Correlation of myosin heavy chains with ATPase staining of skeletal- and cardiac-muscle fibres

Abstract

Different muscle types are known to contain both structurally and functionally different forms of myosin (Margeth et al., 1980). Heterogeneity has been demonstrated in both of the structural elements which make up the myosin molecule, the light chains, which comprise about 20%of the molecule, and the heavy chains, which are the major part of the molecule and contain the site of ATPase activity. Although immunological methods and techniques of enzymic digestion (Whalen et al., 1981) have been used to demonstrate that differences exist between heavy chains, electrophoretic methods show different myosin heavy chains directly (Carrero et al., 1982; Perrie et al., 1982). Perrie at al. (1982) used electrophoresis to show that differences in myosin heavy chains were matched by variations in staining (Padykula & Herman, 1955) for ATPase activity of cryostat sections of muscles from which the myosins have been extracted. We used essentially the same method with a more dilute buffer to reduce the time of electrophoresis and to obtain more compact bands on the gel. It was then possible to separate myosin heavy chains from skeletal muscles into three bands but sections from these muscles were only differentiated into two fibre types by the histochemical ATPase reaction. However, if sections of rat muscle are preincubated at pH4.3 before incubation with ATP (Brooke & Kaiser, 1970), not only does a reversal of the staining of muscle fibres for ATPase activity occur but some fibres stain an intermediate grey between the extremes of black (type 1) and unstained (type 2) fibres (Fig. 1). We found that the three myosin heavy chain bands separated by electrophoresis correlated with the three intensities of ATPase staining of muscle fibres under these conditions. The fastest migrating heavy chain band corresponded to the fibres which stained with greatest intensity (black), the slowest band to the fibres stained an intermediate grey and the middle band to the fibres which hardly stained at all. Although the myosin heavy chain from cardiac muscle was not resolved from the skeletal muscle type of greatest mobility (Fig. I ) , co-electrophoresis did show a widening of the combined band. Subsequently we were able to show that with 3M-urea in the gel the bands were not identical and cardiac myosin was distinct from the skeletal muscle forms. However, although whole myosin from rat ventricle can be resolved into three bands (Lambert et al., 1983), we were only able to show one, apparently identical, myosin heavy chain band from both the auricle and ventricle of rat heart. The separation of three myosin heavy chains from skeletal muscle would seem to be compatible with the results of Whalen et al. (1981) who demonstrated three heavy chain isoenzymes in developmental studies on the rat. Under our electrophoretic conditions, the slowest migrating heavy chain from human skeletal muscle appears to be identical with the single band found in gastrocnemius muscle from the 20week foetus. The use of frozen sections dissolved in 0.1% SDS as electrophoresis samples was originally designed to eliminate the possibility of modification of myosin during the extraction procedure with the production of spurious