Myotube Formation: How We Learned that Skeletal Muscle is Made by Cell Fusion

skeletal muscle cell is an extremely large, elongated cell that contains many nuclei. In the mid-1960s, developmental biologists debated whether each of these cells (often called myotubes) was derived from the fusion of several mononucleate muscle precursor cells (myoblasts) or from a single myoblast that undergoes nuclear division without cytokinesis. Evidence for the fusion of skeletal myoblasts to form multinucleated myotubes came from two independent sources. The critical evidence for myoblast fusion came from chimeric mice. These mice can be formed from the fusion of two early embryos, which regulate to produce a single mouse having two distinct cell populations. Mintz and Baker (1967) fused mouse embryos that produced different types of the enzyme isocitrate dehydrogenase. This enzyme, found in all cells, is composed of two identical subunits. Thus, if myotubes are formed from one cell whose nuclei divide without cytokinesis, one would expect to find two distinct forms of the enzyme, that is, the two parental forms, in the allophenic mouse (Figure 1). But if myotubes are formed by fusion between cells, one would expect to find muscle cells expressing not only the two parental types of enzymes (AA and BB), but also a third class composed of a subunit from each of the parental types (AB). The different forms of isocitrate dehydrogenase can be separated and identified by their electrophoretic mobility. The results clearly demonstrated that although only the two parental types of enzyme were present in all the other tissues of the allophenic mice, the hybrid (AB) enzyme was present in extracts of skeletal muscle tissue. Thus, the myotubes must have been formed from the fusion of numerous myoblasts.

Figure 1
Figure 1   The two possible mechanisms of skeletal muscle formation and how to distinguish between them. Allophenic mice are made from the fusion of mouse embryos from two different strains, each strain making a different form of the enzyme isocitrate dehydrogenase. This enzyme is composed of two subunits; one strain of mouse makes AA isocitrate dehydrogenase (indicated in black) and the other makes BB (color). (A) If the enzymes are made in a single cell, or in multinucleate cells arising from the nuclear divisions within a single cell, the enzyme will be purely AA or BB. (B) If there are two different nuclei in the same cell, however, one might code for B subunits while the other might code for A, with the result that some molecules of the enzyme will be hybrid (AB). Electrophoresis can separate these three types of molecules. The presence of the AB molecule in skeletal muscle cells (but not in other cell types) confirms the fusion model. (After Mintz and Baker, 1967.)

This evidence was important in showing that myoblast fusion actually occurred within the embryo. The analysis of how this fusion takes place was based on such fusion events occurring in culture. Konigsberg (1963) found that myoblasts isolated from chick embryos would proliferate in collagen-coated petri dishes. After about two days, however, these myoblasts stopped dividing and began to fuse with their neighbors to produce extended myotubes synthesizing muscle-specific proteins.

“Myomaker”: The major myoblast cell fusion protein

In 2013, Millay et al. concluded that they had found the major protein involved in myoblast cell fusion. Searching a gene database for genes expressed in myoblasts but not in mature muscle cells, they found a gene that produced a particular cell membrane protein associated with myoblast cells. This protein is found on the myoblast cell membrane, but it is dramatically downregulated after cell fusion. Moreover, not only did over-expression of this protein accelerate myoblast fusion, but its ectopic expression in fibroblasts caused the fibroblast cells to fuse with the myoblasts. Mutating this gene caused a non-functional protein and also the inability of the myoblasts containing it to fuse together. Thus, they named the protein “Myomaker.”

The expression of Myomaker is seen only in the myotomes of somites, developing limb buds, and skeletal muscle precursors. Interestingly, it is re-activated in the satellite cells that help the muscle regenerate after damage. Expression of the Myomaker gene is induced in these satellite cells after injury to the adult mouse muscle. Also, the activity of Myomaker protein is blocked by drugs that interfere with the actin microfilaments, consistent with the observations that the cytoskeleton is critical in the fusion process. It appears that the major protein of mammalian muscle cell fusion has finally been found.

Literature Cited

Konigsberg, I. R. 1963. Clonal analysis of myogenesis. Science 140: 1273-1284.

Millay, D. P., J. R. O’Rourke, L. B. Sutherland, S. Bezprozvannaya, J. M. Shelton, R. Bassel-Duby, E. N. Olson. 2013. Myomaker is a membrane activator of myoblast fusion and muscle formation. Nature 499: 301–305.

Mintz, B. and Baker, W. W. 1967. Normal mammalian muscle differentiation and gene control of isocitrate dehydrogenase synthesis. Proc. Natl. Acad. Sci. USA 58: 592-598.

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