Noninvasive imaging of angiotensin receptors after myocardial infarction

Abstract

Objectives: The purpose of this study was to evaluate the feasibility of noninvasive imaging of angiotensin II (AT) receptor upregulation in a mouse model of post-myocardial infarction (MI) heart failure (HF).

Background: Circulating AT levels do not reflect the status of upregulation of renin-angiotensin axis in the myocardium, which plays a central role in ventricular remodeling and evolution of HF after MI. Appropriately labeled AT or AT receptor blocking agents should be able to specifically target AT receptors by molecular imaging techniques.

Methods: AT receptor imaging was performed in 29 mice at various time points after permanent coronary artery ligation or in controls using a fluoresceinated angiotensin peptide analog (APA) and radiolabeled losartan. The APA was used in 19 animals for intravital fluorescence microscopy on a beating mouse heart. Tc-99m losartan was used for in vivo radionuclide imaging and quantitative assessment of AT receptor expression in 10 mice. After imaging, hearts were harvested for pathological characterization using confocal and 2-photon microscopy.

Results: No or little APA uptake was observed in control animals or within infarct regions on days 0 and 1. Distinct uptake occurred in the infarct area at 1 to 12 weeks after MI; the uptake was at maximum at 3 weeks and reduced markedly at 12 weeks after MI. Ultrasonographic examination demonstrated left ventricular remodeling, and pathologic characterization revealed localization of the APA tracer with collagen-producing myofibroblasts. Tc-99m losartan uptake in the infarct region (0.524 +/- 0.212% injected dose/g) increased 2.4-fold as compared to uptake in the control animals (0.215 +/- 0.129%; p < 0.05).

Conclusions: The present study demonstrates the feasibility of in vivo molecular imaging of AT receptors in the remodeling myocardium. Noninvasive imaging studies aimed at AT receptor expression could play a role in identification of subjects likely to develop heart failure. In addition, such a strategy could allow for optimization of anti-angiotensin therapy in patients after MI.

Figure 1. Structure of Tracers Used for AT Receptor Targeting

Fluorescent angiotensin peptide analog (APA) (A), technetium Tc-99m losartan (B). AT = angiotensin II.

Figure 2. Optical Imaging of AT Receptors

Intravital microscopy images show a 3-week old MI in the murine heart failure model from baseline (A), and 3 min (B), and at 20 min (C) after administration of green fluorescent angiotensin peptide analog (APA), demonstrating gradual increase of the fluorescent APA in infarcted region. Fluorescent uptake was maximized at 20 min after intravenous APA injection. The ex vivo image (D), demonstrates APA uptake in the border zone, as thinned out infarct zone has collapsed and is therefore not visible. Panel E shows APA uptake in smooth muscle cell layer of a small coronary artery (vertical arrows) as well as minimal uptake in thinner veins (diagonal arrows)

Figure 3. Evolution of APA Uptake After MI

Intravital microscopy images demonstrating uptake of fluorescent APA from 6 groups: no infarction, and 0 day, 1 day, 1 week, 3 weeks and 12 weeks after MI (A) The uptake increases from 1 to 3 weeks, and decreases thereafter. Panel B shows the respective echocardio-grams at 1, 3, and 12 weeks, indicating left ventricular dilation and progressive heart failure in these mice with MI.

Figure 4. Pathologic Characterization APA Localization

The remote zone (A) demonstrates no APA uptake in interstitium (*) or cardiomyocytes (arrows) The cells of nonmyocytic origin demonstrate fluorescent APA uptake in the infarct zone (B), with an enlarged image of cellular uptake (inset) These cells in the infarct region (D, green) also stained positively for smooth muscle actin (SMA) (E, red), showing colocalization (F, yellow), indicating AT receptors (ATR) on myofibroblasts. (C) Ex vivo 2-photon microscopy demonstrated fluorescent APA intracellularly within the myofibroblast (green), with collagen surrounding the myofibroblast using second harmonic generation imaging (blue) Abbreviations as in Figure 1.

Figure 5. Noninvasive Imaging of AT Receptors With Radiolabeled Losartan

The micro–SPECT and micro-CT images are shown in a control mouse after technetium Tc-99m losartan administration; no uptake in the heart can be seen (A) in the in vivo and ex vivo images. There is only some liver uptake on the bottom left of the SPECT image. (B) In the 3-week post-MI animal, significant radiolabeled losartan uptake is observed in the anterolateral wall (arrows) The infarct uptake on the in vivo image is confirmed in the ex vivo image. The histogram (C) demonstrates significantly (*) higher uptake in the infarcted region (0.524 ± 0.212% ID/g) as compared to control noninfarcted animals (0.215 ± 0.129% ID/g; p < 0.05). ID = injected dose.