Browsing by Author "Hagemann, Kyle Nicholas"

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    Self-talk vs crosstalk: extracellular particles derived from myoblasts and fibroblasts and their effects on myoblast function and related myogenic regulatory factors
    (Stellenbosch : Stellenbosch University, 2024-03) Hagemann, Kyle Nicholas; Myburgh, Kathryn Helen; McColl, Rhys; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: In the intricate environment of intercellular communication, small extracellular vesicles (EVs) have emerged as mediators, facilitating the transfer of bioactive molecules and influencing recipient cell behaviour. Myoblasts, the precursors of muscle cells, and fibroblasts are both found in the muscle niche. They release small EVs that play a crucial yet incompletely understood role in orchestrating various aspects of muscle biology. This study delves into the interactions between myoblast- and fibroblast-derived extracellular particles (myo-EPs and fibro-EPs) and myoblast function, with a specific focus on uptake, proliferation, the SMAD pathway, and cellular migration. Myoblast (C2C12) and fibroblast (L929) EPs were harvested from conditioned media using differential ultracentrifugation and characterised according to guidelines (MISEV 2018). Their effects on myoblasts were analysed using a combination of light and confocal microscopy, and western blotting. Four conditions were compared: Normal growth media (NGM), EP-depleted media (EP-DM) control, Myo-EP and Fibro-EP. Preferential uptake of myo-EPs into myoblasts after 48 hours was shown (EP-DM control: 0.9396 spots per cell ± 0.6313 spots per cell; Myo-EP: 16.55 spots per cell ± 12.60 spots per cell; Fibro-EP: 9.668 spots per cell ± 4.882 spots per cell). No effect of EPs on proliferation was observed over 48 hours for either Myo-EPs or Fibro-EPs. Both EP types were shown to contain TGF-β, an activator of the SMAD pathway. Follistatin was also found in the myo-EPs. There was no change in the SMAD pathway associated proteins p-SMAD2/3 (NGM: 1; EP-DM: 1.328 ± 0.5896; Myo-EP: 1.149 ± 0.5299; Fibro- EP: 0.8192 ± 0.3035) or SMAD6/7 (NGM: 1; EP-DM control: 1.033 ± 0.2187; Myo- EP: 1.059 ± 0.4896; Fibro-EP: 1.000 ± 0.4976) expression after 48 hours. There was no change in myoD expression (NGM: 1; EP-DM: 1.489 ± 0.5930; Myo-EP: 1.561 ± 0.7696; Fibro-EP: 1.561 ± 0.1693), however myogenin expression was significantly increased in the three conditions compared to the NGM (NGM: 1; EP-DM: 7.397 ± 1.913; Myo-EP: 7.578 ± 2.073; Fibro-EP: 8.024 ± 1.987). Myoblast migration was altered by fibro-EPs, with these myoblasts having a more directionless migration pattern (Rayleigh p-values: NGM: p < 6.90244E-11; EP-DM control: p < 4.58659E-08; Myo-EP: p < 2.1185E-12; Fibro-EP: p < 4.76142E-07). Cellular communication is crucial for normal cell function, and small EVs have come to be known as an integral mediator of this communication. In this study, we indicated preferential uptake of myoblast EPs into myoblasts over fibroblast EPs. Although uptake was shown, these EPs had little effect on myoblast proliferation, as well as SMAD pathway associated proteins within the time course assessed. Cell migration characteristics were also observed, and it was shown that fibroblast EPs decreased the directionality of C2C12 myoblasts.