Matrix Metalloproteinases (MMPs) In Skeletal Muscle

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Biochemistry and Genetics, School of Molecular Sciences, College of Science, Health and Engineering, La Trobe University, Victoria, Australia.

Matrix metalloproteinases, MMPs, are zinc and calcium dependent enzymes, involved in maintaining integrity and homeostasis of the extracellular matrix, ECM, of cells, including skeletal muscle. MMP2 is implicated in motility, differentiation, and regeneration of skeletal muscle fibres through processing extracellular components. MMP9 has been found to be associated with muscle wasting diseases. MMP2 has been reported to also have an intracellular localization in cardiomyocytes, where it may degrade contractile proteins following ischemia reperfusion injury. Any potential intracellular function of MMP2 and MMP9 in skeletal muscle remains obscure. This study determined the absolute amounts of MMP2 and MMP9 in quiescent skeletal muscle. Using mechanically skinned muscle fibres, proportion of MMP2 and MMP9 between intact and skinned muscle fibres, where the surface membrane is fully removed, revealed about 70 percent of total MMP2 and about 50 percent of total MMP9 have an intracellular location. After separation of subcellular compartments of skinned muscle fibres, about 57 percent of intracellular MMP2 was freely diffusible, about 6 percent associated with membrane, and about 37 percent bound within the fibre; all intracellular MMP9 was freely diffusible. SDS based gelatin zymograms detected gelatinolytic activity of MMP2 and MMP9 in skeletal muscle, demonstrating they were predominantly in their pro, inactive, forms in resting skeletal muscle. Gelatin zymograms under native conditions revealed about 10 percent MMP2 became active when exposed to the physiologically relevant concentration of Calcium Ions of 20 micrometre, albeit for the extended duration of 17h. Potential association of MMP2 and MMP9 with muscular dystrophy was investigated using the mdx mouse at 28 and 70 days. These findings indicated MMP2 may play a critical role in necrosis of dystrophic muscle fibres, which was further supported by alteration of MMP2 activity in mdx mice with taurine supplementation, which improves muscle function in these mice. MMP9 may function during the age related growth of skeletal muscle but did not appear crucial for the degeneration/regeneration cycles occurring in the mdx mouse model.