Pre- and post-ischaemic Apelin-13 effect on cardiac function and APJ receptor expression in rat hearts

Apelin is the endogenous ligand for the G-protein-coupled APJ receptor. Its gene encodes a 77 aminoacid preprotein with the active sequence in the C-terminal region. Various fragments of apelin have been isolated and classified according to the aminoacid number. Apelin-13 is reported to be the most active fragment. Apelin and APJ have been found in vascular smooth muscle, endothelial cells and cardiomyocytes. Apelin-13 protects rat hearts against ischaemia-reperfusion injury if given at the concentration of 500 nM after ischaemia, but not before. We investigated: a) whether the protection includes an improved mechanical recovery after ischaemia; b) whether increased Apelin-13 concentrations, given before ischaemia, could induce protection; c) whether the protective effect of Apelin-13 given after ischaemia is due to an ischaemia-induced APJ overexpression. Rats were anaesthetized with ketamine (90 mg/kg) and xylazine (10 mg/kg) and were killed by decapitation. The hearts were excised and perfused with oxygenated buffer. They were divided in 5 groups: i (control): a 30 min global ischaemia was followed by a 120 min reperfusion. ii: Apelin-13 was given after ischaemia at 500 nM concentration. iii and iv: Apelin-13 was given before ischaemia at 500 and 1000 nM concentration respectively. v: Apelin-13 was not given and reperfusion was stopped at 0, 7, 15, 30 or 120 min. APJ expression was evaluated by RT-PCR and Western blot analyses. In Groups i-iv left ventricular pressure (LVP) was measured and infarct size was assessed by nitro-blue tetrazolium. Lactate-dehydrogenase (LDH) was tested in the effluent at fixed intervals during reperfusion. Data are expressed as mean±sem and were statistical analyzed by 1-way ANOVA. RT-PCR and Western blot analyses were performed in triplicate for each sample. In the control group, global ischaemia and reperfusion caused an increase (p<0.001) in diastolic LVP 6±1 to 40±9 mmHg without any change in systolic LVP and with a reduction by about 70% of developed pressure. After ischaemia, Apelin-13 reduced infarct size from 54±3% to 26±4% (p<0.001) and abolished the increase in diastolic LVP. No effect was obtained when Apelin-13 was infused before ischaemia even at 1000 nM. Total LDH released during reperfusion was proportional to the infarct size. Ischaemia increased both APJ transcription and translation within the first 15 min after ischaemia, i.e. after most of reperfusion injury has taken place. It is confirmed that apelin-induced protection is not achieved if the compound is given before ischaemia, even at increased concentration. The protection includes an improved post-ischaemic mechanical recovery consisting in the suppression of the contracture due to ischaemia and reperfusion. Finally, the timing of injury and APJ protein expression does not explain why the compound is effective only if given after ischaemia.