Skeletal muscle is heterogeneous in nature. In rodents, predominantly slow-twitch oxidative (soleus) and fast-twitch glycolytic (psoas and extensor digitorum longus, EDL) contain fibres that differ in both contractile and metabolic processes and consequently respond differently to cellular events and external stresses. Dysferlinopathies have been shown to affect various muscles and gender differentially and it has been suggested that there is a metabolic link in the disease manifestation.
Calibrated western blotting was used to determine the abundance of various proteins in whole muscles and individual muscle fibres.
The amount of dysferlin was similar between EDL, psoas and soleus muscles from male mice, with more present in soleus compared with EDL muscles form female mice. There was no difference in dysferlin between type I or type IIA fibres from either male or female mice. When mitochondrial proteins were examined, these were, as expected, highest in soleus muscle in both male and female mice. There was no correlation between dysferlin and mitochondrial proteins, suggesting that there is no link between metabolic capacity of a muscle and the amount of dysferlin in muscle from mice.
When rat EDL and soleus muscles were compared, there was more dysferlin in the oxidative soleus muscles compared with EDL muscles. Examination of individual fibres showed more dysferlin in type I compared with type IIA fibres but no difference between type IIA, IIB and IIX fibres. The type I and IIa fibre-type dependent expression of dysferlin in rat was opposite to that seen for the mitochondrial protein COXIV, suggesting a negative association between the amount of dysferlin and oxidative capacity, although the linear regression did not reach significance (p=0.07).
Using micro-dissection techniques, we removed the surface membrane from half the length of isolated individual fibre segments from rat muscle, allowing us to compare the dysferlin in an intact region and a ‘skinned’ region of the same muscle fibre. We found ~85% of the total dysferlin was in the skinned fibre, that is the region of muscle that did not have the surface membrane. This indicates that the majority of the dysferlin is likely situated at the t-system as previously shown using immunofluorescence (Roche et al, 2011). We also excised samples that contained only the surface membrane and the dysferlin present was below the lowest point on the calibration curves used, which amounted to ~15% of the total fibre. The extracellular matrix protein, laminin, was detected in these sarcolemmal samples.
Overall, these findings demonstrate the importance of examining muscle at the individual muscle fibre level. Given the disparity in the dysferlin abundance in muscle fibre types dissected from mouse and rat, human research is required to reveal the most appropriate model and muscle selection for analyses of endogenous dysferlin in further research. A further finding is that a major role for dysferlin must be associated with non-sarcolemmal regions of muscle fibres.