Novel Filamin C Myofibrillar Myopathy Variants Cause Different Pathomechanisms and Alterations in Protein Quality Systems

Abstract

Myofibrillar myopathies (MFM) are a group of chronic muscle diseases pathophysiologically characterized by accumulation of protein aggregates and structural failure of muscle fibers. A subtype of MFM is caused by heterozygous mutations in the filamin C (FLNC) gene, exhibiting progressive muscle weakness, muscle structural alterations and intracellular protein accumulations. Here, we characterize in depth the pathogenicity of two novel truncating FLNc variants (p.Q1662X and p.Y2704X) and assess their distinct effect on FLNc stability and distribution as well as their impact on protein quality system (PQS) pathways. Both variants cause a slowly progressive myopathy with disease onset in adulthood, chronic myopathic alterations in muscle biopsy including the presence of intracellular protein aggregates. Our analyses revealed that p.Q1662X results in FLNc haploinsufficiency and p.Y2704X in a dominant-negative FLNc accumulation. Moreover, both protein-truncating variants cause different PQS alterations: p.Q1662X leads to an increase in expression of several genes involved in the ubiquitin-proteasome system (UPS) and the chaperone-assisted selective autophagy (CASA) system, whereas p.Y2704X results in increased abundance of proteins involved in UPS activation and autophagic buildup. We conclude that truncating FLNC variants might have different pathogenetic consequences and impair PQS function by diverse mechanisms and to varying extents. Further studies on a larger number of patients are necessary to confirm our observations.

Keywords: cardiomyopathy; chaperone-assisted selected autophagy; filamin c; myofibrillar myopathy; protein folding; protein quality system.

Figure 1

Genetic and histochemical findings and immunofluorescence studies. (A) Schematic presentation of the FLNc protein with the aminoterminal actin-binding domain (ABD, purple) and 24 Ig-like domains (blue). Domain 20 (green) is colored differentially because it is extended and contains an 82 amino acid long insertion. The locations of the two novel mutations are shown. (B,C) Analysis of muscle biopsy samples from control, patients I:1 (p.Q1662X) and II:2 (p.Y2704X). (B) Cryosections stained with H&E and trichrome (TC) showed fiber diameter variability, fiber splitting, fatty replacement, endomysial fibrosis and increase in central nuclei. These findings were more pronounced in II:2 (p.Y2704X). Scale bars: 50 µm. (C) Immunolocalization of FLNc and muscle damage marker Xin in longitudinal cryosections of control and patient muscle. FLNc and Xin co-localize in protein aggregations (asterisk), macrolesions (arrowheads) and microlesions (arrows) in patients I:1 and II:2, but not in control muscle fibers. Scale bars: 50 µm.

Figure 2

Electron microscopy showing ultrastructural overview of pathologic hallmarks. (left panels) Ultrastructural analysis of muscle biopsy samples from patients I:1 (p.Q1662X) and II:2 (p.Y2704X) showing both typical signs of MFM pathology. Extended areas of myofibrillar disorganization with sarcomeric lesions and (central panels) electron-dense material connecting adjacent Z-discs surrounded by many autophagic vesicles. (right panels) Frequent observation of subsarcolemmal depositions including heterogeneous, granulofilamentous protein aggregates (*) mainly localized at the subsarcolemmal level and in the perinuclear environment. MF: myofibril, AG: aggregates, NC: nucleus, SL: sarcolemma, AV: autophagic compartment, asterisks: degradation area, Scale bar: 1 μm.

Figure 3

Muscle imaging findings in lower extremities.T1-weighted muscle MRI revealed no major alterations in patient I:1 (p.Q1662X), and moderate fatty degenerative changes in posterior thigh muscles (especially semimembranosus and biceps femoris), soleus and anterior tibial muscles in patients II.2 and II:1 (both p.Y2704), which were pronounced in the older patient (II:1).

Figure 4

Cardiac MRI. Phase-sensitive inversion-recovery (PSIR) of patient I:1, Basal (left) and mid-short-axis (middle) images show subepicardial to mid-wall late enhancement (arrowheads) in the lateral wall (star) and interventricular septum (+). Typical example late gadolinium enhancement image with diffuse late enhancement in a 23-year-old control patient with FLNC-cardiomyopathy (p.E2009GfsX29; (right), arrowheads). RVOT: Right ventricular outflow tract, LV: Left ventricle, RV: Right ventricle.

Figure 5

Analysis of the expression of the mutant alleles at protein and mRNA level. (A) RT-PCR analysis of the p.Y2704X patient. Specific primers were used to amplify a 670 bp long fragment of FLNc cDNA containing the mutation. Digestion with restriction enzyme BseGI cuts the normal (arrow) but not the mutant variant (arrow with red ×). In control cDNA, only 500 bp and 170 bp fragments, but no non-digested fragment was detected (670 bp), while the relative intensity measured by densitometry indicated that ~53% of the p.Y2704X cDNA is not digested, and thus mutant. (B) RT-PCR analysis of p.Q1662X patient tissue and cultured myocytes. Specific primers were used to amplify a 137 bp long fragment of FLNc cDNA containing the mutation (arrow on the left). Digestion with restriction enzyme HpyF10VI cuts the normal (arrow) but not the mutant variant (arrow with red×). In control as well as patient cDNA, only digested 112 bp fragments were found (arrow on the right), while no non-digested fragment was detected at 137 bp, indicating that no mutant mRNA is expressed in the patient. The 25 bp fragment is too small to be visible on the gel. (C) Analysis of FLNc protein expression in patient and control skeletal muscle tissue using an antibody specific for a central region of FLNc (d16–20) and its extreme C-terminus (CT). The relative expression level of FLNc when compared to the α-tubulin loading control indicates that in our p.Y2704X patient 84% of the normal FLNc level is expressed. The anti-CT antibody that does not recognize the p.Y2704X variant, detects only 38% of the level in normal vastus lateralis muscle. In p.Q1662X patient tissue, the level of FLNc recognized by both antibodies is decreased to 40–50% of the level in control tissue. (D) Total protein extracts from skeletal muscle biopsies from our p.Q1662X patient and control tissue were analyzed for the expression of FLNc derived from the wildtype allele (~291 kDa) and the mutant allele (~171 kDa, arrow) using an FLNc antibody recognizing the N-terminus (d1–2) of FLNc. Note that no extra band was observed in the extract from patient tissue, indicating no detectable levels of truncated protein expressed from the mutant allele.

Figure 6

Functional characterization of the p.Y2704X mutation. (A) Schematic illustrations of the effect of the p.Y2704X mutation. Topology of filamin C Ig-like domain 24 aligned to the amino acid sequence. The position of the mutation is marked with a red asterisk. Grey and black asterisks in panels A and B mark previously found mutations in this domain. (B) Two-dimensional view of domain 24. The interface between dimerized domains is formed by β-strands C and D form. In p.Y2704X, FLNc strand G is lacking together with strand F’ and a part of strand F. Strand G normally interacts with strand A, which is important for the coherence of the β-sheets comprising an Ig-like domain. (C) Three-dimensional view of an FLNc domain 24-dimer (PDB code: 1V05). β-strands (yellow) are numbered as in panels A and B (A–G for one monomer, and A’–G’ for the second monomer). The premature stop codon in p.Y2704X is shown by a blue asterisk. The part of the domain that is deleted is shown hatched in one of the monomers. (D) Chemical cross-linking experiments using EEF-tagged wild-type (WT) and T7-tagged p.Y2704Xfilamin fragments (domains 23 + 24), or a mixture of both polypeptides (WT + p.2704X as indicated above the panels. Assays were performed in the absence (−) or presence (+) of the cross-linker EGS. Blots were either stained with antibodies detecting the EEF-tag of WT FLNc d23–24 monomers (m) and dimers (d), or the T7-tag of the p.2704 variant as indicated. Note normal dimerization of the wild-type protein, also in the presence of the mutant variant, whereas p.Y2704X FLNc also forms large, aggregated oligomers. (E) Protein stability analysis. Wild-type and p.Y2704X filamin C fragments (d23–24) were treated with the protease thermolysin for 0 to 40 min, as indicated. Samples were analyzed by polyacrylamide gel electrophoresis. The mutant protein was already partially digested after 2 min, and the largest part of the protein after 40 min, while the wild-type variant was still almost completely intact after 40 min. This indicates less stable folding of the mutant protein.

Figure 7

Transcript studies and immunoblot analysis of PQS markers in skeletal muscle tissue. (A) We have analyzed multiple genes involved in autophagy-, proteasome- and unfolded protein response (UPR) -pathways. The mutations differentially affect gene expressions, with upregulation of, e.g., LC3 in the FLNc variant p.W2710X. However, both new mutations do not show strong dysregulation of autophagy markers on the RNA level. In contrast, the patient with the p.Q1662X mutation showed increased HSPA8 expression. (B) Furthermore, upregulation of UPR markers and proteasome-associated genes, compared with non-disease controls (NDC), which was observed in the p.W2710X mutant, was not detected in patients with p.Q1662X or p.Y2704X mutation. (C) Densitometric data of the western blot signals were normalized to the corresponding total protein amount determined from coomassie based total protein stain. Values are shown in relation of the signals of the patients (IBM n = 2, p.Q1662X, p.Y2704X and p.W2710X) and the values of the non-disease controls (n = 2), respectively. Analysis was performed in technical triplicates. (D) Representative protein of interest bands together with the corresponding marker bands are shown. (E) A representative Coomassie-stained SDS-polyacrylamide is displayed to verify the total protein amount for each protein sample. Error bars indicate the deviation of the arithmetic mean of independent experiments.

Figure 8

Immunolocalization studies of UPS markers. (A) Analysis of the distribution of CASA markers BAG3 CHIP, p62 and LC3B in double stained cryosections of muscle biopsies from patient I:1 (p.Q1662X) and (B) II:2 (p.Y2704X) using antibodies against FLNc (green) to localize aggregates and microlesions together with the aforementioned UPS-marker proteins (red). Scale bars: 50 µm.

Figure 9

Overview of FLNC mutations causing isolated myopathy or combined myopathy and cardiomyopathy. Disease-associated variants in FLNC were found throughout the whole protein. In the actin-binding domain (ABD) [43,44], and also in domain 2 [42], domain 4 [45], domain 7 [46,47,48], domain 10 [10,49,50,51,52], domain 15 [53,54], domain 20 [52], domain 21 [55], domain 22 [56], domain 23 [57] and domain 24 [5,51,58,59]. Domain 20 is colored green because it is extended and contains an 82 amino acid long insertion. Note that variants denominated as “variant of unknown significance” (VUS) are not listed. ^§ Recessive mutation; * combination of myopathy and cardiomyopathy; two different mutations at the genetic level. The novel mutations presented in this work are printed red.