Thin Filament Length in Mouse Skeletal Muscle and its Relationship to Differential Splicing of Nebulin

Nebulin is a large actin binding protein (600-800 kDa) which undergoes differential splicing to yield proteins of various sizes in different skeletal muscles. This giant protein has been proposed to function as a molecular ruler to determine the length of the thin filament in skeletal muscle. Using electron microscopy, we measured the length of the thin filament in mouse skeletal muscle. It was found that the length of the thin filament was greatest in the soleus (1.25 ± 0.004, N=136), shortest in the psoas major muscle (1.20 ± 0.011, N=102), and intermediate in the tibialis cranialis muscle (1.22 ± 0.005, N=161). Next, we estimated the molecular weight of nebulin in these tissues to assess if nebulin size correlates with thin filament length. In mice, nebulin size was found to vary between 700kDa in the psoas major, extensor digitus longdum and gastrocnemius to 750kDa in the soleus and diaphragm muscle while intermediate lengths were found in quadriceps and tibialis cranialis muscle. Since there was a positive correlation between thin filament length and nebulin size, we next sought to determine if the differences in nebulin size could be explained by differential splicing of nebulin between mouse skeletal muscles. Using a home-made nebulin exon microarray, we compared the exon composition between these muscles and found that the soleus muscle nebulin isoforms contains M-repeats in the Z-disk (exons 153-155) while nebulin isoforms in other muscle types do not contain these repeats. This data has been confirmed by qPCR. In conclusion, the changes in nebulin size between mouse skeletal muscles can partially be explained by differential splicing of the nebulin gene, and further studies are needed to ascertain in the mouse the correlation between nebulin size and thin filament length.