Matrix metalloproteinases (MMPs) play an essential role in normal and pathological extracellular matrix degradation. Deuterium exchange mass spectrometry (DXMS) was used to localize the binding regions of the broad-spectrum MMP inhibitor doxycycline on the active form of matrilysin (residues 95-267) and to assess alterations in structure induced by doxycycline binding. DXMS analyses of inhibitor-bound versus inhibitor-free forms of matrilysin reveal two primary sites of reduced hydrogen/ deuterium exchange (residues 145-153; residues 193-204) that flank the structural zinc binding site. Equilibrium dialysis studies of doxycycline-matrilysin binding yielded a K d of 73 M with a binding stoichiometry of 2.3 inhibitor molecules per protein, which compares well with DXMS results that show principal reduction in deuterium exchange at two sites. Lesser changes in deuterium exchange evident at the amino and carboxyl termini are attributed to inhibitor-induced structural fluctuations. Tryptophan fluorescence quenching experiments of matrilysin with potassium iodide suggest changes in conformation induced by doxycycline binding. In the presence of doxycycline, tryptophan quenching is reduced by approximately 17% relative to inhibitor-free matrilysin. Examination of the X-ray crystal structure of matrilysin shows that the doxycycline-binding site at residues 193 to 204 is positioned within the structural metal center of matrilysin, adjacent to the structural zinc atom and near both calcium atoms. These results suggest a mode of matrilysin inhibition by doxycycline that could involve interactions with the structural zinc atom and/or calcium atoms within the structural metal center of the protein.

The development of strategies to ameliorate post-myocardial infarction (MI) remodeling and improve function continues to be an area of clinical importance. Use of steroids for this purpose is controversial since the effects of timed treatment on relevant inflammatory, biochemical and structure/function endpoints are unclear. In a previous report, we demonstrated that use of doxycycline pre-treatment improves post-MI remodeling and passive left ventricular (LV) function. However, the effects of timed doxycycline post-MI treatment are unknown. To examine these issues, we performed a study using a rat MI model. Animals were administered one of the following: doxycycline (DOX), the corticosteroid methylprednisolone (MP), or aqueous vehicle. Treatment was given early, short-term (at time of MI to 24 h post-MI) or late, long term (2-7 days post-MI). Animals were sacrificed at 3, 7 or 42 days post-surgery. We assessed LV hemodynamics, pressure-volume, and pressure-scar strains, histomorphometry, inflammation via measurements of myeloperoxidase activity, and matrix metalloproteinase (MMP) activity. Late MP treatment yielded a robust right-shifted pressure-volume curve, which was accompanied by increased scar strains. Late DOX treatment yielded reduced average heart weight and size and preserved scar thickness. DOX treatment did not suppress inflammation, which contrasts with the suppressive effects of MP. Use of early or late MP yielded increased MMP activity in infarcted and non-infarcted regions. Early and late treatment with DOX yielded infarct-associated MMP activity levels comparable to those of vehicle-treated animals. In conclusion, results indicate that late use of MP yields adverse post-MI structure/function outcomes that correlate with suppression of inflammation and increased MMP activity. These observations contrast with those of DOX, in particular, late treatment where improved outcomes were observed in LV structure and were accompanied by the lack of suppression of inflammation.

Activation of matrix metalloproteinases (MMPs) in the heart is known to facilitate cardiac remodeling and progression to failure. We hypothesized that regional dyskinetic wall motion of the left ventricle would stimulate activation of MMPs. Abnormal wall motion at a target site on the anterior lateral wall of the left ventricle was induced by pacing atrial and ventricular sites of five open-chest anesthetized dogs. Changes in shortening at the left ventricular (LV) pacing site and at a remote site at the anterior base of the left ventricle were monitored with piezoelectric crystals. Simultaneous atrial and ventricular pacing resulted in abnormal motion at the LV pacing site, yielding early shortening and late systolic lengthening, whereas the shortening pattern at the remote site remained unaffected. Assessment of global myocardial MMP activity showed a sevenfold increase in substrate cleavage (P < 0.02) at the LV pacing site relative to the remote site. Gelatin zymography revealed increases in 92-kDa MMP-9 activity and 86-kDa MMP-9 activity at the LV pacing site relative to the remote site, whereas MMP-2 activity was unaffected. Abnormal wall motion was associated with increases in collagen degradation (approximately 2-fold; P < 0.03), plasmin activity (approximately 1.5-fold; P < 0.05), nitrotyrosine levels (approximately 20-fold; P = 0.05), and inflammatory infiltrate (approximately 2-fold; P < 0.02) relative to the remote site. Results indicate that regional dyskinesis induced by epicardial activation is sufficient to stimulate significant MMP activity in the heart, suggesting that abnormal wall motion is a stimulus for MMP activation.

Analysis of collagen degradation remains an important but cumbersome task. Traditional methods with dansyl chloride derivitatization of collagen have been used to quantify collagen damage. Fluorescent labeling reagents have been developed that offer advantages such as greater solubility in water and low background emission. One such reagent is o-phthaladehyde (OPA). In this study, we used OPA as a means of detecting small amounts of degraded collagen. Collagen samples isolated from skin or heart were used for OPA conjugation to exposed amino termini ("opalation"). Experiments utilizing small samples aliquoted in microtiter plates were performed to evaluate effects of increasing concentrations of OPA, varying concentrations of collagen, and effects of matrix metalloproteinase (MMP) digestion. Results indicate that within 10 minutes of reaction, OPA can be used to detect relative differences in cleaved vs. uncleaved collagen from skin or heart. Heart samples obtained from regions of high MMP activity correlated with increased OPA fluorescence relative to tissue with lower MMP activity. On the basis of these results, we conclude that OPA has valuable practical advantages for analytical use in detecting cleaved collagen in small tissue samples.