Investigating the non-immune cell mediated mechanisms of skeletal muscle weakness in myositis; developing an investigative platform to study human diseases

Thoma, Anastasia (2022) Investigating the non-immune cell mediated mechanisms of skeletal muscle weakness in myositis; developing an investigative platform to study human diseases. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

Idiopathic inflammatory myopathies, collectively termed as myositis, are a group of heterogeneous rare autoimmune muscle diseases, described by muscle weakness and fatigue leading to poor quality of life. Widely recognised by its immune mediated features, its current treatment focuses on immunosuppressive therapies, which however, seem to be ineffective in treating muscle weakness. Growing evidence support the involvement of non-immune mediated mechanisms including ER stress and mitochondrial impairements, as well as myokines up- or down-regulation, with major histocompatibility complex class I (MHC-I), a prominent myositis feature, being suggested to initiate those events. Thus, there is a great necessity for investigating the non-immune mediated mechanisms underlying myositis pathophysiology, providing the foundation for developing novel targeted therapeutic approaches. This thesis investigated the non-immune mediated mechanisms, focusing on mitochondrial bioenergetics and biodynamics, and reactive oxygen species generation, in three human in vitro models of myositic muscle: (a) pharmacological cell model of ER stress; (b) genetically modified cell model of MHC-I overexpression in presence or absence of type I interferons (IFNs); and (c) clinical cell model of primary human myositis skeletal muscle cell line. Lastly, the downstream mechanisms of the myokines secretome derived from the primary myositis cell line was assessed. Tunicamycin-induced ER stress activation led to an overall increase in mitochondrial respiration, potentially as an adaptive response to stress; however, mitochondrial dysfunction was evident by decreases in respiratory flux control ratios and mitochondrial membrane potential. A similar trend of mitochondrial changes was seen in IBM and DM primary cells, with effects being more drastic in DM compared to IBM primary cells. MHC-I overexpression caused significant depletion in mitochondrial respiration, accompanying by reduced respiratory flux control ratios and mitochondrial membrane potential. Interestingly, those effects seemed to exacerbate in presence of type I IFNs, suggesting the strong combinational effects of MHC-I and type I IFNs. MHC-I-induced effects were similar to those following exposure to myositic conditioned media, where DM conditioned media collected from DM primary cells showed greater mitochondrial respiratory deficiency compared to IBM primary cells-derived conditioned media. An interesting finding was that antioxidant intervention, using Eukarion-134, was able to amend aspects of mitochondrial dysfunction in those models, highlighting the potential role of ROS accumulation in myositis. Overall, this thesis has provided numerous models that can be used for further investigating the mechanisms involved in myositis pathogenesis and identify potential therapeutic targets. The findings suggest the involvement of multiple mechanisms that can be overlapping, but also act independently in inducing mitochondrial impairments, leading to muscle weakness in myositis. Lastly, these studies have indicated a possible role of ROS generation in mediating aspects of ER stress and mitochondrial abnormalities, suggesting that targeting ROS accumulation could be a promising therapeutic strategy for the persistent muscle weakness in individuals with myositis.