Development and Characterisation of Skeletal Muscle Models for Secretome Investigation

Exercise significantly reduces the risk of a spectrum of diseases and is increasingly prescribed as a therapeutic intervention, particularly for the management of chronic conditions including diabetes, cardiovascular diseases, musculoskeletal diseases, and neurological and psychiatric disorders. The skeletal muscle (SkM) secretome encompasses several hundred secreted proteins, forming a conceptual basis for understanding multi-organ crosstalk. During SkM contraction, myokines are secreted as proteins and proteoglycan peptides of approximately 5–35 kDa which mediate the protective and beneficial effects of exercise. Given the pivotal role of myokines, investigating their secretion patterns and responses to exercise-like stimuli is key to enhancing their therapeutic potential. The complexity and redundancy of exercise as a biological stimulus, combined with the challenges of isolating specific myokine effects, necessitates in vitro models. Tissue-engineered SkM models were developed using a C2C12 murine myoblast cell line. From this, a foundational 2D SkM model and a novel tensile-strained 3D SkM construct was developed and characterised by structural and developmental myogenic markers. Histological and immunofluorescence analysis revealed aligned SkM fibres along a tension axis and deformation in the 3D construct. The application of electrical pulse stimulation (EPS) induced contraction and promoted myokine secretion. Nano-liquid chromatography mass spectrometry (nano-LCMS) facilitated the detection and profiling of the low-abundance myokines, validated through multiplex immunoassay (MIA). Untargeted data analysis revealed up to 1210 proteins from a single model. Secretome mining identified a putative novel myokine, serotransferrin (sTf), which contributes to the myokinome and demonstrates the effectiveness of the applied analytical and bioinformatics methodologies. Temporal myokine profiles, both transient and sustained, were observed in stimulated and unstimulated models, persisting up to 72 hours. Additionally, endogenous myokine secretion was observed in the in vitro models, which contributed to a baseline or spontaneous secretion profile. The research presented in this thesis, developed a novel 3D muscle model where EPS stimulation effectively modulated myokine secretion, partially replicating the pleiotropic effects of exercise. Understanding how stimulation duration influences post-contraction myokine release offers valuable insights into the long-term effects of exercise on systemic health, including its role in chronic disease management, tissue regeneration, and sustained metabolic adaptations.