Early matrix metalloproteinase-9 inhibition post-myocardial infarction worsens cardiac dysfunction by delaying inflammation resolution.

Matrix metalloproteinase-9 (MMP-9) is robustly elevated in the first week post-myocardial infarction (MI). Targeted deletion of the MMP-9 gene attenuates cardiac remodeling post-MI by reducing macrophage infiltration and collagen accumulation through increased apoptosis and reduced inflammation. In this study, we used a translational experimental design to determine whether selective MMP-9 inhibition early post-MI would be an effective therapeutic strategy in mice. We enrolled male C57BL/6J mice (3-6months old, n=116) for this study. Mice were subjected to coronary artery ligation. Saline or MMP-9 inhibitor (MMP-9i; 0.03µg/day) treatment was initiated at 3h post-MI and the mice were sacrificed at day (D) 1 or 7 post-MI. MMP-9i reduced MMP-9 activity by 31±1% at D1 post-MI (p<0.05 vs saline) and did not affect survival or infarct area. Surprisingly, MMP-9i treatment increased infarct wall thinning and worsened cardiac function at D7 post-MI. While MMP-9i enhanced neutrophil infiltration at D1 and macrophage infiltration at D7 post-MI, CD36 levels were lower in MMP-9i compared to saline, signifying reduced phagocytic potential per macrophage. Escalation and prolongation of the inflammatory response at D7 post-MI in the MMP-9i group was evident by increased expression of 18 pro-inflammatory cytokines (all p<0.05). MMP-9i reduced cleaved caspase 3 levels at D7 post-MI, consistent with reduced apoptosis and defective inflammation resolution. Because MMP-9i effects on inflammatory cells were significantly different from previously observed MMP-9 null mechanisms, we evaluated pre-MI (baseline) systemic differences between C57BL/6J and MMP-9 null plasma. By mass spectrometry, 34 plasma proteins were significantly different between groups, revealing a previously unappreciated altered baseline environment pre-MI when MMP-9 was deleted. In conclusion, early MMP-9 inhibition delayed inflammation resolution and exacerbated cardiac dysfunction, highlighting the importance of using translational approaches in mice.

Early matrix metalloproteinase-12 inhibition worsens post-myocardial infarction cardiac dysfunction by delaying inflammation resolution.

RATIONALE: Matrix metalloproteinases (MMPs) regulate remodeling of the left ventricle (LV) post-myocardial infarction (MI). MMP-12 has potent macrophage-dependent remodeling properties in the atherosclerotic plaque; however, post-MI roles have not been examined. OBJECTIVE: The goal was to determine MMP-12 post-MI mechanisms. METHODS AND RESULTS: Male C57BL/6J mice (3-6 months old) were subjected to left coronary artery ligation. Saline or the RXP 470.1 MMP-12 inhibitor (MMP-12i; 0.5mg/kg/day) was delivered by osmotic mini-pump beginning 3h post-MI, and mice were sacrificed at day (d)1, 3, 5 or 7 post-MI and compared to d0 controls (mice without MI; n=6-12/group/time). MMP-12 expression increased early post-MI, and contrary to expected, neutrophils were a surprising early cellular source for MMP-12. MMP-12i reduced MMP-12 activity 33 ± 1% at d1 post-MI. Despite similar infarct areas and survival rates, MMP-12i led to greater LV dilation and worsened LV function. At d7 post-MI, MMP-12i prolonged pro-inflammatory cytokine upregulation (IL1r1, IL6ra, IL11, and Cxcr5) and decreased CD44 (both gene and protein levels). Hyaluronan (HA), a CD44 ligand, was elevated at d1 and d7 post-MI with MMP12i, as a result of decreased fragmentation. Because CD44-HA regulates neutrophil removal, apoptosis markers were evaluated. Caspase 3 increased, while cleaved caspase 3 levels decreased in MMP-12i group at d7 post-MI, indicating reduced neutrophil apoptosis. In isolated neutrophils, active MMP-12 directly stimulated CD44, caspase 3, and caspase 8 expression. CONCLUSION: Our results reveal a novel protective mechanism for MMP-12 in neutrophil biology. Post-MI, MMP-12i impaired CD44-HA interactions to suppress neutrophil apoptosis and prolong inflammation, which worsened LV function.

Multiple polymer architectures of human polyhomeotic homolog 3 sterile alpha motif.

The self-association of sterile alpha motifs (SAMs) into a helical polymer architecture is a critical functional component of many different and diverse array of proteins. For the Drosophila Polycomb group (PcG) protein Polyhomeotic (Ph), its SAM polymerization serves as the structural foundation to cluster multiple PcG complexes, helping to maintain a silenced chromatin state. Ph SAM shares 64% sequence identity with its human ortholog, PHC3 SAM, and both SAMs polymerize. However, in the context of their larger protein regions, PHC3 SAM forms longer polymers compared with Ph SAM. Motivated to establish the precise structural basis for the differences, if any, between Ph and PHC3 SAM, we determined the crystal structure of the PHC3 SAM polymer. PHC3 SAM uses the same SAM-SAM interaction as the Ph SAM sixfold repeat polymer. Yet, PHC3 SAM polymerizes using just five SAMs per turn of the helical polymer rather than the typical six per turn observed for all SAM polymers reported to date. Structural analysis suggested that malleability of the PHC3 SAM would allow formation of not just the fivefold repeat structure but also possibly others. Indeed, a second PHC3 SAM polymer in a different crystal form forms a sixfold repeat polymer. These results suggest that the polymers formed by PHC3 SAM, and likely others, are dynamic. The functional consequence of the variable PHC3 SAM polymers may be to create different chromatin architectures.

Using proteomics to uncover extracellular matrix interactions during cardiac remodeling.

The left ventricle (LV) responds to a myocardial infarction with an orchestrated sequence of events that result in fundamental changes to both the structure and function of the myocardium. This collection of responses is termed as LV remodeling. Myocardial ischemia resulting in necrosis is the initiating event that culminates in the formation of an extracellular matrix (ECM) rich infarct scar that replaces necrotic myocytes. While the cardiomyocyte is the major cell type that responds to ischemia, infiltrating leukocytes and cardiac fibroblasts coordinate the subsequent wound healing response. The matrix metalloproteinase family of enzymes regulates the inflammatory and ECM responses that modulate scar formation. Matridomics is the proteomic evaluation focused on ECM, while degradomics is the proteomic evaluation of proteases as well as their inhibitors and substrates. This review will summarize the use of proteomics to better understand matrix metalloproteinase roles in post myocardial infarction LV remodeling.

The history of matrix metalloproteinases: milestones, myths, and misperceptions.

Since the discovery of tadpole collagenase in 1962, the matrix metalloproteinase (MMP) family has emerged as a significant proteinase group with recognized effects on the cardiovascular system. Over the last 40 years, many milestones have been achieved, from the identification of the first MMP, to the generation of the first MMP cDNA clone and null mouse, to the clinical approval of the first MMP inhibitor. Over the years, a few myths and misunderstandings have interwoven into the truths. In this review, we will discuss the major milestones of MMP research, as well as review the misinterpretations and misperceptions that have evolved. Clarifying the confusions and dispelling the myths will both provide a better understanding of MMP properties and functions and focus the cardiovascular field on the outstanding research questions that need to be addressed.