A129. Bradykinin cardioprotection is mediated by receptor internalization and mitoKATP opening

Abstract

Bradykinin (Bk) mimics the cardioprotective effects of ischemic preconditioning (IPC) by a complex series of cellular signaling events that include activation of protein kinase G (PKG), opening of mitochondrial ATP-sensitive K+-channels (mitoKATP), and increased production of reactive oxygen species (ROS). In this study, we investigate the hypothesis that the bradykinin B2 receptor is internalized within a signaling platform that mediates the opening of mitoKATP and cardioprotection. We describe a procedure in which hearts are perfused with Bk in a Langendorff apparatus. Following perfusion, mitochondria are isolated and subjected to purification in a Percoll density gradient. This yields a low-density fraction (henceforth termed the light-layer) and a purified mitochondrial fraction. As determined by western blot analysis, the light-layer contains caveolin-1 and -3, the bradykinin B2 receptor, and PKG as well as other proteins. We find that addition of the light-layer to untreated mitochondria opens mitoKATP similar to KATP openers such as diazoxide, as measured by changes in matrix volume. MitoKATP opening by the light-layer does not require the addition of cyclic-GMP but is sensitive to the mitoKATP blocker 5-hydroxydecanoate, the PKCε peptide antagonist εV1–2, the serine/threonine phosphatase PP2A and the PKG-specific blocker KT5823, indicating that PKG was activated by Bk treatment. Inhibition of the open state was not observed upon addition of the Src kinase inhibitor PP2, the tyrosine phosphatase PTP2B, MEK inhibitor I or the ERK inhibitor 3-(2-Aminoethyl)-5-((4-ethoxyphenyl)methylene)-2,4-thiazolidinedione, HCl. From this data, we conclude that the light-layer from Bk perfused hearts contains a cardioprotective signaling platform that migrates to mitochondria and opens mitoKATP, resulting in a protected state.