Collagen-targeted tracers for molecular imaging of atrial fibrosis and sensitive detection of atrial fibrillation

Figures

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  Fig. 1. Interstitial fibrosis and active matrix metalloproteinases (MMPs) are enhanced in the ventricles and atria of the ‘AF + HF’ mouse model. a Representative images of Picrosirius red-stained coronal heart sections from ‘AF + HF’ double transgenic mouse (lower panels) and a healthy littermate control (upper panels) (6 months). The magnified blue-framed ventricular images (middle) and green-framed atrial images (right) correspond to the colored frames on the whole section (left). A wide spread interstitial fibrosis in both heart compartments – a hallmark of the ‘AF + HF’ mouse model7,20 - is evident. The significant expansion of atrial collagen, together with atrial fibrillation (AF), makes the ‘AF + HF’ mouse model suitable for molecular imaging studies of atrial disease. b, c Gelatin zymograms (left panels), as well as DQ™-gelatin-based MMP activity assays (curves in the middle panels and quantification of reaction rates in the right panels) on atrial (b) and left ventricle (LV) (c) heart lysates from ‘AF + HF’ and control mice (males; 28–29 weeks). An increase in the MMP-2 content and activity is observed in both heart chambers. These conditions could create an optimal target for…

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  Fig. 2. Collagen type I and type IV are upregulated in the hearts of ‘AF + HF’ mice. Gene expression microarray analysis demonstrating upregulation of the collagen IV alpha chains α3, α4 and α5 (a) and the collagen I α1 and α2 chains (b) in fibrotic atria of ‘AF + HF’ double transgenic mice relative to non-fibrotic control mice at 4.5-5 months of age. c Representative images of collagen IV-stained atrial tissue sections from control and ‘AF + HF’ mice (28–30 weeks; left panels), and quantification of the collagen-occupied area (middle), and mean collagen signal intensity (right) in these sections. This analysis confirms an increase in collagen IV in ‘AF + HF’ mouse atria. The ‘AF + HF’ mouse therefore contains the relevant types of cardiac collagens to suit molecular imaging with EP-3533 and the T-peptide tracer. n = 4 in (a, b), n = 3–4 in (c). Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by two-tailed Student’s t test.

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  Fig. 3. Ex vivo imaging of atrial fibrosis in ‘AF + HF’ mice with the collagen I-targeted cyclic peptide probe EP-3533. a A representative ex vivo near-infrared scan and b quantification of mean fluorescence intensity demonstrating the biodistribution of Cy5.5-conjugated EP-3533 (0.5 mg/kg) in perfused organs of double transgenic ‘AF + HF’ mice (29–31 weeks) and littermate controls at 4 h post-intravenous administration. Tracer uptake is similar between transgenic and control animals in all organs, except the heart (both in ventricles and atria). The prominent renal uptake indicates a renal clearance pathway. c A high-resolution, near-infrared ex vivo scan of an ‘AF + HF’ mouse heart (arbitrary color scale) and d a sub-analysis of the cardiac data from b showing robust tracer uptake by fibrotic atria, especially in the upper right chamber (RA). No significant differences between sexes are observed. Mean ± SD. n = 6 for control and n = 10 for ‘AF + HF’ (male mice in pink/red, female mice in gray). In (b) ****p < 0.0001 comparing the kidney to all other organs within each genotype; ##p < 0.01 comparing to the liver within each genotype, or as indicated, by two-way ANOVA with Sidak’s…

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  Fig. 4. Ex vivo imaging with the T-peptide probe targeting MMP-2-digested collagen IV demonstrates atrial fibrosis in ‘AF + HF’ mice. a A representative ex vivo near-infrared scan and b quantification of the mean fluorescence intensities demonstrating the biodistribution of Cy5.5-conjugated T-peptide (0.5 mg/kg; 4 h post-intravenous administration) in perfused organs of double transgenic ‘AF + HF’ mice (28–30 weeks) and littermate controls. Tracer uptake is high in the liver, lung and kidney. The fibrotic atria are particularly enhanced to the levels seen in the liver. Significant uptake is observed in both the fibrotic heart compartments (ventricles and atria) relative to control. c A sub-analysis of the cardiac data alone showing robust tracer uptake by the fibrotic atria of ‘AF + HF’ mice relative to both the healthy atria and diseased ventricles. Mean ± SD. n = 9 for control and n = 8 for ‘AF + HF’ (male mice in pink/red, female mice in gray). In (a) and (b) **p < 0.01, ***p < 0.001, ****p < 0.0001 by two-way ANOVA with Sidak’s post-hoc; #p < 0.05 and exact p-values by two-tailed Student’s t tests. In (c) δδδδp < 0.0001 by repeated-measure 2-way ANOVA with Sidak’s post-hoc.

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  Fig. 5. T-peptide uptake in the fibrotic atria of ‘AF + HF’ mice is specific. Comparison of the biodistribution in all major organs (a) and a sub-analysis of the cardiac accumulation (b) of Cy5.5-conjugated T-peptide (0.5 mg/kg; 0.258 µM/kg) vs. Cy5.5-labeled, mutated S-peptide (0.466 mg/kg; 0.258 µM/kg) in ‘AF + HF’ double transgenic mice (28–30 weeks). Perfused organs were scanned ex vivo on a near-infrared scanner 4 h post-intravenous tracer administration. The S-peptide signal was adjusted by the S-peptide/T-peptide brightness per mole slope ratio obtained from standard curves (Supplementary Figure 2). The T-peptide cardiac uptake is higher than the S-peptide in both ventricles and atria of ‘AF + HF’ animals, indicating its specificity. Mean ± SD. n = 5 per group (male mice in red squares and green triangles, female mice in gray). In (a) *p < 0.05, ***p < 0.001, ****p < 0.0001 by 2-way ANOVA with Sidak’s post-hoc; #p < 0.05, ##p < 0.01 by multiple unpaired t-test adjusted for multiple comparisons by Holm–Sidak post-hoc. In (b) *p < 0.05, ****p < 0.0001 by two-way ANOVA with Sidak’s post-hoc; δδδp < 0.001, δδδδp < 0.0001 by repeated-measure 2-way ANOVA with Sidak’s post-hoc, as…

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  Fig. 6. The T-peptide enhances collagen IV-containing structures in the atria of ‘AF + HF’ mice with distinct separation from intact collagen. Co-immunostaining for Cy5.5-labeled T-peptide (red) and intact collagen IV (green) in atrial (a) and ventricular (b) coronal sections of non-transgenic (control) and ‘AF + HF’ transgenic mouse hearts. The tracer labeling was performed in vivo by intravenous injection of fluorescent T-peptide (0.5 mg/kg; 4 h), followed by collagen IV immunohistochemistry ex vivo. Cell nuclei are depicted in blue (Hoechst). a In transgenic atria, the T-peptide strongly binds the thick fibrotic endocardium that envelopes the hypertrophic pectinate muscles (arrow heads), with some penetration into the atrial tissue (asterisks). The peptide staining pattern of fine filaments differs from the continuous pattern of intact collagen IV, and there is clear partitioning between the structures (right panels). b In ventricles, the T-peptide depicts the basement membrane of larger blood vessels, which are also positive for intact collagen IV (arrow heads); yet, while the two stains overlap, they do not co-localize. The right panels are magnifications of the framed areas in the…

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  Fig. 7. PET imaging with the T-peptide demonstrates cardiac fibrosis in ‘AF + HF’ mice in vivo. a Representative coronal, axial and sagittal PET/CT images of a control mouse (left) and an ‘AF + HF’ transgenic mouse (right) showing cumulative cardiac accumulation of 64Cu-T-peptide-MeCOSar27 (0.25 mg/kg; 25.86 ± 2.16 MBq) in the transgenic, but not control heart. The multi-bed PET acquisition was performed between 30 and 45 min post-tracer administration. b Quantification of the cumulative cardiac PET signal (in kBq/cc; acquisition parameters as in (a)) comparing ‘AF + HF’ mice to non-transgenic littermate controls (28–30 weeks). Strong enhancement of the transgenic (fibrotic) heart is observed relative to controls. Mean ± SD. n = 4 for control and n = 5 for ‘AF + HF’ (male mice in red, female mice in gray). ##p < 0.01 by t-test. Scale bars as indicated on the image. CT computed tomography, kBq/cc kilobecquerel per millilitre, MeCOSar 5-(8-methyl-3,6,10,13,16,19-hexaaza-bicyclo[6.6.6]icosan-1-ylamino)-5-oxopentanoic acid, PET positron emission tomography.