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Research Article

Alterations in mRNA and Protein Levels of Metalloproteinases-2,-9 and-14 and Tissue Inhibitor of Metalloproteinase-2 post- Traumatic Skeletal Muscle Injury

¹Arteriocyte Medical Systems, Inc. ²U.S. Army Research Institute of Environmental Medicine (USARIEM), Natick, MA ³Georgia State University

Submitted 15 May 2009 ; revised 4 September 2009 ; accepted in final form 25 September 2009

This study characterizes the temporal relationship of membrane-type -1 matrix metalloproteinase (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2) expression in skeletal muscle following injury. Tibialis anterior (TA) muscles from 60 mice were exposed and injured by applying a cold steel probe (-79 °C) to the muscle for 10 s. Thereafter, TA muscles from uninjured and injured legs were collected at 3, 10, 24, 48, and 72 h post-injury for analysis of local MT1-MMP, TIMP-2, and matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9) mRNA and protein content via qRT-PCR, immunoblotting, zymography and immunofluorescence. All data are expressed as fold change of injured leg vs. uninjured leg. MT1-MMP mRNA levels were decreased significantly at 48 and 72 h post-injury by ~9- and 21-fold, respectively (p<0.01). Both TIMP-2 and MMP-2 mRNA expression significantly decreased in the injured leg by ~4- to 10-fold at 10-72 h post-injury (p<0.01). MMP-9 mRNA expression was significantly increased at 10, 24 and 48 h post-injury by 6- (p<0.05), 25-, and 12-fold (p<0.01), respectively. Protein content of latent (63 kDa) MT1-MMP was decreased at 48 and 72 h post-injury by ~2-fold (P<0.01). Content of the soluble (50 kDa) fragment of MT1-MMP was significantly increased by ~17-, 25- and 67-fold at 24 (p<0.05), 48 and 72 h (P<0.01) post-injury, respectively. TIMP-2 protein levels diminished from 3 to 48 h post-injury by 1.5-1.8-fold (P<0.01), prior to returning to baseline levels at 72 h post-injury. Zymography revealed visual increases in gelatinase activity in molecular weight regions corresponding to MMP-9 and MMP-2. In conclusion, skeletal muscle injury initiates a sequence of events in the MT1-MMP proteolytic cascade resulting in elevated levels of the soluble (50 kDa) fragment of MT1-MMP, which could enhance pericellular ECM remodeling.

Extracellular Matrix; Freeze Injury; MT1-MMP; shedding