Skeletal muscle is heterogeneous in nature, with slow, oxidative (type I) fibres and fast, oxidative or glycolytic (type II) fibres being present in many muscles. These fibres differ in both contractile and metabolic processes and consequently respond differently to cellular events and external stresses. The fibre types of the muscle fibres are determined by the presence of the predominant myosin heavy chain (MHC I, IIa, IIx/b) isoform. Dysferlinopathies have been shown to affect various muscles and gender differentially. This project will address muscle heterogeneity in males and females and will provide insight into disease mechanisms.
The abundance of dysferlin protein is fibre type specific when examining predominantly slow-twitch oxidative (soleus) and fast-twitch glycolytic (extensor digitorum longus, EDL) muscles in both rat and mouse skeletal muscle. Surprisingly, however, the dysferlin abundance is highest in EDL muscle in mice, but highest in soleus muscle in rats. This project will provide an in-depth analysis of the fibre type abundance of dysferlin in rat and mouse muscle fibres using quantitative western blotting, ie. calibration curves used. Alongside dysferlin, other proteins important in metabolic regulation and in contractile function will be analysed in the same samples to observe potential relationships.
The project will use micro-dissection techniques to (i) isolate individual fibre segments, and (ii) to isolate the surface membrane (sarcolemma) of a segment of muscle fibre. Samples will then be quantitively analysed by western blotting for the proteins present, in particular dysferlin. This will be in both individual muscle fibres with known fibre type assignment and the pure sarcolemma portion which will be stated as the amount of dysferlin present relative to the rest of the fibre. The biochemical measures importantly analyse a muscle fibre in its entirety but separated into two portions and so the proportion of dysferlin at the sites will be known in absolute relative terms. Further, intact and skinned fibre segment preparations will be assessed using immunofluorescence, validating the location of dysferlin.