Selective inhibition of a step of myotube formation with wheat germ agglutinin in a murine myoblast cell line, C2C12.

Abstract

Myoblast cells, C2C12, which is an established cell line from satellite cells of skeletal muscle of C3H mouse, start to fuse and form multinucleated cells (myotubes) and begin to express creatine phosphokinase and myosin, when culture medium is changed from the growth medium to the differentiation medium. Among the 12 lectins that we tested, wheat germ agglutinin apparently suppressed the myotube development judged by phase-contrast microscopy, but did not affect the induction of creatine phosphokinase activity. The addition of N-acetylglucosamine or N,N',N"-triacetylchitotriose, which is a specific ligand for wheat germ agglutinin, to the differentiation medium, recovered this apparently suppressive effect of wheat germ agglutinin on the myotube development. Tachypleus tridentatus (Japanese horseshoe crab) lectin that specifically recognizes N-acetylneuraminic acid, one of the sialic acids, showed no effect on the myotube development. It was suggested that wheat germ agglutinin suppressed the process through recognizing N-acetylglucosamine containing sugar. Surprisingly, even in the presence of wheat germ agglutinin, the ratio of mononucleated cell numbers to the genomic DNA content, which represents the fusion level, decreased after incubation in the differentiation medium, indicating that even when wheat germ agglutinin was present in the medium, cell fusion, which is the initial step of the myotube formation, occurred. Immunostaining with anti-skeletal muscle myosin antiserum confirmed that the myosin expressing cells actually fused and formed multinucleated cells. Their shape, however, was thin compared to that in the absence of wheat germ agglutinin. We propose that the membrane fusion step to form myotubes is composed of two distinct steps in C2C12; one fusion step is to form long and thin myotubes from mononucleated cells and the other one is to develop fat myotubes. Wheat germ agglutinin specifically inhibits the latter fusion step.