New insights into the protein aggregation pathology in myotilinopathy by combined proteomic and immunolocalization analyses.

INTRODUCTION: Myofibrillar myopathies are characterized by progressive muscle weakness and impressive abnormal protein aggregation in muscle fibers. In about 10 % of patients, the disease is caused by mutations in the MYOT gene encoding myotilin. The aim of our study was to decipher the composition of protein deposits in myotilinopathy to get new information about aggregate pathology. RESULTS: Skeletal muscle samples from 15 myotilinopathy patients were included in the study. Aggregate and control samples were collected from muscle sections by laser microdissection and subsequently analyzed by a highly sensitive proteomic approach that enables a relative protein quantification. In total 1002 different proteins were detected. Seventy-six proteins showed a significant over-representation in aggregate samples including 66 newly identified aggregate proteins. Z-disc-associated proteins were the most abundant aggregate components, followed by sarcolemmal and extracellular matrix proteins, proteins involved in protein quality control and degradation, and proteins with a function in actin dynamics or cytoskeletal transport. Forty over-represented proteins were evaluated by immunolocalization studies. These analyses validated our mass spectrometric data and revealed different regions of protein accumulation in abnormal muscle fibers. Comparison of data from our proteomic analysis in myotilinopathy with findings in other myofibrillar myopathy subtypes indicates a characteristic basic pattern of aggregate composition and resulted in identification of a highly sensitive and specific diagnostic marker for myotilinopathy. CONCLUSIONS: Our findings i) indicate that main protein components of aggregates belong to a network of interacting proteins, ii) provide new insights into the complex regulation of protein degradation in myotilinopathy that may be relevant for new treatment strategies, iii) imply a combination of a toxic gain-of-function leading to myotilin-positive protein aggregates and a loss-of-function caused by a shift in subcellular distribution with a deficiency of myotilin at Z-discs that impairs the integrity of myofibrils, and iv) demonstrate that proteomic analysis can be helpful in differential diagnosis of protein aggregate myopathies.

Xin-deficient mice display myopathy, impaired contractility, attenuated muscle repair and altered satellite cell functionality.

AIM: Xin is an F-actin-binding protein expressed during development of cardiac and skeletal muscle. We used Xin-/- mice to determine the impact of Xin deficiency on different aspects of skeletal muscle health, including functionality and regeneration. METHODS: Xin-/- skeletal muscles and their satellite cell (SC) population were investigated for the presence of myopathic changes by a series of histological and immunofluorescent stains on resting uninjured muscles. To further understand the effect of Xin loss on muscle health and its SCs, we studied SCs responses following cardiotoxin-induced muscle injury. Functional data were determined using in situ muscle stimulation protocol. RESULTS: Compared to age-matched wild-type (WT), Xin-/- muscles exhibited generalized myopathy and increased fatigability with a significantly decreased force recovery post-fatiguing contractions. Muscle regeneration was attenuated in Xin-/- mice. This impaired regeneration prompted an investigation into SC content and functionality. Although SC content was not different, significantly more activated SCs were present in Xin-/- vs. WT muscles. Primary Xin-/- myoblasts displayed significant reductions (approx. 50%) in proliferative capacity vs. WT; a finding corroborated by significantly decreased MyoD-positive nuclei in 3 days post-injury Xin-/- muscle vs. WT. As more activated SCs did not translate to more proliferating myoblasts, we investigated whether Xin-/- SCs displayed an exaggerated loss by apoptosis. More apoptotic SCs (TUNEL+/Pax7+) were present in Xin-/- muscle vs. WT. Furthermore, more Xin-/- myoblasts were expressing nuclear caspase-3 compared to WT at 3 days post-injury. CONCLUSION: Xin deficiency leads to a myopathic condition characterized by increased muscle fatigability, impaired regeneration and SC dysfunction.

Differential proteomic analysis of abnormal intramyoplasmic aggregates in desminopathy.

Desminopathy is a subtype of myofibrillar myopathy caused by desmin mutations and characterized by protein aggregates accumulating in muscle fibers. The aim of this study was to assess the protein composition of these aggregates. Aggregates and intact myofiber sections were obtained from skeletal muscle biopsies of five desminopathy patients by laser microdissection and analyzed by a label-free spectral count-based proteomic approach. We identified 397 proteins with 22 showing significantly higher spectral indices in aggregates (ratio >1.8, p<0.05). Fifteen of these proteins not previously reported as specific aggregate components provide new insights regarding pathomechanisms of desminopathy. Results of proteomic analysis were supported by immunolocalization studies and parallel reaction monitoring. Three mutant desmin variants were detected directly on the protein level as components of the aggregates, suggesting their direct involvement in aggregate-formation and demonstrating for the first time that proteomic analysis can be used for direct identification of a disease-causing mutation in myofibrillar myopathy. Comparison of the proteomic results in desminopathy with our previous analysis of aggregate composition in filaminopathy, another myofibrillar myopathy subtype, allows to determine subtype-specific proteomic profile that facilitates identification of the specific disorder. BIOLOGICAL SIGNIFICANCE: Our proteomic analysis provides essential new insights in the composition of pathological protein aggregates in skeletal muscle fibers of desminopathy patients. The results contribute to a better understanding of pathomechanisms in myofibrillar myopathies and provide the basis for hypothesis-driven studies. The detection of specific proteomic profiles in different myofibrillar myopathy subtypes indicates that proteomic analysis may become a useful tool in differential diagnosis of protein aggregate myopathies.

Distal myopathy with upper limb predominance caused by filamin C haploinsufficiency.

OBJECTIVE: In this study, we investigated the detailed clinical findings and underlying genetic defect in 3 presumably related Bulgarian families displaying dominantly transmitted adult onset distal myopathy with upper limb predominance. METHODS: We performed neurologic, electrophysiologic, radiologic, and histopathologic analyses of 13 patients and 13 at-risk but asymptomatic individuals from 3 generations. Genome-wide parametric linkage analysis was followed by bidirectional sequencing of the filamin C (FLNC) gene. We characterized the identified nonsense mutation at cDNA and protein level. RESULTS: Based on clinical findings, no known myopathy subtype was implicated in our distal myopathy patients. Light microscopic analysis of affected muscle tissue showed no specific hallmarks; however, the electron microscopy revealed changes compatible with myofibrillar myopathy. Linkage studies delineated a 9.76 Mb region on chromosome 7q22.1-q35 containing filamin C (FLNC), a gene previously associated with myofibrillar myopathy. Mutation analysis revealed a novel c.5160delC frameshift deletion in all patients of the 3 families. The mutation results in a premature stop codon (p.Phe1720LeufsX63) that triggers nonsense-mediated mRNA decay. FLNC transcript levels were reduced in muscle and lymphoblast cells from affected subjects and partial loss of FLNC in muscle tissue was confirmed by protein analysis. CONCLUSIONS: The FLNC mutation that we identified is distinct in terms of the associated phenotype, muscle morphology, and underlying molecular mechanism, thus extending the currently recognized clinical and genetic spectrum of filaminopathies. We conclude that filamin C is a dosage-sensitive gene and that FLNC haploinsufficiency can cause a specific type of myopathy in humans.

A novel autosomal dominant limb-girdle muscular dystrophy (LGMD 1F) maps to 7q32.1-32.2.

In 2001, the authors described the clinical features of a genetically distinct autosomal dominant limb-girdle muscular dystrophy (LGMD; LGMD 1F). Using a genome-wide screen with more than 400 microsatellite markers, the authors identified a novel LGMD disease locus at chromosome 7q32.1-32.2. Within this chromosomal region, filamin C, a gene encoding actin binding protein highly expressed in muscle, was an obvious candidate gene; however, the authors did not detect any defects in filamin C or its protein product.

Filamin C accumulation is a strong but nonspecific immunohistochemical marker of core formation in muscle.

Filamin C is the muscle isoform of a group of large actin-crosslinking proteins. On the one hand, filamin C is associated with the Z-disk of the myofibrillar apparatus and binds to myotilin; on the other hand, it interacts with the sarcoglycan complex at the sarcolemma. Filamin C may be involved in reorganizing the cytoskeleton in response to signalling events and in muscle it may, in addition, fulfill structural functions at the Z-disk. An examination of biopsies from patients with multi-minicore myopathy, central core myopathy and neurogenic target fibers with core-like target formations (TF) revealed strong reactivity of all the cores and target formations with two different anti-filamin C antibodies. In all three conditions, the immunoreactivity in the cores for filamin C was considerably stronger than that for desmin. Only for alphaB-crystallin were comparable levels of immunoreactivity detected. There was no difference in intensity for filamin C between the three pathological conditions. Thus, filamin C along with alphaB-crystallin is a strong and robust, but nonspecific marker of core formation. The reason why filamin C accumulates in cores is unclear at present, but we postulate that it may be critically involved in the chain of events eventually leading to myofibrillar degeneration.

Case challenge: painful swelling of the temporomandibular joint.

A 59-year old woman was referred by her dentist to an Oral Medicine practice for evaluation of acute pain and swelling in her right temporomandibular joint (TMJ).

Indications for a novel muscular dystrophy pathway. gamma-filamin, the muscle-specific filamin isoform, interacts with myotilin.

gamma-Filamin, also called ABP-L, is a filamin isoform that is specifically expressed in striated muscles, where it is predominantly localized in myofibrillar Z-discs. A minor fraction of the protein shows subsarcolemmal localization. Although gamma-filamin has the same overall structure as the two other known isoforms, it is the only isoform that carries a unique insertion in its immunoglobulin (Ig)-like domain 20. Sequencing of the genomic region encoding this part of the molecule shows that this insert is encoded by an extra exon. Transient transfections of the insert-bearing domain in skeletal muscle cells and cardiomyocytes show that this single domain is sufficient for targeting to developing and mature Z-discs. The yeast two-hybrid method was used to identify possible binding partners for the insert-bearing Ig-like domain 20 of gamma-filamin. The two Ig-like domains of the recently described alpha-actinin-binding Z-disc protein myotilin were found to interact directly with this filamin domain, indicating that the amino-terminal end of gamma-filamin may be indirectly anchored to alpha-actinin in the Z-disc via myotilin. Since defects in the myotilin gene were recently reported to cause a form of autosomal dominant limb-girdle muscular dystrophy, our findings provide a further contribution to the molecular understanding of this disease.

Association of plectin with Z-discs is a prerequisite for the formation of the intermyofibrillar desmin cytoskeleton.

Plectin is a high-molecular mass protein (approximately 500 kd) that binds actin, intermediate filaments, and microtubules. Mutations of the plectin gene cause a generalized blistering skin disorder and muscular dystrophy. In adult muscle, plectin is colocalized with desmin at structures forming the intermyofibrillar scaffold and beneath the plasma membrane. To study the involvement of plectin in myofibrillogenesis, we analyzed the spatial and temporal expression patterns of plectin in cultured differentiating human skeletal muscle cells and its relationship to desmin intermediate filaments during this process. Northern and Western blot analyses demonstrated that at least two different plectin isoforms are expressed at all developmental stages from proliferating myoblasts to mature myotubes. Using immunocytochemistry, we show that the localization of plectin dramatically changes from a network-like distribution into a cross-striated distribution during maturation of myocytes. Double immunofluorescence experiments revealed that desmin and plectin are colocalized in premyofibrillar stages and in mature myotubes. Interestingly, plectin was often found to localize to the periphery of Z-discs during the actual alignment of neighboring myofibrils, and an obvious cross-striated plectin staining pattern was observed before desmin was localized in the Z-disc region. We conclude that the association of plectin with Z-discs is an early event in the lateral alignment of myofibrils that precedes the formation of the intermyofibrillar desmin cytoskeleton.

A functional knock-out of titin results in defective myofibril assembly.

Titin, also called connectin, is a giant muscle protein that spans the distance from the sarcomeric Z-disc to the M-band. Titin is thought to direct the assembly of sarcomeres and to maintain sarcomeric integrity by interacting with numerous sarcomeric proteins and providing a mechanical linkage. Since severe defects of such an important molecule are likely to result in embryonic lethality, a cell culture model should offer the best practicable tool to probe the cellular functions of titin. The myofibroblast cell line BHK-21/C13 was described to assemble myofibrils in culture. We have now characterized the sub-line BHK-21-Bi, which bears a small deletion within the titin gene. RNA analysis revealed that in this mutant cell line only a small internal portion of the titin mRNA is deleted. However, western blots, immunofluorescence microscopy and immunoprecipitation experiments showed that only the N-terminal, approx. 100 kDa central Z-disc portion of the 3 MDa titin protein is expressed, due to the homozygous deletion in the gene. Most importantly, in BHK-21-Bi cells the formation of thick myosin filaments and the assembly of myofibrils are impaired, although sarcomeric proteins are expressed. Lack of thick filament formation and of ordered actin-myosin arrays was confirmed by electron microscopy. Myogenisation induced by transfection with MyoD yielded myofibrils only in myotubes formed from wild type and not from mutant cells, ruling out that a principal failure in myogenic commitment of the BHK-21-Bi cells might cause the observed effects. These experiments provide the first direct evidence for the crucial role of titin in both thick filament formation as a molecular ruler and in the coordination of myofibrillogenesis.

Characterization of muscle filamin isoforms suggests a possible role of gamma-filamin/ABP-L in sarcomeric Z-disc formation.

Filamin, also called actin binding protein-280, is a dimeric protein that cross-links actin filaments in the cortical cytoplasm. In addition to this ubiquitously expressed isoform (FLN1), a second isoform (ABP-L/gamma-filamin) was recently identified that is highly expressed in mammalian striated muscles. A monoclonal antibody was developed, that enabled us to identify filamin as a Z-disc protein in mammalian striated muscles by immunocytochemistry and immunoelectron microscopy. In addition, filamin was identified as a component of intercalated discs in mammalian cardiac muscle and of myotendinous junctions in skeletal muscle. Northern and Western blots showed that both, ABP-L/gamma-filamin mRNA and protein, are absent from proliferating cultured human skeletal muscle cells. This muscle specific filamin isoform is, however, up-regulated immediately after the induction of differentiation. In cultured myotubes, ABP-L/gamma-filamin localises in Z-discs already at the first stages of Z-disc formation, suggesting that ABP-L/gamma-filamin might play a role in Z-disc assembly.

Genomic structure and fine mapping of the two human filamin gene paralogues FLNB and FLNC and comparative analysis of the filamin gene family.

The genomic structure of the filamin gene paralogues FLNB and FLNC was determined and related to FLNA. FLNB consists of 45 exons and 44 introns and spans approximately 80 kb of genomic DNA. FLNC is divided into 48 exons and 47 introns and covers approximately 29.5 kb of genomic DNA. A previously unknown intron was found in FLNA. The comparison of all three filamin gene paralogues revealed a highly conserved exon-intron structure with significant differences in the exons 32 of all paralogues encoding the hinge I region, as well as the insertion of a novel exon 40A in FLNC only. Gene organization does not correlate with the domain structures of the respective proteins. To improve candidate gene cloning approaches, FLNB was precisely mapped at 3p14 in an interval of 0.81 cM between WI3771 and WI6691 and FLNC at 7q32 in an interval of 2.07 cM between D7S530 and D7S649.

p0071, a member of the armadillo multigene family, is a constituent of sarcomeric I-bands in human skeletal muscle.

p0071 is a member of the armadillo gene family that is expressed in a wide variety of mammalian tissues and cell types with a prominent cell-cell contact association in epithelial cells. Here, we report the expression and localization patterns of p0071 in differentiating human skeletal muscle cells and in normal and diseased human skeletal muscle tissues. Northern blots revealed expression of p0071 mRNA in adult skeletal muscle tissue. RT-PCR analysis and Western blotting experiments identified two differentially spliced isoforms of p0071. The balance between these isoforms shifted during in vitro differentiation of isolated muscle cells from predominant expression of the short variant to a preponderance of the larger variant from day 6 onwards. Immunolocalization studies in mature skeletal muscle tissue revealed that p0071 is a constituent of myofibrils with a distinct localization at the level of sarcomeric N2-lines. During myofibrillogenesis, p0071 was not detected in non-striated nascent myofibrils, but became apparent shortly after the development of compact Z-discs in early myotubes. Furthermore, we studied the expression of p0071 in a wide variety of neuromuscular disorders by indirect immunofluorescence. Here, the myofibrillar staining of p0071 was preserved in all the disease entities included in our study. Our results provide the first evidence that a member of the armadillo multigene family is a constituent of the contractile apparatus in human skeletal muscle. The localization of p0071 at the level of I-bands and the timepoint of its integration into developing myofibrils suggest a possible role in the organization of thin filaments.

Thick filament assembly occurs after the formation of a cytoskeletal scaffold.

The development of myofibrils involves the formation of contractile filaments and their assembly into the strikingly regular structure of the sarcomere. We analysed this assembly process in cultured human skeletal muscle cells and in rat neonatal cardiomyocytes by immunofluorescence microscopy using antibodies directed against cytoskeletal and contractile proteins. In particular, the question in which temporal order the respective proteins are integrated into developing sarcomeres was addressed. Although sarcomeric myosin heavy chain is expressed as one of the first myofibrillar proteins, its characteristic A band arrangement is reached at a very late stage. In contrast, titin, then myomesin and finally C-protein (MyBP-C) gradually form a regularly arranged scaffold on stress fiber-like structures (SFLS), on non-striated myofibrils (NSMF) and on nascent striated myofibrils (naSMF). Immediately subsequent to the completion of sarcomere cytoskeleton formation, the labeling pattern of myosin changes from the continuous staining of SFLS to the periodic staining characteristic for mature myofibrils. This series of events can be seen most clearly in the skeletal muscle cell cultures and--probably due to a faster developmental progression less well in cardiomyocytes. We therefore conclude that the correct assembly of a cytoskeletal scaffold is a prerequisite for correct thick filament assembly and for the integration of the contractile apparatus into the myofibril.

Immunogold EM reveals a close association of plectin and the desmin cytoskeleton in human skeletal muscle.

Plectin is a multifunctional cytoskeletal linker protein with an intermediate filament-binding site and sequence elements with high homology to actin-binding domains. Mutations of the human plectin gene as well as the targeted inactivation of its murine analog cause a generalized blistering skin disorder and muscular dystrophy, thus implying its essential role in cells that are exposed to mechanical stress. In the present study we report the characterization of two new domain-specific plectin antibodies as well as ultrastructural localization of plectin in normal human skeletal muscle. Using immunogold electron microscopy, we localized plectin at three prominent sites: 1) Plectin is found at regularly spaced intervals along the cytoplasmic face of the plasma membrane. 2) It is distinctly localized at filamentous bridges between Z-lines of peripheral myofibrils and the sarcolemma and 3) at structures forming the intermyofibrillar scaffold. At the latter two locations, plectin and desmin were found to colocalize. Our ultrastructural analysis suggests that plectin may have a central role in the structural and functional organization of the intermediate filament cytoskeleton in mature human skeletal muscle.

Expression of sarcomeric proteins and assembly of myofibrils in the putative myofibroblast cell line BHK-21/C13.

The expression and organization patterns of several myofibrillar proteins were analysed in the putative myofibroblast cell line BHK-21/C13. Although this cell line originates from renal tissue, the majority of the cells express titin. In these cells, titin is, under standard culture conditions, detected in myofibril-like structures (MLSs), where it alternates with non-muscle myosin (NMM). Expression of sarcomeric myosin heavy chain (sMyHC) is observed in a small minority of cells, while other sarcomeric proteins, such as nebulin, myosin binding protein C (MyBP-C), myomesin and M-protein are not expressed at all. By changing the culture conditions in a way equal to conditions that induce differentiation of skeletal muscle cells, a process reminiscent of sarcomerogenesis in vitro is induced. Within one day after the switch to a low-nutrition medium, myofibrillar proteins can be detected in a subset of cells, and after two to five days, all myofibrillar proteins examined are organized in typical sarcomeric patterns. Frequently, cross-striations are visible with phase contrast optics. Transfection of these cells with truncated myomesin fragments showed that a specific part of the myomesin molecule, known to contain a titin-binding site, binds to MLSs, whereas other parts do not. These results demonstrate that this cell line could serve as a powerful model to study the assembly of myofibrils. At the same time, its transfectability offers an invaluable tool for in vivo studies concerning binding properties of sarcomeric proteins.

Structural basis for activation of the titin kinase domain during myofibrillogenesis.

The giant muscle protein titin (connectin) is essential in the temporal and spatial control of the assembly of the highly ordered sarcomeres (contractile units) of striated muscle. Here we present the crystal structure of titin's only catalytic domain, an autoregulated serine kinase (titin kinase). The structure shows how the active site is inhibited by a tyrosine of the kinase domain. We describe a dual mechanism of activation of titin kinase that consists of phosphorylation of this tyrosine and binding of calcium/calmodulin to the regulatory tail. The serine kinase domain of titin is the first known non-arginine-aspartate kinase to be activated by phosphorylation. The phosphorylated tyrosine is not located in the activation segment, as in other kinases, but in the P + 1 loop, indicating that this tyrosine is a binding partner of the titin kinase substrate. Titin kinase phosphorylates the muscle protein telethonin in early differentiating myocytes, indicating that this kinase may act in myofibrillogenesis.