Adenosine A2A Receptors Are Upregulated in Peripheral Blood Mononuclear Cells from Atrial Fibrillation Patients

Figures

• 

  Figure 1. A2AR expression in human right atrium. (A) Representative immunoblot showing the expression of A2AR in right atrium from non-dilated sinus rhythm (ndSR) and atrial fibrillation (AF) patients; membranes from human atrium were analysed by SDS-PAGE (10 μg of protein/lane) and immunoblotted using goat anti-A2AR and rabbit anti-α-actinin antibodies (see Methods). (B) Relative quantification of A2AR density; the immunoblot protein bands corresponding to A2AR and α-actinin from ndSR (n = 11) and AF (n = 12) patients were quantified by densitometric scanning; values were normalized to the respective amount of α-actinin in each lane to correct for protein loading. (C) Relative expression of A2AR transcripts in human atrium. Mean ± SEM from ndSR (n = 11) and AF (n = 9) patients. **** p < 0.0001 and ** p < 0.01, Student t test.

• 

  Figure 2. ADA activity and adenosine content in human plasma. (A) ADA activity in plasma from ndSR (n = 24), dSR (n = 22) and AF (n = 23) patients was determined using the Dyazime kit. Two outliers were removed from the dSR group by the ROUT method (see Materials and Methods). (B) Adenosine content in plasma from ndSR (n = 15), dSR (n = 15) and AF (n = 13) patients was determined by MS-HPLC. One outlier was removed from the AF group by the ROUT method (see Materials and Methods). (C) Correlation between ADA activity and adenosine content in ndSR (yellow, n = 18) and AF (red, n = 14) patients. The correlation coefficients (r) were calculated 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.

• 

  Figure 3. A2AR detection in living cells using a fluorescent ligand. HEK-293 cells permanently expressing 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.

• 

  Figure 4. Detection of A2AR in human PBMCs. (A) The specific MRS7396 binding to PBMCs from ndSR (yellow, n = 16), dSR (orange, n = 12) and AF (red, n = 14)) patients was obtained by subtracting the nonspecific binding (i.e., 100 nM MRS7396 in the presence of 100 nM of ZM241385) and represented as ∆FL-1 (specific binding). (B) Relative expression of A2AR mRNA in PMBCs. Mean ± SEM of ndSR (n = 10), DSR (n = 7) and AF (n = 9) patients. **** p < 0.0001, one-way ANOVA with Tukey’s post-hoc test.

• 

  Figure 5. Correlation of atrial A2AR density and peripheric adenosinergic system. Correlation between the relative A2AR density in the right atrium determined by immunoblot (Integrated density, see Figure 1B) and the specific MRS7396 binding (∆FL-1) to PBMCc (A), adenosine content (C) and ADA activity (E) from ndSR (yellow) and AF (red) patients. Also, the correlation between 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 (two-tailed correlation) test.