No abstract

Filaminopathy is a subtype of myofibrillar myopathy caused by mutations in FLNC, the gene encoding filamin C, and histologically characterized by pathologic accumulation of several proteins within skeletal muscle fibers. With the aim to get new insights in aggregate composition, we collected aggregates and control tissue from skeletal muscle biopsies of six myofibrillar myopathy patients harboring three different FLNC mutations by laser microdissection and analyzed the samples by a label-free mass spectrometry approach. A total of 390 proteins were identified, and 31 of those showed significantly higher spectral indices in aggregates compared with patient controls with a ratio >1.8. These proteins included filamin C, other known myofibrillar myopathy associated proteins, and a striking number of filamin C binding partners. Across the patients the patterns were extremely homogeneous. Xin actin-binding repeat containing protein 2, heat shock protein 27, nebulin-related-anchoring protein, and Rab35 could be verified as new filaminopathy biomarker candidates. In addition, further experiments identified heat shock protein 27 and Xin actin-binding repeat containing protein 2 as novel filamin C interaction partners and we could show that Xin actin-binding repeat containing protein 2 and the known interaction partner Xin actinbinding repeat containing protein 1 simultaneously associate with filamin C. Ten proteins showed significant lower spectral indices in aggregate samples compared with patient controls (ratio <0.56) including M-band proteins myomesin-1 and myomesin-2. Proteomic findings were consistent with previous and novel immunolocalization data. Our findings suggest that aggregates in filaminopathy have a largely organized structure of proteins also interacting under physiological conditions. Different filamin C mutations seem to lead to almost identical aggregate compositions. The finding that filamin C was detected as highly abundant protein in aggregates in filaminopathy indicates that our proteomic approach may be suitable to identify new candidate genes among the many MFM patients with so far unknown mutation. Molecular & Cellular Proteomics

Total Xin deficiency leads to topographical ID alterations, premature fibrosis and subtle changes in contractile behaviour; this is a milder cardiac phenotype than that observed in XinAB(-/-) mice, which still can express XinC. Together with the finding that XinC is detected solely in cardiomyopathic human tissues, this suggests that its expression is responsible for the stronger dominant phenotype in XinAB(-/-) mice. Furthermore, it indicates that XinC may be involved in the development of human cardiac hypertrophy.

The striated muscle–specific actin-binding proteins Xin and Xirp2 are identified as novel ligands of the SH3 domains of the thin filament ruler nebulin and nebulette. The interaction is spatially restricted to structures associated with myofibril development or remodeling, indicating a role for these proteins in myofibril assembly and repair.

Aims: Target skeletal muscle fibresdefined by different concentric areas in oxidative enzyme stainingcan occur in patients with neurogenic muscular atrophy. Here, we used our established hypothesis-free proteomic approach with the aim of deciphering the protein composition of targets. We also searched for potential novel interactions between target proteins.Methods: Targets and control areas were laser microdissected from skeletal muscle sections of 20 patients with neurogenic muscular atrophy. Samples were analysed by a highly sensitive mass spectrometry approach, enabling relative protein quantification.The results were validated by immunofluorescence studies. Protein interactions were investigated by yeast two-hybrid assays, coimmunoprecipitation experiments and bimolecular fluorescence complementation.Results: More than 1000 proteins were identified. Among these, 55 proteins were significantly over-represented and 40 proteins were significantly under-represented in targets compared to intraindividual control samples. The majority of over-represented proteins were associated with the myofibrillar Z-disc and actin dynamics, followed by myosin and myosin-associated proteins, proteins involved in protein biosynthesis and chaperones.Under-represented proteins were mainly mitochondrial proteins. Functional studies revealed that the LIM domain of the over-represented protein LIMCH1 interacts with isoform A of Xin actin-binding repeat-containing protein 1 (XinA). Conclusions:In particular, proteins involved in myofibrillogenesis are over-represented in target structures, which indicate an ongoing process of sarcomere assembly and/or