Target formation in muscle fibres indicates reinnervation - A proteomic study in muscle samples from peripheral neuropathies.

AIMS: Target skeletal muscle fibres - defined by different concentric areas in oxidative enzyme staining - can 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 remodelling within this specific area of the muscle fibres. We speculate that target structures are the result of reinnervation processes in which filamin C-associated myofibrillogenesis is tightly regulated by the BAG3-associated protein quality system.

A combined laser microdissection and mass spectrometry approach reveals new disease relevant proteins accumulating in aggregates of filaminopathy patients.

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 actin-binding 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.

Intestinal stroma guides monocyte differentiation to macrophages through GM-CSF.

Stromal cells support epithelial cell and immune cell homeostasis and play an important role in inflammatory bowel disease (IBD) pathogenesis. Here, we quantify the stromal response to inflammation in pediatric IBD and reveal subset-specific inflammatory responses across colon segments and intestinal layers. Using data from a murine dynamic gut injury model and human ex vivo transcriptomic, protein and spatial analyses, we report that PDGFRA+CD142-/low fibroblasts and monocytes/macrophages co-localize in the intestine. In primary human fibroblast-monocyte co-cultures, intestinal PDGFRA+CD142-/low fibroblasts foster monocyte transition to CCR2+CD206+ macrophages through granulocyte-macrophage colony-stimulating factor (GM-CSF). Monocyte-derived CCR2+CD206+ cells from co-cultures have a phenotype similar to intestinal CCR2+CD206+ macrophages from newly diagnosed pediatric IBD patients, with high levels of PD-L1 and low levels of GM-CSF receptor. The study describes subset-specific changes in stromal responses to inflammation and suggests that the intestinal stroma guides intestinal macrophage differentiation.

Identification of Xin-repeat proteins as novel ligands of the SH3 domains of nebulin and nebulette and analysis of their interaction during myofibril formation and remodeling.

The Xin actin-binding repeat-containing proteins Xin and XIRP2 are exclusively expressed in striated muscle cells, where they are believed to play an important role in development. In adult muscle, both proteins are concentrated at attachment sites of myofibrils to the membrane. In contrast, during development they are localized to immature myofibrils together with their binding partner, filamin C, indicating an involvement of both proteins in myofibril assembly. We identify the SH3 domains of nebulin and nebulette as novel ligands of proline-rich regions of Xin and XIRP2. Precise binding motifs are mapped and shown to bind both SH3 domains with micromolar affinity. Cocrystallization of the nebulette SH3 domain with the interacting XIRP2 peptide PPPTLPKPKLPKH reveals selective interactions that conform to class II SH3 domain-binding peptides. Bimolecular fluorescence complementation experiments in cultured muscle cells indicate a temporally restricted interaction of Xin-repeat proteins with nebulin/nebulette during early stages of myofibril development that is lost upon further maturation. In mature myofibrils, this interaction is limited to longitudinally oriented structures associated with myofibril development and remodeling. These data provide new insights into the role of Xin actin-binding repeat-containing proteins (together with their interaction partners) in myofibril assembly and after muscle damage.

Complete loss of murine Xin results in a mild cardiac phenotype with altered distribution of intercalated discs.

AIMS: Xin is a striated muscle-specific F-actin binding protein that has been implicated in cardiomyopathies. In cardiomyocytes, Xin is localized at intercalated discs (IDs). Mice lacking only two of the three Xin isoforms (XinAB(-/-) mice) develop severe cardiac hypertrophy. To further investigate the function of Xin variants in the mammalian heart, we generated XinABC(-/-) mice deficient in all Xin isoforms. METHODS AND RESULTS: XinABC(-/-) mice showed a very mild phenotype: heart weight, heart weight to tibia length ratios, and cardiac dimensions were not altered. Increased perivascular fibrosis was only observed in hearts of young XinABC(-/-) mice. Striking differences were revealed in isolated cardiomyocytes: XinABC(-/-) cells demonstrated a significantly increased number of non-terminally localized ID-like structures. Furthermore, resting sarcomere length was increased, sarcomere shortening, peak shortening at 0.5-1 Hz, and the duration of shortening were decreased, and shortening and relengthening velocities were accelerated at frequencies above 4 Hz in XinABC(-/-) cardiomyocytes. ECG showed a significantly shorter HV interval and a trend towards shorter QRS interval in XinABC(-/-) mice, suggesting a faster conduction velocity of the ventricular-specific conduction system. In human cardiac tissue, expression of XinC protein was detected solely in samples from patients with cardiac hypertrophy. CONCLUSION: 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.

Unusual splicing events result in distinct Xin isoforms that associate differentially with filamin c and Mena/VASP.

Filamin c is the predominantly expressed filamin isoform in striated muscles. It is localized in myofibrillar Z-discs, where it binds FATZ and myotilin, and in myotendinous junctions and intercalated discs. Here, we identify Xin, the protein encoded by the human gene 'cardiomyopathy associated 1' (CMYA1) as filamin c binding partner at these specialized structures where the ends of myofibrils are attached to the sarcolemma. Xin directly binds the EVH1 domain proteins Mena and VASP. In the adult heart, Xin and Mena/VASP colocalize with filamin c in intercalated discs. In cultured cardiomyocytes, the proteins also localize in the nonstriated part of myofibrils, where sarcomeres are assembled and an extensive reorganization of the actin cytoskeleton occurs. Unusual intraexonic splicing events result in the existence of three Xin isoforms that associate differentially with its ligands. The identification of the complex filamin c-Xin-Mena/VASP provides a first glance on the role of Xin in the molecular mechanisms involved in developmental and adaptive remodeling of the actin cytoskeleton during cardiac morphogenesis and sarcomere assembly.