SSA - Understanding skeletal muscle plasticity.

Lead Research Organisation: University of Leeds
Department Name: Astbury Centre

Abstract

Skeletal muscle is a plastic organ, in that muscle fibres respond and change to many external influences by remodelling their cytoskeleton. The initial changes that occur in vivo can be recapitulated in cutured skeletal muscle cells, allowing us to test early signalling pathways that define how muscle adapts to change. These changes are important to understand, as they provide insight into health ageing, and the effects of exercise on muscle maintenance.
The aim of this project is to understand the changes that occur in vivo, using model systems that either overload muscle, or change stimulation to the muscle, through investigations both using the in vivo system, and the in vitro culture system. By determining how muscle changes and adapts as a result of stimulation/overload, we hope to learn useful rules that we can apply to the culture system, to improve systems for in vitro muscle culture, including improved culture surfaces, and stimulation regimes.
Our overall aims are to:
Explore how satellite cells (muscle stem cells) are stimulated to contribute to muscle responses to load and stimulation in vivo, including isolation of the satellite cells, and characterisation of their properties. The student will learn how to perform in vivo manipulations, how to isolate muscles and analyse them by serial sectioning and light microscopy, and how to isolate and analyse satellite cells.
Analyse available RNAseq data for muscle fibres, to determine which factors contribute to their responses to load or changes in stimulation, and subsequent fibre remodelling. The student will learn how to handle RNAseq data, and how to analyse this type of data to make predictions.
Test the roles of these factors both in vivo and in the in in vitro culture system, to understand how they contribute to skeletal muscle fibre remodelling. The student will learn how to culture muscle cells, and perform genetic manipulations to test effects of specific factors on differentiation, and will develop novel approaches to tissue culture to improve differentiation in culture

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M011151/1 01/10/2015 30/09/2023
1940655 Studentship BB/M011151/1 01/10/2017 30/09/2021 Louise Richardson
 
Description I've explored new ways to culture skeletal muscle cells, advancing on the conventional method of culture on hard glass surfaces. I cultured cells on soft surfaces, made with a silicone material of the same elasticity as natural skeletal muscle. Compared to standard glass surfaces, cells on the soft surfaces divided and grew into mature skeletal muscle cells (myotubes) more efficiently. This was ascertained by carrying out a proliferation assay and fusion index to find rates of division and fusion with other cells, respectively. I also used antibodies to probe for proteins that are expressed by the cells that aid in the development of immature muscle cells into adult, and I found that these proteins were expressed in greater abundance by cells on the soft surfaces. I am currently in the middle of analysing some sequencing data from cells grown on both surfaces, to compare gene expression profiles. I will then see if this compliments the findings from the antibody staining investigations.

I am also looking at muscle plasticity using mouse models. To replicate stimulated muscle growth (as seen from e.g. lifting weights in a gym), I performed surgery to remove the tibialis anterior in the leg, forcing the synergistic extensor digitorum longus (EDL) to work harder in its absence. This causes a mild hypetrophy of the EDL. I looked at the individual muscle fibres from loaded and unloaded EDL, and I found a greater overall number of muscle stem cells (satellite cells) on loaded fibres. This suggests greater division of the stem cells, to create more new muscle cells, aiding the growth of the muscle. When probing the fibres with antibodies, I found greater numbers of satellite cells expressing proteins that are involved in muscle development in loaded EDL. Additionally, the cross-sectional area of the fibres is increased too.
Exploitation Route The findings warrant discussion of the use of modified cell culture surfaces as standard, as opposed to glass. Further research can be carried out to modify the special silicone surfaces further, experimenting with different coatings, and perhaps adding micropatterned grooves to further emulate the texture of skeletal muscle in vivo.

If I find any particular genes that have been over or underexpressed on modified vs conventional surfaces, there can be explored further by other researchers. I will be exploring pathways of interest to an extent with gene ontology analysis, however more detail can be obtained with immunofluorescence, knockout experiments etc.
Sectors Pharmaceuticals and Medical Biotechnology