Adenosine receptor transcriptomic profiling in leukocytes and cardiac fibroblast of patients with valvular disease undergoing prosthetic implantation.

Purpose: Adenosine is a potent extracellular messenger produced in high con- centrations under metabolically unfavourable conditions. It restores tissue home- ostasis through the interaction with its G-protein coupled membrane receptors (A1R, A2aR, A2bR and A3R), all with a cardioprotective role. ARs are expressed on multiple cardiac cells, including fibroblasts, endothelial cells, smooth muscle cells and leukocytes however the modulation of these receptors is still not fully understood. Aim of this study was to assess changes of transcriptomic profiling of A1R, A2aR, A2bR and A3R in human leukocytes of patients with HF due to valvu- lar disease (HF-V) as compared to healthy subjects (C) and in cardiac fibroblast (CF) obtained by human atrium (auricle) fragments of patients undergoing car- diac surgery for prosthetic implantation. A human CF atrial cell line (NHCF-A) was used as control (cc). Methods: Total RNA was extracted from leukocytes of C (n=7) and of Vlv pts (n=6, NYHA III-IV) with PAXgene Blood RNA Kit and by acid guanidinium thiocyanate-phenol-chloroform from CF (n=13). To avoid changes in the original phenotype, the experiments were conducted at the third passage in fibroblast cultures. Real-time PCR was performed and optimized for each AR primer. CD39 and CD73 mRNA expression was also evaluated. Results: For each receptor analyzed higher levels of mRNA expression were observed in patients with respect to C both at peripheral and cardiac level: Leuko- cytes A1R: C=1.97±0.72, HF-V=10.9±3.27, p=0.0098; A2aR: C=0.45±0.15, HF-V=3.19±0.94, p=0.0057; A2bR: C=0.43±0.16, HF-V=5.00±2.98, p=0.05; A3R: C=1.39±0.26, HF-V=2.9±0.71, p=0.04; CD39:C=0.33±0.05, HF- V=2.37±0.40 p<0.0001; CD73:C=1.34±0.38, HF-V=6.77±1.91, p=0.01. CF A1R: cc=0.33±0.004, CF=4.24±1.72; A2aR: cc=1.01±0.006, CF=2.77±0.67; A2bR: cc=0.07±0.03, CF=3.04±1.3; A3R: cc=0.74±0.26, CF=2.9±0.71, p=0.04; CD39 cc=0.66±0.002 CF=9.6±3.06; CD73: cc=0.32±0.01, CF=4.71±2.22. In the subgroup of patients (n=4) in which simultaneous cardiac biopsy and blood sample were obtained, AR leukocytes transcriptomic profile mimed that of CF showing the previously reported trend. Conclusions: These results demonstrated that all ARs are activated both in pe- ripheral leukocytes and at tissue cardiac level in HF-V pts. These preliminary data show, for the first time, that human circulating blood cells may express the same alterations occurring in the heart, thus suggesting their application for the study of the adenosinergic system in greater populations and in different pathologic conditions.