Reperfusion kinase phosphorylation is essential but not sufficient in the mediation of pharmacological preconditioning: Characterisation in the bi-phasic profile of early and late protection

Objective: Pharmacological preconditioning (PPC) triggers early (ePPC) and delayed protection (dPPC), occurring within 1 h or after 24 h following the preconditioning stimulus, respectively, through recruitment of protein kinase signalling. Angiotensin II (ATII) is a recognised trigger of PPC, recruiting kinases and transcription factors known to be involved in both phases of protection. Our objectives were to determine whether ATII is capable of triggering dPPC and whether recruitment of pro-survival kinases, Akt and extracellular signal-regulated kinase (ERK), following the injurious ischaemic insult is essential for the mediation of PPC. Methods: In a mouse Langendorff model of ischaemia/reperfusion injury, we undertook to determine whether ATII triggers both ePPC and dPPC. Western blot analysis was used to determine kinase phosphorylation at reperfusion, and kinase inhibitors wortmannin and PD98059 were used to ascertain the significance of kinase regulation. Results: We demonstrated that ATII triggered PPC with attenuation of infarction at 1 and 24 h (19 +/- 4% and 25 +/- 4% versus control, 35 +/- 4% of risk zone, p < 0.05), consistent with the ePPC and dPPC time-course. This bi-phasic protection was associated with significant post-ischaemic phosphorylation of both Akt and ERK within the first 5 min of reperfusion. Akt and ERK phosphorylation was increased following ePPC by 4.5 +/- 0.5 and 1.9 +/- 0.6 fold, respectively (p < 0.001), and dPPC by 24 +/- 2.0 and 2.1 +/- 0.1 fold, respectively (p < 0.001). Both wortmannin and PD98059 administered during reperfusion ameliorated the phosphorylation of Akt and ERK and abrogated the resistance to infarction resulting from both ePPC and dPPC (33 +/- 3% and 35 +/- 4%, respectively, versus controls 33 +/- 4% and 33 +/- 5%, p = NS). There was no evidence of augmented phosphorylation of either p38 kinase or INK at either time point. Conclusion: We demonstrate that PPC results in a clearly delineated time-course of bi-phasic protection against injurious ischemic injury that is correlated with reperfusion kinase phosphorylation of both Akt and ERK. These data indicate a novel mechanism of early and particularly delayed preconditioning. (c) 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.