Replicating myoblasts express a muscle-specific phenotype

Abstract

During the terminal stage of skeletal myogenesis, myoblasts stop replicating, fuse to form multinucleate fibers, and express the genes that encode the proteins that convey contractile capacity. Because of this dramatic shift in proliferative state, morphology, and gene expression, it has been possible to readily identify and quantitate terminally differentiating myoblasts. In contrast, it is not clear whether the proliferating cells that give rise to postmitotic myoblasts are equally distinct in their phenotype and in fact whether distinct stages in skeletal myogenesis precede the onset of terminal differentiation. To address these questions, monoclonal antibodies and immunofluorescence microscopy were used to determine that replicating myoblasts from newborn rats do express a muscle-specific phenotype. To identify replicating cells, incorporation of 5-bromo-2'-deoxyuridine (BrdUrd) into DNA was assayed by using anti-BrdUrd antibody. The developmentally regulated, muscle-specific, integral membrane protein H36 and the intermediate-filament protein desmin were scored as markers of the myogenic phenotype. The percentage of BrdUrd+ (i.e., proliferative) cells among H36+ and desmin+ myoblasts was equal to the percentage of BrdUrd+ cells in the entire population, indicating that the expression of H36 and desmin is uniformly characteristic of replicating myoblasts. Inhibition of protein synthesis before and during growth in BrdUrd did not alter the frequency of desmin and H36 immunofluorescence in BrdUrd+ cells. Thus, desmin and H36 were present in the replicating myoblasts prior to the onset of growth in BrdUrd. These results were confirmed using H36+ cells selected by flow cytometry: these purified H36+ myoblasts replicate, express desmin, and differentiate. Similar results were obtained with mouse myoblasts. Desmin expression in these mammalian cells differs from that in chicken embryo myoblasts: only a small proportion of replicating chicken embryo myoblasts express desmin. That replicating mammalian myoblasts have a muscle-specific phenotype serves to define a distinct stage in myogenic development and a specific cell in the myogenic lineage. Further, it implies that there is a regulatory event activated during myogenesis that precedes terminal differentiation and that is required for expression of those genes whose products distinguish the replicating myoblast.