Activation and localization of matrix metalloproteinase-2 and -9 in the skeletal muscle of the muscular dystrophy dog (CXMDJ).
BACKGROUND
Matrix metalloproteinases (MMPs) are key regulatory molecules in the formation, remodeling and degradation of all extracellular matrix (ECM) components in both physiological and pathological processes in various tissues. The aim of this study was to examine the involvement of gelatinase MMP family members, MMP-2 and MMP-9, in dystrophin-deficient skeletal muscle. Towards this aim, we made use of the canine X-linked muscular dystrophy in Japan (CXMDJ) model, a suitable animal model for Duchenne muscular dystrophy.


METHODS
We used surgically biopsied tibialis cranialis muscles of normal male dogs (n = 3) and CXMDJ dogs (n = 3) at 4, 5 and 6 months of age. Muscle sections were analyzed by conventional morphological methods and in situ zymography to identify the localization of MMP-2 and MMP-9. MMP-2 and MMP-9 activity was examined by gelatin zymography and the levels of the respective mRNAs in addition to those of regulatory molecules, including MT1-MMP, TIMP-1, TIMP-2, and RECK, were analyzed by semi-quantitative RT-PCR.


RESULTS
In CXMDJ skeletal muscle, multiple foci of both degenerating and regenerating muscle fibers were associated with gelatinolytic MMP activity derived from MMP-2 and/or MMP-9. In CXMDJ muscle, MMP-9 immunoreactivity localized to degenerated fibers with inflammatory cells. Weak and disconnected immunoreactivity of basal lamina components was seen in MMP-9-immunoreactive necrotic fibers of CXMDJ muscle. Gelatinolytic MMP activity observed in the endomysium of groups of regenerating fibers in CXMDJ did not co-localize with MMP-9 immunoreactivity, suggesting that it was due to the presence of MMP-2. We observed increased activities of pro MMP-2, MMP-2 and pro MMP-9, and levels of the mRNAs encoding MMP-2, MMP-9 and the regulatory molecules, MT1-MMP, TIMP-1, TIMP-2, and RECK in the skeletal muscle of CXMDJ dogs compared to the levels observed in normal controls.


CONCLUSIONS
MMP-2 and MMP-9 are likely involved in the pathology of dystrophin-deficient skeletal muscle. MMP-9 may be involved predominantly in the inflammatory process during muscle degeneration. In contrast, MMP-2, which was activated in the endomysium of groups of regenerating fibers, may be associated with ECM remodeling during muscle regeneration and fiber growth.
Fukushima
Kazuhiro
K
Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan. fuku@hsp.md.shinshu-u.ac.jp <fuku@hsp.md.shinshu-u.ac.jp>
Nakamura
Akinori
A
Ueda
Hideho
H
Yuasa
Katsutoshi
K
Yoshida
Kunihiro
K
Takeda
Shin'ichi
S
Ikeda
Shu-ichi
S
D000818:Animals; D004195:Disease Models, Animal; D004285:Dogs; D016189:Dystrophin / Q000235:genetics; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D007150:Immunohistochemistry; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000201:enzymology* / Q000473:pathology; D009137:Muscular Dystrophy, Animal / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D009220:Myositis / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D012038:Regeneration; D020133:Reverse Transcriptase Polymerase Chain Reaction
D016189:Dystrophin; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9