Barrett, Philip Laurence; (2022) Nrg-1 effects on C2C12 differentiation: Towards generating Intrafusal muscle fibres in vitro. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Intrafusal fibres are a specialised population in skeletal muscle, found within the muscle spindle. These fibres have a mechano-sensory capacity, they detect and mediate static and dynamic muscle stretch and monitor muscle position, forming a key contribution to proprioception. Impairment of proprioception and associated dysfunction of the muscle spindle is linked with multiple neuromuscular diseases, aging and nerve injuries. To date, there is a paucity of publications focussed on de novo generation and characterisation of intrafusal muscle fibres in vitro and current skeletal muscle models lack integration of the afferent functions of the muscle spindle. It was hypothesised that de novo intrafusal myotubes could be generated and characterised from differentiated C2C12 myoblasts, utilising addition of recombinant Neuregulin 1, an essential molecule for intrafusal fibre development. Intrafusal bag myotubes have a distinctive fusiform shape and were characterised using novel morphological parameters, immunofluorescent microscopy and western blot analysis, directed against an extensive list of putative intrafusal specific markers, as identified in vivo. Nrg-1 supplementation resulted in a 5-fold increase in intrafusal bag myotubes, increased expression of the intrafusal specific transcription factor, Egr3 and significantly altered the expression of myosin heavy chains. Nrg-1 also upregulated proliferation of C2C12s, resulting in increased nuclei per image field of view. Following this, siRNA was employed to knockdown Nrg-1 mediated Egr3 expression and Mitomycin-c was applied to mitigate Nrg-1 mediated proliferation. Results indicate generation of intrafusal bag like morphologies occurs independently of Nrg-1 initiated Egr3 expression and proliferation in vitro. Key assays from the 2D work were replicated in a biomimetic 3D collagen system. Bag fibre morphology and myonuclei clustering were detected and myotubes displayed evidence of increased maturity, however, the expression patterns for intrafusal specific markers mimicked the 2D results. Force outputs following electrical field stimulation of 3D constructs were not indicative of an intrafusal fibre phenotype, casting doubt over the suitability of a monocellular C2C12 model for intrafusal fibres. This research provides the most in-depth characterisation and the first tissue engineering approach toward generating de novo intrafusal skeletal muscle. Future iterations of this model will provide platforms for developmental or disease state studies, pre-clinical screening, or clinical applications, which will likely provide novel therapeutic strategies to enhance patient care.

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