Cardiac matrix metalloproteinase-2 expression independently induces marked ventricular remodeling and systolic dysfunction.

Although enhanced cardiac matrix metalloproteinase (MMP)-2 synthesis has been associated with ventricular remodeling and failure, whether MMP-2 expression is a direct mediator of this process is unknown. We generated transgenic mice expressing active MMP-2 driven by the alpha-myosin heavy chain promoter. At 4 mo MMP-2 transgenic hearts demonstrated expression of the MMP-2 transgene, myocyte hypertrophy, breakdown of Z-band registration, lysis of myofilaments, disruption of sarcomere and mitochondrial architecture, and cardiac fibroblast proliferation. Hearts from 8-mo-old transgenic mice displayed extensive myocyte disorganization and dropout with replacement fibrosis and perivascular fibrosis. Older transgenic mice also exhibited a massive increase in cardiac MMP-2 expression, representing recruitment of endogenous MMP-2 synthesis, with associated expression of MMP-9 and membrane type 1 MMP. Increases in diastolic [control (C) 33 +/- 3 vs. MMP 51 +/- 12 microl; P = 0.003] and systolic (C 7 +/- 2 vs. MMP 28 +/- 14 microl; P = 0.003) left ventricular (LV) volumes and relatively preserved stroke volume (C 26 +/- 4 vs. MMP 23 +/- 3 microl; P = 0.16) resulted in markedly decreased LV ejection fraction (C 78 +/- 7% vs. MMP 48 +/- 16%; P = 0.0006). Markedly impaired systolic function in the MMP transgenic mice was demonstrated in the reduced preload-adjusted maximal power (C 240 +/- 84 vs. MMP 78 +/- 49 mW/microl(2); P = 0.0003) and decreased end-systolic pressure-volume relation (C 7.5 +/- 1.5 vs. MMP 4.7 +/- 2.0; P = 0.016). Expression of active MMP-2 is sufficient to induce severe ventricular remodeling and systolic dysfunction in the absence of superimposed injury.

Cardiac ischemia-reperfusion injury induces matrix metalloproteinase-2 expression through the AP-1 components FosB and JunB.

Matrix metalloproteinase-2 (MMP-2) is a central component of the response to injury in the heart. During ischemia, MMP-2 influences ventricular performance and is a determinant of postinfarction remodeling. Elevation of MMP-2 during reperfusion after ischemia suggests that new protein is synthesized, but the molecular regulation of MMP-2 generation during ischemia-reperfusion (I/R) injury has not been studied. Using the MMP-2 promoter linked to a beta-galactosidase reporter in transgenic mice, we investigated the transcriptional regulation and cellular sources of MMP-2 in isolated, perfused mouse hearts subjected to acute global I/R injury. I/R injury induced a rapid activation of MMP-2 promoter activity with the appearance of beta-galactosidase antigen in cardiomyocytes, fibroblasts, and endothelial cells. Activation of intrinsic MMP-2 transcription and translation was confirmed by real-time PCR and quantitative Western blot analyses. MMP-2 transcription and translation were inhibited by perfusion with 1.0 mM hydroxyl radical scavenger N-(-2-mercaptopropionyl)-glycine. Nuclear extracts demonstrated increased abundance of two activator proteins-1 (AP-1) components JunB and FosB following I/R injury. Immunohistochemical staining localized JunB and FosB proteins to the nuclei of all three cardiac cell types following I/R injury, consistent with enhanced nuclear transport of these transcription factors. Chromatin immunoprecipitation (ChIP) of the AP-1 binding site in the intrinsic murine MMP-2 promoter yielded only JunB under control conditions, whereas ChIP following I/R injury recovered both JunB and FosB, consistent with a change in occupancy from JunB homodimers in controls to JunB/FosB heterodimers following I/R injury. We conclude that enhanced MMP-2 transcription and translation following I/R injury are mediated by induction, via oxidant stress, of discrete AP-1 transcription factor components.

Cardiac transgenic matrix metalloproteinase-2 expression directly induces impaired contractility.

OBJECTIVE: Matrix metalloproteinase-2 (MMP-2) plays a major role in dysfunctional ventricular remodeling following myocardial injury induced by ischemia/reperfusion and heart failure. To directly assess the role of MMP-2 in the absence of superimposed injury, we generated cardiac-specific, constitutively active MMP-2 transgenic mice. METHODS: Morphologic and functional studies were carried out using both intact and demembranated (skinned) right ventricular trabeculae dissected from hearts of 8-month-old MMP-2 transgenic mice and wild-type controls (WT). RESULTS: Electron micrographs showed that compared to WT, MMP-2 myocardium had no gross, ultrastructural changes (no myocyte dropout or gross fibrosis). However, MMP-2 myocardium contained fibroblasts with abundant rough endoplasmic reticulum, consistent with an activated synthetic phenotype, suggesting extracellular matrix remodeling in MMP-2 trabeculae. Consistent with remodeling, mechanical studies found increased stiffness of intact unstimulated trabeculae (increasing sarcomere lengths from 2 to 2.3 microm caused a greater rise of passive muscle force for MMP-2 trabeculae versus WT). With electrical stimulation, MMP-2 trabeculae generated substantially less active force at all sarcomere lengths. Moreover, inotropic responses to increases of bath [Ca2+], pacing frequency, and isoproterenol were all significantly reduced versus WT trabeculae. Skinned fiber assessment of myofilament function revealed that maximum Ca2+-activated force of skinned MMP-2 trabeculae was reduced to approximately 50% of WT, suggesting a myofilament contraction defect. CONCLUSION: Cardiac-specific, constitutively active MMP-2 expression leads to impaired contraction and diminished responses to inotropic stimulation. These findings indicate that MMP-2 can directly impair ventricular function in the absence of superimposed injury.

A functional activating protein 1 (AP-1) site regulates matrix metalloproteinase 2 (MMP-2) transcription by cardiac cells through interactions with JunB-Fra1 and JunB-FosB heterodimers.

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 1beta. 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 -1433 to -1342 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 -1410 and -1362 bp that included a potential activating protein 1 (AP-1)-binding sequence, C(-1394)CTGACCTCC. Site-specific mutagenesis of the core TGAC sequence (indicated in bold) eliminated the transactivating activity within the -1410 to -1362 bp sequence. Electrophoretic mobility shift assays (EMSAs) using the -1410 to -1362 bp oligonucleotide and rat cardiac fibroblast nuclear extracts demonstrated specific nuclear-protein binding that was eliminated by cold competitor oligonucleotide, but not by the AP-1-mutated oligonucleotide. Antibody-supershift EMSAs of nuclear extracts from normoxic rat cardiac fibroblasts demonstrated Fra1 and JunB binding to the -1410 to -1362 bp oligonucleotide. Nuclear extracts isolated from hypoxic rat cardiac fibroblasts contained Fra1, JunB and also included FosB. Co-transfection of cardiac fibroblasts with Fra1-JunB and FosB-JunB expression plasmids led to significant increases in transcriptional activity. These studies demonstrate that a functional AP-1 site mediates MMP-2 transcription in cardiac cells through the binding of distinctive Fra1-JunB and FosB-JunB heterodimers. The synthesis of MMP-2 is widely considered, in contrast with many members of the MMP gene family, to be independent of the AP-1 transcriptional complex. This report is the first demonstration that defined members of the Fos and Jun transcription-factor families specifically regulate this gene under conditions relevant to critical pathophysiological processes.