Leucine/glutamic acid/lysine protein 1 is localized to subsets of myonuclei in bovine muscle fibers and satellite cells

Abstract

Skeletal muscle growth is accomplished chiefly through the actions of satellite cells, a heterogeneous population that includes the adult muscle stem cell. Located adjacent to a mature muscle fiber, satellite cells typically reside in a quiescent state. Little information exists detailing satellite cell regulation of reversible G(0). One member of the mitosin family of centromere proteins, LEK1 (leucine/glutamic acid/lysine protein 1), is present in the nucleus of nondividing mouse satellite cells. The objective of this study was to evaluate LEK1 as a marker of quiescent bovine satellite cells (BSC) in vitro and in vivo. The BSC were isolated from young bull calves (< or =7 d) and cultured in vitro for up to 9 d before fixation and immunostaining for LEK1. Results demonstrated that all myogenic cells contain the protein, with immunostaining primarily within the nucleus and immediate perinuclear region. Immunocytochemical detection of LEK1 in cryosections of mature cows revealed that the protein was present in a fraction of satellite cells and muscle fiber nuclei. Approximately 20% of Pax7-expressing satellite cells contained LEK1. An equivalent percentage of myonuclei, as defined by nuclei within a dystrophin boundary, contained nuclear LEK1. To gain insight into the functional role of LEK1, BSC were transiently transfected with plasmids coding for putative dominant inhibitory LEK1 proteins [DeltaLEK1(991) and DeltaLEK1(911)] and evaluated for cell proliferation. Both forms of DeltaLEK1 inhibited (P < 0.05) BSC proliferation, as indicated by a decrease in Ki67 immunopositive cells. In C2C12 myoblasts, DeltaLEK1(911) inhibited (P < 0.05) myoblast determination protein 1 (MyoD)-directed muscle gene transcriptional activity; DeltaLEK1(991) had no effect on TnI-Luc transcription. By contrast, both DeltaLEK1 fusion proteins inhibited myogenin expression in BSC without disrupting myoblast fusion. These results provide evidence that LEK1 serves to coordinate proliferation and differentiation in myogenic cells. Coupling the immunostaining pattern and functional data, we propose that LEK1 may serve as a useful marker for satellite cells that are preparing to fuse into adjacent fibers as well as an indicator of recently added myonuclei.