Duchenne muscular dystrophy (DMD) is the most common lethal X-linked recessive disease, presenting with progressive muscular atrophy and weakness. DMD is caused by mutations in the
DMD gene that encodes the cytoskeletal protein dystrophin. Dystrophin and the dystrophin-associated protein complex provide a crucial structural link between the extracellular matrix (ECM) and the intracellular actin cytoskeleton [
1]. Dystrophin deficiency affects the sarcolemma-ECM interaction, resulting in sarcolemmal instability [
2,
3]. Histopathological hallmarks in DMD include degeneration, necrosis, and insufficient regeneration of muscle fibers, suggesting that constitutive ECM remodeling takes place in DMD skeletal muscles. Although the cycles of degeneration and regeneration of muscle fibers continues throughout postnatal development, regeneration gradually slows and the balance is eventually tipped in favor of degeneration in DMD [
4].
ECM is a three-dimensional network of macromolecules, and transmits signals from cells to the ECM and vice versa, mediating cell adhesion, migration, proliferation, differentiation and survival [
5]. Muscle fibers are embedded in connective tissue organized into three interconnected sheaths: (1) Epimysium is a collagenous tissue that surrounds whole muscle; (2) Perimysium is smaller bundles of collagen fibers extended inward from epimysium, separates muscle fibers into fascicles or bundles; (3) Endomysium encloses the individual muscle fibers, including basal lamina, capillaries, fine nerve branches, fibroblasts, and macrophages [
6]. The basal lamina, which consists of ECM components such as type IV collagen, laminin, and proteoglycans, maintains the physiological integrity of the muscle fibers and has a role in muscle fiber repair after injury or excessive exercise [
7]. In the last decade, matrix metalloproteinases (MMPs) have been shown to degrade all ECM components [
8]. MMPs, a group of zinc-dependent endopeptidases, are thought to play a central role in the modulation of ECM functions [
9]. MMPs are commonly induced by cytokine signals as inactive zymogens (pro-forms), that require processing of a prodomain by other MMPs or serine proteinases to attain full activity. Their activities are inhibited by endogenous MMP inhibitors (tissue inhibitors of metalloproteinases; TIMPs-1, -2, -3, and -4) [
10,
11]. Membranous type metalloproteinases (MT-MMPs) have recently been discovered as a subgroup of membrane-anchored metalloproteinases. Membranous type metalloproteinase-1 (MT1-MMP) is associated with pro matrix metalloproteinase type 2 (pro MMP-2) and TIMP-2 to form a trimolecular complex, that activates pro MMP-2. [
12‐
16]. Reversion-inducing-cysteine-rich protein with Kazal Motifs (RECK) is a new class of membrane-anchored inhibitor of MMP-2, matrix metalloproteinase type 9 (MMP-9), and MT1-MMP [
17]. RECK has been described as a tumor and metastasis suppressor, as well as an angiogenesis suppressor and regulator of ECM integrity [
18,
19]. MMP activity contributes to a variety of physiological processes, such as embryonic development, organ morphogenesis, cell migration, apoptosis, angiogenesis, cartilage remodeling, bone growth, and wound healing [
8,
10,
20]. On the other hand, loss of the exquisite regulation of MMPs leads to extensive ECM degradation, resulting in various diseases such as tumor progression or metastasis, cerebrovascular diseases, cardiovascular diseases, rheumatoid arthritis, and lung diseases. Therefore, MMP inhibitors can be useful prospective agents for the prevention and treatment of these diseases [
21‐
27].
With respect to muscular disorders, particular attention has been paid to a subgroup of MMPs termed 'gelatinases,' comprising MMP-2 (also called gelatinase A, or 72-kDa type IV collagenase) and MMP-9 (also called gelatinase B, or 92-kDa type V collagenase). These enzymes contain three repeats of a gelatin-binding type II fibronectin domain inserted into their catalytic domain [
28]. Besides gelatin, they degrade various ECM components including denatured type I, II, and III collagen, native IV and V collagen, laminin, elastin, proteoglycan, and fibronectin [
28,
29]. In some inflammatory myopathies, MMP-9 is expressed primarily by invading T lymphocytes, and is implicated in the pathogenesis [
30‐
32]. MMP-2 is up-regulated in DMD skeletal muscle, and is expressed by mesenchymal fibroblastic cells [
33]. Up-regulation of MMP-2 and MMP-9 has also been observed in skeletal muscle of dystrophin-deficient
mdx mice [
34,
35], an animal model of DMD. Moreover, it has been reported that MMP-2 and MMP-9 are able to process beta-dystroglycan and disrupt the link between the ECM and the cell membrane via the dystroglycan complex in the skeletal muscle from DMD and sarcoglycanopathy patients [
36‐
38].
We hypothesized that MMP-2 and/or MMP-9 might also play an important role in the pathogenesis of muscular dystrophies, involving ECM remodeling during the cycle of muscle fiber degeneration and regeneration. We evaluated the expression, activation, and immunolocalization of MMP-2 and MMP-9, as well as of regulatory molecules MT1-MMP, TIMP-1, TIMP-2 and RECK, in the dystrophin-deficient skeletal muscle of the canine X-linked muscular dystrophy in Japan (CXMD
J) model of DMD, which shows more prominent skeletal muscle involvement than
mdx mice [
39].