Overexpression of the adenosine A2A receptor in murine heart assessed by PET imaging with [18F]FLUDA


Overexpression of the adenosine A2A receptor in murine heart assessed by PET imaging with [18F]FLUDA

Gündel, D.; Lai, T. H.; Dukic-Stefanovic, S.; Toussaint, M.; Deuther-Conrad, W.; Teodoro, R.; Boknik, P.; Gergs, U.; Neumann, J.; Brust, P.

Introduction
The impact of adenosine A2A receptors (A2AR) in heart diseases is discussed diversely and only a few preclinical models are available. Therefore, we generated a transgenic mouse (TG) model with heart-specific overexpression of the human A2AR (hA2AR). The overexpressed hA2AR exhibited a cardioprotective, but proarrhythmic function, and A2AR agonists exerted a positive inotropic effect in vitro [1, 2]. Here, we show, that our new radioligand [18F]FLUDA can be used to assess hA2AR overexpression in murine heart by PET and to proof specific A2AR binding in human atrial samples by autoradiography.

Methods
FVB/N mice overexpressing the hA2AR under control of a α-MHC promotor were studied. Receptor autoradiography was used to determine Kd and Bmax of [18F]FLUDA in hearts of WT and TG mice (n=3). A 90 min dynamic µPET acquisition was performed to determine the biodistribution of [18F]FLUDA (7.4±3.4 MBq i.v.; Am=23-154 GBq/µmol) in wildtype (WT) and TG (n=3-5) after preinjection of vehicle or the A2AR antagonist tozadenant (2.5 mg/kg bw). The PET images were attenuation corrected by a T1-weighted MRI and list mode data were reconstructed (dynamic, 3D-OSEM algorithm). For activity quantification in tissues VOIs were determined by MRI images (Fig. 2A). In addition, the impact of 1µM FLUDA on the force of contraction (FOC) in isolated electrically driven atrial preparation was investigated.

Results
In vitro autoradiography of [18F]FLUDA in cryosections of hearts (Fig. 1) revealed a KD of 5.9 ± 1.6 nM and Bmax of 455 ± 78 fmol/mg protein in TG, whereas in WT the receptor density was too low for exact quantification. Using ZM241385 as competitor 36.3 ± 5.3 % specific binding of [18F]FLUDA was demonstrated in normal human atrial samples. PET/MR analyses revealed a 26 % (p<0.05) higher SUV ratio (SUVR) of myocard/blood (Fig.2B) in TG vs. WT mice. In TG mice the SUVR was reduced by tozadenant pre-treatment by 6 % (p<0.05) while no change in WT was observed. In electrically driven atrial preparations of TG (but not WT), FLUDA reduced the increase of FOC after CGS 2180 stimulation by about 20% (p<0.05), indicating a functional antagonism of FLUDA at the human cardiac A2AR.

Conclusion
FVB/N mice overexpressing the hA2AR are a useful model for non-invasive investigation of this receptor function with [18F]FLUDA PET. The demonstration of specific radiotracer binding in the human heart provides evidence that [18F]FLUDA might be a suitable radiotracer for assessment the A2AR status of human heart with PET. Our next steps will focus on the disease-related changes of the A2AR expression in human atrial samples with [18F]FLUDA.

Acknowledgments
The authors thank the European Regional Development Fund and Sächsische Aufbaubank (SAB) for financial support (project no. 100226753).

Keywords: [18F]FLUDA, adenosine, A2AR, heart

References
1. Boknik P, Drzewiecki K, Eskandar J, et al (2018) Phenotyping of mice with heart specific overexpression of A2A-adenosine receptors: Evidence for cardioprotective effects of A2A-adenosine receptors. Front Pharmacol 9:1–12. https://doi.org/10.3389/fphar.2018.00013
2. Boknik P, Drzewiecki K, Eskandar J, et al (2019) Evidence for arrhythmogenic effects of A2A-adenosine receptors. Front Pharmacol 10:1–12. https://doi.org/10.3389/fphar.2019.01051


Fig. 1: Representative autoradiography of 20 µm cryosections of a FVB/N mouse heart overexpressing the human adenosine A2AR (A2AR-TG), showing the total binding of [18F]FLUDA (left) and the non-homologous competition of [18F]FLUDA with the A2AR specific antagonist ZM241385 (right).


Fig. 2: A) Representative MR image (left), PET image (middle) and merged overlay (right) of one slice in the short axis orientation of TG mouse heart used for the determination of VOIs.; B) standardized uptake value ratio (SUVR) of myocard over blood under baseline (veh – vehicle) and blocking conditions (toz- tozadenant) in wildtype (WT) and myocard specific hA2AR overexpressing mice (TG); n=3-5, *p≤0.05 WT vs. TG and #p≤0.05 veh vs. toz.

Keywords: [18F]FLUDA; adenosine; A2AR; heart

  • Lecture (Conference) (Online presentation)
    15th European Molecular Imaging Meeting, 24.-27.03.2020, Thessaloniki, Griechenland

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