Phenotyping of Mice with Heart Specific Overexpression of A2A-Adenosine Receptors: Evidence for Cardioprotective Effects of A2A-Adenosine Receptors

FIGURE 1. (A) Radioligand binding studies in membrane fraction of A2A-AR-overexpressing animals. The experiments were performed as described in Section “Materials and Methods.” The binding of [3H]-labeled A2A-AR-agonist CGS 21680 could be almost completely blocked by addition of A2A-AR-antagonist ZM 241385, whereas A1-AR-antagonist PSB36 and A3-AR-antagonist MRS 1220 did not affect the binding of [3H]-CGS 21680. No specific binding was detectable in membrane fraction from WT animals. (B) Immunohistological staining of ventricular tissue from A2A-AR-overexpressing and wild type (WT) animals. A2A-AR was visualized using a monoclonal antibody against A2A-AR and goat anti-mouse antibody conjugated to Alexa Fluor 594 (red, upper row). The nuclei were counterstained using DAPI (blue). Note staining of cardiomyocytes from A2A-AR animals, mainly in the vicinity of the sarcolemma. Lower row: negative controls with omission of the primary antibody. ⋆p < 0.05 vs. [3H]-CGS 21680 alone.

FIGURE 2. (A,B) cAMP content in isolated ventricular cardiomyocytes from WT and A2A-AR-overexpressing animals. cAMP content was determined by Biotrak direct Enzymimmunoassay. Note concentration dependent increase of cAMP content by A2A-AR agonist CGS 21680 in A2A-AR cardiomyocytes (A); this effect could be completely blocked by A2A-AR antagonist ZM 241385. The effect of β-adrenoceptor agonist isoproterenol on cAMP content did not differ between WT and A2A-AR (B). (C,D) Phosphorylation of phospholamban at serine-16 (pPLB). (E,F) Phosphorylation of the inhibitory subunit of troponin (pTnI). The phosphorylation state of PLB and TnI was assessed by phosphorylation specific antibodies. The signals obtained using phosphorylation specific antibodies were normalized to the corresponding protein expression. Note concentration dependent increase of pPLB (C) and pTnI (E) by A2A-AR agonist CGS 21680 in A2A-AR cardiomyocytes; these effects were abolished by A2A-AR antagonist ZM 241385. The effects of isoproterenol on protein phosphorylation were comparable in WT and A2A-AR (D,F). ⋆p < 0.05 vs. Ctr; #p < 0.05 vs. WT.

FIGURE 3. (A) Force of contraction (FOC) in isolated electrically driven left atria. (B) Beating frequency (BF) in isolated spontaneously beating right atria. The contraction experiments were performed in the presence of A1-AR antagonist DPCPX (1 μM). Note the higher basal BF and the increase of FOC and BF by A2A-AR agonist CGS 21680 in atria of A2A-AR overexpressing animals. These effects were abolished in the presence of A2A-AR antagonist ZM 241385. ⋆p < 0.05 vs. Ctr; #p < 0.05 vs. WT; +p < 0.05 vs. CGS 21680 alone.

FIGURE 4. Hemodynamic parameters assessed by left ventricular catheterization in anesthetized WT and A2A-AR overexpressing animals as described in Section “Materials and Methods.” Effects of dobutamine on beating frequency (BF, A) and maximum rate of left ventricular pressure development (+dP/dt, B). Note the higher basal +dP/dt and the lack of dobutamine effect on +dP/dt in A2A-AR overexpressing animals. ⋆p < 0.05 vs. Ctr; #p < 0.05 vs. WT.

FIGURE 5. Hemodynamic parameters assessed by Doppler echocardiography in anesthetized WT and A2A-AR overexpressing animals. Note the increased basal beating frequency (A) and interventricular systolic septum thickness (IVSs, B) in A2A-AR overexpressing animals. The effects of β-adrenoceptor agonist isoproterenol on both parameters were comparable between WT and A2A-AR overexpressing animals. ⋆p < 0.05 vs. Ctr; #p < 0.05 vs. WT.

FIGURE 6. Comparison of 20 min (A), 40 min (B), and 60 min (C) of global ischemia in isolated Langendorff-perfused hearts from WT and A2A-AR overexpressing animals. Maximum rate of left ventricular pressure development (+dP/dt) is presented as % of basal value. ⋆p < 0.05 vs. WT.

FIGURE 7. Hemodynamic parameters during global ischemia with subsequent reperfusion assessed in isolated Langendorff-perfused hearts from WT and A2A-AR overexpressing animals. (A) Maximum rate of left ventricular pressure development (+dP/dt) as % of basal value with and without the A2A-AR antagonist SCH 442416 (SCH, 100 nM). (B) Left ventricular end diastolic pressure with and without the A2A-AR antagonist SCH 442416 (SCH, 100 nM). (C) Effect of A2A-AR agonist CGS 21680 (1 μM) administered during reperfusion on +dP/dt in A2A-AR overexpressing hearts. Note the higher basal +dP/dt, markedly prolonged time to ischemic contracture as well as the additional effect of A2A-AR agonist CGS 21680 on the +dP/dt in A2A-AR overexpressing hearts during reperfusion. ⋆p < 0.05 vs. WT; #p < 0.05 vs. A2A-AR.