In vivo migration of transplanted myoblasts requires matrix metalloproteinase activity

Muscle cell migration and extracellular matrix remodeling are essential aspects of muscle development and regeneration. In this study, using a new technique to assess in vivo myoblast migration, we have confirmed previous results showing that the C(2)C(12) myoblast cell line exhibits a higher migratory capacity than primary myoblasts. To test the hypothesis that matrix metalloproteinases (MMPs) are required for the migration of C(2)C(12) myoblasts, we determined whether a synthetic metalloproteinase inhibitor, BB94 (Batimastat), inhibited this process in vivo. Pretreatment with BB94 for 3 days decreased the C(2)C(12) migration at 2 days after cell injection. Since MMP expression is thus necessary for myoblast migration, we have undertaken the identification and characterization of the MMPs expressed by the C(2)C(12) cell line. An RT-PCR assay was used to determine the pattern of MMP mRNA expression by the C(2)C(12) cell line. The proteolytic activities of the MMPs secreted in the culture medium were also assessed by gelatin zymography. The results showed that MMP2 (gelatinase A, 72-kDa type IV collagenase) and MT1-MMP transcripts were expressed by this cell line; however, only MMP2 was secreted and was able to be activated in the extracellular environment. This cell line failed to express MMP9 (gelatinase B, 92-kDa type IV collagenase), stromelysine 2, or stromelysine 3. Our observation that the membrane type MMP (MT1-MMP) transcript is also expressed by the C(2)C(12) suggests that the MMP2 proform (pro-MMP2), may be activated by the MT1-MMP. This possibility is supported by our observation that the pretreatment of C(2)C(12) with concanavalin A (which is known to induce the expression of MT1-MMP) resulted in the processing of pro-MMP2 to its mature form, in a dose-dependent manner. Overexpression and activation of MMP2 in normal myoblasts showed significant increased migration of mouse myoblasts in vivo. Our finding that MMP2 and MT1-MMP gene are coexpressed by C(2)C(12) myoblasts could account for the high migratory capacity of C(2)C(12). Together these results supported the importance of MMP2 and its activation by MT1-MMP for myoblast migration.