. This article presents for the first time a comprehensive analysis of the dysferlin mutational spectrum based on all compiled DYSF disease‐causing mutations reported in the literature to date, and using the main bioinformatics tools offered in UMD‐DYSF. ©2011 Wiley‐Liss, Inc. Hum Mutat 33:E2317–E2331, 2012. © 2012 Wiley Periodicals, Inc.     

Impaired calcium calmodulin kinase signaling and muscle adaptation response in the absence of calpain 3

Mutations in the non-lysosomal, cysteine protease calpain 3 (CAPN3) result in the disease limb girdle muscular dystrophy type 2A (LGMD2A). CAPN3 is localized to several subcellular compartments, including triads, where it plays a structural, rather than a proteolytic, role. In the absence of CAPN3, several triad components are reduced, including the major Ca(2+) release channel, ryanodine receptor (RyR). Furthermore, Ca(2+) release upon excitation is impaired in the absence of CAPN3. In the present study, we show that Ca-calmodulin protein kinase II (CaMKII) signaling is compromised in CAPN3 knockout (C3KO) mice. The CaMK pathway has been previously implicated in promoting the slow skeletal muscle phenotype. As expected, the decrease in CaMKII signaling that was observed in the absence of CAPN3 is associated with a reduction in the slow versus fast muscle fiber phenotype. We show that muscles of WT mice subjected to exercise training activate the CaMKII signaling pathway and increase expression of the slow form of myosin; however, muscles of C3KO mice do not exhibit these adaptive changes to exercise. These data strongly suggest that skeletal muscle's adaptive response to functional demand is compromised in the absence of CAPN3. In agreement with our mouse studies, RyR levels were also decreased in biopsies from LGMD2A patients. Moreover, we observed a preferential pathological involvement of slow fibers in LGMD2A biopsies. Thus, impaired CaMKII signaling and, as a result, a weakened muscle adaptation response identify a novel mechanism that may underlie LGMD2A and suggest a pharmacological target that should be explored for therapy.     

Genetic Manipulation of Dysferlin Expression in Skeletal Muscle : Novel Insights into Muscular Dystrophy

Mutations in the gene DYSF, which codes for the protein dysferlin, underlie Miyoshi myopathy and limb-girdle muscular dystrophy 2B in humans and produce a slowly progressing skeletal muscle degenerative disease in mice. Dysferlin is a Ca²⁺-sensing, regulatory protein that is involved in membrane repair after injury. To assess the function of dysferlin in healthy and dystrophic skeletal muscle, we generated skeletal muscle–specific transgenic mice with threefold overexpression of this protein. These mice were phenotypically indistinguishable from wild-type, and more importantly, the transgene completely rescued the muscular dystrophy (MD) disease in Dysf-null A/J mice. The dysferlin transgene rescued all histopathology and macrophage infiltration in skeletal muscle of Dysf^−/− A/J mice, as well as promoted the rapid recovery of muscle function after forced lengthening contractions. These results indicate that MD in A/J mice is autonomous to skeletal muscle and not initiated by any other cell type. However, overexpression of dysferlin did not improve dystrophic symptoms or membrane instability in the dystrophin-glycoprotein complex–lacking Scgd (δ-sarcoglycan) null mouse, indicating that dysferlin functionality is not a limiting factor underlying membrane repair in other models of MD. In summary, the restoration of dysferlin in skeletal muscle fibers is sufficient to rescue the MD in Dysf-deficient mice, although its mild overexpression does not appear to functionally enhance membrane repair in other models of MD.The muscular dystrophies (MD) are a diverse group of genetic muscle wasting disorders that typically result in premature death due to cardiac or respiratory failure.¹ Most characterized mutations in humans that cause MD result from alterations in structural proteins that connect the underlying contractile proteins to the basal lamina, providing rigidity to the skeletal muscle cell membrane, or in proteins that directly stabilize or repair the cell membrane.^1,2 For example, loss of dystrophin in Duchenne’s MD or mutations in other components of the dystrophin-glycoprotein complex (DGC) leads to a fundamental alteration in the physical properties of the sarcolemma, permitting contraction-induced microtears and the unregulated exchange of ions such as Ca²⁺, leading to necrosis and degeneration of myofibers.^1,2 The DGC is a multisubunit complex organized at the sarcolemma that links the underlying contractile proteins to the extracellular matrix, providing the sarcolemma with cytoskeletal support and protecting it from damage incurred during the contractile cycle. The DGC contains structural proteins such as dystrophin, dystroglycans, sarcoglycans, dystrobrevin, sarcospan, and syntrophins, as well as a number of signaling proteins.^1,2 That membrane instability and aberrant repair capacity underlie myofiber degeneration in MD was further suggested by the observation that mutations in the putative membrane repair protein dysferlin cause limb girdle muscular dystrophy 2B and Miyoshi myopathy.³ Limb girdle muscular dystrophy 2B/Miyoshi myopathy typically presents in early adulthood or the late teen years, and muscle biopsies from these patients show a striking inflammatory response.³Dysferlin is a 230 kDa, Ca²⁺ sensitive protein that participates in membrane resealing events following injury, but does not directly interact with components of the DGC. Mice lacking dysferlin (Dysf) exhibit progressive disease in skeletal muscle and cardiac tissue despite having a functional DGC, which is characterized by myofiber necrosis, cycles of degeneration and regeneration, inflammation, and adipocyte replacement.^4,5 Lack of dysferlin also results in the accumulation of vesicles and structural membrane defects as analyzed by electron microscopy, suggesting a role in normal membrane turnover and recycling.^4,6Disease in dystrophic skeletal muscle is dramatically influenced by the inflammatory response, achieved mainly by infiltration of cytotoxic T-lymphocytes and macrophages.^7,8 It has been suggested that Dysf-null inflammatory cells may initiate the disease process in the muscles of Dysf-null mice and humans, as these immune cells appear to be hyperactive and abnormal.^9,10,11 For example, macrophages and dendritic-T cell activation markers are elevated in the SJL mouse model for Dysf deficiency and in human limb girdle muscular dystrophy 2B.⁹ Thus, it is unclear whether disease due to dysferlin deficiency is primarily due to an autonomous effect in immune cells or skeletal muscle fibers. To address this issue, we generated transgenic mice that express dysferlin specifically in skeletal muscle and used them to evaluate the necessity of dysferlin within myofibers to initiate muscle disease in Dysf null A/J mice. Moreover, we assessed the ability of increased dysferlin expression to alleviate pathology in a MD model that lacks a component of the DGC, Scgd (δ-sarcoglycan).     

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.     

The calpain system and diabetes

Diabetes mellitus is recognized as a clinical syndrome that is characterized by hyperglycemia due to deficiency of insulin. The global prevalence of diabetes has been estimated to increase from 4% (1995) to 5.4% by the year 2025. Insulin dependent diabetes mellitus (IDDM/Type-1) in human, generating hyperglycemia due to insulin deficiency as a consequence of destructing beta cells in the pancreatic islets. Non-insulin dependent diabetes mellitus (NIDDM/Type-II), is a multifactorial, exact biochemical and genetic defect which has not yet been elucidated completely. Calpains seem to play a role in NIDDM and IDDM. Positional cloning experiments revealed that there is a NIDDM susceptibility to calpain 10 (CAPN10). Increased calpain activity and leukocyte trafficking were noticed in the microcirculation in ZDF (Zuker diabetic fatty) rats. Exercise and low body weight play a significant role in reducing calpains expression or elevating the calpains degradation in the skeletal muscle of NIDDM rats. Numerous investigations have been reported that non-coding polymorphisms in CAPN10 proteins might be involved in the NIDDM. Calpain and its mRNA presence had been reported in tissues from many mammalian species. CAPN10 and other calpains seem to be linked to glucose metabolism, insulin secretion and action pathways. This review will give an overview of the role of calpain in NIDDM and IDDM.     

Novel role of calpain-3 in the triad-associated protein complex regulating calcium release in skeletal muscle

Calpain-3 (CAPN3) is a non-lysosomal cysteine protease that is necessary for normal muscle function, as mutations in CAPN3 result in an autosomal recessive form of limb girdle muscular dystrophy type 2A. To elucidate the biological roles of CAPN3 in skeletal muscle, we performed a search for potential substrates and interacting partners. By yeast-two-hybrid analysis we identified the glycolytic enzyme aldolase A (AldoA) as a binding partner of CAPN3. In co-expression studies CAPN3 degraded AldoA; however, no accumulation of AldoA was observed in total extracts from CAPN3-deficient muscles suggesting that AldoA is not an in vivo substrate of CAPN3. Instead, we found CAPN3 to be necessary for recruitment of AldoA to one specific location, namely the triads, which are structural components of muscle responsible for calcium transport and excitation-contraction coupling. Both aldolase and CAPN3 are present in the triad-enriched fraction and are able to interact with ryanodine receptors (RyR) that form major calcium release channels. Levels of triad-associated AldoA and RyR were decreased in CAPN3-deficient muscles compared with wild-type. Consistent with these observations we found calcium release to be significantly reduced in fibers from CAPN3-deficient muscles. Together, these data suggest that CAPN3 is necessary for the structural integrity of the triad-associated protein complex and that impairment of calcium transport is a phenotypic feature of CAPN3-deficient muscle.     

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.     

A single c.1715G>C calpain 3 gene variant causes dominant calpainopathy with loss of calpain 3 expression and activity

Recessively inherited limb girdle muscular dystrophy (LGMD) type 2A is the most common LGMD worldwide. Here, we report the first single missense variant in CAPN3 causing dominantly inherited calpainopathy. A 43‐year‐old proband, his father and two sons were heterozygous for a c.1715G>C p.(Arg572Pro) variant in CAPN3. Affected family members had at least three of the following; muscle pain, a LGMD2A pattern of muscle weakness and wasting, muscle fat replacement on magnetic resonance imaging, myopathic muscle biopsy, and elevated creatine kinase. Total calpain 3 protein expression was 4 ± 3% of normal. In vitro analysis of c.1715G>C and the previously described c.643_663del variant indicated that the mutant proteins lack autolytic and proteolytic activity and decrease the quantity of wild‐type CAPN3 protein. Our findings suggest that dominantly inherited calpainopathy is not unique to the previously reported c.643_663del mutation of CAPN3, and that dominantly inherited calpainopathy should be considered for other single variations in CAPN3.