Increased Density of Endogenous Adenosine A2A Receptors in Atrial Fibrillation: From Cellular and Porcine Models to Human Patients

Abstract

Adenosine, an endogenous nucleoside, plays a critical role in maintaining homeostasis during stressful situations, such as energy deprivation or cellular damage. Therefore, extracellular adenosine is generated locally in tissues under conditions such as hypoxia, ischemia, or inflammation. In fact, plasma levels of adenosine in patients with atrial fibrillation (AF) are elevated, which also correlates with an increased density of adenosine A_2A receptors (A_2ARs) both in the right atrium and in peripheral blood mononuclear cells (PBMCs). The complexity of adenosine-mediated effects in health and disease requires simple and reproducible experimental models of AF. Here, we generate two AF models, namely the cardiomyocyte cell line HL-1 submitted to Anemonia toxin II (ATX-II) and a large animal model of AF, the right atrium tachypaced pig (A-TP). We evaluated the density of endogenous A_2AR in those AF models. Treatment of HL-1 cells with ATX-II reduced cell viability, while the density of A_2AR increased significantly, as previously observed in cardiomyocytes with AF. Next, we generated the animal model of AF based on tachypacing pigs. In particular, the density of the key calcium regulatory protein calsequestrin-2 was reduced in A-TP animals, which is consistent with the atrial remodelling shown in humans suffering from AF. Likewise, the density of A_2AR in the atrium of the AF pig model increased significantly, as also shown in the biopsies of the right atrium of subjects with AF. Overall, our findings revealed that these two experimental models of AF mimicked the alterations in A_2AR density observed in patients with AF, making them attractive models for studying the adenosinergic system in AF.

Keywords: A2AR density; AF patients; HL-1 cells; adenosine A2AR receptor; atrial fibrillation; tachypaced pig.

Figure 1

Effect of ATX-II on the viability of HL-1 cells. Cells were incubated in the absence (circles in grey columns,) or presence of ATX-II (30 nM) (squares in red columns) for 24 h before cell viability, cell damage and ROS production were monitored by MTT tetrazolium reduction (left panel), PI (middle panel), and DCF (right panel) assays, respectively (see Methods). The results are shown as percentage of viable cells, cell damage and ROS production in cells treated with saline (ATX-II -) and expressed as mean ± SEM of three independent experiments, each performed in triplicate. * p < 0.05, *** p < 0.001, Student’s t-test.

Figure 2

Effect of ATX-II on the density of A_2AR in HL-1 cells. HL-1 cells were incubated in the absence (circles in grey colum) or presence of ATX-II (30 nM) (squares in redcolumn) for 24 h before membrane extracts were analysed by SDS-PAGE (20 μg of protein/lane) and immunoblotted using mouse anti-A_2AR and rabbit anti-α-actinin antibodies. A representative immunoblot is shown (lower panel). Immunoblot protein bands corresponding to A_2AR and α-actinin from vehicle (n = 5) and ATX-II (n = 5) treated cells were quantified by densitometric scanning. Values were normalized to the respective amount of α-actinin in each lane to correct for protein loading. The results are shown as percentage of the relative density of A_2AR in cells treated with saline (ATX-II -; dashed lane) and expressed as mean ± SEM (n = 5). * p < 0.05, Student’s t-test.

Figure 3

Expression of calcium-handling proteins in the atrium of the porcine model of AF. (A) Representative immunoblots showing the density of SERCA2a, NCX-1, and Csq-2 proteins in the right atrium of control (SHAM) and tachypaced (A-TP) pigs. The atrial membrane extract of SHAM and A-TP pigs was analysed by SDS-PAGE (10 μg of protein/lane) and immunoblotted using antibodies against SERCA2a, NCX-1, and Csq-2 (see Methods). (B) Relative quantification of SERCA2a, NCX-1, and Csq-2 density. Immunoblot protein bands corresponding to SERCA2, NCX-1, and Csq-2 and α-actinin from SHAM (n = 4) and A-TP pigs (n = 6) were quantified by densitometric scanning. The values were normalized to the respective amount of α-actinin in each lane to correct for protein loading. The results are shown as percentages of the relative density of each calcium-handling protein in A-TP animals relative to control pigs (SHAM; dashed line) and are expressed as mean ± SEM (n = 6). * p < 0.05 one-way ANOVA with Dunnett’s post hoc test compared to control pigs (SHAM; dashed line).

Figure 4

Expression of A_2AR in the atrium of the porcine model of AF. (A) Representative immunoblot showing the density of A_2AR in the right atrium of control (SHAM, circles) and tachypaced (A-TP, squares) pigs. The atrial membrane extract of SHAM and A-TP pigs was analysed by SDS-PAGE (10 μg of protein/lane) and immunoblotted using mouse anti-A_2AR and rabbit anti-α-actinin antibodies (see Methods). (B) Relative quantification of A_2AR density. The immunoblot protein bands corresponding to A_2AR and α-actinin from SHAM (n = 4) and A-TP pigs (n = 6) were quantified by densitometric scanning. Values were normalized to the respective amount of α-actinin in each lane to correct for protein loading. The results are shown as percentages of the relative density of A_2AR in SHAM pigs (dashed lane) and expressed as mean ± SEM. * p < 0.05, Student’s t-test.

Figure 5

Expression A_2AR in human heart atria of AF patients. (A) Representative immunoblot showing the expression of the A_2A receptor in the right atria of non-dilated sinus rhythm (ndSR, circles) and atrial fibrillation (AF, squeres) patients. Membrane extracts from human atrium were analysed by SDS-PAGE (10 μg of protein/lane) and immunoblotted using rabbit anti-A₁R, mouse anti-A_2AR and rabbit anti-α-actinin antibodies (see Material and Methods). (B) Relative quantification of A_2AR density. The immunoblot protein bands corresponding to A_2AR and α-actinin from ndSR (n = 11) and AF (n = 9) subjects were quantified by densitometric scanning. Values were normalized to the respective amount of α-actinin in each lane to correct for protein loading. The results are shown as percentage of the relative density of A_2AR in ndRS (dashed lane) and expressed as mean ± SEM. *** p < 0.001, Student’s t-test.