A novel cardioprotective function of adenosine A1 and A3 receptors during prolonged simulated ischemia

Abstract

The possible cardioprotective roles of adenosine A1 and A3 receptors were investigated in a cardiac myocyte model of injury. The adenosine A3 receptor is a novel cardiac receptor capable of mediating potentially important cardioprotective functions. Prolonged hypoxia with glucose deprivation was used to simulate ischemia and to induce injury in cardiac ventricular myocytes cultured from chick embryos 14 days in ovo. When present during the prolonged hypoxia, the adenosine A3 agonists N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) and 2-chloro-N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (CI-IB-MECA) caused a dose-dependent reduction in the extent of hypoxia-induced injury as manifested by a decrease in the amount of creatine kinase released and the percentage of myocytes killed. The adenosine A1 agonists 2-chloro-N6-cyclopentyladenosine (CCPA), N6-cyclohexyladenosine, and adenosine amine congener were also able to cause a decrease in the extent of myocyte injury. The A1 receptor-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine blocked the cardioprotective effect of the A1 but not of the A3 agonists. Conversely, the selective A3 antagonists MRS-1191 and MRS-1097 blocked the protection induced by CI-IB-MECA but had minimal effect on that caused by CCPA. Thus the cardioprotective effects of A1 and A3 agonists were mediated by their respective receptors. This study defines a novel cardioprotective function of the cardiac A3 receptor and provides conclusive evidence that activation of both A1 and A3 receptors during hypoxia can attenuate myocyte injury.

Fig. 1

Effect of adenosine A₃ receptor agonists on hypoxia-induced myocyte injury. Cardiac ventricular myocytes were cultured from chick embryos 14 days in ovo, and myocyte injury was induced as described in METHODS. N⁶-(3-iodobenzyl)adenosine-5′-N-methyluronamide (IB-MECA) and 2-chloro-N⁶-(3-iodobenzyl)adenosine-5′-N-methyluronamide (Cl-IB-MECA) were added to medium at concentrations indicated in presence of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1 μM) during hypoxia. Percentage of myocytes injured (open symbols) and amount of creatine kinase (CK; solid symbols) released were determined after hypoxia. Data are means of 4 experiments. At 1 and 10 nM Cl-IB-MECA or IB-MECA, levels of CK released and percentages of cells killed were significantly lower than those determined in myocytes exposed to hypoxia only in absence of any adenosine agonist or antagonist [P < 0.01 by analysis of variance (ANOVA) and t-test].

Fig. 2

Effect of adenosine A₃-receptor antagonists on 2-chloro-N⁶-cyclopentyladenosine (CCPA)- and Cl-IB-MECA-induced cardioprotective effects. Cultured ventricular myocytes were prepared, and extent of hypoxia-induced myocyte injury was determined as described in METHODS. A₃ antagonist MRS-1191 was present at concentrations indicated with CCPA (10 nM) or Cl-IB-MECA (10 nM) during 90-min hypoxic period. Percentage of myocytes killed (A) and amount of CK released (B) were determined after prolonged hypoxia. Data are means ± SE of 3 experiments. In presence of Cl-IB-MECA and 30 nM, 300 nM, and 3 μM MRS-1191, levels of CK released and percentages of cells killed were significantly higher than those determined in myocytes exposed to Cl-IB-MECA only (P < 0.01 by ANOVA and t-test).

Fig. 3

Effect of adenosine A₁ agonists on cardiac myocyte injury. Cultured ventricular myocytes were prepared, and extent of hypoxia-mediated myocyte injury was determined as described in METHODS. CHA, N⁶-cyclohexyladenosine; ADAC, adenosine amine congener. Adenosine A₁-receptor agonists were added to medium at concentrations indicated in absence or presence of A₁-receptor antagonist DPCPX during prolonged hypoxia. Percentage of cells killed was determined after hypoxic exposure and removal of A₁-receptor agonists and antagonist. Data are means of 4 experiments. At 1 and 10 nM concentrations of A₁ agonists, percentages of myocytes killed were significantly lower than those obtained in presence of either of the 2 A₁-agonist concentrations and DPCPX (1 μM) (P < 0.01 by ANOVA and t-test).