Abstract 15210: Differential Phosphorylation of Members of the Mitogen Activated Protein Kinase and Apoptosis Pathways in Response to Ischemic Preconditioning in Rat Myocardium
Despite being first described 26 years ago, the protective mechanisms of ischemic preconditioning (IPC) are still largely unknown. A response to conditioning stimuli is rapidly mounted, and thus phosphorylation, a rapid, transient protein modifier, has been hypothesised to be a major mediator. Single protein studies have suggested mitogen-activated protein kinase (MAPK) mediated signaling plays an important role in IPC. A large scale investigation of protein phosphorylation has yet to define the extent of MAPK signal cascades and accessory networks to modulate IPC. Langendorff perfused myocardium underwent the following protocols in triplicate; Non-ischemic control, IPC alone (3 × 2I/2R), IPC + ischemia / reperfusion (I/R) injury (IPC + 30I/30R) or I/R alone. Myocardial tryptic peptides were labelled with isobaric tags to allow side-by-side relative quantitation prior to phosphopeptide enrichment. Enriched phosphopeptides were identified and quantitated by liquid chromatography coupled to tandem mass spectrometry. Preconditioned hearts recovered to 93.7 ± 6.7% versus 62.6 ± 5.6% in I/R. Analysis of significantly regulated phosphopeptides in IPC revealed additional proteins involved in both the MAPK and Apoptosis pathways than previously observed. STMN1 showed 2-fold increase in phosphorylation when IPC preceded I/R, which would otherwise show 1.2 fold increase, whilst DUSP27 showed a slight decrease with IPC as opposed to a 10 fold increase without. Our results demonstrate differential phosphorylation of a number of classically IPC associated proteins (MAPK 1/3/14, MAP2K4, AKT1). This untargeted study quantified changes in phosphorylation to additional proteins (TJP1, BAG3, MYOZ2) previously hypothesized but un observed (PAK2) and novel proteins previously unassociated with IPC (LAMTOR1, STMN1, DUSP27). By taking a global approach, the current study identified novel phosphosites on MAPK and apoptotic proteins that are differentially regulated in IPC (Bcl2l13). This phosphoproteomic study has identified the extent to which IPC regulates MAPK signaling, including classical modulators, important accessory proteins and novel targets of phosphorylation which target additional downstream networks, independent of MAPK pathways.
- © 2012 by American Heart Association, Inc.