Animal models
The animal experiment was approved and carried out under the guidance of the animal ethics committee of Tongji Medical College, HUST. Fifty Sprague-Dawley (SD) rats (190 ± 20 g) with no specific pathogenicity were assigned to two groups: ISO-induced HF model group (n = 25) and the control group (n = 25). In the ISO-induced HF model group, rats were intraperitoneally injected with ISO (Aladdin, 5 mg/kg/day) for 14 days [20]. Rats in the control group received intraperitoneal injections of the same volume of physiological saline over the same period.
Echocardiographic assessment of cardiac function
Routine transthoracic echocardiography was performed every week before and after isoprenaline administration by two experienced operators (S He and J Wang), to verify the progress of the model and assess the left ventricular structural and functional parameters of rats. Before transthoracic echocardiography, rats were anesthetized with 1.5%–2% isoflurane to induce sedation and immobility in a supine position and at room temperature. We used Vivid E95 ultrasound machine (GE Medical Systems, Milwaukee, Wisconsin) equipped with a 12S probe at 9.0 MHz and EchoPAC workstation. At the level of the left ventricle papillary muscles, M-mode images guided by two-dimensional images of parasternal short-axis views were acquired. Left ventricular ejection fraction (LVEF), left ventricular fraction shortening (LVFS), systole left ventricle internal dimension (LVIDs), diastole left ventricle internal dimension (LVIDd), systole interventricular septal thickness (IVSs), diastole interventricular septal thickness (IVSd), systole posterior wall thickness (LVPWs), diastole posterior wall thickness (LVPWd), left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), stroke volume (SV), left ventricular mass (LVM) were measured.
Preparation of 68Ga-FAPI and micro-PET/CT imaging of rats
The methods of synthesis and labeling of 68Ga-FAPI refer to those reported in the literature [21]. The experimental and control group rats were injected with 68Ga-FAPI (18.5 ± 0.7 MBq) via the tail vein on certain days (0, 7, 14, 21 and 28 d after the first injection of ISO). The microPET/CT (TransPET Discoverist 180, Raycan Technology Co., Ltd, Suzhou, China) imaging was performed after 45 min post injection with an acquisition time of 15 min. The microPET/CT images were reconstructed with the ordered-subset expectation maximization three-dimensional/maximum a posteriori probability algorithm, and then the analysis of images was done using inveon research workplace.
Tissue biodistribution and autoradiography
At 0 (before modeling), 7, 14, 21 and 28 d, experimental and control groups (n = 3 per group) sacrificed at 30 min after injected with 68Ga-FAPI (18.3 ± 0.5 MBq). The organs of interest were harvested and weighed. Radioactivity was quantified using the γ-counter (PerkinElmer, USA). The heart was then serially sectioned according to Figure 1a (section thickness: 2 mm) and repeat the biodistribution measurements. The biodistribution result was expressed as a percentage of the injected dose per gram of tissue (% ID/g).
Phosphor-autoradiography (ARG) was performed on the sectioned tissue immediately afterwards. Tissue slices were exposed on the phosphor screen for 15 min, followed by scanning in the Storage Phosphor Imaging System (PerkinElmer Cyclone Plus, USA) to acquire ARG images.
Histology examination
The tissues were collected for hematoxylin-eosin (H&E) and Masson's trichrome staining. Each specimen was fixed with 4% paraformaldehyde and embedded in paraffin. Immunofluorescence (IF) was applied to identify FAP expression. The fixed tissue sections were treated in methanol at −20 °C for 15 min, in a serum-free blocking agent for 1 h at room temperature, and then incubated with primary antibodies for 1 h. The tissue sections were incubated in the secondary antibodies (FAP antibody, 1:200 dilution, Abcam) for 40–60 min, and mounted with Mounting Medium with 4',6-diamidino-2-phenylindole (DAPI). Image J was used to analyze the images of pathological tissues. For Masson's trichrome staining, the Collagen Volume Fraction (CVF) of myocardial tissues was calculated based on the formula: CVF = Collagen area / Total observed area × 100 %. The mean gray values of the FAP protein from IF were analyzed by Image J too.
Enzyme-linked immunosorbent assay
Blood samples of rats from experimental and control groups at different time were collected from the abdominal aorta with ethylenediaminetetraacetic acid (EDTA)-K2 vacuum blood-sampling tubes and centrifuged for 15 min at 3000 × g to obtain plasma. Plasma C-reactive protein (CRP), interleukin 6 (IL-6), cardiac troponin I (cTn-I), tumor necrosis factor alpha (TNF-α), angiotensin II (Ang-II) and B-type natriuretic peptide (BNP) levels were measured with commercially available enzyme-linked immunoassay (ELISA) kits (RuixinBiotech, Inc., China), according to the manufacturer’s protocol.
Recruitment of HF patients
This study was approved by the Clinical Research Ethics Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (NO. 20210617-01) and registered at the Clinical Trail (NCT04982458). Patients diagnosed with HF were recruited as part of this study. The inclusion criteria were shown as follows, 1) symptoms and signs of HF, 2) evidence of cardiac abnormalities, 3) age ≥ 18 y, 4) the consent of the patients, and 5) complete clinical data. Exclusion criteria were as follows: 1) acute coronary syndrome, 2) acute systemic disease or infection, uncontrolled metabolic disease, severe hepatic and renal dysfunction, 3) pregnant and lactating women. The patients had undergone both 13N-NH3 myocardial perfusion imaging (MPI) and 68Ga-FAPI PET scans within one day. The cardiac 68Ga-FAPI uptake from the subjects without cardiac disease (n = 20, clinically confirmed) were retrospectively collected for controls.
68Ga-FAPI and 13N-NH3 imaging protocol and image analysis in HF patients
For 13N-NH3 MPI, PET acquisition (Discovery VCT, GE Healthcare) was performed immediately after intravenous injection of 13N-NH3 (370–740 MBq) with an acquisition time of 10 min. Two hours after completing 13N-NH3 MPI, 68Ga-FAPI (1.8–2.2 MBq/kg) was injected intravenously. 68Ga-FAPI imaging was performed for 20 min at 45 min time points according to the previous method [22]. Attenuation-corrected PET images were reconstructed using ordered-subset expectation maximization iterative method. The gated data were reconstructed by volume image protocol (VIP) replay. 13N-NH3 perfusion and 68Ga-FAPI imaging were further processed using Emory Cardiac Toolbox in Xeleris Workstation (2.0, GE Healthcare). The PET indices of 68Ga-FAPI (maximum standardized uptake value [SUVmax], mean standardized uptake value [SUVmean] and SUVmax of myocardium / cardiocoelomic SUVmean [Normalized-SUVmax]) were acquired by Advanced Workstation (AW4.6, GE Healthcare).
Statistical analysis
Data were expressed as mean ± standard deviations (SD). Statistical analysis was performed using analysis of variance (ANOVA) and Student’s t-test with commercial software (GraphPad Prism 8 and SPSS 13.0). P < 0.05 indicates statistical significance.