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.

ATOH8: a novel marker in human muscle fiber regeneration.

Regenerating muscle fibers emerge from quiescent satellite cells, which differentiate into mature multinuclear myofibers upon activation. It has recently been found that ATOH8, a bHLH transcription factor, is regulated during myogenic differentiation. In this study, expression and localization of ATOH8, the other well-described regeneration markers, vimentin, nestin and neonatal myosin, and the satellite cell marker Pax7 were analyzed on protein level in human myopathy samples by immunofluorescence studies. On mRNA level, expression levels of ATOH8 and vimentin were studied by quantitative real-time PCR. ATOH8 is expressed in activated satellite cells and proliferating myoblasts of human skeletal muscle tissue. Quantitative analyses of ATOH8+, Pax7+, vimentin+, nestin+ and neonatal myosin+ muscle fibers showed the highest amount of regenerating muscle fibers in inflammatory myopathies, followed by muscular dystrophy. The relative co-expression of ATOH8 with the above-mentioned markers did not vary among the disorders. These results show that the novel regeneration marker ATOH8 contributes to muscle cell differentiation in healthy and diseased human muscle tissue.

Two novel nebulin variants in an adult patient with congenital nemaline myopathy.

Congenital myopathies are clinically and genetically heterogeneous disorders, which often remain genetically undiagnosed for many years. Here we present a 40-year old patient with an almost lifelong history of a congenital myopathy of unknown cause. Muscle biopsy in childhood revealed mild myopathic features and rods. Clinical examination on presentation at the age of 40 revealed a facial weakness, atrophy and weakness of the arm muscles and distal leg muscles with mild contractures of the foot flexors and the right elbow. Subsequently, the nebulin gene was identified as a putative candidate gene by linkage analyses, but sequence analysis only revealed one heterozygous splice site mutation in intron 73 (c.10872+1G>T). Therefore, "Next Generation Sequencing" was performed, which revealed a second pathogenic variant in exon 145 (c.21622A>C). Compound-heterozygous carrier status was confirmed via sequence analysis of the index patient's parents. Whole body muscle MRI showed a muscle involvement as previously described in nebulin-associated myopathies. Based on biopsy material, genetic analyses and muscle MRI, we identified two novel, compound-heterozygous variants in the nebulin gene after a 30 year clinical history, which cause a classical childhood type of nemaline myopathy.