and Use Committees of St. Jude Children’s Research Hospital and the University of Tennessee Health Sciences Center. The A2AR KO mice and WT littermates were obtained from a subcolony of the original lines generated and characterized by Ledent et al.. Analysis of cardiac tissue confirms ablation of A2AR transcript without compensatory shifts in other adenosine receptors. Mice were bred and maintained onsite at St. Jude Children’s Research Hospital, with standard laboratory food and water available ad libitum. Animal genotype was confirmed by PCR analysis of tail snips. All animals were originated from the same breeding series and were matched for age and weight. Male and female mice were used with equal representation in each experimental group. Both A2AR KO and WT littermate mice were randomly allocated to receive either an intraperitoneal injection of 20 mg/kg LPS isolated from E. coli or an equal volume of sterile saline vehicle. Blood was sampled at 12 and 24 h of LPS challenge to assess shifts in blood cell 
counts and circulating cytokines/biomarkers, as detailed by us previously. Mice were monitored for the development of symptoms of illness or distress during the initial 12 h post-injection and every 4 h thereafter; observation of Purinergic Signalling 13:2749 29 any combination of symptoms prompted immediate euthanasia. All efforts were made to minimize animal 946128-88-7 biological activity suffering and distress. No analgesic or anaesthetic was administered other than for euthanasia immediately on evidence of illness/ distress. We initially tested responses PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19802338 to 24 h LPS challenge in both young and old WT and A2AR KO mice. However, absent overt symptoms of illness/distress, there was nonetheless significant mortality in aged LPStreated mice, as detailed in the “Results” section and in Supplementary material. Cause of death in older animals was not determined. As a result of mortality, detailed molecular interrogation of cardiac gene expression, together with analyses of cardiac function in isolated hearts and haematological/biomarker assessment is necessarily constrained to the young group of mice. Langendorff perfusion and tissue sampling After 24 h of LPS challenge, mice were anaesthetised with sodium pentobarbital, a thoracotomy performed and hearts excised into ice-cold KrebsHenseleit solution for Langendorff perfusion, as detailed previously, or sampling of ventricular tissue for RNA preparation. Briefly, for perfusions, the aorta was immediately cannulated and hearts perfused at 80 mmHg with modified Krebs-Henseleit solution containing: NaCl, 120; NaHCO3, 25; KCl, 4.7; CaCl2, 2.5; MgCl2, 1.2; KH2PO4, 1.2; D-glucose, 15 and EDTA, 0.5. Perfusion fluid was maintained at 37 C and bubbled with a mix of 95 % O2/5 % CO2 at 37 C to provide a pH of 7.4. Ventricular function was monitored via a fluid-filled plastic film balloon in the left ventricle, connected to a P23 XL pressure transducer. Coronary flow was monitored via a flow-probe in the aortic perfusion line, connected to a T206 flowmeter. Functional data were recorded at 1 KHz on MacLab data acquisition system. Statistical comparisons of cardiovascular and blood parameters between groups were made via analysis of variance, with a Newman-Keuls post hoc test for specific comparisons. A P < 0.05 was considered indicative of significance in all tests. RNA preparation and microarray hybridisation Ventricular tissue was isolated, frozen in liquid N2 and stored at -80 C until analysis. Tissue was subsequently hom