Sporadic inclusion body myositis (sIBM) is the most commonly acquired myopathy in middle-aged and elderly people. The muscle histology is characterized by both inflammation and degeneration, including sarcoplasmic aggregation of TDP-43. Cylindromatosis (CYLD) is a deubiquitinating enzyme that targets Lys63-linked ubiquitin chains and negatively regulates signal transduction pathways, such as NF-κB signalling pathways. We examined localization of CYLD as well as phosphorylated TDP-43, phosphorylated p62, and Lys63-linked ubiquitin in muscle tissues of sIBM patients and muscle-specific wild-type TDP-43 transgenic (TDP-43 TG) mice. We investigated whether overexpression of CYLD can affect muscle toxicity in the cell models treated by endoplasmic reticulum (ER) stress inducers tunicamycin and thapsigargin. CYLD expressed with phosphorylated TDP-43, phosphorylated p62, and Lys63-linked ubiquitin in the nuclear and perinuclear regions of muscle fibres of wild-type TDP-43 TG mice and the degenerative myofibres of sIBM patients with rimmed vacuoles and endomysial cellular infiltration. Although expression levels of CYLD decreased and cell viability was reduced in cells treated with ER stress inducers, wild-type CYLD, but not the catalytic mutant, substantially improved cell viability based on the deubiquitinase activity. Dysregulation of CYLD may reinforce myodegeneration in the pathophysiology of sIBM by attenuating autophagic clearance of protein aggregates. Regulating CYLD in muscle fibres might serve as a novel therapeutic strategy for sIBM treatment.

Contractile activity is a fundamental property of skeletal muscles. We describe the establishment of a "feeder-supported in vitro exercise model" using human-origin primary satellite cells, allowing highly-developed contractile myotubes to readily be generated by applying electrical pulse stimulation (EPS). The use of murine fibroblasts as the feeder cells allows biological responses to EPS in contractile human myotubes to be selectively evaluated with species-specific analyses such as RT-PCR. We successfully applied this feeder-supported co-culture system to myotubes derived from primary satellite cells obtained from sporadic inclusion body myositis (sIBM) patients who are incapable of strenuous exercise testing. Our results demonstrated that sIBM myotubes possess essentially normal muscle functions, including contractility development, de novo sarcomere formation, and contraction-dependent myokine upregulation, upon EPS treatment. However, we found that some of sIBM myotubes, but not healthy control myotubes, often exhibit abnormal cytoplasmic TDP-43 accumulation upon EPS-evoked contraction, suggesting potential pathogenic involvement of the contraction-inducible TDP-43 distribution peculiar to sIBM. Thus, our "feeder-supported in vitro exercise model" enables us to obtain contractile human-origin myotubes, potentially utilizable for evaluating exercise-dependent intrinsic and pathogenic properties of patient muscle cells. Our approach, using feeder layers, further expands the usefulness of the "in vitro exercise model".

Myositis is characterised by muscle inflammation and weakness. Although generally thought to be driven by a systemic autoimmune response, increasing evidence suggests that intrinsic changes in the muscle might also contribute to the pathogenesis. Long non-coding RNAs (lncRNAs) are a family of novel genes that regulate gene transcription and translation. To determine the potential role of lncRNAs, we employed next generation sequencing to examine the transcriptome in muscle biopsies obtained from two histologically distinct patient populations, inclusion body myositis (IBM) and anti-Jo-1-associated myositis (Jo-1). 1287 mRNAs and 1068 mRNAs were differentially expressed in the muscle from Jo-1 and IBM patients, respectively. Pathway analysis showed the top canonical pathway in both Jo-1 and IBM was oxidative phosphorylation and mitochondrial dysfunction. We identified 731 known and 325 novel lncRNAs in the muscles biopsies. Comparison with controls showed 55 and 46 lncRNAs were differentially expressed in IBM and Jo-1 myositis, respectively. Of these, 16 lncRNAs were differentially expressed in both IBM and Jo-1 myositis and included upregulated H19, lncMyoD and MALAT1. Given that these are known to regulate muscle proliferation and differentiation, we speculate that changes in lncRNAs might contribute to the phenotypic changes in Jo-1 and IBM myositis.

Sporadic inclusion body myositis (sIBM) is an idiopathic inflammatory myopathy with invasion of CD8 T cells in muscle and aggregation of proteins in the sarcoplasm. TDP-43 and p62 are two proteins that aggregate in affected muscle, and have been suggested as specific markers for sIBM over other inflammatory myopathies. TDP-43 is also mislocalised from the nucleus to the sarcoplasm in sIBM. It is not clear if inflammation precedes protein aggregation in sIBM. This study investigated if exposure to cytotoxic inflammatory cells caused TDP-43 and p62 aggregation or TDP-43 mislocalisation in cultured myotubes. TALL-104 coculture was highly cytotoxic to myotubes after 24 h. Secretion of IFNγ and TNFα were higher in cocultures compared to monocultured TALL-104 cells, indicating activation. TALL-104 cells attached to and infiltrated myotubes. There was no effect of TALL-104 coculture on TDP-43 or p62 sarcoplasmic aggregate size or frequency. However, there was decreased localisation of TDP-43 to the nucleus with TALL-104 coculture compared to control. In an in vitro setting, cytotoxic immune cells did not cause TDP-43 or p62 sarcoplasmic aggregation, suggesting cellular cytotoxicity may not trigger aggregation of these proteins. However TALL-104 coculture influenced TDP-43 localisation, suggesting cytotoxic immune cells may contribute to TDP-43 localisation shifts which is observed in sIBM.