Is intercellular communication via gap junctions required for myoblast fusion?

Fusion of myoblasts to form syncitial muscle cells results from a complex series of sequential events including cell alignment, cell adhesion and cell communication. The aim of the present investigation was to assess whether intercellular communication through gap junctions would be required for subsequent membrane fusion. The presence of the gap junction protein connexin 43 at areas of contact between prefusing rat L6 myoblasts was established by immunofluorescent staining. These myoblasts were dye-coupled, as demonstrated by the use of the scrape-loading/dye transfer technique. L6 myoblast dye coupling was reversibly blocked by heptanol in short term experiments as well as after chronic treatment. After a single addition of 3.5 mM heptanol, gap junctions remained blocked for up to 8 hours, then this inhibitory effect decreased gradually, likely because the alcohol was evaporated. Changing heptanol solutions every 8 hours during the time course of L6 differentiation resulted in a lasting drastic inhibition of myoblast fusion. We further investigated the effect of heptanol and of other uncoupling agents on the differentiation of primary cultures of embryonic chicken myoblasts. These cells are transiently coupled by gap junctions before myoblast fusion and prolonged application of heptanol, octanol and 18-beta-glycyrrhetinic acid also inhibited their fusion. The effect of heptanol and octanol was neither due to a cytotoxic effect nor to a modification of cell proliferation. Moreover, heptanol treatment did not alter myoblast alignment and adhesion. Taken together these observations suggest that intercellular communication might be a necessary step for myoblast fusion.