A cooperative cardioprotective effect of adenosine A1 and A2a receptor agonism in ischemia-reperfusion damage in the isolated perfused mouse heart
Adenosine A1 agonist (CPA) ameliorates necrotic damage in a cardiac cell line after simulated ischemia by a cooperative effect on adenosine A2A/2B receptors1. In the present study, we investigated whether this cooperative effect was seen in mouse hearts using a Langendorff preparation with an ischemia-reperfusion protocol. Langendorff isolated perfused CD1 A2A knockout (KO) and wild-type (WT) mouse hearts were subjected to no-flow global ischemia (30 min) and reperfusion (60 min) at constant pressure (80 mmHg). Cardiac troponin I (cTn I) and lactate dehydrogenase (LDH) release were measured by collecting cardiac effluent before ischemia and at the end of reperfusion. Adenosine A1 and A2A cooperative cardioprotective signalling was assessed by extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. Both wild-type and A2AKO mice were treated with adenosine A1 agonist CPA (100nM) added at reperfusion, either in presence of adenosine A1 antagonist (DPCPX, 1μM) or adenosine A2A antagonist (ZM241385, 50nM) or adenosine A2B antagonist (MRS1754, 100nM). A significant increase in the infarct size was observed in A2AKO compare to WT mice (26.75 ± 1.78 and 18.69 ± 5.21 % of area at risk respectively, p<0.05). CPA treatment significantly reduced infarct size in WT animals. Treatment with ZM241385 worsened left ventricular developed pressure (LVDP) recovery in WT but, as expected, not in A2AKO mice (72.02 ± 22.50% and 17.60 ± 2.83% change from baseline for CPA vs. 5.48 ± 5.48 and 41.86 ± 19.34% change from base line for CPA + ZM241385 in WT and A2AKO mice at 70 min respectively, p<0.05). In contrast, MRS1754 worsened LVDP recovery in both A2A KO and WT mice (72.02 ± 22.50% change from baseline for CPA vs. 5.06 ± 3.68% change from baseline for CPA + MRS1754 in WT mice at 70 min, p<0.05). Conversely, adenosine A1 antagonist (DPCPX) caused a bigger reduction in contractility (dP/dtmax), in A2A KO compare to WT mice. In WT mice CPA and CPA + CGS21680 significantly reduced cTn I level (2.84 ± 0.59 and 4.97 ± 0.08 ng/ml vs. 11.99 ± 1.06 ng/ml in control, p<0.05). In A2AKO CPA + CGS21680 did not reduced the cTn I release. In WT mice LDH release reduced significantly (4.73 ± 0.04, 6.68 ± 0.51% cytotoxicity in CPA and CPA + CGS21680 groups vs. 14.03 ± 1.92% cytotoxicity in control group, p<0.05), however in A2AKO mice no significant difference was observed. In WT mice CPA treatment increased ERK1/2 phosphorylation (1.53 ± 0.26 pERK/ERK1/2 in CPA vs. 0.83 ± 0.06 pERK/ERK1/2 in control, p<0.05), which was significantly reversed by DPCPX and ZM241385 (0.92 ± 0.16 and 0.83 ± 0.05 pERK/ERK1/2 respectively, p<0.05). Overall the beneficial cooperative effects of CPA were inhibited by DPCPX, ZM241385 and MRS1754. In conclusion, the data suggests that in wild-type mice both adenosine A2A and A2B receptor antagonists worsen functional recovery in the presence of adenosine A1 agonist. However, in the A2A KO mice, an adenosine A2B antagonist worsened functional recovery but as expected the adenosine A2A antagonist did not. In the isolated perfused mouse heart, A1-mediated cardioprotection requires a cooperative activation of A2 receptors, presumably via endogenous adenosine.
1. Urmaliya et al, Cardiovasc Pharmacol 53(5):424-433 (2009)