Enhanced synthesis of a specific matrix metalloproteinase, MMP-2, has been demonstrated in experimental models of ventricular failure and in cardiac extracts from patients with ischaemic cardiomyopathy. Cultured neonatal rat cardiac fibroblasts and myocytes were used to analyse the determinants of MMP-2 synthesis, including the effects of hypoxia. Culture of rat cardiac fibroblasts for 24 h in 1 % oxygen enhanced MMP-2 synthesis by more than 5-fold and augmented the MMP-2 synthetic responses of these cells to endothelin-1, angiotensin II and interleukin 1β. A series of MMP-2 promoter-luciferase constructs were used to map the specific enhancer element(s) that drive MMP-2 transcription in cardiac cells. Deletion studies mapped a region of potent transactivating function within the 91 bp region from k1433 to k1342 bp, the activity of which was increased by hypoxia. Oligonucleotides from this region were cloned in front of a heterologous simian-virus-40 (SV40) promoter and mapped the enhancer activity to a region between k1410 and k1362 bp that included a potential activating protein 1 (AP-1)-binding sequence, C −"$*% CTGACCTCC. Site-specific mutagenesis of the core TGAC sequence (indicated in bold) eliminated the transactivating activity within the k1410 to k1362 bp sequence. Electrophoretic mobility shift assays (EMSAs) using the k1410

No abstract

Migration of cardiac fibroblasts is implicated in infarct healing and ventricular remodeling. Activation of matrix metalloproteinases induced by three-dimensional type I collagen, the principal component of the myocardial interstitium, is hypothesized to be essential for this migration. By utilizing primary cultures of cardiac fibroblasts and collagen lattice models, we demonstrated that type I collagen induced MMP-2 activation, and cells undergoing a change from isometric tension to mechanical unloading were associated with increased levels of total and active MMP-2 species. The collageninduced MMP-2 activation coincided with up-regulated cellular levels of both membrane type 1-matrix metalloproteinase (MT1-MMP) and TIMP-2. A fraction of cellular membrane prepared from cells embedded in the collagen lattice containing active MT1-MMP and TIMP-2 was capable of activating pro-MMP-2, and exogenous TIMP-2 had a biphasic effect on this membrane-mediated MMP-2 activation. Interestingly, the presence of 43-kDa MT1-MMP species in a fraction of intracellular soluble proteins prepared from monolayer cells but not cells embedded in the lattices indicates that MT1-MMP metabolizes differently under the two different culture conditions. Treatment of cells embedded in the lattice with furin inhibitor attenuated pro-MT1-MMP processing and MMP-2 activation and impeded cell migration and invasion. These results suggest that the migration and invasion of cardiac fibroblasts is furin-dependent and that the active species of MT1-MMP and MMP-2 may be involved in both events.Cardiac fibrosis, a consequence of infarct healing and ventricular remodeling, is characterized by hyperplasia of ␣-smooth muscle actin expressing fibroblast-like cells and deposition of excessive extracellular matrix (ECM) 1 proteins by these cells in the myocardium. These cells are thought to originate from cardiac fibroblasts, which are recruited from the interstitium of the heart following myocardial injury (1). In vitro cardiac fibroblasts grown as a monolayer have been proved to be migratory cells (2, 3). However, cardiac fibroblasts in vivo reside in the interstitial fibrillar collagen network; the migratory/invasive potential of cardiac fibroblasts, by which the cells detach from the collagen network where they normally reside in vivo and then penetrate basement membrane surrounding necrotic myocytes, has not been investigated previously. Type I collagen, the principal component of the interstitial fibrillar collagen network, is a known matrix effector for MMP-2 activation in various cell types including neonatal rat cardiac fibroblasts (4). However, the mode of MMP-2 activation induced by three-dimensional type I collagen remains largely undefined (5-11). Furthermore, reorganization of the cytoskeleton subsequent to changes in mechanical tension in the collagen lattice has been reported to underlie three-dimensional type I collagen-induced MMP-2 activation in human skin fibroblasts (12) but not rat capillary endothelial cells (8), raising the possibility ...

No abstract

The antiarrhythmic effects of preconditioning can be demonstrated in isolated rat hearts perfused at constant flow with an artificial medium and this protection is lost following treatment with Bordetella pertussis toxin 48 h previously.