Homozygous expression of the myofibrillar myopathy-associated p.W2710X filamin C variant reveals major pathomechanisms of sarcomeric lesion formation.
ABSTRACT: Filamin C (FLNc) is mainly expressed in striated muscle cells where it localizes to Z-discs, myotendinous junctions and intercalated discs. Recent studies have revealed numerous mutations in the FLNC gene causing familial and sporadic myopathies and cardiomyopathies with marked clinical variability. The most frequent myopathic mutation, p.W2710X, which is associated with myofibrillar myopathy, deletes the carboxy-terminal 16 amino acids from FLNc and abolishes the dimerization property of Ig-like domain 24. We previously characterized "knock-in" mice heterozygous for this mutation (p.W2711X), and have now investigated homozygous mice using protein and mRNA expression analyses, mass spectrometry, and extensive immunolocalization and ultrastructural studies. Although the latter mice display a relatively mild myopathy under normal conditions, our analyses identified major mechanisms causing the pathophysiology of this disease: in comparison to wildtype animals (i) the expression level of FLNc protein is drastically reduced; (ii) mutant FLNc is relocalized from Z-discs to particularly mechanically strained parts of muscle cells, i.e. myotendinous junctions and myofibrillar lesions; (iii) the number of lesions is greatly increased and these lesions lack Bcl2-associated athanogene 3 (BAG3) protein; (iv) the expression of heat shock protein beta-7 (HSPB7) is almost completely abolished. These findings indicate grave disturbances of BAG3-dependent and -independent autophagy pathways that are required for efficient lesion repair. In addition, our studies reveal general mechanisms of lesion formation and demonstrate that defective FLNc dimerization via its carboxy-terminal domain does not disturb assembly and basic function of myofibrils. An alternative, more amino-terminally located dimerization site might compensate for that loss. Since filamins function as stress sensors, our data further substantiate that FLNc is important for mechanosensing in the context of Z-disc stabilization and maintenance.
Project description:Filamin C is mainly expressed in striated muscle cells where it localizes to Z-discs, myotendinous junctions and intercalated discs. Recent studies have revealed numerous mutations in the FLNC gene causing familiar and sporadic myopathies and cardiomyopathies with marked clinical variability. The most frequent myopathic mutation, p.W2710X, which is associated with myofibrillar myopathy, deletes the carboxy-terminal 16 amino acids from filamin C and abolishes the dimerization property of Ig-like domain 24. We previously characterized "knock-in" mice heterozygous for this mutation (p.W2711X), and have now investigated homozygous mice using protein and mRNA expression analyses, mass spectrometry, and extensive immunolocalization and ultrastructural studies. Although the latter mice display a relatively mild myopathy under normal conditions, our analyses identified major mechanisms causing the pathophysiology of this disease: (i) the expression level of filamin C protein is drastically reduced; (ii) mutant filamin C is relocalized from Z-discs to particularly mechanically strained parts of muscle cells, i.e. myotendinous junctions and myofibrillar lesions; (iii) the number of lesions is greatly increased and these lesions lack BAG3; (iv) the expression of HSPB7 is almost completely abolished. These findings indicate grave disturbances of BAG3-dependent and -independent autophagy pathways that are required for efficient lesion repair. In addition, our studies reveal general mechanisms of lesion formation and demonstrate that defective filamin C dimerization via its carboxy-terminal domain does not disturb assembly and basic function of myofibrils. An alternative dimerization site might compensate for that loss. Since filamins function as stress sensors, our data further substantiate that filamin C is important for mechanosensing in the context of Z‑disc stabilization and maintenance.
Project description:The term filaminopathy was introduced after a truncating mutation in the dimerization domain of filamin C (FLNc) was shown to be responsible for a devastating muscle disease. Subsequently, the same mutation was found in patients from diverse ethnical origins, indicating that this specific alteration is a mutational hot spot. Patients initially present with proximal muscle weakness, while distal and respiratory muscles become affected with disease progression. Muscle biopsies of these patients show typical signs of myofibrillar myopathy, including disintegration of myofibrils and aggregation of several proteins into distinct intracellular deposits. Highly similar phenotypes were observed in patients with other mutations in Ig-like domains of FLNc that result in expression of a noxious protein. Biochemical and biophysical studies showed that the mutated domains acquire an abnormal structure causing decreased stability and eventually becoming a seed for abnormal aggregation with other proteins. The disease usually presents only after the fourth decade of life possibly as a result of ageing-related impairments in the machinery that is responsible for disposal of damaged proteins. This is confirmed by mutations in components of this machinery that cause a highly similar phenotype. Transfection studies of cultured muscle cells reflect the events observed in patient muscles and, therefore, may provide a helpful model for testing future dedicated therapeutic strategies. More recently, FLNC mutations were also found in families with a distal myopathy phenotype, caused either by mutations in the actin-binding domain of FLNc that result in increased actin-binding and non-specific myopathic abnormalities without myofibrillar myopathy pathology, or a nonsense mutation in the rod domain that leads to RNA instability, haploinsufficiency with decreased expression levels of FLNc in the muscle fibers and myofibrillar abnormalities, but not to the formation of desmin-positive protein aggregates required for the diagnosis of myofibrillar myopathy.
Project description:Myofibrillar myopathy (MFM) is a human disease that is characterized by focal myofibrillar destruction and pathological cytoplasmic protein aggregations. In an extended German pedigree with a novel form of MFM characterized by clinical features of a limb-girdle myopathy and morphological features of MFM, we identified a co-segregating, heterozygous nonsense mutation (8130G-->A; W2710X) in the filamin c gene (FLNC) on chromosome 7q32.1. The mutation is the first found in FLNC and is localized in the dimerization domain of filamin c. Functional studies showed that, in the truncated mutant protein, this domain has a disturbed secondary structure that leads to the inability to dimerize properly. As a consequence of this malfunction, the muscle fibers of our patients display massive cytoplasmic aggregates containing filamin c and several Z-disk-associated and sarcolemmal proteins.
Project description:A homozygous disruption or genetic mutation of the bag3 gene causes progressive myofibrillar myopathy in mouse and human skeletal and cardiac muscle disorder while mutations in the small heat shock protein ?B-crystallin gene (CRYAB) are reported to be responsible for myofibrillar myopathy. Here, we demonstrate that BAG3 directly binds to wild-type ?B-crystallin and the ?B-crystallin mutant R120G, via the intermediate domain of BAG3. Peptides that inhibit this interaction in an in vitro binding assay indicate that two conserved Ile-Pro-Val regions of BAG3 are involved in the interaction with ?B-crystallin, which is similar to results showing BAG3 binding to HspB8 and HspB6. BAG3 overexpression increased ?B-crystallin R120G solubility and inhibited its intracellular aggregation in HEK293 cells. BAG3 suppressed cell death induced by ?B-crystallin R120G overexpression in differentiating C2C12 mouse myoblast cells. Our findings indicate a novel function for BAG3 in inhibiting protein aggregation caused by the genetic mutation of CRYAB responsible for human myofibrillar myopathy.
Project description:Linkage analysis of the dominant distal myopathy we previously identified in a large Australian family demonstrated one significant linkage region located on chromosome 7 and encompassing 18.6 Mbp and 151 genes. The strongest candidate gene was FLNC because filamin C, the encoded protein, is muscle-specific and associated with myofibrillar myopathy. Sequencing of FLNC cDNA identified a c.752T>C (p.Met251Thr) mutation in the N-terminal actin-binding domain (ABD); this mutation segregated with the disease and was absent in 200 controls. We identified an Italian family with the same phenotype and found a c.577G>A (p.Ala193Thr) filamin C ABD mutation that segregated with the disease. Filamin C ABD mutations have not been described, although filamin A and filamin B ABD mutations cause multiple musculoskeletal disorders. The distal myopathy phenotype and muscle pathology in the two families differ from myofibrillar myopathies caused by filamin C rod and dimerization domain mutations because of the distinct involvement of hand muscles and lack of pathological protein aggregation. Thus, like the position of FLNA and B mutations, the position of the FLNC mutation determines disease phenotype. The two filamin C ABD mutations increase actin-binding affinity in a manner similar to filamin A and filamin B ABD mutations. Cell-culture expression of the c.752T>C (p.Met251)Thr mutant filamin C ABD demonstrated reduced nuclear localization as did mutant filamin A and filamin B ABDs. Expression of both filamin C ABD mutants as full-length proteins induced increased aggregation of filamin. We conclude filamin C ABD mutations cause a recognizable distal myopathy, most likely through increased actin affinity, similar to the pathological mechanism of filamin A and filamin B ABD mutations.
Project description:Filamin C (FLNC) variants are associated with cardiac and muscular phenotypes. Originally, FLNC variants were described in myofibrillar myopathy (MFM) patients. Later, high-throughput screening in cardiomyopathy cohorts determined a prominent role for FLNC in isolated hypertrophic and dilated cardiomyopathies (HCM and DCM). FLNC variants are now among the more prevalent causes of genetic DCM. FLNC-associated DCM is associated with a malignant clinical course and a high risk of sudden cardiac death. The clinical spectrum of FLNC suggests different pathomechanisms related to variant types and their location in the gene. The appropriate functioning of FLNC is crucial for structural integrity and cell signaling of the sarcomere. The secondary protein structure of FLNC is critical to ensure this function. Truncating variants with subsequent haploinsufficiency are associated with DCM and cardiac arrhythmias. Interference with the dimerization and folding of the protein leads to aggregate formation detrimental for muscle function, as found in HCM and MFM. Variants associated with HCM are predominantly missense variants, which cluster in the ROD2 domain. This domain is important for binding to the sarcomere and to ensure appropriate cell signaling. We here review FLNC genotype-phenotype correlations based on available evidence.
Project description:Mutations in FLNC cause two distinct types of myopathy. Disease associated with mutations in filamin C rod domain leading to expression of a toxic protein presents with progressive proximal muscle weakness and shows focal destructive lesions of polymorphous aggregates containing desmin, myotilin and other proteins in the affected myofibres; these features correspond to the profile of myofibrillar myopathy. The second variant associated with mutations in the actin-binding domain of filamin C is characterized by weakness of distal muscles and morphologically by non-specific myopathic features. A frameshift mutation in the filamin C rod domain causing haploinsufficiency was also found responsible for distal myopathy with some myofibrillar changes but no protein aggregation typical of myofibrillar myopathies. Controversial data accumulating in the literature require re-evaluation and comparative analysis of phenotypes associated with the position of the FLNC mutation and investigation of the underlying disease mechanisms. This is relevant and necessary for the refinement of diagnostic criteria and developing therapeutic approaches. We identified a p.W2710X mutation in families originating from ethnically diverse populations and re-evaluated a family with a p.V930_T933del mutation. Analysis of the expanded database allows us to refine clinical and myopathological characteristics of myofibrillar myopathy caused by mutations in the rod domain of filamin C. Biophysical and biochemical studies indicate that certain pathogenic mutations in FLNC cause protein misfolding, which triggers aggregation of the mutant filamin C protein and subsequently involves several other proteins. Immunofluorescence analyses using markers for the ubiquitin-proteasome system and autophagy reveal that the affected muscle fibres react to protein aggregate formation with a highly increased expression of chaperones and proteins involved in proteasomal protein degradation and autophagy. However, there is a noticeably diminished efficiency of both the ubiquitin-proteasome system and autophagy that impairs the muscle capacity to prevent the formation or mediate the degradation of aggregates. Transfection studies of cultured muscle cells imitate events observed in the patient's affected muscle and therefore provide a helpful model for testing future therapeutic strategies.
Project description:A homozygous disruption or genetic mutation of the bag3 gene, a member of the Bcl-2-associated athanogene (BAG) family proteins, causes cardiomyopathy and myofibrillar myopathy that is characterized by myofibril and Z-disc disruption. However, the detailed disease mechanism is not yet fully understood.bag3(-/-) mice exhibit differences in the extent of muscle degeneration between muscle groups with muscles experiencing the most usage degenerating at an accelerated rate. Usage-dependent muscle degeneration suggests a role for BAG3 in supporting cytoskeletal connections between the Z-disc and myofibrils under mechanical stress. The mechanism by which myofibrillar structure is maintained under mechanical stress remains unclear. The purpose of the study is to clarify the detailed molecular mechanism of BAG3-mediated muscle maintenance under mechanical stress.To address the question of whether bag3 gene knockdown induces myofibrillar disorganization caused by mechanical stress, in vitro mechanical stretch experiments using rat neonatal cardiomyocytes and a short hairpin RNA-mediated gene knockdown system of the bag3 gene were performed. As expected, mechanical stretch rapidly disrupts myofibril structures in bag3 knockdown cardiomyocytes. BAG3 regulates the structural stability of F-actin through the actin capping protein, CapZ?1, by promoting association between Hsc70 and CapZ?1. BAG3 facilitates the distribution of CapZ?1 to the proper location, and dysfunction of BAG3 induces CapZ ubiquitin-proteasome-mediated degradation. Inhibition of CapZ?1 function by overexpressing CapZ?2 increased myofibril vulnerability and fragmentation under mechanical stress. On the other hand, overexpression of CapZ?1 inhibits myofibrillar disruption in bag3 knockdown cells under mechanical stress. As a result, heart muscle isolated from bag3(-/-) mice exhibited myofibrillar degeneration and lost contractile activity after caffeine contraction.These results suggest novel roles for BAG3 and Hsc70 in stabilizing myofibril structure and inhibiting myofibrillar degeneration in response to mechanical stress. These proteins are possible targets for further research to identify therapies for myofibrillar myopathy or other degenerative diseases.
Project description:Cbl-associated protein (CAP) is an adaptor protein that interacts with both signaling and cytoskeletal proteins. Here, we characterize the expression, localization and potential function of CAP in striated muscle. CAP is markedly induced during myoblast differentiation, and colocalizes with vinculin during costamerogenesis. In adult mice, CAP is enriched in oxidative muscle fibers, and it is found in membrane anchorage complexes, including intercalated discs, costameres, and myotendinous junctions. Using both yeast two-hybrid and proteomic approaches, we identified the sarcomeric protein filamin C (FLNc) as a binding partner for CAP. When overexpressed, CAP recruits FLNc to cell-extracellular matrix adhesions, where the two proteins cooperatively regulate actin reorganization. Moreover, overexpression of CAP inhibits FLNc-induced cell spreading on fibronectin. In dystrophin-deficient mdx mice, the expression and membrane localization of CAP is increased, concomitant with the elevated plasma membrane content of FLNc, suggesting that CAP may compensate for the reduced membrane linkage of the myofibrils due to the loss of the dystroglycan-sarcoglycan complex in these mice. Thus, through its interaction with FLNc, CAP provides another link between the myofibril cytoskeleton and the plasma membrane of muscle cells, and it may play a dynamic role in the regulation and maintenance of muscle structural integrity.
Project description:Myofibrillar myopathies (MFM) are characterised by focal myofibrillar destruction and accumulation of myofibrillar elements as protein aggregates. They are caused by mutations in the DES, MYOT, CRYAB, FLNC, BAG3, DNAJB6 and ZASP genes as well as other as yet unidentified genes. Previous studies have reported changes in mitochondrial morphology and cellular positioning, as well as clonally-expanded, large-scale mitochondrial DNA (mtDNA) deletions and focal respiratory chain deficiency in muscle of MFM patients. Here we examine skeletal muscle from patients with desmin (n?=?6), ZASP (n?=?1) and myotilin (n?=?2) mutations and MFM protein aggregates, to understand how mitochondrial dysfunction may contribute to the underlying mechanisms causing disease pathology. We have used a validated quantitative immunofluorescent assay to study respiratory chain protein levels, together with oxidative enzyme histochemistry and single cell mitochondrial DNA analysis, to examine mitochondrial changes. Results demonstrate a small number of clonally-expanded mitochondrial DNA deletions, which we conclude are due to both ageing and disease pathology. Further to this we report higher levels of respiratory chain complex I and IV deficiency compared to age matched controls, although overall levels of respiratory deficient muscle fibres in patient biopsies are low. More strikingly, a significantly higher percentage of myofibrillar myopathy patient muscle fibres have a low mitochondrial mass compared to controls. We concluded this is mechanistically unrelated to desmin and myotilin protein aggregates; however, correlation between mitochondrial mass and muscle fibre area is found. We suggest this may be due to reduced mitochondrial biogenesis in combination with muscle fibre hypertrophy.