Apelin protects the heart against ischemia-reperfusion injury via epidermal growth factor receptor (EGFR) transactivation

Apelin peptide is a ligand of the G protein coupled receptor (GPCR) APJ. Apelin-13 (Ape) is the most active isoform on the cardiovascular system, where it exerts positive inotropy and vasodilation. It also mimics ischemic postconditioning (IPost) against ischemia–reperfusion (I/R) injury. Protection is caused by NO after phosphatidylinositol 3-kinase (PI3K)-Akt activation. Various GPCRs activate PI3K-Akt via epidermal growth factor receptor (EGFR) transactivation by ligand-dependent and -independent pathways, the former due to the shedding of epidermal growth factor (EGF)-like ligands by matrix metalloproteinase (MMP), the latter to the nonreceptor tyrosine kinase Src. We investigated the main steps of the pathway of Apeinduced cardioprotection focusing the attention on a possible role of EGFR transactivation. Materials and methods: Rat hearts underwent 30 min of global ischemia and 2 h of reperfusion. Left ventricular pressure (LVP) was measured from a balloon in the left ventricle. After reperfusion infarct size (IS) was measured. EGFR phosphorylation was assessed on hearts undergone 10 min of reperfusion with western blot. These inhibitors were used: GM6001 to prevent EGF-like shedding by MMP, AG1478 to inhibit EGFR, PP2 to inhibit Src, LY294002, ODQ and 5HD to inhibit PI3K, guanylyl cyclase (GC) and mitochondrial ATP-dependent K+ channels (K+mito) respectively.Results: Global ischemia caused an increase in left ventricular diastolic pressure (LVDP) to about 50 mm Hg. Ape limited (p < 0.001) this increase to about 30 mm Hg. All inhibitors prevented this effect. I/R reduced the recovery of developed LVP to about 35% only of the baseline. The recovery was improved by Ape (75% of baseline p < 0.001) an effect which was partially prevented by MMP and EGFR inhibition and abolished by PP2 and other inhibitors. Ape reduced IS from 64 ± 4% to 30 ± 3% (p < 0.001). This effect was abolished by all inhibitors. As shown by preliminary data, Ape increased EGFR phosphorylation (p < 0.05). This increase was prevented by inhibition of Src and EGFR, but not by prevention of MMP shedding of EGF-like ligands. Conclusions: The results show that EGFR mediates Ape-induced protection. The protection is abolished by inhibition of the various steps from PI3K activation to the opening of K + mito via NO–cGMP pathway, a cascade which is also involved in IPost. However, while the limitation of IS and of LVDP increase requires both liganddependent and -independent transactivations of EGFR, the recovery of developed LVP is mainly mediated by the ligand-independent pathway. It may be argued the ligand-independent pathway plays a critical role in the overall cardioprotection.