Myeloperoxidase and plasminogen activator inhibitor 1 play a central role in ventricular remodeling after myocardial infarction

Abstract

Left ventricular (LV) remodeling after myocardial infarction (MI) results in LV dilation, a major cause of congestive heart failure and sudden cardiac death. Ischemic injury and the ensuing inflammatory response participate in LV remodeling, leading to myocardial rupture and LV dilation. Myeloperoxidase (MPO), which accumulates in the infarct zone, is released from neutrophils and monocytes leading to the formation of reactive chlorinating species capable of oxidizing proteins and altering biological function. We studied acute myocardial infarction (AMI) in a chronic coronary artery ligation model in MPO null mice (MPO(-/-)). MPO(-/-) demonstrated decreased leukocyte infiltration, significant reduction in LV dilation, and marked preservation of LV function. The mechanism appears to be due to decreased oxidative inactivation of plasminogen activator inhibitor 1 (PAI-1) in the MPO(-/-), leading to decreased tissue plasmin activity. MPO and PAI-1 are shown to have a critical role in the LV response immediately after MI, as demonstrated by markedly delayed myocardial rupture in the MPO(-/-) and accelerated rupture in the PAI-1(-/-). These data offer a mechanistic link between inflammation and LV remodeling by demonstrating a heretofore unrecognized role for MPO and PAI-1 in orchestrating the myocardial response to AMI.

Figure 1.

Hematoxylin and eosin staining of representative cross sections from the mid-ventricle of (A and C) WT and (B and D) MPO^−/− 3 d after LAD ligation. The figures represent (A and B) the infarct zone and (C and D) the non-infarcted posterior wall. The insets of A and B show higher power images from the infarct zone revealing dead enucleated myocytes in the MPO^−/− and inflammatory infiltrate in the WT infarct zones. Bar, 50 μM.

Figure 2.

Quantification of CD45 immunoreactive cells within the infarct zone of WT and MPO^−/− 3 d after LAD ligation (n = 6) in each group. Data represent mean ± SD of five fields per animal.

Figure 3.

Echocardiographic analysis of LV size in WT and MPO^−/− as a function of time after AMI. (A) Anterior wall thickness, (B) posterior wall thickness, and (C) LVEDD before and 3 and 21 d after AMI in WT (○, n = 8 at each time point) and MPO^−/− (•, n = 8 at 0 and 3 d and n = 7 at 21 d after AMI) mice. There were no significant differences in measured parameters 3 d after AMI. At 21 d the anterior and posterior walls were thicker (P < 0.05 and P < 0.01, respectively) and the ventricle was less dilated (P < 0.01) in the MPO^−/− mice. Data represent mean ± SD. (D) Representative m-mode recording from a WT and MPO^−/− animal 21 d after AMI. *, P < 0.05, WT versus MPO^−/−.

Figure 4.

Protease activation within the infarct zone of WT and MPO^−/−. (A) Representative casein and (B) gelatin zymograms of protein extracts from the infarct zones of two representative WT and two representative MPO^−/− mice 3 d after AMI. Quantification of the density of (C) plasmin activity band and the (D) MMP-2 (open bars) and proMMP-9 (black bars) bands in the infarct zone of WT (n = 6) and MPO^−/− (n = 6) mice 3 d after AMI. No significant differences in plasmin activity in non-infarct myocardium between strains were seen, and no MMP activity was seen in non-infarcted tissue from either strain.

Figure 5.

PAI-1 activity 1 d after AMI in WT and MPO^−/−. (A) Representative reverse casein zymograms for PAI-1 activity in protein extracts from the infarct zones of two representative WT and two MPO^−/− mice with no MI (filled bars) or one representative MPO^−/−. (B) PAI-1 activity in WT and MPO^−/− 1 d after AMI (open bars) as quantified in arbitrary units from reverse zymograms (n = 4 animals per group per time point). *, P < 0.02, WT versus MPO^−/− 1 d after AMI. (C) Inhibition of PAI-1 activity (%) in the infarct zone relative to PAI-1 activity in strain-matched non-infarcted tissue as measured by two-stage indirect plasmin-based assay for PAI-1 in WT (n = 6) and MPO^−/− (n = 6) myocardium 1 d after LAD ligation. Data represent mean ± SD. *, P < 0.01.

Figure 6.

Collagen content in the infarct zone was quantified as the percent area that was birefringent under illumination with polarized light after staining with picrosirius red. Collagen content was quantified from five areas within the infarct zone from three frozen sections in three animals per time point in both the WT and MPO^−/−. Data represents mean ± SD. *, P < 0.02, WT versus MPO at given time point.

Figure 7.

Effect of MPO-mediated oxidation on PAI-1 activity in vitro and in vivo. (A) Inhibition of PAI-1 activity as a function of increasing hydrogen peroxide concentration in the presence of 25 nM human MPO. PAI-1 activity was quantified as residual urokinase activity after incubation with MPO-H₂O₂-Cl–treated PAI-1. Zero percent PAI-1 inhibition was defined as urokinase activity after incubation with PAI-1 treated with H₂O₂ or MPO alone and 100% PAI-1 inhibition was defined as urokinase activity in the absence of PAI-1. Data represent mean ± SD of three samples per H₂O₂ concentration. (B) Level of protein-bound 3-chlorotyrosine in PAI-1 as measured by gas chromatography-mass spectrometry after (gray bars) treatment with MPO-H₂O₂-Cl⁻ at the indicated H2O2 concentrations, or in non-infarcted myocardium or in the infarct zone of WT (n = 7) and MPO^−/− (n = 4) 1 and 3 d after LAD ligation. Data represent mean ± SD.

Figure 8.

Survival of WT (solid line, n = 17), MPO^−/− (dotted line, n = 10), and PAI-1^−/− (dashed line, n = 6) as a function of time after LAD ligation. No animals died in any group between days 15 and 21.