Spatiotemporal induction of matrix metalloproteinase-9 transcription after discrete myocardial injury.

Radiofrequency (RF) ablation of the myocardium causes discrete sites of injury. RF scars can expand, altering the extracellular matrix (ECM) structure and the continuity of the electrical syncytium of the adjacent myocardium. Matrix metalloproteinases (MMPs), such as MMP-9, contribute to ECM remodeling. However, whether and to what degree transcriptional induction of MMP-9 occurs after myocardial RF injury and the association with electrical conduction patterns after RF injury remains unexplored. This study examined MMP-9 gene promoter (M9PROM) activation after myocardial RF injury using mice in which the M9PROM was fused to a ß-galactosidase (ß-gal) reporter. RF lesions (0.5-mm probe, 80°C, 30 s) were created on the left ventricular (LV) epicardium of M9PROM mice (n=62) and terminally studied at 1 h, 1 d, 3 d, 7 d, 14 d, and 28 d after RF injury. M9PROM activation was localized through ß-gal staining. The RF scar area and the area of ß-gal staining were measured and normalized to LV area (planimetry). RF scar size increased from 1 h post-RF-injury values by 7 d and remained higher at 28 d. M9PROM activation became evident at 3 d and peaked at 7 d. Electrical conduction was measured (potentiometric dye mapping) at 7 d after RF injury. Heterogeneities in action potentials and electrical impulse propagation coincident with M9PROM activation were observed after RF injury. For example, conduction proximal to the RF site was slower than that in the remote myocardium (0.15±0.02 vs. 0.83±0.08 mm/ms, P<0.05). Thus, a unique spatiotemporal pattern of MMP-9 transcriptional activation occurred after discrete myocardial injury, which was associated with the development of electrical heterogeneity. Therefore, these findings suggest that changes in a key determinant of extracellular matrix remodeling, in addition to changes in myocardial structure, can contribute to arrhythmogenesis around the region of myocardial injury.

Matrix metalloproteinase-7 affects connexin-43 levels, electrical conduction, and survival after myocardial infarction.

BACKGROUND: Matrix metalloproteinases (MMPs) contribute to left ventricular remodeling after myocardial infarction (MI). Specific causative roles of particular MMPs, however, remain unclear. MMP-7 is abundant in cardiomyocytes and macrophages, but MMP-7 function after MI has not been defined. METHODS AND RESULTS: Wild-type (WT; n=55) and MMP-7-null (MMP-7-/-; n=32) mice underwent permanent coronary artery ligation for 7 days. MI sizes were similar, but survival was greatly improved in MMP-7-/- mice. The survival difference could not be attributed to differences in left ventricular dilation because end-diastolic volumes increased similarly. ECG analysis revealed a prolonged PR interval in WT but not in MMP-7-/- post-MI mice. Post-MI conduction velocity, determined by optically mapping electrical wavefront propagation, decreased to 78+/-6% of control for WT and was normalized in MMP-7-/- mice. In WT mice, slower conduction velocity correlated with a 53% reduction in the gap junction protein connexin-43. Direct binding of MMP-7 to connexin-43, determined by surface plasmon resonance technology, occurred in a dose-dependent manner. Connexin-43 processing by MMP-7 was confirmed by in silico and in vitro substrate analyses and MMP-7 infusion induced arrhythmias in vivo. CONCLUSIONS: MMP-7 deletion results in improved survival and myocardial conduction patterns after MI. This is the first report to implicate MMP-7 in post-MI remodeling and to demonstrate that connexin-43 is a novel MMP-7 substrate.

Selective spatiotemporal induction of matrix metalloproteinase-2 and matrix metalloproteinase-9 transcription after myocardial infarction.

Myocardial remodeling after myocardial infarction (MI) is associated with increased levels of the matrix metalloproteinases (MMPs). Levels of two MMP species, MMP-2 and MMP-9, are increased after MI, and transgenic deletion of these MMPs attenuates post-MI left ventricular (LV) remodeling. This study characterized the spatiotemporal patterns of gene promoter induction for MMP-2 and MMP-9 after MI. MI was induced in transgenic mice in which the MMP-2 or MMP-9 promoter sequence was fused to the beta-galactosidase reporter, and reporter level was assayed up to 28 days after MI. Myocardial localization with respect to cellular sources of MMP-2 and MMP-9 promoter induction was examined. After MI, LV diameter increased by 70% (P < 0.05), consistent with LV remodeling. beta-Galactosidase staining in MMP-2 reporter mice was increased by 1 day after MI and increased further to 64 +/- 6% of LV epicardial area by 7 days after MI (P < 0.05). MMP-2 promoter activation occurred in fibroblasts and myofibroblasts in the MI region. In MMP-9 reporter mice, promoter induction was detected after 3 days and peaked at 7 days after MI (53 +/- 6%, P < 0.05) and was colocalized with inflammatory cells at the peri-infarct region. Although MMP-2 promoter activation was similarly distributed in the MI and border regions, activation of the MMP-9 promoter was highest at the border between the MI and remote regions. These unique findings visually demonstrated that activation of the MMP-2 and MMP-9 gene promoters occurs in a distinct spatial relation with reference to the MI region and changes in a characteristic time-dependent manner after MI.

Time-dependent changes in myocardial structure following discrete injury in mice deficient of matrix metalloproteinase-3.

Myocardial scars from radiofrequency (RF) ablation can increase in size in the post-injury period, resulting in remodeling of the extracellular matrix (ECM). The matrix metalloproteinases (MMPs) contribute to adverse myocardial remodeling following injury. However, the role of specific MMP types in RF scar enlargement remains unclear. One MMP type, MMP-3, degrades a wide range of ECM substrates and can activate other MMPs. This project examined LV remodeling in wild type (WT) and MMP-3 deficient (mmp-3-/-) mice following RF injury. RF lesions (0.5 mm probe, 80 degrees C, 30 s) were created on the LV epicardium of WT (C57/BL6) and mmp-3-/- mice and were terminally studied at 1 h, 3, 7, and 28 days post-RF (n=10 each). Heart mass indexed to tibial length (mg/mm) was similar in the WT and mmp-3-/- mice at 1 h (8.1+/-0.3 vs. 7.6+/-0.3), but lower in the mmp-3-/- mice at 28 days post-RF (11.9+/-0.4 vs. 10.5+/-0.4, P<0.05). Scar volumes were greater in the mmp-3-/- mice at 3 days, but similar in the two groups at 28 days. Immunohistochemical localization showed fewer macrophages and lymphocytes at the scar border at 3 days in the mmp-3-/- hearts, but similar staining for these cells in WT and mmp-3-/- hearts at 7 and 28 days post-RF. Post-RF, the early increase in scar volume was accelerated in mmp-3-/- mice and associated with abnormal inflammatory cell infiltration/migration to the area of injury. These findings define a mechanistic role for MMP-3 in RF scar expansion and provide a temporal window during which interruption of MMP-3 activation may impair post-RF myocardial wound healing.

Proteomic analysis of mouse mammary terminal end buds identifies axonal growth cone proteins.

Ductal morphogenesis in the mouse mammary gland occurs mainly postnatally and is driven by specialized structures at the ends of the developing ducts, the terminal end buds (TEBs), which later regress once ductal growth is complete. To identify proteins that are specifically associated with migration of TEBs we developed a novel method of isolating TEBs, which eliminated the mammary stroma. The protein expression profile of the TEBs was then compared with that of isolates taken from the 4th inguinal mammary gland of adult virgin mice using two-dimensional (2-D) gel electrophoresis and mass spectrometry (MS) analysis (matrix-assisted laser desorption/ionization and quadrupole time of flight). Following construction of an integrated protein expression database, 44 protein features which showed differential expression levels between the two sets were chosen for MS analysis. Of these, 24 gave protein annotations whereas the other 20 produced unidentified peptides. Fourteen unequivocal proteins were identified from these 24, whereas the remaining 10 matched more than one protein within a single 2-D gel feature. Several of the identified proteins were associated with the cytoskeleton and have previously been reported in axonal growth cones, suggesting that they may influence cell shape and motility within the advancing TEBs, in a similar fashion to migrating axons.