Fiber type-specific immunostaining of the Na+,K+-ATPase subunit isoforms in skeletal muscle: age-associated differential changes.

The expression of the Na+,K+-ATPase α and β subunit isoforms in rat skeletal muscle and its age-associated changes have been shown to be muscle-type dependent. The cellular basis underlying these findings is not completely understood. In this study, we examined the expression of Na+,K+-ATPase isoforms in individual fiber types and tested the hypothesis that, with age, the changes in the expression of the isoforms differ among individual fibers. We utilized immunohistochemical techniques to examine the expression of the subunit isoforms at the individual fiber levels. Immunofluorescence staining of the subunit isoforms in both white gastrocnemius (GW) and red gastrocnemius (GR) revealed a predominance of staining on the sarcolemmal membrane. Compared to the skeletal muscle of 6-month-old rats, there were substantial increases in the levels of α1, β1, and β3 subunit isoforms, and decreases in the levels of α2 and β2 in 30-month-old rats. In addition, we found distinct patterns of staining for the α1, α2, β1, and β2 isoforms in tissue sections from young and aged rats. Muscle fiber-typing was performed to correlate the pattern of staining with specific fiber types. Staining for α1 and α2 isoforms in the skeletal muscle of young rats was generally evenly distributed among the fibers of GW and GR, with the exception of higher α1 levels in slow-twitch oxidative Type I fibers of GR. By contrast, staining for the β1 and β2 isoforms in the mostly oxidative fibers and the mostly glycolytic fibers, respectively, was almost mutually exclusive. With age, there was a fiber-type selective qualitative decrease of α2 and β2 in Type IIB fibers, and increase of β1 in Type IIB fibers and β2 in Type IID fibers of white gastrocnemius. These results provide, at the individual fiber level, a cellular basis for the differential expression of the Na+,K+-ATPase subunit isoforms in the muscle groups. The data further indicate that the aged-associated changes in expression of the subunit isoforms occur in both a fiber-type specific as well as an across fiber-type manner. Because of the differing biochemical properties of the subunit isoforms, these changes add another layer of complexity in our understanding of the adaptation of the Na-pump in skeletal muscle with advancing age.