Inefficient Skeletal Muscle Repair in Inhibitor of Differentiation Knockout Mice Suggests a Crucial Role for BMP Signaling During Adult Muscle Regeneration

The bone morphogenetic protein (BMP) pathway is known to be involved in limb myogenesis during development, but whether it is involved in postnatal muscle regeneration is unclear. We have found that adult inhibitor of differentiation (Id)-mutant (Id1(+/-)Id3(-/-)) mice display delayed and reduced skeletal muscle regeneration after injury compared with either wild-type littermates or Id3-null mice. Immunoblotting of wild-type muscle lysates revealed that, not only were Id1 and Id3 highly upregulated within 24 h after injury, but other upstream components of the BMP pathway were as well, including the BMP receptor type II and phosphorylated Smad1/5/8 (pSmad1/5/8). Inhibition of BMP signaling in injured skeletal muscle by Noggin injection reduced pSmad1/5/8, Id1, and Id3 protein levels. The mouse myoblast-derived cell line C2C12 also expressed Id1, Id3, BMP receptor type II, and pSmad1/5/8 during proliferation, but all were reduced upon differentiation into myotubes. In addition, these cells secreted mature BMP-4, and BMP signaling could be inhibited with exogenous Noggin, causing a reduction in pSmad1/5/8, Id1, and Id3 levels. Confocal immunofluorescence microscopy revealed that activated Pax7(+) myoblasts coexpressed nuclear pSmad1/5/8, Id1, and Id3 in injured mouse skeletal muscle sections. Although we did not observe differences in the numbers of quiescent Pax7(+) satellite cells in adult uninjured hindlimb muscles, we did observe a significant reduction in the number of proliferating Pax7(+) cells in the Id-mutant mice after muscle injury compared with either wild-type or Id3-null mice. These data suggest a model in which BMP signaling regulates Id1 and Id3 in muscle satellite cells, which directs their proper proliferation before terminal myogenic differentiation after skeletal muscle injury in postnatal animals.