Adenosine receptor-mediated cardioproctection

Ischemic heart disease is the most common type of cardiovascular disease. The Australian Institute of Health and Welfare reported that there were 22,983 deaths (17% of all deaths in Australia) due to ischemic heart disease that were recorded in 2006. Substantial evidence accumulated from human data and animal models suggests that adenosine released under conditions of physiological stress such as hypoxia and ischemia-reperfusion injury plays an important cardioprotective role via activation of adenosine receptors (four subtypes: A1, A2A, A2B and A3) and reduces the extent of cardiac cell death. The present thesis describes a body of work investigating cardioprotection induced by adenosine receptor and synergistic cooperative interactions between adenosine A1 and A2 receptors during ischemia-reperfusion injury using a cardiac cell ischemia and Langendorff perfused isolated mouse/rat heart models. Chapter 2 describes the development of a suitable pharmacological model to mimic the in-vivo ischemic condition using H9c2(2-1) cardiac cells. This was used for preliminary pharmacological evaluation of novel compounds synthesized by the Department of Medicinal Chemistry in our faculty. Exposure of cardiac cells to ischemic buffer for 12 hrs significantly increased the number of nonviable cells, the effect reduced in the presence of adenosine A1 and A3 receptor agonists N6cyclopentyladenosine (CPA) and N6-(3-Iodobenzyl)adenosine-5′-N-methyluronamide (IB-MECA), and novel adenosine A1 and A3 agonists VCP28, VCP102 and VCP103, VCP438, VCP439, VCP485, VCP486, VCP487 respectively. A novel, highly selective adenosine A1 partial agonist VCP28 have shown the cardioprotection at lower nanomolar concentration, which is further investigated in the isolated rat heart model in chapter 5. Chapter 3 demonstrate the interactions between A1 and A2 receptors in the presence and absence of endogenous adenosine in a cardiac cell ischemia model. In a cardiac cell ischemia model necrosis was assessed using propidium iodide (5μM) staining or apoptosis using AnnexinV-PE flowcytometry. CPA (100 nM) and IB-MECA (100 nM) reduced the proportion of non-viable cells to 30.87 ± 2.49% and 35.18 ± 10.30% respectively (% of SI group, P<0.05, n=3-5). In the absence of endogenous adenosine, using the introduction of adenosine deaminase into the media, the protective effect of CPA was reduced, whilst the efficacy of IB-MECA was unchanged. The protective effects of CPA and IB-MECA were abrogated in the presence of their respective antagonists DPCPX and MRS1191, whilst A2A and A2B agonists had no significant effect. CPA-mediated protection was abrogated in the presence of both A2A (ZM241385) and A2B (MRS1754) antagonists. In the absence of endogenous adenosine significant protection was observed with CPA in presence of A2A or A2B agonists (CGS21680 or LUF5834). Apoptosis was not significantly reduced by CPA or IB-MECA. The data demonstrate that cardioprotection induced by adenosine A1 receptor agonist in a cardiac cell ischemia model involves co-operative activation of adenosine A2A and A2B receptors by endogenous adenosine. Chapter 4 looks at the synergistic cooperative interaction between adenosine A1 and A2 receptors in the intact heart – in ischemia-reperfused isolated mouse hearts when CPA was given briefly at reperfusion. Adenosine A2A receptor knockout (A2AKO) and wild-type (WT) mouse hearts (n=9-11) were subjected to global ischemia (30 min) and reperfusion (60 min) in presence of CPA or CGS21680 for the initial 15 min of reperfusion. In WT hearts, CPA (100 nM) significantly (P<0.05) improved contractility (dP/dtmax, 52.69 ± 6.22 vs. 23.94 ± 4.93% of baseline), left ventricular developed pressure (LVDP), end diastolic pressure (EDP), reduced infarct size (7.86 ± 1.73 vs. 23.94 ± 6.62% area at risk), decreased lactate dehydrogenase efflux and increased pERK1/2 signalling. ZM241385 and MRS1754 abolished CPA-mediated cardioprotection in WT groups, similar to the DPCPX. In A2AKO hearts, CPA did not improve functional recovery and pERK1/2 signaling. In this clinically relevant model of pharmacological intervention, pERK1/2-dependent A1-mediated cardioprotection requires a cooperative activation of A2 receptors, presumably via endogenous adenosine. Chapter 5 describes the further evaluation of compound identified in chapter four, the novel, highly selective adenosine A1 receptor partial agonist N6-(2,2,5,5-tetramethylpyrrolidin-1-yloxyl-3-ylmethyl)adenosine (VCP28) in a cardiac cell ischemia model and in an isolated rat heart ischemia-reperfusion model. In the cardiac cell ischemic model, CPA and VCP28 (100 nM) treatment during ischemia significantly reduced the proportion of nonviable cells (30.88 ± 2.49, 16.17 ± 3.77% of SI group, respectively, P<0.05, n= 5-6) and lactate dehydrogenase efflux. In isolated rat hearts, CPA and VCP28 treatment briefly at reperfusion significantly (n= 6-8, P<0.05) improved postischemic contractility (81.69 ± 10.96, 91.07 ± 19.87 % of baseline, respectively), LVDP, EDP and reduced infarct size. DPCPX abolished the cardioprotective effects of CPA and VCP28 in cardiac cell ischemia and isolated rat hearts models. The data described in this chapter demonstrate that the A1 partial agonist VCP28 has equal cardioprotective effects to the full agonist CPA at concentrations that have no effect on isolated rat heart rate. In conclusion, studies described in the present thesis provide compelling evidence that endogenous adenosine released during ischemic condition increase cardioprotection of A1 agonist, and cooperatively activate adenosine A2A and A2B receptors in a cardiac cell ischemia and isolated mouse heart ischemia reperfusion models. Of note, the synergistic “cooperative A1 and A2 ischemic protection” involve downstream activation of ERK1/2 phosphorylation signalling in isolated mouse hearts. Novel and highly selective adenosine A1 and A3 receptor agonists were screened and the A1 partial agonist VCP28 was shown to provide cardioprotection when administered during ischemia (pharmacological preconditioning) in a cardiac cell ischemia model and during reperfusion (pharmacological postconditioning) with greater efficacy and potency than the same compound was able to activate pathways mediating acute cardiac effects, such as minimal bradycardic effect, in isolated rat heart model. This thesis demonstrates that adenosine A1 receptor-mediated cardioprotection during ischemia-reperfusion damage is dependent on cooperative activation of adenosine A2 receptor by endogenous adenosine.