The role of MKP-1 in autophagy, apoptosis and necrosis during ischaemia/reperfusion injury in the heart

[摘要] ENGLISH ABSTRACT: Ischaemic heart disease is a leading cause of death worldwide and is alsolargely contributing to deaths in Africa. Better treatment or even prevention ofischaemia/reperfusion injury in the heart, necessitates a better understandingof the molecular pathways and mechanisms of cell death. Three types of celldeath can occur in the diseased myocardium. Type I, better known asapoptotic cell death, is characterised by cell shrinkage and chromatincondensation, type II, known as autophagic cell death, is characterised byintracellular accumulation of double membranes vacuoles and type III,necrotic cell death, is characterised by cellular swelling and loss ofmembrane integrity. Many signaling pathways are activated duringischaemia/reperfusion injury which include the mitogen activated proteinkinases (MAPKs), such as extracellular signal-regulated protein kinase(ERK), c-Jun NH2-terminal protein kinase (JNK) and p38 MAPK. Thesekinases are dephosphorylated by appropriate phosphatases. MAPKphosphatase-1 (MKP-1), a dual specificity phosphatase, inactivates theMAPKs by dephosphorylating specific Thr/Tyr residues. Upregulation ofMKP-1 during ischaemia/reperfusion injury has been shown to becardioprotective, however no knowledge regarding a role of MKP-1 inautophagy exists. Therefore the aim of this study is to investigate the role ofMKP-1 in autophagy, apoptosis and necrosis during simulatedischaemia/reperfusion injury in the heart.METHOD: H9C2 cells (rat cardiomyocytes) were cultured under standard conditions.Upon reaching 75-80% confluency, cells were treated for 30 min duringnormoxic conditions with dexamethasone, to induce MKP-1 expression, orsanguinarine, to inhibit MKP-1 induction. Thereafter, they were exposed to 3hrs simulated ischaemia (induced by an ischaemic buffer and 5% CO2/1%O2) in the presence of the above mentioned treatments. Cells were thenallowed to reperfuse for 30 min in the presence of dexamethasone orsanguinarine. Samples were analysed after simulated ischaemia and afterreperfusion. Cell viability was measured by MTT assay. Propidium iodide andHoechst staining were used to assess morphological markers of apoptosisand necrosis. LDH release during reperfusion was assessed as indicator ofnecrotic cell death. LysoTracker®Red was used to visualise the autophagicflux occurring during ischaemia/reperfusion in the cell. Flow cytometry wasused to quantify cells stained with acridine orange as indicator for autophagy.Autophagic and apoptotic protein markers as well as MAPK and MKP-1activity were analysed by Western Blotting.RESULTS: Our results indicate a clear relationship between MKP-1 induction,autophagy and cell survival during simulated ischaemia/reperfusion (SI/R).MKP-1 inhibition during SI/R resulted in decreased autophagy activityaccompanied by significant apoptotic and necrotic cell death. Increased MKP-1 induction, on the other hand, during SI/R resulted in increased levelsof autophagy activity and subsequent attenuation of apoptotic and necroticcell death. p38 MAPK phosphorylation was significantly higher while MKP-1was inhibited and significantly lower while MKP-1 was induced. This stronglyindicates that upregulation of MKP-1, known to attenuateischaemia/reperfusion injury, has an important role in cell survival duringischaemia/reperfusion injury in the heart, through its involvement in theregulation of autophagic activity as a stress response against apoptotic ornecrotic cell death.

[发布机构] Stellenbosch University