Myeloperoxidase Inhibition Improves Ventricular Function and Remodeling After Experimental Myocardial Infarction

Abstract

PF-1355 is an oral myeloperoxidase (MPO) inhibitor that successfully decreased elevated MPO activity in mouse myocardial infarction models. Short duration PF-1355 treatment for 7 days decreased the number of inflammatory cells and attenuated left ventricular dilation. Cardiac function and remodeling improved when treatment was increased to 21 days. Better therapeutic effect was further achieved with early compared with delayed treatment initiation (1 h vs. 24 h after infarction). In conclusion, PF-1355 treatment protected a mouse heart from acute and chronic effects of MI, and this study paves the way for future translational studies investigating this class of drugs in cardiovascular diseases.

Keywords: CNR, contrast to noise ratio; EDV, end-diastolic volume; EF, ejection fraction; IRI, ischemia reperfusion injury; LAR, lesion activation ratio; Ly-6C, lymphocyte antigen 6C; MI, myocardial infarction; MPO, myeloperoxidase; MPO-Gd, bis-5-hydroxytryptamide-diethylenetriaminepentaacetate-gadolinium; MPO−/−, myeloperoxidase knock out; inflammation; myeloperoxidase; myocardial infarction; oxidative stress; treatment.

Graphical abstract

Figure 1

Day 2 MPO Inhibition With PF-1355 (A) Mice with permanent coronary ligation were administered with oral 50 mg/kg PF-1355 (2-[6-(2,5-dimethoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl]acetamide) twice daily and compared with vehicle-treated control animals (n = 4 to 7 per group). On day 2 post–myocardial infarction (MI), hearts were processed for extracellular protein fractions (ECF), intracellular protein fractions (ICF), and myeloperoxidase (MPO) activity was performed with antibody capture assay with 10-acetyl-3,7-dihydroxyphenoxazine, reported as relative fluorescence units (RFU)/s/μg of protein. (B to D) In vivo MPO inhibition as measured by bis-5-hydroxytryptamide-diethylenetriaminepentaacetate-gadolinium imaging. (B) Representative T₁-weighted midventricular slices showing pre-contrast, early (15 min), and delayed (60 min) contrast-enhanced images; dotted areas represent the infarct areas. (C) Lesion activation ratio (LAR) of untreated (n = 4) and PF-1355–treated groups (n = 15); bar graphs represent 60-min CNR values. (D) CNR values plotted as a function of time show a decrease in enhancement over time compared with untreated control subjects. (E) There is no significant difference in infarct area between groups at day 2 post-MI. Data plotted as mean ± SEM; *p < 0.05; **p < 0.01; ∗∗∗p < 0.001.

Figure 2

Comparison Between Vehicle and PF-1355–Treated Groups on Day 7 (A) Both MPO- and CD11b-positive areas, plotted as percent of high power field (HPF), are decreased in treated mice (n = 5/group; scale bar: 100 μm). (B) Collagen I and CD31 staining at day 7 do not show differences at this early healing phase (n = 5/group; scale bar: 100 μm). (C) Midventricular cardiac sections show decreased ventricular thinning as early as day 7 post-MI in treated mice (scale bar: 2 mm). Arrows point to the ventricular wall containing infarct tissue that is at risk of ventricular thinning. (D) Flow cytometry analysis representing heart neutrophils, lymphocyte antigen 6C (Ly-6C)^high monocytes, and Ly-6C^low monocytes from 7-day-old infarcts, plotted as percent cells/total leukocytes, defined as CD45⁺ cells. Both neutrophil and Ly-6C^high monocyte percentages were decreased in the treated group with sparing of Ly-6C^low monocytes (n = 4 to 5 mice/group). Data plotted as mean ± SEM. *p < 0.05. Abbreviations as in Figure 1.

Figure 3

Presence of MPO-Containing Inflammatory Cells in Infarcts During Late Remodeling Phase (A) Immunostaining on days 7 and 14 post-MI reveals higher CD11b and MPO staining areas as compared with no MI (n = 3 to 5/group). Low-magnification midventricular sections of day 7 and 14 infarcts show extensive left ventricular thinning and remodeling (scale bar: 2 mm). Inset: high-magnification histology (scale bar: 100 μm) shown at days 7 and 14. (B) Percentage of MPO- and CD11b-positive areas per high power field (HPF). (C) Dot plots showing increased CD11b positive cells/leukocytes (blue boxes) in hearts of 7- and 15-day-old infarcts as compared with hearts without infarcts. Neutrophils (red boxes) and Ly-6C^high monocytes (green boxes) were also significantly elevated at both time points. Data plotted as mean ± SEM. *p < 0.05; **p < 0.01. Abbreviations as in Figures 1 and 2.

Figure 4

Prolonged PF-1355 Treatment Improves Cardiac Function and Remodeling Infarcted mice were divided into 3 groups: untreated (MI-no Tx, n = 6), treated for 21 days (MI-21 days Tx, n = 5), and treated for first 7 days only and then treatment stopped until day 21 for imaging (MI-7 days Tx, n = 8). (A to C) Representative midventricular magnetic resonance images obtained at day 21 after MI. Videos obtained from the same mice at day 2 and day 21 are shown in the Supplemental Video 1. (D) There is significant improvement in ejection fraction (EF) in the 21-day treatment group as compared with untreated infarcts; however, the 7-day treatment group did not show significant improvement. (E) Bar graphs representing end-diastolic volume (EDV). (F) Left ventricular mass is less in the 21-day treatment group representing decreased remote myocardial hypertrophy and improved remodeling. Data plotted as mean ± SEM. *p < 0.05; ***p < 0.001. MI = myocardial infarction; Tx = treatment.

Supplemental Video 1 Top row: Day 2 post-MI imaging of representative mice with similar sized infarcts (MPO-Gd enhanced areas marked with arrows) for all three groups. Bottom row: Follow-up day 21 MRIs in the same mice demonstrating infarct wall thickness and motion improved with longer treatment periods compared to the untreated control mice. Arrows in the follow-up day 21 imaging are only illustrative and point to the myocardial areas that earlier contained infarcts on day 2 imaging. The same zoom factor was applied to all the movies.

Figure 5

Therapeutic Benefit of MPO Inhibitor Treatment Is Maximized by Initiating Treatment Close to the Event Mice were subjected to coronary artery occlusion. MPO inhibitor treatment (50 mg/kg, twice daily) was initiated at 1 h (MI early Tx_i, n = 15) or 24 h (MI delayed Tx_i, n = 13) post-surgery. Vehicle treatment started at 1 h as control (MI vehicle, n = 11). Cardiac function was evaluated by echocardiography. (A) Representative left ventricular M-mode images. (B to E) Bar graphs represent ejection fraction (EF), fractional shortening (FS), stroke volume, and left ventricular end-systolic volume (ESV). Data plotted as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Tx_i = treatment initiation; other abbreviations as in Figures 1, 2, and 3.