Expression of matrix metalloproteinases 2 and 9 in regenerating skeletal muscle: a study in experimentally injured and mdx muscles.

Matrix metalloproteinases (MMPs) cooperatively degrade all components of the extracellular matrix (ECM). Remodeling of ECM during skeletal muscle degeneration and regeneration suggests a tight regulation of matrix-degrading activity during muscle regeneration. In this study, we investigated the expression of MMP-2 and MMP-9, in normal muscles and their regulation during regeneration process. We further investigated their secretion by C2C12 myogenic cell line. Two models of muscle degeneration-regeneration were used: (1) normal muscles in which necrosis was experimentally induced by cardiotoxin injection; (2) mdx muscles which exhibit recurrent signs of focal myofiber necrosis followed by successful regeneration. MMPs were studied by zymography; their free activity was quantified using 3H-labeled gelatin substrate and mRNA expression was followed by Northern hybridization. Muscle degeneration-regeneration was analyzed by conventional morphological methods and in situ hybridization was performed on muscle sections to identify the cells expressing these MMPs. Results show that MMP-2, but not MMP-9 expression, is constitutive in normal muscles. Upon injury, the active form of MMP-2 is transiently increased, whereas MMP-9 is induced within 24 h and remains present for several days. Quantitative assays of free gelatinolytic activity show a progressive and steady increase that culminates at 7 days postinjury and slowly returns to normal levels. In adult mdx mice, both pro and active forms of MMP-2 and MMP-9 are expressed. Northern blot results support these findings. Zymography of C2C12-conditioned medium shows that myogenic cells produce MMP-2. By in situ hybridization we localized MMP-9 mRNA in inflammatory cells and putative activated satellite cells in injured muscles. Our data allow the correlation of the differential expression of pro and/or active forms of MMP-2 and MMP-9 with different stages of the degeneration-regeneration process: MMP-9 expression is related to the inflammatory response and probably to the activation of satellite cells, whereas MMP-2 activation is concomitant with the regeneration of new myofibers.

Matrix metalloproteinases MMP-2 and MMP-9 in denervated muscle and injured nerve.

Nerve crush or axotomy results in a transient or longterm denervation accompanied by remodelling in nerve, muscle and neuromuscular junctions. These changes include an increased turnover of several extracellular matrix molecules and proliferation of Schwann cells in injured nerves. Given the role of matrix degrading metalloproteinases MMP-2 and MMP-9 (gelatinases-type IV collagenases) in extracellular matrix remodelling, we investigated their regulation and activation in denervated muscles and injured nerves in mice. For this, immunofluorescence using MMP-2 and MMP-9 antibodies was carried concomitantly with gelatin zymography and quantification of gelatinase activity using [3H]-gelatin substrate. Results show that in normal mouse muscles MMP-2 and MMP-9 are localized at the neuromuscular junctions, in Schwann cells and the perineurium of the intramuscular nerves. In denervated mouse muscles, MMP-2 immunolabelling persists at the neuromuscular junctions but decreases in the nerves whereas MMP-9 immunolabelling persists at the neuromuscular junctions but is enhanced in degenerated intramuscular nerves. Denervated muscles did not show any significant change of gelatinolytic activity or expression pattern, while injured nerves exhibited a transient increase of MMP-9 and activation of MMP-2. In conclusion, this study demonstrates that MMP-2 and MMP-9 are expressed at mouse neuromuscular junctions and that their localization and expression pattern appear not to be modified by denervation. Their modulation in injured nerves suggests they are involved in axonal degeneration and regeneration.