Summary                         

Changes in the amount and ratio of COL1 to COL3 alter the biomechanics of the heart, impair cardiac function, and drive heart failure (HF), which affects 64 million people globally with enormous societal and healthcare costs. Despite evidence that collagen type III (COL3) content changes during myocardial fibrosis, in vivo imaging of COL3 has not been achieved. Here, we discovered the first imaging probe that binds to COL3 with high affinity and specificity, by screening candidate peptide-based probes. Characterisation of the probe showed favourable magnetic and biodistribution properties. The probe’s potential for in vivo molecular cardiac magnetic resonance imaging was evaluated in a murine model of myocardial infarction. Using the new probe, we were able to map and quantify, previously undetectable, spatiotemporal changes in COL3 after myocardial infarction and monitor response to treatment. This innovative probe provides a promising tool to non-invasively study the unexplored roles of COL3 in cardiac fibrosis and other cardiovascular conditions marked by changes in COL3.

In vivo detection of myocardial fibrosis remains challenging. To date, invasive biopsy still remains the gold-standard method. Cardiac magnetic resonance (CMR) has emerged as the non-invasive imaging modality of choice to diagnose myocardial fibrosis, but T1 mapping and changes in ECV using nontargeted gadolinium agents can also be caused by other factors and thus may not be specific to fibrosis. CMR does not directly measure fibrosis and cannot differentiate between collagen subtypes. Therefore, development and optimisation of molecular probes for specific imaging of collagen types are needed to detect fibrosis and monitor therapeutic response. Two peptides were identified as potential COL3 binders:  Collagen Binding Peptide 1 (CBP1) and CBP2.

Results from nanoScan® PET/CT

In this study, the first COL3-binding probe suitable for molecular MRI was developed. Using this probe, changes in the natural turnover of COL3 noninvasively in a murine model of myocardial infarction (MI) was detected for the first time. It was also shown that the newly developed COL3 probe can detect changes in COL3 turnover following MI after treatment with the angiotensin-converting enzyme (ACE) inhibitor, enalapril.

In vivo biodistribution imaging was carried out using a positron emission tomography/computed tomography (PET/CT) scanner (NanoPET/CT scanner; Mediso, Hungary). Male apolipoprotein E knockout (ApoE-/-) were anesthetised with 4% isoflurane mixed with 1% medical oxygen and maintained at 2% isoflurane for the PET/CT experiment. Mice were injected intravenously (i.v.) with either ⁶⁸Ga-DOTAMA-CBP1 or ⁶⁸GaDOTAMA-CBP2 (12MBq/mouse for both probes). To verify anatomical structures, CT images were obtained after the PET imaging with a 55 kVp voltage. PET images were acquired continuously up to 90 min postinjection with a 256 × 256 matrix size and a resolution of 1 mm/pixel. PET and CT images were reconstructed using the manufacturer software with a voxel size of 0.4 mm and analysed with a VivoQuant 2.5 software (Invicro, Boston, MA, USA). The data acquired with the CBP1 and CBP2 imaging probes were reconstructed in 15-minute bins up to 90 min. The software was used for region-of-Interest (ROI) analysis and results were reported as %ID/g for each organ.

Results show:

• In vivo biodistribution experiments of the ⁶⁸Ga-DOTAMA-CBP1 and ⁶⁸Ga-DOTAMA-CBP2 probes in mice showed fast blood clearance, renal excretion, and no significant unspecific uptake in tissues (Fig. 3a).
• Quantification of the injected dose showed a fast first phase clearance from the blood with most of the probe eliminated within 15–30 minutes and a slower elimination phase between 30–90 min (Fig. 3b,c). Ex vivo gamma counting biodistribution experiments used to precisely quantify radioactivity in tissues confirmed the in vivo observations (Fig. 3d), indicating that both probes are excreted by renal clearance. These findings highlight the probes’ potential for targeted imaging and in vivo applications without unspecific retention in tissues and a safe elimination profile.

Fig. 3 | In vivo PET/CT pharmacokinetics and biodistribution using ⁶⁸Gallium labelled probes. a Fused PET and CT images demonstrate the clearance pathway and segmentation of the organs for quantitative analysis. b, c Quantification of injected dose of the ⁶⁸Ga-DOTAMA-CBP1 (b) and the ⁶⁸Ga-DOTAMA-CBP2 (c) probes in the kidney, liver, and blood over time shows favourable pharmacokinetics. d Ex vivo radioactivity measurements show high kidney uptake and low uptake in all other tissues. T time post-injection (minutes).

Also the probe demonstrated strong affinity against COL3 and favourable properties for in vivo MR imaging. The probe uncovered previously undetectable changes in COL3 remodelling post-MI and allowed monitoring of therapeutic response to an ACE inhibitor. This probe may provide a non-invasive tool to investigate the unexplored roles of COL3 in cardiac fibrosis.

Full article on nature.com