Adenosine receptors in Atrial Fibrillation

Abstract

Atrial fibrillation (AF) is the most common arrhythmia affecting 1 of every 20 persons older than 65 years and 9% of the octogenarians, and extensive research efforts have been undertaken to identify molecular, electrophysiological, and clinical mechanisms that contribute to the induction and maintenance of this arrhythmia. In spite of this, treatment of this arrhythmia remains deficient or inefficient, with frequent re-incidence of the arrhythmia after treatment because knowledge of the complex pathophysiology of the disease remains incomplete. However, dysfunctional calcium handling is gaining strength as a key contributor to the induction of cardiac arrhythmia. In the literature is described those pathological changes in adenosine A2A receptor (A2AR) expression and activation promote arrhythmogenic calcium release from the sarcoplasmic reticulum (SR) in atrial myocytes from patients with AF. Accordingly, this proposal aims to test the general working hypothesis postulating that adenosine receptors constitute a new source of therapeutical targets and biomarkers for prevention, risk-stratification, and treatment of atrial fibrillation. In the present work we analysed the A2AR expression and mRNA content from AF patients to determine its AF phenotype. Moreover, we analysed the impact of AF in peripheral tissues. Thus, A2AR expression was measured in peripheral blood mononuclear cells (PBMCs) from AF patients, finding its enhanced expression. In addition, ADA and adenosine plasma content was also altered in AF patients. Concomitantly, these results suggest a huge imbalance in adenosinergic signalling that is not hitting just cardiac tissue but also peripherical ones. Secondly, we aimed to develop new tools to use in the AF research. In our case were the HL-1 cells for in vitro assays and pigs for animal modelling. We compared the results on adenosine 1 receptor (A1R) and A2AR expression and heterodimerization of the induced AF phenotype in the two models with AF human patients. Interestingly, we found the same alterations (enhanced expression and reduced heterodimer levels) in all of them. These results point to a usefulness of these two models in the AF research. Moreover, this was the first time that heterodimer A1R-A2AR was detected in a protein:protein interaction assay. Nevertheless, further research is needed to understand the role and the impact of this imbalance during AF events. Also, we have found the same alterations in calcium handling related proteins in pig and humans with AF. We found a reduction in calsequestrin CSQ and PLB expression in both models, which probably contributes to a loss of calcium buffering in

AF. In the last chapter, we aimed to develop new pharmacological approaches y precluding A2AR activation or enhancing A1R activity, which will probably restore the normal beating in the heart during the arrythmia. We successfully blocked A2AR activity by the SCH442416 full antagonist. Also, we enhanced A1R activity with CPA agonist and T62 PAM. In addition, we measured the impact on the heterodimerization in a heterologous system by transfected A1R-A2AR HEK cells. Our results shown that T-62 and SCH442416 does not affect heterodimer formation, being potentially useful in the AF treatment. Finally, we developed the same drugs from the optopharmacological point of view, in order to avoid side-off target effects. We have successfully developed the four light-sensitive compounds, which mimic the effect of the classical drugs under light conditions. This opens a new field to treat AF and other diseases. In conclusion, the present work has deepened into the AF understanding and treatment. We have revealed new insights related with AF through the ADA and Adenosine alterations found in plasma. Also, we have revealed new impacts of AF in surrounding cells that offer a new course to study or prevent and detect AF. We have additionally provided evidence for the use of relevant tools to research about AF in in vitro and animal models by using HL-1 cells and pigs. That will facilitate the future analysis of AF causes, effects, and treatment. In addition, novel findings of AF impacts have been found in the three systems through the heterodimeric A1-A2AR reduced formation and the alterations in CSQ, PLB and A1R expression. Finally, we showed new potential approaches to pharmacologically tackle this disease by antagonizing A2AR or potentiate A1R activity. In addition, we have demonstrated that the emerging field of optopharmacology could give us the chance to go further the classical pharmacology and possibly be useful for the development of light- dependent treatments in the near future.