Persistent Microvascular Obstruction After Myocardial Infarction Culminates in the Confluence of Ferric Iron Oxide Crystals, Proinflammatory Burden, and Adverse Remodeling

Abstract

Background: Emerging evidence indicates that persistent microvascular obstruction (PMO) is more predictive of major adverse cardiovascular events than myocardial infarct (MI) size. But it remains unclear how PMO, a phenomenon limited to the acute/subacute period of MI, drives adverse remodeling in chronic MI setting. We hypothesized that PMO resolves into chronic iron crystals within MI territories, which in turn are proinflammatory and favor adverse remodeling post-MI.

Methods and results: Canines (n=40) were studied with cardiac magnetic resonance imaging to characterize the spatiotemporal relationships among PMO, iron deposition, infarct resorption, and left ventricular remodeling between day 7 (acute) and week 8 (chronic) post-MI. Histology was used to assess iron deposition and to examine relationships between iron content with macrophage infiltration, proinflammatory cytokine synthesis, and matrix metalloproteinase activation. Atomic resolution transmission electron microscopy was used to determine iron crystallinity, and energy-dispersive X-ray spectroscopy was used to identify the chemical composition of the iron composite. PMO with or without reperfusion hemorrhage led to chronic iron deposition, and the extent of this deposition was strongly related to PMO volume (r>0.8). Iron deposits were found within macrophages as aggregates of nanocrystals (≈2.5 nm diameter) in the ferric state. Extent of iron deposits was strongly correlated with proinflammatory burden, collagen-degrading enzyme activity, infarct resorption, and adverse structural remodeling (r>0.5).

Conclusions: Crystallized iron deposition from PMO is directly related to proinflammatory burden, infarct resorption, and adverse left ventricular remodeling in the chronic phase of MI in canines. Therapeutic strategies to combat adverse remodeling could potentially benefit from taking into account the chronic iron-driven inflammatory process.

Keywords: cytokines; hemorrhage; inflammation; iron ischemia-reperfusion injury; myocardial infarction.

© 2016 American Heart Association, Inc.

Figures

Figure 1. Chronic iron deposition in reperfused myocardial infarctions

Representative in-vivo raw and processed LGE and T2*-weighted images from Reperfused canines acquired in both acute and chronic phases post-MI are shown. Arrows point to the sites of MI and iron deposition on LGE and T2*-weighted images respectively. Corresponding ex-vivo histological sections stained with TTC, EMT and Perls stain are also shown. Scale bars in the microscopic histology images correspond to 50µm. Note the significant chronic iron deposition in the PMO+/T2*− group, despite the absence of acute reperfusion hemorrhage. Perls stain confirmed the presence of chronic iron deposition (blue deposits pointed at by the arrows) in the PMO+/T2*+ and PMO+/T2*− groups, but not in the PMO−/T2*− group. EMT stains showed significant fibrosis in the infarcted territory compared to a very mild diffuse fibrosis in the remote territory which were not visually evident on LGE. Asterisks in the T2*-weighted images point to the sites of off-resonance artifacts that were manually excluded in the final analysis.

Figure 2. Chronic iron deposition in non-reperfused myocardial infarctions

Representative in-vivo raw and processed LGE and T2*-weighted images from Non-reperfused canines acquired in both acute and chronic phases post-MI are shown. Arrows point to the sites of MI and iron deposition on LGE and T2*-weighted images respectively. Corresponding ex-vivo histological sections stained with TTC, EMT, and Perls stain are also shown. Scale bars in the microscopic histology images correspond to 50µm. Note the significant chronic iron deposition in the NR-PMO+/T2*+ group as observed on the in-vivo T2*-weighted images. Perls stain confirmed the presence of chronic iron deposition (blue deposits pointed at by the arrows) in the NR-PMO+/T2*+ group, but not in the NR-PMO−/T2*− group. Similar to the reperfused MI, EMT stains showed significant fibrosis in the infarcted territory compared to a very mild diffuse fibrosis in the remote territory, which were not visually evident on LGE. Asterisks in the T2*-weighted images point to the sites of off-resonance artifacts that were manually excluded in the final analysis.

Figure 3. PMO, and iron volumes in reperfused and non-reperfused myocardial infarctions

Median PMO volume (%LV, A), iron volume (%LV, B), and relationships between PMO volume with acute and chronic iron volumes (C) are shown from canines with reperfused MIs (PMO+/T2*+: n=9; PMO+/T2*−: n=4; PMO−/T2*−: n=4). Similarly, median NR-PMO volume (%LV, D), iron volume (%LV, E), and relationships between PMO volume with acute and chronic iron volumes (F) are shown from canines with non-reperfused MIs (NR-PMO+/T2*+: n=15; NR-PMO−/T2*−: n=1).

Figure 4. Transmission Electron Microscopy Images of Crystalline Deposits within Macrophages Found in the Territories of Chronic Myocardial Infarction

Panel A shows a longitudinal section of the macrophage cell with pronounced intracellular electron-dense material deposits (arrows). Panels B and C show enlarged area of a typical nodular pattern of material deposition. Panel D shows that the nodules are composed of clusters of highly crystalline nanoparticles with an approximate diameter of 2.5 nm (Panel E).

Figure 5. Physicochemical Characterization of Crystalline Iron within Macrophages

Panel A shows atomic resolution STEM image of a representative nanocrystalline particle from a Fe nodular cluster in a macrophage intracellular space. Notice the highly ordered pattern of aligned atomic columns. Panel B shows the EDS spectrum of the nodular material with the strong Fe presence. Panel C shows a SAED obtained from the Fe nodules revealing an exact fit with the pattern of a 6-line ferrihydrite. The respective values of diffraction rings are: 1) 0.150 nm, 2) 0.176 nm, 3) 0.214 nm, 4) 0.226 and 5) 0.256 nm.

Figure 6. Relationship between pro-inflammatory burden and chronic iron deposition

Representative contiguous ex-vivo histology sections stained with EMT, Perls, and monoclonal antibodies for Mac387, CD163, IL-1β, TNF-α and MMP-9 are shown from canines with reperfused and non-reperfused MIs with and without T2* losses (T2*+ and T2*- respectively) as observed in ex-vivo T2*-weighted images. Note significant co-localization of Mac387+ cells, TNF-α activity, and MMP-9 activity with chronic iron deposits. Strong linear relationships (n=20) of the area of iron (measured from Perls stain) were observed with area of Mac387+ cells (A), area of CD163+ cells (B), area of IL-1β activity (C), area of TNF-α activity (D), and area of MMP-9 activity (E). Arrows indicate the presence of specific markers of interest, which are zoomed in (insets).

Figure 7. Relationship between iron volume and infarct remodeling in reperfused and non-reperfused myocardial infarctions

Median iron volume as a fraction of infarct volume in acute and chronic phases of infarctions is shown in panels A (reperfused MI) and C (non-reperfused MI). The relationship between infarct resorption as a function of acute and chronic iron volumes are shown in panel B (non-reperfused MI) and D (non-reperfused MI). Sample sizes for the different reperfused and non-reperfused groups are as follows: i) Reperfused MIs – PMO+/T2*+: n=9; PMO+/T2*−: n=4; PMO−/T2*−: n=4, ii) Non-Reperfused MIs – NR-PMO+/T2*+: n=15; NR-PMO−/T2*−: n=1.

Figure 8. Relationship between Iron Volume and LV Structural Remodeling in reperfused and non-reperfused myocardial infarctions

Median EDSI from reperfused MIs (A) and non-reperfused MIs (E), as well as ΔEDSI from reperfused MIs (B) and non-reperfused MIs (F). Significant linear relationships of ΔEDSI with both acute and chronic infarct volumes were observed in both reperfused (C) and non-reperfused MIs (G). Similarly, significant linear relationships of ΔEDSI with acute and chronic iron volumes were evident in both reperfused (D) and non-reperfused (H) MIs. Sample sizes for the different reperfused and non-reperfused groups are as follows: i) Reperfused MIs – PMO+/T2*+: n=9; PMO+/T2*−: n=4; PMO−/T2*−: n=4, ii) Non-Reperfused MIs – NR-PMO+/T2*+: n=15; NR-PMO−/T2*−: n=1.