Loss of FilaminC (FLNc) results in severe defects in myogenesis and myotube structure.
ABSTRACT: FilaminC (FLNc) is the muscle-specific member of a family of actin binding proteins. Although it interacts with many proteins involved in muscular dystrophies, its unique role in muscle is poorly understood. To address this, two models were developed. First, FLNc expression was stably reduced in C2C12 myoblasts by RNA interference. While these cells start differentiation normally, they display defects in differentiation and fusion ability and ultimately form multinucleated "myoballs" rather than maintain elongated morphology. Second, a mouse model carrying a deletion of last 8 exons of Flnc was developed. FLNc-deficient mice die shortly after birth, due to respiratory failure, and have severely reduced birth weights, with fewer muscle fibers and primary myotubes, indicating defects in primary myogenesis. They exhibit variation in fiber size, fibers with centrally located nuclei, and some rounded fibers resembling the in vitro phenotype. The similarity of the phenotype of FLNc-deficient mice to the filamin-interacting TRIO null mice was further confirmed by comparing FLNc-deficient C2C12 cells to TRIO-deficient cells. These data provide the first evidence that FLNc has a crucial role in muscle development and maintenance of muscle structural integrity and suggest the presence of a TRIO-FLNc-dependent pathway in maintaining proper myotube structure.
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: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:The importance of kyphoscoliosis peptidase (KY) in skeletal muscle physiology has recently been emphasised by the identification of novel human myopathies associated with KY deficiency. Neither the pathogenic mechanism of KY deficiency nor a specific role for KY in muscle function have been established. However, aberrant localisation of filamin C (FLNC) in muscle fibres has been shown in humans and mice with loss-of-function mutations in the KY gene. FLNC turnover has been proposed to be controlled by chaperone-assisted selective autophagy (CASA), a client-specific and tension-induced pathway that is required for muscle maintenance. Here, we have generated new C2C12 myoblast and zebrafish models of KY deficiency by CRISPR/Cas9 mutagenesis. To obtain insights into the pathogenic mechanism caused by KY deficiency, expression of the co-chaperone BAG3 and other CASA factors was analyzed in the cellular, zebrafish and ky/ky mouse models. Ky-deficient C2C12-derived clones show trends of higher transcription of CASA factors in differentiated myotubes. The ky-deficient zebrafish model (kyyo1/kyyo1 ) lacks overt signs of pathology, but shows significantly increased bag3 and flnca/b expression in embryos and adult muscle. Additionally, kyyo1/kyyo1 embryos challenged by swimming in viscous media show an inability to further increase expression of these factors in contrast with wild-type controls. The ky/ky mouse shows elevated expression of Bag3 in the non-pathological exterior digitorum longus (EDL) and evidence of impaired BAG3 turnover in the pathological soleus. Thus, upregulation of CASA factors appears to be an early and primary molecular hallmark of KY deficiency.
Project description:OBJECTIVE:To identify novel dilated cardiomyopathy (DCM) causing genes, and to elucidate the pathological mechanism leading to DCM by utilizing zebrafish as a model organism. BACKGROUND:DCM, a major cause of heart failure, is frequently familial and caused by a genetic defect. However, only 50% of DCM cases can be attributed to a known DCM gene variant, motivating the ongoing search for novel disease genes. METHODS:We performed whole exome sequencing (WES) in two multigenerational Italian families and one US family with arrhythmogenic DCM without skeletal muscle defects, in whom prior genetic testing had been unrevealing. Pathogenic variants were sought by a combination of bioinformatic filtering and cosegregation testing among affected individuals within the families. We performed function assays and generated a zebrafish morpholino knockdown model. RESULTS:A novel filamin C gene splicing variant (FLNC c.7251+1 G>A) was identified by WES in all affected family members in the two Italian families. A separate novel splicing mutation (FLNC c.5669-1delG) was identified in the US family. Western blot analysis of cardiac heart tissue from an affected individual showed decreased FLNC protein, supporting a haploinsufficiency model of pathogenesis. To further analyze this model, a morpholino knockdown of the ortholog filamin Cb in zebrafish was created which resulted in abnormal cardiac function and ultrastructure. CONCLUSIONS:Using WES, we identified two novel FLNC splicing variants as the likely cause of DCM in three families. We provided protein expression and in vivo zebrafish data supporting haploinsufficiency as the pathogenic mechanism leading to DCM.
Project description:Phosphorodiamidate morpholino oligomer (PMO)-mediated exon skipping is among the more promising approaches to the treatment of several neuromuscular disorders including Duchenne muscular dystrophy. The main weakness of this approach arises from the low efficiency and sporadic nature of the delivery of charge-neutral PMO into muscle fibers, the mechanism of which is unknown. In this study, to test our hypothesis that muscle fibers take up PMO more efficiently during myotube formation, we induced synchronous muscle regeneration by injection of cardiotoxin into the tibialis anterior muscle of Dmd exon 52-deficient mdx52 and wild-type mice. Interestingly, by in situ hybridization, we detected PMO mainly in embryonic myosin heavy chain-positive regenerating fibers. In addition, we showed that PMO or 2'-O-methyl phosphorothioate is taken up efficiently into C2C12 myotubes when transfected 24-72 h after the induction of differentiation but is poorly taken up into undifferentiated C2C12 myoblasts suggesting efficient uptake of PMO in the early stages of C2C12 myotube formation. Next, we tested the therapeutic potential of PMO for laminin-?2 chain-null dy(3K)/dy(3K) mice: a model of merosin-deficient congenital muscular dystrophy (MDC1A) with active muscle regeneration. We confirmed the recovery of laminin-?2 chain and slightly prolonged life span following skipping of the mutated exon 4 in dy(3K)/dy(3K) mice. These findings support the idea that PMO entry into fibers is dependent on a developmental stage in myogenesis rather than on dystrophinless muscle membranes and provide a platform for developing PMO-mediated therapies for a variety of muscular disorders, such as MDC1A, that involve active muscle regeneration.
Project description:BACKGROUND:Restrictive cardiomyopathy (RCM) is a rare cardiomyopathy characterized by impaired diastolic ventricular function resulting in a poor clinical prognosis. Rarely, heritable forms of RCM have been reported, and mutations underlying RCM have been identified in genes that govern the contractile function of the cardiomyocytes. METHODS AND RESULTS:We evaluated 8 family members across 4 generations by history, physical examination, electrocardiography, and echocardiography. Affected individuals presented with a pleitropic syndrome of progressive RCM, atrioventricular septal defects, and a high prevalence of atrial fibrillation. Exome sequencing of 5 affected members identified a single novel missense variant in a highly conserved residue of FLNC (filamin C; p.V2297M). FLNC encodes filamin C-a protein that acts as both a scaffold for the assembly and organization of the central contractile unit of striated muscle and also as a mechanosensitive signaling molecule during cell migration and shear stress. Immunohistochemical analysis of FLNC localization in cardiac tissue from an affected family member revealed a diminished localization at the z disk, whereas traditional localization at the intercalated disk was preserved. Stem cell-derived cardiomyocytes mutated to carry the effect allele had diminished contractile activity when compared with controls. CONCLUSION:We have identified a novel variant in FLNC as pathogenic variant for familial RCM-a finding that further expands on the genetic basis of this rare and morbid cardiomyopathy.
Project description:Stac3 was identified as a nutritionally regulated gene from an Atlantic salmon subtractive hybridization library with highest expression in skeletal muscle. Salmon Stac3 mRNA was highly correlated with myogenin and myoD1a expression during differentiation of a salmon primary myogenic culture and was regulated by amino acid availability. In zebrafish embryos, stac3 was initially expressed in myotomal adaxial cells and in fast muscle fibers post-segmentation. Morpholino knockdown resulted in defects in myofibrillar protein assembly, particularly in slow muscle fibers, and decreased levels of the hedgehog receptor patched. The function of Stac3 was further characterized in vitro using the mammalian C2C12 myogenic cell line. Stac3 mRNA expression increased during the differentiation of the C2C12 myogenic cell line. Knockdown of Stac3 by RNAi inhibited myotube formation, and microarray analysis revealed that transcripts involved in cell cycle, focal adhesion, cytoskeleton, and the pro-myogenic factors Igfbp-5 and Igf2 were down-regulated. RNAi-treated cells had suppressed Akt signaling and exogenous insulin-like growth factor (Igf) 2 was unable to rescue the phenotype, however, Igf/Akt signaling was not blocked. Overexpression of Stac3, which results in increased levels of Igfbp-5 mRNA, did not lead to increased differentiation. In synchronized cells, Stac3 mRNA was most abundant during the G(1) phase of the cell cycle. RNAi-treated cells were smaller, had higher proliferation rates and a decreased proportion of cells in G(1) phase when compared with controls, suggesting a role in the G(1) phase checkpoint. These results identify Stac3 as a new gene required for myogenic differentiation and myofibrillar protein assembly in vertebrates.
Project description:BACKGROUND:Filamin C (FLNC) mutation was reported as a cause of HCM, with a high probability of sudden cardiac death. However, the mutation profile of FLNC, and its relationship with phenotypic expression in HCM, remains to be elucidated. METHODS:In this study, FLNC gene was sequenced in 540 HCM patients and 307 healthy controls. RESULTS:We found that 39 (7.2%) patients carried FLNC mutations, with a similar frequency to that of controls (4.2%, p = 0.101). Pedigree analysis showed that mutations were not well segregated with HCM. The baseline characteristics between HCM patients, with and without mutations, were comparable. FLNC mutations did not increase the risk for either all-cause mortality (HR 0.746, 95% CI 0.222-2.295, p = 0.575) or cardiac mortality (HR 0.615, 95% CI 0.153-1.947, p = 0.354) in HCM patients during a follow-up of 4.7 ± 3.2 years. Moreover, there was no significant difference in survival free from sudden cardiac arrest (HR 0.721, 95% CI 0.128-3.667, p = 0.660) and heart failure (HR 0.757, 95% CI 0.318-1.642, p = 0.447). CONCLUSIONS:FLNC mutations were common in both HCM patients and healthy population. The pathogenicity of FLNC mutations detected in HCM patients and its association with the clinical outcomes should be cautiously interpreted.
Project description:A novel FLNC c.5161delG (p.Gly1722ValfsTer61) mutation was identified in two members of a French family affected by distal myopathy and in one healthy relative. This FLNC c.5161delG mutation is one nucleotide away from a previously reported FLNC mutation (c.5160delC) that was identified in patients and in asymptomatic carriers of three Bulgarian families with distal muscular dystrophy, indicating a low penetrance of the FLNC frameshift mutations. Given these similarities, we believe that the two FLNC mutations alone can be causative of distal myopathy without full penetrance. Moreover, comparative analysis of the clinical manifestations indicates that patients of the French family show an earlier onset and a complete segregation of the disease. As a possible explanation of this, the two French patients also carry a OBSCN c.13330C>T (p.Arg4444Trp) mutation. The p.Arg4444Trp variant is localized within the OBSCN Ig59 domain that, together with Ig58, binds to the ZIg9/ZIg10 domains of titin at Z-disks. Structural and functional studies indicate that this OBSCN p.Arg4444Trp mutation decreases titin binding by ~15-fold. On this line, we suggest that the combination of the OBSCN p.Arg4444Trp variant and of the FLNC c.5161delG mutation, can cooperatively affect myofibril stability and increase the penetrance of muscular dystrophy in the French family.
Project description:BACKGROUND:Skeletal muscle contributes to roughly 40% of lean body mass, and its loss contributes to morbidity and mortality in a variety of pathogenic conditions. Significant insights into muscle function have been made using cultured cells, in particular, the C2C12 myoblast line. However, differentiation of these cells in vitro typically yields immature myotubes relative to skeletal muscles in vivo. While many efforts have attempted to improve the maturity of cultured myotubes, including the use of bioengineered substrates, lack of molecular characterization has precluded their widespread implementation. This study characterizes morphological, molecular, and transcriptional features of C2C12 myotubes cultured on crosslinked, micropatterned gelatin substrates fabricated using previously established methods and compares them to myotubes grown on unpatterned gelatin or traditional plasticware. METHODS:We used immunocytochemistry, SDS-PAGE, and RNAseq to characterize C2C12 myotubes grown on micropatterned gelatin hydrogels, unpatterned gelatin hydrogels, and typical cell culture substrates (i.e., plastic or collagen-coated glass) across a differentiation time course. The ability to form aligned sarcomeres and myofilament protein concentration was assessed. Additionally, the transcriptome was analyzed across the differentiation time course. RESULTS:C2C12 myotubes grown on micropatterned gelatin hydrogels display an increased ability to form aligned sarcomeres as well as increased contractile protein content relative to myotubes cultured on unpatterned gelatin and plastic. Additionally, genes related to sarcomere formation and in vivo muscle maturation are upregulated in myotubes grown on micropatterned gelatin hydrogels relative to control myotubes. CONCLUSIONS:Our results suggest that growing C2C12 myotubes on micropatterned gelatin hydrogels accelerates sarcomere formation and yields a more fully matured myotube culture. Thus, the use of micropatterned hydrogels is a viable and simple approach to better model skeletal muscle biology in vitro.