International Journal of Molecular Sciences Article Adenosine A2A Receptors Are Upregulated in Peripheral Blood Mononuclear Cells from Atrial Fibrillation Patients Héctor Godoy-Marín 1,2 , Romain Duroux 3 , Kenneth A. Jacobson 3 , Concepció Soler 2,4 , Hildegard Colino-Lage 5,6 , Veronica Jiménez-Sábado 6,7 , José Montiel 8 , Leif Hove-Madsen 5,6,7, * and Francisco Ciruela 1,2, * 1 Pharmacology Unit, Department of Pathology and Experimental Therapeutics, Faculty of Medicine and Health Sciences, Institute of Neurosciences, University of Barcelona, 08907 L’Hospitalet de Llobregat, Spain; hectorgodoymarin13@gmail.com 2 Neuropharmacology & Pain Group, Neuroscience Program, Institut d’Investigació Biomèdica de Bellvitge, IDIBELL, 08907 L’Hospitalet de Llobregat, Spain; concepciosoler@ub.edu 3 Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA; romain.duroux@gmail.com (R.D.); kennethj@niddk.nih.gov (K.A.J.) 4 Immunology Unit, Department of Pathology and Experimental Therapeutics, Faculty of Medicine and Health Sciences, Institute of Neurosciences, University of Barcelona, 08907 L’Hospitalet de Llobregat, Spain 5 Barcelona Biomedical Research Institute, IIBB-CSIC, 08036 Barcelona, Spain; HColino@santpau.cat 6 Biomedical Research Institute Sant Pau, IIB Sant Pau, 08025 Barcelona, Spain; VJimenezS@santpau.cat 7 CIBERCV, 28029 Madrid, Spain 8  Department Cardiac Surgery, Hospital de la Santa Creu i Sant Pau, 08036 Barcelona, Spain;  JMontiel@santpau.cat * Correspondence: leif.hove@iibb.csic.es (L.H.-M.); fciruela@ub.edu (F.C.) Citation: Godoy-Marín, H.; Duroux, R.; Jacobson, K.A.; Soler, C.; Colino-Lage, H.; Jiménez-Sábado, V.; Abstract: Atrial fibrillation (AF) is the most common form of cardiac arrhythmia seen in clinical Montiel, J.; Hove-Madsen, L.; Ciruela, practice. While some clinical parameters may predict the transition from paroxysmal to persistent AF, F. Adenosine A2A Receptors Are the molecular mechanisms behind the AF perpetuation are poorly understood. Thus, oxidative stress, Upregulated in Peripheral Blood calcium overload and inflammation, among others, are believed to be involved in AF-induced atrial Mononuclear Cells from Atrial remodelling. Interestingly, adenosine and its receptors have also been related to AF development Fibrillation Patients. Int. J. Mol. Sci. and perpetuation. Here, we investigated the expression of adenosine A2A receptor (A2AR) both 2021, 22, 3467. https://doi.org/ in right atrium biopsies and peripheral blood mononuclear cells (PBMCs) from non-dilated sinus 10.3390/ijms22073467 rhythm (ndSR), dilated sinus rhythm (dSR) and AF patients. In addition, plasma adenosine content and adenosine deaminase (ADA) activity in these subjects were also determined. Our results Academic Editor: Katia Varani revealed increased A2AR expression in the right atrium from AF patients, as previously described. Received: 6 March 2021 Interestingly, increased levels of adenosine content and reduced ADA activity in plasma from AF Accepted: 24 March 2021 patients were detected. An increase was observed when A2AR expression was assessed in PBMCs Published: 27 March 2021 from AF subjects. Importantly, a positive correlation (p = 0.001) between A2AR expression in the right atrium and PBMCs was observed. Overall, these results highlight the importance of the A2AR Publisher’s Note: MDPI stays neutral in AF and suggest that the evaluation of this receptor in PBMCs may be potentially be useful in with regard to jurisdictional claims in monitoring disease severity and the efficacy of pharmacological treatments in AF patients. published maps and institutional affil- iations. Keywords: adenosine A2A receptor; atrial fibrillation; adenosine deaminase; peripheral blood mononuclear cells; adenosine Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. 1. Introduction This article is an open access article Atrial fibrillation (AF) is one of the most common cardiac arrhythmias, which is distributed under the terms and currently thought to affect approximately 2% of the world population. Its prevalence conditions of the Creative Commons increases with age, affecting around 0.14% of people under 49 years old and increasing Attribution (CC BY) license (https:// to 10-17% in people aged 80 or older [1]. This arrhythmia is known to cause an irregular creativecommons.org/licenses/by/ and often abnormal fast heart rate and increases the risk of stroke, heart failure and other 4.0/). Int. J. Mol. Sci. 2021, 22, 3467. https://doi.org/10.3390/ijms22073467 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 3467 2 of 12 heart-related complications. The mechanisms underlying AF are complex and partially unresolved, but they involve electrical and structural remodelling (fibrosis) [2] that chiefly provokes a desynchronized activation of the atrium due to a shortening of the action potential wavelength, thus, facilitating fast and irregular beating [3]. AF episodes typically occur when arrhythmogenic substrates, such as fibrosis and inflammation coincide [4]. Indeed, inflammation has been associated with AF-pathological processes, as well as oxidative stress or thrombogenesis [5]. Therefore, it has been shown that the prevalence and prognosis of AF are highly associated with plasma levels of inflammatory biomarkers. Interestingly, as in any other inflammatory event, lymphocytes are recruited to the heart environment and play a key role in both stimulating and inhibiting inflammatory responses during the initiation, maintenance, and progression of AF. Therefore, the neutrophils to lymphocyte ratio becomes an actual outcome predictor in AF [6]. Notably, lymphocytes are also affected by the atrium inflammatory environment, which courses with an increased adenosine production and release, thus, inducing plastic changes in the lymphocytes during this process [7]. Adenosine is a purine nucleoside generated mostly by ATP catabolism and regulated by adenosine deaminase (ADA), adenosine kinase (ADK) and S-adenosyl-l-homocysteine hydrolase (SAHase) [8]. Adenosine levels are dramatically increased during inflamma- tion [9,10], hypoxia [11], ischemia [12] and β-adrenergic receptor stimulation [13], and its release into the heart induces profound alterations in cardiomyocyte physiology, which may eventually evolve into AF development [14,15]. Since extracellular actions of adeno- sine are exerted through G protein-coupled adenosine receptors (ARs), namely A1 R, A2A R, A2B R and A3 R, some of these have been related to AF physiopathology. For instance, it has been demonstrated that A2A R density is increased in AF patients, leading to an abnormal calcium handling in the cardiomyocyte and triggering heart disfunctions [16,17]. Therefore, it is well-accepted that A2A R overexpression and activation by selective agonists may favour the occurrence of cardiac arrhythmias [18]. Conversely, the activation of A1 R by intrinsic adenosine modifies atrial electrophysiology and promotes AF [19]. Accordingly, it has been postulated that adenosine shows a Janus-faced cardiac behaviour: Protection against ischemic damage but induction of cardiac arrhythmias [20]. Here, we set out to assess the status of the adenosinergic system in AF by analysing the expression of A2A Rs both in right atrium biopsies and peripheral blood mononuclear cells (PBMCs) from AF patients. In addition, the adenosine levels and adenosine deaminase (ADA) activity in plasma from the same patients were evaluated. 2. Results Adenosinergic System in AF We previously demonstrated that AF patients showed an increased expression of A2A R in right atrial tissue [17], thus, suggesting a participation of this receptor in the development of AF. Now, we aimed at investigating a potential cardiac-peripheric connection of the adenosinergic system in AF. First, we confirmed a significant increase of A2A R density (p < 0.0001) in the right atrium from AF patients when compared to non-AF subjects (i.e., non-dilated sinus rhythm, ndSR) (Figure 1A,B) (see Table 1 for cardiac diseases related patients information). Indeed, a concomitant significant (p = 0.0207) increase in A2A R mRNA expression in right atrial tissue from these patients was also found (Figure 1C), consistent with the immunoblot results (Figure 1). RyR-inhibitors 0 0 0 ARA-II 5 4 2 Ca2+ antagonist 6 3 4 Sintrom 3 1 16 Int. J. Mol. Sci. 2021, 22, 3467 AAS 17 15 6 3 of 12 Statins 17 18 15 Figure 1. A2A R expression Figure in human1. Aright 2AR expression atrium. (A)in Representative human right atrium. (A) Representative immunoblot showing theimmunoblot expression showing of A2A R inthe expression of A 2AR in right atrium from non-dilated sinus rhythm (ndSR) and atrial fibrillation right atrium from non-dilated sinus rhythm (ndSR) and atrial fibrillation (AF) patients; membranes from human atrium (AF) patients; membranes from human atrium were analysed by SDS-PAGE (10 μg of pro- were analysed by SDS-PAGE (10 µg of protein/lane) and immunoblotted using goat anti-A2A R and rabbit anti-α-actinin tein/lane) and immunoblotted using goat anti-A2AR and rabbit anti-α-actinin antibodies (see Meth- antibodies (see Methods). (B) Relative quantification of A2A R density; the immunoblot protein bands corresponding to ods). (B) Relative quantification of A2AR density; the immunoblot protein bands corresponding to A2A R and α-actinin from ndSR A2AR(nand = 11) and AFfrom α-actinin (n = ndSR 12) patients (n = 11)were and quantified AF (n = 12)by densitometric patients scanning; were quantified byvalues were densitometric normalized to the respective amount of α-actinin in each lane to correct for protein loading. (C) Relative expression scanning; values were normalized to the respective amount of α-actinin in each lane to correct of for A2A R transcripts in human atrium. protein Mean ± SEM loading. from ndSR (C) Relative (n = 11)of expression and AF A2A (n = 9) patients. R transcripts **** patrium. in human and **± pSEM < 0.0001Mean < 0.01, from Student t test. ndSR (n = 11) and AF (n = 9) patients. **** p < 0.0001 and ** p < 0.01, Student t test. **** p < 0.0001 and ** p < 0.01 Student t test. Table 1. Patients’ clinical information. Next, we interrogated whether adenosine homeostasis was also altered in these pa- ndSR dSR tients. To this end, ADA activity and adenosine content in plasma from patientsAF with, and Number of patients 28 24 without, AF were determined. Interestingly, a significant 38% reduction in ADA 27 activity Weight (mean±SD) (Kg) 83.18 ± 9.86 71.5 ± 10.5 75.5 ± 11.386 was found Heightin(mean plasma ±SD)from (cm) AF patients when 169.4 ± 9.8compared to ndSR 160.8 ± 8.7subjects (p = 0.0286, 163.8 ± 8.9 F(2, 66) = 3.753) (Figure Age (mean2A). Yet, no significant ±SD) alteration 63.1 ± 9.7 in ADA activity 73.5 ± 8.4 was found 72.9in ± patients 9.4 Sex (male/female) 23/5 17/7 14/13 Body Surface (mean ± SD) (m2 ) 1.93 ± 0.14 1.75 ± 0.15 1.81 ± 0.15 LA diameter index (mean ± SD) 1.99 ± 0.17 2.6 ± 0.2 2.65 ± 0.55 LV diameter index (mean ± SD) 2.7 ± 0.5 2.83 ± 0.54 2.82 ± 0.42 Tabaquism (y/n/ex) 9/12/7 3/18/3 6/18/3 Enolism (y/n/ex) 0/28/0 1/23/0 1/26/0 Hipertension 25 22 20 Diabetes 11 12 9 Dislipemia 15 18 15 Drugs IECAs 16 12 12 Beta-bloquers 12 9 16 β1 3 2 8 β2 0 0 1 α 0 0 0 RyR-inhibitors 0 0 0 ARA-II 5 4 2 Ca2+ antagonist 6 3 4 Sintrom 3 1 16 AAS 17 15 6 Statins 17 18 15 Next, we interrogated whether adenosine homeostasis was also altered in these pa- tients. To this end, ADA activity and adenosine content in plasma from patients with, and without, AF were determined. Interestingly, a significant 38% reduction in ADA activ- ity was found in plasma from AF patients when compared to ndSR subjects (p = 0.0286, F(2, 66) = 3.753) (Figure 2A). Yet, no significant alteration in ADA activity was found in patients displaying atrial dilation (i.e., dilated sinus rhythm, dSR) (p = 0.1312) but not AF (Figure 2A). Subsequently, when the plasma adenosine content was evaluated, the Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 12 Int. J. Mol. Sci. 2021, 22, 3467 4 of 12 displaying atrial dilation (i.e., dilated sinus rhythm, dSR) (p = 0.1312) but not AF (Figure 2A). Subsequently, when the plasma adenosine content was evaluated, the levels in pa- tients levelswith AF were in patients ~2.5-fold with higher AF were when compared ~2.5-fold higher whento ndSR and dSR compared subjects to ndSR and(p dSR < 0.0001, sub- Fjects = 44.66) (2, 40) (p (Figure < 0.0001, 2B). F(2, 40) Indeed, = 44.66) a negative (Figure correlation 2B). Indeed, (r = −0.4087, a negative p = 0.0202) correlation −0.4087, (r = between ADA activity p = 0.0202) and adenosine between contentand ADA activity within ndSR and adenosine AF patients content within was ndSRfound and (Figure 2C). AF patients was found (Figure Collectively, 2C). Collectively, these results theseconcomitantly suggested that results suggested to anthat concomitantly increased to an in- A2AR expression creased in right A 2A R expression atrium, AF patientsin right alsoatrium, AF patients show reduced ADA also show reduced activity ADA activity and increased and adenosine increased content in adenosine plasma. content in plasma. Figure 2. ADA activity andFigure 2. ADA adenosine activity content and adenosine in human plasma.content (A) ADA in activity human plasma. in plasma(A)from ADA activity ndSR (n =in24), plasma dSR fromwas (n = 22) and AF (n = 23) patients ndSR (n = 24), dSR determined (n =the using 22)Dyazime and AF kit. (n =Two 23) patients was determined outliers were removed from using thethedSRDyazime group bykit. Two outliers the ROUT method (see Materials were removed and Methods). from the (B) Adenosine dSR group content by the in plasma ROUT from ndSRmethod (seedSR (n = 15), Materials (n = 15)and andMeth- AF ods). (B) Adenosine content in plasma from ndSR (n = 15), dSR (n = 15) and AF (n = 13) patients (n = 13) patients was determined by MS-HPLC. One outlier was removed from the AF group by the ROUT method (see was determined by MS-HPLC. One outlier was removed from the AF group by the ROUT method Materials and Methods). (C) Correlation between ADA activity and adenosine content in ndSR (yellow, n = 18) and AF (red, (see Materials and Methods). (C) Correlation between ADA activity and adenosine content in n = 14) patients. The correlation ndSRcoefficients (yellow, n =(r)18) were andcalculated AF (red, nusing = 14) the Pearson’s patients. two-tailed correlation The correlation coefficientstest. The results (r) were calculated are expressed as mean ± SEM. * p < 0.05 and **** p < 0.0001, one-way ANOVA with Tukey’s post-hoc test. using the Pearson’s two-tailed correlation test. The results are expressed as mean ± SEM. * p < 0.05 and **** p < 0.0001, one-way ANOVA with Tukey’s post-hoc test. Once the adenosine content and ADA activity in plasma from AF subjects was altered, we next Oncequestioned whether the adenosine this dysregulation content and ADA activitymay eventually in plasma have from any impact on AF subjects A2Aal- was R density in PBMCs from these patients. To this end, we used a fluorescent tered, we next questioned whether this dysregulation may eventually have any A R antago- 2Aimpact on nist, A namely MRS7396 [21], to monitor A2A RTo 2AR density in PBMCs from these patients. density in PBMCs. this end, we used First, we validated a fluorescent the A2AR an- specificity of MRS7396 A R staining in HEK293-A RSNAP cells by displacing its bind- 2A 2A tagonist, namely MRS7396 [21], to monitor A2AR density in PBMCs. First, we validated the ing with ZM241385, specificity of MRS7396 a selective A2A Rinantagonist A2AR staining HEK293-A (Figure 3). Interestingly, when PBMCs 2ARSNAP cells by displacing its binding from a healthy subject were stained with MRS7396 we observed with ZM241385, a selective A2AR antagonist (Figure 3). Interestingly, similar results when PBMCs(Figure 3). from Therefore, the antagonist ZM241385 was able to displace the binding of a healthy subject were stained with MRS7396 we observed similar results (Figure 3). the fluorescent A2A R ligand. Therefore, the antagonist ZM241385 was able to displace the binding of the fluorescent Subsequently, we stained PBMCs from ndSR, dSR and AF patients with MRS7396. A2AR ligand. PBMCs were further gated by flow cytometry based on morphology and the MRS7396 staining was determined in the absence or presence ZM241385 to define the A2A R specific binding in PBMCs from ndSR, dSR and AF patients (Figure 4A). The A2A R density in PBMCs from AF patients was significantly increased (~3.7-fold) when compared to both ndSR and dSR patients (p < 0.0001, F(2, 39) = 23.55) (Figure 4A). Next, we assessed A2A R mRNA expression in PBMCs from ndSR, dSR and AF patients through RT-qPCR analysis A three-fold significant increase in A2A R mRNA expression in PBMCs from AF patients was found (p < 0.0001, F(2, 24) = 14.64) (Figure 4B). Int. Int. J.J. Mol. Mol. Sci. 2021, 22, Sci. 2021, 22, 3467 x FOR PEER REVIEW 55 of of 12 12 Figure 3. A2AR detection in living cells using a fluorescent ligand. HEK-293 cells permanently ex- pressing A2AR and human peripheral blood mononuclear cells (PBMC) were incubated with 50 nM MRS7396 (MRS, red) in the absence or presence of 100 nM of ZM241385 (ZM). For live nuclear staining cells were incubated with 1μM Hoechst 33342 (blue). Scale bar: 20 μm. Subsequently, we stained PBMCs from ndSR, dSR and AF patients with MRS7396. PBMCs were further gated by flow cytometry based on morphology and the MRS7396 staining was determined in the absence or presence ZM241385 to define the A2AR specific binding in PBMCs from ndSR, dSR and AF patients (Figure 4A). The A2AR density in Figure PBMCs 3. A 2AR detection from A in living AF patients wascells using a fluorescent significantly increasedligand. HEK-293 (~3.7-fold) cells compared when permanently toex- both Figure 3. 2A R detection in living cells using a fluorescent ligand. HEK-293 cells permanently pressing ndSR A and2AR and human peripheral blood mononuclear cells (PBMC) were incubated with 50 dSR patients (p < 0.0001, F (2, 39) = 23.55) (Figure 4A). Next, we assessed A2AR expressing A2A R and human peripheral blood mononuclear cells (PBMC) were incubated with 50 nM nM MRS7396 (MRS, red) in the absence or presence of 100 nM of ZM241385 (ZM). For live nuclear mRNA expression in PBMCs from ndSR, dSR and AF patients through MRS7396 (MRS, red) in the absence or presence of 100 nM of ZM241385 (ZM). For live nuclear RT-qPCR analysis staining cells were incubated with 1μM Hoechst 33342 (blue). Scale bar: 20 μm. A three-fold staining significant cells were incubatedincrease with 1µMinHoechst A2AR mRNA expression 33342 (blue). in 20 Scale bar: PBMCs µm. from AF patients was found (p < 0.0001, F(2, 24) = 14.64) (Figure 4B). Subsequently, we stained PBMCs from ndSR, dSR and AF patients with MRS7396. PBMCs were further gated by flow cytometry based on morphology and the MRS7396 staining was determined in the absence or presence ZM241385 to define the A2AR specific binding in PBMCs from ndSR, dSR and AF patients (Figure 4A). The A2AR density in PBMCs from AF patients was significantly increased (~3.7-fold) when compared to both ndSR and dSR patients (p < 0.0001, F(2, 39) = 23.55) (Figure 4A). Next, we assessed A2AR mRNA expression in PBMCs from ndSR, dSR and AF patients through RT-qPCR analysis A three-fold significant increase in A2AR mRNA expression in PBMCs from AF patients was found (p < 0.0001, F(2, 24) = 14.64) (Figure 4B). Figure4.4. Detection Figure Detection of of AA2A2A RRininhuman human PBMCs. PBMCs.(A) (A) TheThe specific MRS7396 specific binding MRS7396 to PBMCs binding from to PBMCs ndSR (yellow, n = 16), dSR (orange, n = 12) and AF (red, n = 14)) patients was obtained from ndSR (yellow, n = 16), dSR (orange, n = 12) and AF (red, n = 14)) patients was obtained by by subtract- ing the nonspecific binding (i.e., 100 nM MRS7396 in the presence of 100 nM of ZM241385) and subtracting the nonspecific binding (i.e., 100 nM MRS7396 in the presence of 100 nM of ZM241385) represented as ∆FL-1 (specific binding). (B) Relative expression of A2AR mRNA in PMBCs. Mean ± and represented as ∆FL-1 (specific binding). (B) Relative expression of A2A R mRNA in PMBCs. SEM of ndSR (n = 10), DSR (n = 7) and AF (n = 9) patients. **** p < 0.0001, one-way ANOVA with Mean ± SEM Tukey’s test.(n = 10), DSR (n = 7) and AF (n = 9) patients. **** p < 0.0001, one-way ANOVA of ndSR post-hoc with Tukey’s post-hoc test. Importantly, a positive correlation between A2AR density in PBMCs and in right Importantly, a positive correlation between A2A R density in PBMCs and in right atrium of the same patients was observed (r = 0.7813, p = 0.001) (Figure 5A), thus indicating atrium of the same patients was observed (r = 0.7813, p = 0.001) (Figure 5A), thus indicating that changes Figure of peripheral 4. Detection of A2AR in humanA2AR PBMCs. density (A) mayThemirror specificchanges MRS7396occurring binding toin the diseased PBMCs from that changes of peripheral A2A R density may mirror changes occurring in the diseased ndSR (yellow, atrium. n = 16), dSR In addition, (orange, ncorrelation a positive = 12) and AF (red, n = plasma between 14)) patients was obtained adenosine by and content subtract- A2AR atrium. ing In addition, the nonspecific a positive binding (i.e., correlation 100 nM MRS7396 between plasma adenosine in the presence of 100 content andand nMrof= ZM241385) A2A R density in PBMCs and in right atrium (r = 0.7650, p < 0.0001 and 0.7922, p = 0.0007, density representedin PBMCs as ∆FL-1and in right (specific atrium binding). (B) (r = 0.7650, Relative p < 0.0001 expression of A2Aand r = 0.7922, R mRNA p = Mean in PMBCs. 0.0007, ± respectively) SEM of ndSR (n was found = 10), DSR(Figure 5B,C). (n = 7) and AF Finally, no correlation (n = 9) patients. between **** p < 0.0001, plasma one-way ADA activity ANOVA with and A2Apost-hoc Tukey’s R density test.in PBMCs and in right atrium was observed (Figure 5D,E). Overall, these results suggested a relationship between atrial and PBMCs A2A R expression, which might be related to adenosine Importantly, content. a positive correlation between A2AR density in PBMCs and in right atrium of the same patients was observed (r = 0.7813, p = 0.001) (Figure 5A), thus indicating that changes of peripheral A2AR density may mirror changes occurring in the diseased atrium. In addition, a positive correlation between plasma adenosine content and A2AR density in PBMCs and in right atrium (r = 0.7650, p < 0.0001 and r = 0.7922, p = 0.0007, Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 12 respectively) was found (Figure 5B,C). Finally, no correlation between plasma ADA activ- Int. J. Mol. Sci. 2021, 22, 3467 ity and A2AR density in PBMCs and in right atrium was observed (Figure 5D,E). Overall, 6 of 12 these results suggested a relationship between atrial and PBMCs A2AR expression, which might be related to adenosine content. Figure Figure 5. Correlation of atrial A2A R 5. density Correlation and of atrial A2Aadenosinergic peripheric R density andsystem. peripheric adenosinergic Correlation between system. Correlation the relative A2A Rbe- tween the relative A R density in the right atrium determined by immunoblot (Integrated density in the right atrium determined by immunoblot (Integrated density, see Figure 1B) and the specific MRS7396 binding 2A den- (∆FL-1) to PBMCc (A), adenosine content (C) and ADA activity (E) from ndSR (yellow) and AF (red) patients. Also, the (C) sity, see Figure 1B) and the specific MRS7396 binding (∆FL-1) to PBMCc (A), adenosine content and ADA correlation between the specific MRS7396activity (E) from binding ndSRto(yellow) (∆FL-1) PBMCc and and AF the (red) patients. adenosine Also,(B) content theand correlation between ADA activity (D) the specific MRS7396 binding (∆FL-1) to PBMCc and the adenosine content (B) and ADA activity (D) from ndSR (yellow) and AF (red) patients was assessed. The correlation coefficient (r) was calculated using the Pearson from ndSR (yellow) and AF (red) patients was assessed. The correlation coefficient (r) was calcu- (two-tailed correlation) test. lated using the Pearson (two-tailed correlation) test. 3.3.Discussion Discussion Whilecardiac While cardiacAA RsRs 2A2A playplay a key a key rolerole in coronary in coronary flowflow and force and force generation, generation, they they have have been also associated with some cardiopathological conditions, been also associated with some cardiopathological conditions, such as the genesis of ar- such as the genesis of arrhythmias [17,22]. Here, we assessed the status of the peripheric rhythmias [17,22]. Here, we assessed the status of the peripheric adenosinergic system in adenosinergic system in the the mostmost common common typetype of cardiac of cardiac arrhythmia arrhythmia seenseen in clinical in clinical practice, practice, namelynamely the AF.theWeAF. We hypothesized that the cardiac and peripherial adenosinergic hypothesized that the cardiac and peripherial adenosinergic systems may interact in AF. systems may interact in AF. Indeed, Indeed, immunoblot immunoblot and and RT-qPCR RT-qPCR analysis analysis of cardiac of cardiac A2AR A2A R revealed revealed onceonceagain again an an in- increased receptor density and RNA expression in the in right creased receptor density and RNA expression in the in right atrium biopsies from AF pa- atrium biopsies from AF patients, in agreement with previous work [16]. Interestingly, the expression of A2A R tients, in agreement with previous work [16]. Interestingly, the expression of A2AR in in PMBCs from AF patients was increased when compared to ndSR and dSR subjects, PMBCs from AF patients was increased when compared to ndSR and dSR subjects, thus, thus, mirroring that observed in right atrium. In addition, the adenosine levels in plasma mirroring that observed in right atrium. In addition, the adenosine levels in plasma from from AF patients were also increased and correlated with A2A R density in PBMCs and AF patients were also increased and correlated with A2AR density in PBMCs and in right in right atrium. Collectively, these results point towards the existence of a potential atrium. Collectively, these results point towards the existence of a potential cardiac-pe- cardiac-peripheral connection with the adenosinergic system in both health and disease ripheral connection with the adenosinergic system in both health and disease and high- and highlight the importance of the A R in AF. light the importance of the A2AR in AF.2A Importantly, signalling through A2A R regulates PBMCs function [23], and increased Importantly, signalling through A2A R regulates PBMCs function [23], and increased levels of A2A R have been found in lymphocytes from patients with immunological diseases, levels of A2AR have been found in lymphocytes from patients with immunological dis- including systemic lupus erythematosus [24], amyotrophic lateral sclerosis [25], multiple eases, including systemic lupus erythematosus [24], amyotrophic lateral sclerosis [25], sclerosis [26] and rheumatoid arthritis [27]. In addition, A2A R density in lymphocytes multiple sclerosis also increased in[26] and rheumatoid patients with coronary arthritis [27]. artery In addition, disease A2AR [28] and densityheart chronic in lympho- failure cytes also increased in patients with coronary artery disease (CHF) [29]. This link between A2A R and PBMCs is now revealed for AF, although [28] and chronic heart failure the (CHF) [29]. This link between A 2AR and PBMCs is now revealed for AF, although the spe- specific mechanism underlying that remodelling of A2A R has not yet been determined. cific mechanism Moreover, underlying the altered levelsthat remodelling of adenosine and ofADA A2AR activity has not yet been in AF determined. raises the question More- of over, the altered levels of adenosine and ADA activity in AF raises whether the increased adenosine levels in AF are a cause or consequence of the disease. If the question of whether the increased peripheric adenosine adenosine levels in AF augmentation are a AF, triggers cause thenoritconsequence of theifdisease. might be cogitated If pe- a reduction in ripheric adenosine augmentation triggers AF, then it might be ADA activity is behind AF pathophysiology. Interestingly, in CHF a reduction in ADA and cogitated if a reduction in ADA ADA2activity activityishas behind beenAF pathophysiology. reported Interestingly, [30], thus, attenuating theinpathologic CHF a reduction consequencesin ADA of and CHFADA2 activityincreased as putative has beenlevels reported [30], thus, would of adenosine attenuating the pathologic compensate consequences the cardiodepressant of CHF of effects asincreased putative increased TNFα in CHF levelspatients of adenosine would [30]. Yet, herecompensate a lowering of the cardiodepressant ADA activity might effects of increased TNFα in CHF patients [30]. Yet, here a lowering be expected in patients at risk of AF, i.e., dSR, but this is not observed (although of ADA activity ADAmightlevels be expected in patients at risk of AF, i.e., dSR, but this is not observed appear to be slightly reduced and adenosine levels slightly increase in dSR). Alternatively, (although ADA it can be speculated that the reduction in ADA activity is one step further in the different events that ends in an AF or can be just another risk-factor for the AF, as well as high blood pressure, age, or obesity. Conversely, the higher adenosine levels in plasma from AF patients might reflect excessive energy consumption in atria with myocytes beating at abnormally high frequencies. The higher adenosine levels can be sufficiently elevated Int. J. Mol. Sci. 2021, 22, 3467 7 of 12 to preclude a correct ADA catabolism, and thus, to induce an abnormal adenosinergic signalling, which could trigger the observed PBMC and myocytic A2A Rs remodelling. Interestingly, we found a correlation between adenosine levels and A2A R expression. Elevated adenosine levels and A2A R expression observed in patients with AF supports the notion that excessive A2A R activation contributes to a higher incidence of spontaneous calcium release-induced electrical activity and irregular beating reported in atrial myocytes from patients with AF [17,31], but what mechanisms are connecting adenosine levels and A2A R expression? On one hand, a possible explanation is a positive regulatory loop between adenosine and A2A R where adenosine is released to the environment due to excessive and abnormal beating and a consequence of the energy spent on it. This release would induce a positive turnover of A2A R expression, which could be necessary to keep the heart in a fast-beating condition. On the other hand, adenosine levels are probably a consequence of AF, which leave us the possibility that A2A R enhanced expression is the first step on the AF progression. This leads to abnormal heart beating due to a disturbance in calcium homeostasis and triggering higher adenosine levels. This would suggest a causative correlation, where the A2A R expression is altered prior to the adenosine content. In addition, there could be other, and even more complex, reasons behind the increased A2A R density and adenosine levels in AF, which might promote both simultaneously, without any apparently relationship between them. Overall, endogenous adenosine plays a key role on central cardiovascular regulation and it has profound modulatory effects through systemic mechanisms of autonomic regu- lation [32]. Thus, it has been suggested that adenosine may provide a connection between the local mechanisms of blood flow regulation and the systemic processes of autonomic cardiovascular modulation [32]. Hence, upon increased metabolic demand (exercise) and decreased energy supply (ischemia) interstitial myocardial levels of adenosine rise and activate sensory afferent (metaboreceptors) triggering a sympathetic systemic vasoconstric- tion, thus increasing blood pressure, and improving perfusion pressure. Indeed, while this systemic vasoconstriction would be harmful to the ischemic organ, a local adenosine A1 R-dependent anti-adrenergic vasodilatory effects will protect the myocardium, whereas still benefited by the improved perfusion pressure. Thus, further research is needed to reveal the mechanism underlying the precise interplay between A2A R and A1 R in AF. 4. Materials and Methods 4.1. Reagents The following reagents were used: adenosine deaminase (ADA, Roche Diagnostics, Mannheim, Germany), MRS7396 [21,33], ZM241385 (Tocris, Bristol, UK), Hoechst 33342 (Thermo Fisher Scientific, Rockford, IL, USA). The antibodies used were: mouse anti-A2A R (sc-32261, Santa Cruz Biotechnology Inc., Dallas, TX, USA), rabbit anti-α-actinin (sc-17829; Santa Cruz Biotechnology Inc.), horseradish peroxidase (HRP)-conjugated rabbit anti- mouse IgG (Pierce Biotechnology, Rockford, IL, USA) and HRP-conjugated goat anti-rabbit IgG (Pierce Biotechnology, Rockford, IL, USA). 4.2. Human Samples (Subject Demographics) Human whole blood Samples (4 mL) from 28 non-dilated sinus rhythm (ndSR), 24 di- lated sinusal rhythm (dSR) and 27 atrial fibrillation (AF) patients (Hospital Sant Pau Dos de Maig, Barcelona) (Table 1) were collected into lithium heparin tubes. In addition, atrial heart tissue from 11 ndSR and 12 AF patients undergoing cardiac surgery and stored im- mediately at −80 ◦ C. Key clinical and echocardiographic data, as well as pharmacological treatments of these patients, included in the statistical analysis are summarized in Table 1. Patients undergoing mitral valve replacement or repair were not included in this study to avoid potentially confounding the effects of mitral valve disease. Each patient gave written consent for a sample of the right atrial appendix to be given that would have otherwise been discarded during the surgical intervention. The study was approved by the Ethics Int. J. Mol. Sci. 2021, 22, 3467 8 of 12 Committee at Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, and the investigation conforms to the principles outlined in the Declaration of Helsinki. 4.3. Membrane Preparation Right atrial samples were pulverized in liquid nitrogen congelation and sonicated (Brandson Sonifier 250, ICN Hubber S.A.) in 500 µL of ice-cold 10 mM Tris HCl, pH 7.4 buffer containing a protease inhibitor cocktail (Roche Molecular Systems, Belmont, CA, USA). The sonicated tissue was further homogenized using a Polytron (VDI 12, VWR, Barcelona, Spain) for three periods of 10 s each. The homogenate was centrifuged at 12,000 × g at 4 ◦ C for 30 min. The membranes were dispersed in 50 mM Tris HCl (pH 7.4) and 10 mM MgCl2 , washed, and resuspended in the same medium as described previ- ously [34]. Protein concentration was determined using the BCA protein assay kit (Thermo Fisher Scientific Inc., Rockford, IL, USA) and 10 µg of protein was used for immunoblotting. 4.4. Gel Electrophoresis and Immunoblotting Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS/PAGE) was per- formed using 10% polyacrylamide gels. Proteins were transferred to Hybond® -LFP polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Chicago, IL, USA) using the Trans-Blot® TurboTM transfer system (Bio-Rad, Hercules, CA, USA) at 200 mA/membrane for 30 min. PVDF membranes were blocked with 5% (wt/vol) dry non-fat milk in phosphate-buffered saline (PBS; 8.07 mM Na2 HPO4 , 1.47 mM KH2 PO4 , 137 mM NaCl, 0.27 mM KCl, pH 7.2) containing 0.05% Tween-20 (PBS-T) during 1h at 20 ◦ C before being immunoblotted using mouse anti-A2A R (0.5 µg/mL) and rabbit anti-α-actinin (0.5 µg/mL) antibodies in blocking solution overnight at 4 ◦ C. PVDF membranes were washed with PBS-T three times (5 min each) before incubation with either a HRP-conjugated rabbit anti-mouse IgG (1/10,000) or HRP-conjugated goat anti-rabbit IgG (1/30,000) in block- ing solution at 20 ◦ C during 2 h. After washing the PVDF membranes with PBS-T three times (5 min each), the immunoreactive bands were developed using a chemiluminescent detection kit (Thermo Fisher Scientific) and detected with an Amersham Imager 600 (GE Healthcare Europe, Barcelona, Spain). 4.5. RT-qPCR Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples by density centrifugation using Lymphoprep (Lymphoprep, Palex Medical, Madrid, Spain). PBMCs were stored at −80 ◦ C in Triazol until use. Cell RNA was purified using RNeasy Plus Universal Mini Kit (Qiagen, Hilden, Germany). Heart tissue was pulverized upon liquid nitrogen congelation and sonicated in 100 µL of Tris (pH 7.4) buffer and stored with 1mL of TriPure reagent (TriPure isolation Reagent, Roche) at −80 ◦ C. Subsequently, the RNA was separated from DNA/proteins using TriPure (Sigma-Aldrich, St. Louis, MO, USA) and following manufacturer’s instructions. RNA yield was determined using a NanoDrop® ND-1000 UV-Vis spectrophotometer (Thermo Fisher Scientific, Rockford, IL, USA). Reverse transcription was performed using 0.5–1 µg of total RNA obtained from each sample using StaRT Reverse Transcription Kit (AnyGenes, Paris, France) according to the manufacturer’s instructions. To quantify A2 AR mRNA expression, we used TaqMan single tube assay probe (ADORA2A Hs00169123_m1, Thermo Fisher Scientific, Rockford, IL, USA). The TBP (TATA-box binding protein) probe (TBP Hs00427620, Thermo Fisher Scientific, Rockford, IL, USA) was used to normalize mRNA expression from both PBMC’s and heart samples. Reaction conditions were done following manufacturer’s instructions in a 384-well plate (MicroAmp Optical 384-well plate; Applied Biosystems, Foster City, CA, USA). The reaction was carried out in a total volume of 10 µL per reaction. The reaction mix included 5 µL TaqMAn Fast Advanced Master Mix, 0.5 µL TaqMan Assay Probe, 3.5 µL of Nuclease Free Water and 1µL of cDNA template (100 ng cDNA total amount) and were done in 7900HT Advanced Real-Time PCR instrument (Applied Biosystems). Amplification protocol started with 95 ◦ C for 10 min, 42 cycles of 95 ◦ C (10 sec) and 60 ◦ C Int. J. Mol. Sci. 2021, 22, 3467 9 of 12 (30 sec), a final cycle of 95 ◦ C (10 sec), and finishing with a 60 ◦ C (30 sec) step. The results are presented relative to those for the housekeeping gene TBP using the ∆Cq method. Data were analysed with Expression Suit Software v1.2 (Thermo Fisher Scientific, Rockford, IL, USA). 4.6. Sample Preparation and HPLC-MS/MS Determination of Ribonucleosides Quantitative analysis of ribonucleosides (i.e., adenosine) was carried out as previously described [29,35,36]. In brief, we used liquid chromatography technique coupled to tandem mass spectrometry (LC-MS/MS) using an Agilent 1290 Infinity UHPLC chromatograph (Santa Clara, CA, USA) coupled to a 6500 QTRAP mass spectrometer (ABSciex, Framing- ham, MA, USA) equipped with Ion Drive Turbo V ion source operating in positive ion mode. The column used was a Discovery HS F5-3 150 × 2.1 mm 3µm (Supelco, Bellefonte, CA, USA) at 40 ◦ C; autosampler temperature, 4 ◦ C; injection volume, 10µL; flow rate, 0.6 mL min−1 . Mobile phase was A) Ultrapure water with 0.1% HCOOH and B) Acetoni- trile with 0.1 %HCOOH. The gradient program was as follows (t, %B): (0, 1), (0.2, 1), (3, 20), (3.5, 95), (6, 95), (6.5, 1), (10, 1). Mass spectrometry detection was performed by using the multiple reaction monitoring (MRM) mode using the following parameters: ion spray volt- age, +5500 V; source temperature, 600 ◦ C; curtain gas, 20 psi; ion source gas 1 and gas 2, 50, and 20 respectively; collision-activated dissociation gas, High; entrance potential, (+/-)10 V. The MRM transitions for adenosine were 268.3/136.1 (Declustering potential DP 65V and collision energy CE25V) for quantitative purposes and 268.3/119.1 (DP 60V, CE 25V) for confirmation purposes dA-d3 was used as internal standard with a transition 230.9/115 (DP 20V, CE 15V). The calibration curve was constructed with adenosine standard solutions between 0.03–8 ng/mL diluted in water TCA30%. Linear regression was adjusted (1/x or 1/x2 ) to have accuracies between 80-120% for all the adenosine standards. Analyst 1.6.2 Software was used for data acquisition and MultiQuant 3.0.1 for data processing both from ABSciex (Framingham, MA, USA). 4.7. Adenosine Deaminase Activity Determination Plasma from each patient’s blood was isolated by density centrifugation using Lym- phoprep (Lymphoprep, Palex) and stored at −80 ◦ C until use. ADA was determined using a commercial colorimetric ADA assay kit (Dyazime Laboratories Inc., Poway, CA, USA) and following the manufacture’s indications. Adding the reagents of the kit you produce a colorimetric reaction based on quinone dye. Therefore, ADA activity in each plasma sample (5 µL) was determined in triplicate by measuring the absorbance at 550 nm using a POLARstar Omega plate reader (BMG Labtech, Ortenberg, Germany). 4.8. Cell Culture Human embryonic kidney (HEK)-293T stable cell line permanently expressing the A2A RSNAP construct [37] were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich) supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/mL streptomycin, 100 mg/mL penicillin and 5% (v/v) fetal bovine plasma at 37 ◦ C and 5% CO2 . 4.9. Flow Cytometry and Confocal Imaging A2 ARSNAP expressing HEK-293T cells and human PBMCs (4 × 105 cells) were incu- bated with 50 nM MRS7396 in presence or absence of 100 nM ZM241385 (Tocris Biosciences, Bristol, UK) in 300 µL of complete DMEM, or RPMI, respectively, under constant rotation (25 rpm, WiseMix Rotator, Witeg Labortechnik, Wertheim, Germany) during 16 h at 4 ◦ C. Subsequently, cells were centrifuged at 500× g for 5 min at 4 ◦ C and rinsed once with ice-cold Hank’s balanced salt solution (HBSS; Sigma-Aldrich, St. Louis, MO, USA) and immediately used for flow cytometric measurements or confocal imaging. Flow cytometry measurements were conducted as previously described [38]. In brief, for single MRS7396 binding detection a total of 104 cells (events) were analysed using a BD FACSCanto II flow cytometer (Becton Dickinson, Heidelberg, Germany) with excitation at Int. J. Mol. Sci. 2021, 22, 3467 10 of 12 488 nm and emission at 647 nm. Samples were maintained in the dark and at 4 ◦ C during the analysis to avoid photobleaching and ligand dissociation. PBMCc were selected by size and shape, excluding the death cells, triplets and doublets, while the mean fluorescence intensity per event was obtained in the FL-1 channel in log mode. Data were collected using FacsDiva Software v6 1.3 (Becton Dickinson, Ashland, OR, USA) and the analysis was made using the FlowJo v10.1 software (Becton Dickinson, Ashland, OR, USA). For confocal imaging cells were seeded with DMEM or RPMI medium containing 1 µg/mL of Hoechst 33342 (Thermo Fisher Scientific) in an 8-well treated plate (Ibitreat 15µ-Slide 8 well Plate, Ibidi, Gräfelfing, Germany) for 20 min at 4 ◦ C. Confocal images were captured using ZEISS microscope (ZEISS LSM 880 Confocal Laser Scanning Micro- scope, Zeiss, Oberkochen, Germany). Analysis was done with ZEN software (Zen 2 blue edition, Zeiss). 4.10. Data and Statistical Analysis Data are represented as mean ± standard error of mean (SEM) with statistical signifi- cance set at p < 0.05. The number of samples/subjects (n) in each experimental condition is indicated in the corresponding figure legend. Outliers were assessed by the ROUT method [39], thus, subjects were excluded assuming a Q value of 1% in GraphPad Prism 9 (San Diego, CA, USA). Data normality was assessed by the Shapiro-Wilk normality test (p < 0.05). Comparisons among experimental groups was performed by Student’s t test or one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons post-hoc test using GraphPad Prism 9, as indicated. The correlation coefficients (r) were calculated using the Pearson two-tailed correlation test. Author Contributions: Study conception and design: F.C. and L.H-M. Project supervision: F.C. Data collection: H.G-M, R.D., H.C.-L, V.J-S. and J.M. Data analysis and interpretation: H.G-M., R.D.,C.S. and F.C. Drafting: L.H-M, K.A.J. and F.C. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by Ministerio de Ciencia, Innovación y Universidades–Agencia Estatal de Investigación-FEDER-UE (SAF2017-87349-R MICIU/AEI/FEDER/UE and SAF207-88019- C3-1R MICIU/AEI/FEDER/UE) and ISCIII/FEDER (PIE14/00034 and CIBERCV-RD12/0042/0002), Generalitat de Catalunya (2017SGR1604 and 2017SGR1769), Fundació la Marató de TV3 (Grant 20152030/31) and NIDDK Intramural Res. (ZIADK31117). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee at Hospital de la Santa Creu i Sant Pau, Barcelona, Spain (protocol code AZAR-AF_2015, approved 30/11/2015). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Not Applicable. Acknowledgments: We thank Centres de Recerca de Catalunya (CERCA) Programme/Generalitat de Catalunya for IDIBELL institutional support. We thank Esther Castaño and Benjamín Torrejón from the CCiT-Bellvitge Campus of the University of Barcelona. Conflicts of Interest: The authors declare no conflict of interest. References 1. Zoni-Berisso, M.; Lercari, F.; Carazza, T.; Domenicucci, S. Epidemiology of atrial fbrillation: European perspective. Clin. Epidemiol. 2014, 6, 213–220. [CrossRef] [PubMed] 2. Ravens, U. Antiarrhythmic therapy in atrial fibrillation. Pharmacol. Ther. 2010, 128, 129–145. [CrossRef] 3. Staerk, L.; Sherer, J.A.; Ko, D.; Benjamin, E.J.; Helm, R.H. Atrial Fibrillation: Epidemiology, Pathophysiology, and Clinical Outcomes. Circ. Res. 2017, 120, 1501–1517. [CrossRef] 4. Harada, M.; Van Wagoner, D.R.; Nattel, S. Role of inflammation in Atrial fibrillation pathophysiology and management. Circ. J. 2015, 79, 495–502. [CrossRef] [PubMed] 5. Korantzopoulos, P.; Letsas, K.P.; Tse, G.; Fragakis, N.; Goudis, C.A.; Liu, T. Inflammation and atrial fibrillation: A comprehensive review. J. Arrhythmia 2018, 34, 394–401. [CrossRef] [PubMed] Int. J. Mol. Sci. 2021, 22, 3467 11 of 12 6. Berkovitch, A.; Younis, A.; Grossman, Y.; Segev, S.; Kivity, S.; Sidi, Y.; Beinart, R.; Goldenberg, I.; Maor, E. Relation of Neutrophil to Lymphocyte Ratio to Risk of Incident Atrial Fibrillation. Am. J. Cardiol. 2019, 123, 396–401. [CrossRef] [PubMed] 7. Gessi, S.; Varani, K.; Merighi, S.; Fogli, E.; Sacchetto, V.; Benini, A.; Leung, E.; Mac-Lennan, S.; Borea, P.A. Adenosine and lymphocyte regulation. Purinergic Signal. 2007, 3, 109–116. [CrossRef] 8. Newby, A.C.; Worku, Y.; Holmquist, C.A. Adenosine formation. Evidence for a direct biochemical link with energy metabolism. Adv. Myocardiol. 1985, 6, 273–284. 9. Haskó, G.; Linden, J.; Cronstein, B.; Pacher, P. Adenosine receptors: Therapeutic aspects for inflammatory and immune diseases. Nat. Rev. Drug Discov. 2008, 7, 759–770. [CrossRef] 10. Idzko, M.; Ferrari, D.; Eltzschig, H.K. Nucleotide signalling during inflammation. Nature 2014, 509, 310–317. [CrossRef] [PubMed] 11. Eltzschig, H.K.; Faigle, M.; Knapp, S.; Karhausen, J.; Ibla, J.; Rosenberger, P.; Odegard, K.C.; Laussen, P.C.; Thompson, L.F.; Colgan, S.P. Endothelial catabolism of extracellular adenosine during hypoxia: The role of surface adenosine deaminase and CD26. Blood 2006, 108, 1602–1610. [CrossRef] 12. Grenz, A.; Homann, D.; Eltzschig, H.K. Extracellular adenosine: A safety signal that dampens hypoxia-induced inflammation during ischemia. Antioxid. Redox Signal. 2011, 15, 2221–2234. [CrossRef] 13. Sumi, Y.; Woehrle, T.; Chen, Y.; Yao, Y.; Li, A.; Junger, W.G. Adrenergic receptor activation involves ATP release and feedback through purinergic receptors. Am. J. Physiol. Cell Physiol. 2010, 299, C1118–C1126. [CrossRef] 14. Li, N.; Csepe, T.A.; Hansen, B.J.; Sul, L.V.; Kalyanasundaram, A.; Zakharkin, S.O.; Zhao, J.; Guha, A.; Van Wagoner, D.R.; Kilic, A.; et al. Adenosine-induced atrial fibrillation. Circulation 2016, 134, 486–498. [CrossRef] [PubMed] 15. Ishihara, T.; Takasugi, N.; Naruse, G.; Okura, H. Adenosine-induced atrial fibrillation arising from arrhythmogenic right atrial appendage. J. Cardiovasc. Electrophysiol. 2020, 31, 234–235. [CrossRef] [PubMed] 16. Hove-Madsen, L.; Prat-Vidal, C.; Llach, A.; Ciruela, F.; Casadó, V.; Lluis, C.; Bayes-Genis, A.; Cinca, J.; Franco, R. Adenosine A2A receptors are expressed in human atrial myocytes and modulate spontaneous sarcoplasmic reticulum calcium release. Cardiovasc. Res. 2006, 72, 292–302. [CrossRef] [PubMed] 17. Llach, A.; Molina, C.E.; Prat-Vidal, C.; Fernandes, J.; Casad, V.; Ciruela, F.; Llus, C.; Franco, R.; Cinca, J.; Hove-Madsen, L. Abnormal calcium handling in atrial fibrillation is linked to up-regulation of adenosine A2A receptors. Eur. Heart J. 2011, 32, 721–729. [CrossRef] 18. Boknik, P.; Drzewiecki, K.; Eskandar, J.; Gergs, U.; Hofmann, B.; Treede, H.; Grote-Wessels, S.; Fabritz, L.; Kirchhof, P.; Fortmüller, L.; et al. Evidence for arrhythmogenic effects of A2A-adenosine receptors. Front. Pharmacol. 2019, 10, 1051. [CrossRef] 19. Soattin, L.; Lubberding, A.F.; Bentzen, B.H.; Christ, T.; Jespersen, T. Inhibition of Adenosine Pathway Alters Atrial Electrophysiol- ogy and Prevents Atrial Fibrillation. Front. Physiol. 2020, 11, 493. [CrossRef] [PubMed] 20. Guieu, R.; Deharo, J.-C.; Maille, B.; Crotti, L.; Torresani, E.; Brignole, M.; Parati, G. Adenosine and the Cardiovascular System: The Good and the Bad. J. Clin. Med. 2020, 9, 1366. [CrossRef] 21. Duroux, R.; Ciancetta, A.; Mannes, P.; Yu, J.; Boyapati, S.; Gizewski, E.; Yous, S.; Ciruela, F.; Auchampach, J.A.; Gao, Z.-G.; et al. Bitopic fluorescent antagonists of the A2A adenosine receptor based on pyrazolo[4,3-: E] [1,2,4]triazolo[1,5- c] pyrimidin-5-amine functionalized congeners. Medchemcomm 2017, 8, 1659–1667. [CrossRef] 22. Boknik, P.; Eskandar, J.; Hofmann, B.; Zimmermann, N.; Neumann, J.; Gergs, U. Role of cardiac A2A receptors under normal and pathophysiological conditions. Front. Pharmacol. 2021, 11, 627838. [CrossRef] 23. Gessi, S.; Varani, K.; Merighi, S.; Ongini, E.; Borea, P.A. A(2A) adenosine receptors in human peripheral blood cells. Br. J. Pharmacol. 2000, 129, 2–11. [CrossRef] 24. Bortoluzzi, A.; Vincenzi, F.; Govoni, M.; Padovan, M.; Ravani, A.; Borea, P.A.; Varani, K. A2A adenosine receptor upregulation correlates with disease activity in patients with systemic lupus erythematosus. Arthritis Res. Ther. 2016, 18, 1–11. [CrossRef] 25. Vincenzi, F.; Corciulo, C.; Targa, M.; Casetta, I.; Gentile, M.; Granieri, E.; Borea, P.A.; Popoli, P.; Varani, K. A2A adenosine receptors are up-regulated in lymphocytes from amyotrophic lateral sclerosis patients. Amyotroph. Lateral Scler. Front. Degener. 2013, 14, 406–413. [CrossRef] 26. Vincenzi, F.; Corciulo, C.; Targa, M.; Merighi, S.; Gessi, S.; Casetta, I.; Gentile, M.; Granieri, E.; Borea, P.A.; Varani, K. Multiple sclerosis lymphocytes upregulate A2A adenosine receptors that are antiinflammatory when stimulated. Eur. J. Immunol. 2013, 43, 2206–2216. [CrossRef] 27. Vincenzi, F.; Padovan, M.; Targa, M.; Corciulo, C.; Giacuzzo, S.; Merighi, S.; Gessi, S.; Govoni, M.; Borea, P.A.; Varani, K. A2A Adenosine Receptors Are Differentially Modulated by Pharmacological Treatments in Rheumatoid Arthritis Patients and Their Stimulation Ameliorates Adjuvant-Induced Arthritis in Rats. PLoS ONE 2013, 8, e54195. [CrossRef] [PubMed] 28. Gariboldi, V.; Vairo, D.; Guieu, R.; Marlingue, M.; Ravis, E.; Lagier, D.; Mari, A.; Thery, E.; Collart, F.; Gaudry, M.; et al. Expressions of adenosine A2A receptors in coronary arteries and peripheral blood mononuclear cells are correlated in coronary artery disease patients. Int. J. Cardiol. 2017, 230, 427–431. [CrossRef] 29. Varani, K.; Laghi-Pasini, F.; Camurri, A.; Capecchi, P.L.; Maccherini, M.; Diciolla, F.; Ceccatelli, L.; Lazzerini, P.E.; Ulouglu, C.; Cattabeni, F.; et al. Changes of peripheral A2A adenosine receptors in chronic heart failure and cardiac transplantation. FASEB J. 2003, 17, 280–282. [CrossRef] [PubMed] 30. Khodadadi, I.; Vahedi, M.S.; Abdi, M.; Daneshkhah, N.; Rahbari, R.; Menbari, S.; Ahmadi, D.; Ahmadi, A.; Lahoorpour, F.; Hakhamaneshi, M.S.; et al. Evaluation of adenosine deaminase (ADA) isoenzymes activity and tumor necrosis factor-α (TNFα) concentration in chronic heart failure. EXCLI J. 2014, 13, 58–66. [PubMed] Int. J. Mol. Sci. 2021, 22, 3467 12 of 12 31. Molina, C.E.; Llach, A.; Herraiz-Martínez, A.; Tarifa, C.; Barriga, M.; Wiegerinck, R.F.; Fernandes, J.; Cabello, N.; Vallmitjana, A.; Benitéz, R.; et al. Prevention of adenosine A2A receptor activation diminishes beat-to-beat alternation in human atrial myocytes. Basic Res. Cardiol. 2016, 111, 1–15. [CrossRef] [PubMed] 32. Biaggioni, I. Adenosine Receptors and Autonomic Regulation. In Primer on the Autonomic Nervous System; Academic Press: Cambridge, MA, USA, 2012; pp. 95–97. 33. Lanznaster, D.; Massari, C.M.; Marková, V.; Šimková, T.; Duroux, R.; Jacobson, K.A.; Fernández-Dueñas, V.; Tasca, C.I.; Ciruela, F. Adenosine A1-A2A Receptor-Receptor Interaction: Contribution to Guanosine-Mediated Effects. Cells 2019, 8, 1630. [CrossRef] [PubMed] 34. Fernández-Dueñas, V.; Gómez-Soler, M.; Valle-León, M.; Watanabe, M.; Ferrer, I.; Ciruela, F. Revealing Adenosine A2A-Dopamine D2 Receptor Heteromers in Parkinson’s Disease Post-Mortem Brain through a New AlphaScreen-Based Assay. Int. J. Mol. Sci. 2019, 20, 3600. [CrossRef] [PubMed] 35. Morató, X.; Gonçalves, F.Q.; Lopes, J.P.; Jauregui, O.; Soler, C.; Fernández-Dueñas, V.; Cunha, R.A.; Ciruela, F. Chronic adenosine A 2A receptor blockade induces locomotor sensitization and potentiates striatal LTD IN GPR37-deficient mice. J. Neurochem. 2019, 148, 796–809. [CrossRef] 36. Maille, B.; Marlinge, M.; Vairo, D.; Mottola, G.; Koutbi, L.; Deharo, P.; Gastaldi, M.; Gaudry, M.; Guiol, C.; Bottone, S.; et al. Adenosine plasma level in patients with paroxysmal or persistent atrial fibrillation and normal heart during ablation procedure and/or cardioversion. Purinergic Signal. 2019, 15, 45–52. [CrossRef] 37. Fernández-Dueñas, V.; Taura, J.J.; Cottet, M.; Gómez-Soler, M.; López-Cano, M.; Ledent, C.; Watanabe, M.; Trinquet, E.; Pin, J.-P.; Luján, R.; et al. Untangling dopamine-adenosine receptor-receptor assembly in experimental parkinsonism in rats. Dis. Model. Mech. 2015, 8, 57–63. [CrossRef] 38. Fernández-Dueñas, V.; Gomez-Soler, M.; Morato, X.; Nunez, F.; Das, A.; Kumar, T.S.; Jauma, S.; Jacobson, K.A.; Ciruela, F. Dopamine D2 receptor-mediated modulation of adenosine A2A receptor agonist binding within the A2AR/D2R oligomer framework. Neurochem. Int. 2013, 63, 42–46. [CrossRef] 39. Motulsky, H.J.; Brown, R.E. Detecting outliers when fitting data with nonlinear regression—A new method based on robust nonlinear regression and the false discovery rate. BMC Bioinform. 2006, 7, 123. [CrossRef]