The role of MMP-14 and MMP-2 in mediating myoblast fusion.

Abstract

Satellite cells are muscle precursor cells that have the ability to self-renew, proliferate and differentiate into myoblasts that eventually elongate and fuse to form myotubes which are vital for regeneration and repair of muscle. Satellite cells reside in a niche, between the sarcolemma of the muscle fiber and the basal lamina, which consists of mostly collagen IV, proteoglycans and laminin. Matrigel is a gelatinous protein mixture that consists primarily of collagen IV and laminin and therefore resembles the basal lamina. Matrix Metalloproteinases (MMPs) are zinc endopeptidases, proteolytic peptidases which break peptide bonds within their substrates. MMP-14 (membrane bound) also known as membrane-type 1 matrix metalloproteinase (MT1-MMP) is one of the major matrix metalloproteinases (MMPs) involved in muscle repair and regeneration, together with MMP-2 (secreted). MMP-2 is a secreted gelatinase A, which is activated by MMP-14. MMP-2 is also known to be activated by nitric oxide (NO), therefore allowing active MMP-2 to release growth factors such as Hepatocyte Growth Factor (HGF) from the extracellular matrix (ECM). There are two forms of MMP-2, intracellular MMP-2 and extracellular (secreted) MMP-2. Secreted MMP-2 contains a peptide signal that helps direct it outside the cell, while intracellular MMP-2 lacks this feature and is therefore retained within the cell. Intracellular MMP-2 activity is known to be a major cause of muscular atrophy. Secreted MMP-2 is known to degrade ECM components, facilitating satellite cell mobility and release of growth factors such as HGF, aiding in muscle regeneration. MMP-2 can cleave collagen IV due to the presence of a fibronectin-like domain within its catalytic domain; this is not the case with MMP-14. MMP-14 and MMP-2 together degrade collagens, fibronectin, laminin-2/4 and other adhesion molecules. This clears the path for the myoblast to align and fuse to form myotubes which then finally align to form mature muscle fibers. The levels of MMP-14 and MMP-2 must be regulated; low levels can cause muscular dystrophy. The current study analysed expression levels, activity and role of MMP-14 and MMP-2 in C2C12 myoblast differentiation. C2C12 myoblasts first proliferated (Day 0), then aligned and elongated (Days 1-2) and then finally fused into myotubes (Days 3-5) during differentiation. MMP-14 and MMP-2 protein levels were high during the elongation period and also during fusion of C2C12 myoblasts. MMP-14 was localised at the focal adhesions, where actin filaments terminate during myoblast proliferation and fusion. ii Inhibition of MMPs using BB94 (10 µM) was observed to significantly reduce C2C12 myoblasts fusion. Secreted MMP-2 seems to play a vital role in the C2C12 differentiation, as activity was seen during myogenesis; when neutralised with an antibody, an 18% decrease in fusion was observed. Matrigel promoted an increase of MMP-2 expression within the cell during fusion (day 5 of differentiation), while no intracellular MMP-2 protein was observed at day 2 of differentiation. Levels of secreted MMP-2 increased significantly from day 2 to day 5 of differentiation; however, the presence of Matrigel significantly reduced levels of secreted MMP-2 detected in conditioned media at day 5 compared to uncoated conditions. The decrease is, in part, due to the fact that MMP-2 was found to bind to Matrigel. In conclusion, MMP-14 and MMP-2 play an important role in C2C12 myoblast elongation and fusion. This study provides further insight into the role of MMPs in myogenesis and lays the foundation for future work.