The role of Protein Phosphatase 2A (PP2A) in myocardial ischaemia/reperfusion injury

[摘要] ENGLISH ABSTRACT: Ischaemic heart disease is a major contributor to global morbidity and mortality rates. Manoeuvressuch as ischaemic preconditioning confer cardioprotection against ischaemia/reperfusion (I/R)injury by activating several intracellular signalling pathways. These pathways have been definedsolely in terms of the kinases involved, despite the realization in recent years that proteinphosphatase activity also contributes significantly to the attributes of the propagated signal. Proteinphosphatase 2A (PP2A) is a heteromultimeric enzyme involved in an array of phosphatasereactions. We hypothesized that PP2A is an important participant in the myocardial response to I/Rby regulating intracellular signalling.This project aimed to (i) characterize PP2A during myocardial I/R; (ii) determine the importance ofits contribution to the cellular response to I/R; and (iii) investigate its role in the signalling pathwaysmediated by PKB/Akt, GSK-3β, ERK p42/p44 and p38 MAPK.Two models were used to characterize PP2A during I/R: (i) H9c2 cells exposed to simulatedischaemia (SI) buffer in conjunction with hypoxia (0.5% O2) for a maximum of 2 hours, followed byreoxygenation in standard growth medium for up to 30 minutes; and (ii) isolated working rat heartsexposed to a maximum of 20 minutes global ischaemia and 10 minutes reperfusion. In bothmodels samples were collected at several time points during I/R for Western blotting analysis.PP2A-C (the catalytic subunit) accumulated in the nucleus during early ischaemia, but laterredistributed to the cytosol. At the end of ischaemia there was an elevation of PP2A-C relative toPP2A-A in the unfractionated whole cell preparation concomitant with an increase in the inhibitoryphosphorylation of PP2A-C.The impact of PP2A activity was evaluated by either inhibiting PP2A using okadaic acid (OA, 10nM) or activating it by administering FTY720 (1 μM) in an isolated working rat heart model exposedto either 35 minutes of regional ischaemia (RI) with infarct size (IFS) as primary end-point, or 20minutes global ischaemia (GI) with functional recovery as end-point. The results showed that thepre-ischaemic administration of OA or FTY720 reduced or exacerbated IFS respectively, indicatingthat PP2A activation during I/R favours cell death.OA and FTY720 were also employed to assess the contribution of PP2A to intracellular signallingin an isolated working rat heart exposed to I/R. Samples were collected at several timepoints andanalyzed using Western Blotting. Pre-ischaemic administration of OA enhanced thephosphorylation of PKB/Akt, ERK p42/p44 and GSK-3β at the onset of reperfusion, while FTY720given before ischaemia reduced the phosphorylation of GSK-3β, p38 MAPK and PKB/Akt at theend of ischaemia and onset of reperfusion.In summary, PP2A is part of an early nuclear-based response to ischaemia, while long-termischaemia induces an increase in PP2A-C. A portion of this PP2A-C is stored in an inactive form,while an active portion acts as a regulator of the pro-survival signalling components PKB/Akt, GSK-3β and ERK p42/p44 at the end of ischaemia and the onset of reperfusion. PP2A is therefore animportant component of the myocardial response to I/R by regulating pro-survival signalling.

[发布机构] Stellenbosch University