The role of MKP-1 in autophagy, apoptosis and necrosis during ischaemia/reperfusion injury in the heart
Date
2010-12
Authors
Vermeulen, Michelle
Journal Title
Journal ISSN
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Abstract
ENGLISH ABSTRACT: Ischaemic heart disease is a leading cause of death worldwide and is also
largely contributing to deaths in Africa. Better treatment or even prevention of
ischaemia/reperfusion injury in the heart, necessitates a better understanding
of the molecular pathways and mechanisms of cell death. Three types of cell
death can occur in the diseased myocardium. Type I, better known as
apoptotic cell death, is characterised by cell shrinkage and chromatin
condensation, type II, known as autophagic cell death, is characterised by
intracellular accumulation of double membranes vacuoles and type III,
necrotic cell death, is characterised by cellular swelling and loss of
membrane integrity. Many signaling pathways are activated during
ischaemia/reperfusion injury which include the mitogen activated protein
kinases (MAPKs), such as extracellular signal-regulated protein kinase
(ERK), c-Jun NH2-terminal protein kinase (JNK) and p38 MAPK. These
kinases are dephosphorylated by appropriate phosphatases. MAPK
phosphatase-1 (MKP-1), a dual specificity phosphatase, inactivates the
MAPKs by dephosphorylating specific Thr/Tyr residues. Upregulation of
MKP-1 during ischaemia/reperfusion injury has been shown to be
cardioprotective, however no knowledge regarding a role of MKP-1 in
autophagy exists. Therefore the aim of this study is to investigate the role of
MKP-1 in autophagy, apoptosis and necrosis during simulated
ischaemia/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 during
normoxic conditions with dexamethasone, to induce MKP-1 expression, or
sanguinarine, to inhibit MKP-1 induction. Thereafter, they were exposed to 3
hrs simulated ischaemia (induced by an ischaemic buffer and 5% CO2/1%
O2) in the presence of the above mentioned treatments. Cells were then
allowed to reperfuse for 30 min in the presence of dexamethasone or
sanguinarine. Samples were analysed after simulated ischaemia and after
reperfusion. Cell viability was measured by MTT assay. Propidium iodide and
Hoechst staining were used to assess morphological markers of apoptosis
and necrosis. LDH release during reperfusion was assessed as indicator of
necrotic cell death. LysoTracker®Red was used to visualise the autophagic
flux occurring during ischaemia/reperfusion in the cell. Flow cytometry was
used to quantify cells stained with acridine orange as indicator for autophagy.
Autophagic and apoptotic protein markers as well as MAPK and MKP-1
activity 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 activity
accompanied by significant apoptotic and necrotic cell death. Increased MKP-1 induction, on the other hand, during SI/R resulted in increased levels
of autophagy activity and subsequent attenuation of apoptotic and necrotic
cell death. p38 MAPK phosphorylation was significantly higher while MKP-1
was inhibited and significantly lower while MKP-1 was induced. This strongly
indicates that upregulation of MKP-1, known to attenuate
ischaemia/reperfusion injury, has an important role in cell survival during
ischaemia/reperfusion injury in the heart, through its involvement in the
regulation of autophagic activity as a stress response against apoptotic or
necrotic cell death.
AFRIKAANSE OPSOMMING: Iskemiese hartsiekte is een van die grootste oorsake van sterftes wêreldwyd en dra ook beduidend by tot sterftes in Afrika. Om iskemiese hartsiektes te behandel of selfs te voorkom, is 'n goeie begrip van die molekulêre paaie wat betrokke is tydens iskemie/herperfusie, noodsaaklik. Drie tipes seldood kom tydens patologiese toestande in die hart voor. Tipe I, ook bekend as apoptotiese seldood, word gekenmerk deur selkrimping en kromatien kondensasie, tipe II, ook bekend as autofagiese seldood word gekenmerk deur intrasellulêre opeenhoping van dubbelmembraan vakuole en tipe III, bekend as nekrotiese seldood, word deur sellulêre swelling en verlies van membraan integriteit gekenmerk. Iskemie/herperfusie lei tot die aktivering van seintransduksiepaaie wat die MAPKs, soos p38, ERK en JNK insluit. Hierdie kinases word deur die gepaste fosfatases gedefosforileer. MKP-1, 'n dubbele spesifieke fosfatase, deaktiveer MAPKs deur hul Thr/Tyr eenhede te defosforileer. Alhoewel daar al voorheen getoon is dat verhoogte MKP-1 ‘n beskermende funksie in die hart tydens iskemie/herperfusie het, is daar nog geen bewyse vir ‘n rol van MKP-1 tydens autofagie nie. Die doel van hierdie studie is dus om die rol van MKP-1 in autofagie, apoptose en nekrose te ondersoek tydens gesimuleerde iskemie/herperfusie in die hart. METODE: H9C2 selle (rot ventrikulêre hartselle) is onder standaard toestande gekweek. Wanneer die selle 75-80% konfluensie bereik het, is dit behandel met dexamethasone of sanguinarine onder standaard toestande vir 30 min. Daarna is selle blootgestel aan 3 ure iskemie, in die teenwoordigheid van dexamethasone of sanguinarine. Selle is dan toegelaat om vir 30 min te herperfuseer, weer in die teenwoordigheid van dexamethasone of sanguinarine. Monsters is na iskemie en herperfusie geneem vir analise. Selvatbaarheid is gekwantifiseer deur ‘n MTT bepaling. Morfologiese merkers van seldood is bepaal met behulp van propidium iodide en Hoechst kleuringsmetodes. Laktaatdehidrogenase (LDH) vrystelling tydens herperfusie is as merker van nekrose gebruik. Autofagie is gevisualiseer deur gebruik te maak van LysoTracker®Red kleuring tydens iskemie en herperfusie. Akridienoranje is gebruik om suur kompartemente te kleur. Vloeisitometrie is as kwantifiseringstegniek vir autofagie gebruik. Western Blotting is gebruik om uitdrukking van merkerproteïene van autofagie en apoptose sowel as MAPK en MKP-1 aktiwiteit tydens iskemie/reperfisie te bepaal. RESULTATE: Ons resultate toon ‘n verband tussen MKP-1 induksie, autofagie en seloorlewing gedurende gesimuleerde iskemie/herperfusie (SI/R) aan. MKP- 1 inhibisie gedurende SI/R het tot ‘n afname in autofagie gelei tesame met ‘n beduidende toename in apoptotiese en nekrotiese seldood. Verhoogde MKP-1 induksie gedurende SI/R, daarteenoor, het autofagiese aktiwiteit verhoog, gepaardgaande met ‘n verlaging in apoptose en nekrose. p38 MAPK fosforilasie was beduidend hoër tydens MKP-1 inhibisie en laer met MKP-1 induksie. Hierdie resultate toon dat MKP-1 ‘n belangrike rol in seloorlewing speel tydens iskemie/herperfusiesskade in die hart, deur sy deelname in die regulering van autofagiese aktiwiteit as ‘n stres reaksie teen apoptotiese en nekrotiese seldood.
AFRIKAANSE OPSOMMING: Iskemiese hartsiekte is een van die grootste oorsake van sterftes wêreldwyd en dra ook beduidend by tot sterftes in Afrika. Om iskemiese hartsiektes te behandel of selfs te voorkom, is 'n goeie begrip van die molekulêre paaie wat betrokke is tydens iskemie/herperfusie, noodsaaklik. Drie tipes seldood kom tydens patologiese toestande in die hart voor. Tipe I, ook bekend as apoptotiese seldood, word gekenmerk deur selkrimping en kromatien kondensasie, tipe II, ook bekend as autofagiese seldood word gekenmerk deur intrasellulêre opeenhoping van dubbelmembraan vakuole en tipe III, bekend as nekrotiese seldood, word deur sellulêre swelling en verlies van membraan integriteit gekenmerk. Iskemie/herperfusie lei tot die aktivering van seintransduksiepaaie wat die MAPKs, soos p38, ERK en JNK insluit. Hierdie kinases word deur die gepaste fosfatases gedefosforileer. MKP-1, 'n dubbele spesifieke fosfatase, deaktiveer MAPKs deur hul Thr/Tyr eenhede te defosforileer. Alhoewel daar al voorheen getoon is dat verhoogte MKP-1 ‘n beskermende funksie in die hart tydens iskemie/herperfusie het, is daar nog geen bewyse vir ‘n rol van MKP-1 tydens autofagie nie. Die doel van hierdie studie is dus om die rol van MKP-1 in autofagie, apoptose en nekrose te ondersoek tydens gesimuleerde iskemie/herperfusie in die hart. METODE: H9C2 selle (rot ventrikulêre hartselle) is onder standaard toestande gekweek. Wanneer die selle 75-80% konfluensie bereik het, is dit behandel met dexamethasone of sanguinarine onder standaard toestande vir 30 min. Daarna is selle blootgestel aan 3 ure iskemie, in die teenwoordigheid van dexamethasone of sanguinarine. Selle is dan toegelaat om vir 30 min te herperfuseer, weer in die teenwoordigheid van dexamethasone of sanguinarine. Monsters is na iskemie en herperfusie geneem vir analise. Selvatbaarheid is gekwantifiseer deur ‘n MTT bepaling. Morfologiese merkers van seldood is bepaal met behulp van propidium iodide en Hoechst kleuringsmetodes. Laktaatdehidrogenase (LDH) vrystelling tydens herperfusie is as merker van nekrose gebruik. Autofagie is gevisualiseer deur gebruik te maak van LysoTracker®Red kleuring tydens iskemie en herperfusie. Akridienoranje is gebruik om suur kompartemente te kleur. Vloeisitometrie is as kwantifiseringstegniek vir autofagie gebruik. Western Blotting is gebruik om uitdrukking van merkerproteïene van autofagie en apoptose sowel as MAPK en MKP-1 aktiwiteit tydens iskemie/reperfisie te bepaal. RESULTATE: Ons resultate toon ‘n verband tussen MKP-1 induksie, autofagie en seloorlewing gedurende gesimuleerde iskemie/herperfusie (SI/R) aan. MKP- 1 inhibisie gedurende SI/R het tot ‘n afname in autofagie gelei tesame met ‘n beduidende toename in apoptotiese en nekrotiese seldood. Verhoogde MKP-1 induksie gedurende SI/R, daarteenoor, het autofagiese aktiwiteit verhoog, gepaardgaande met ‘n verlaging in apoptose en nekrose. p38 MAPK fosforilasie was beduidend hoër tydens MKP-1 inhibisie en laer met MKP-1 induksie. Hierdie resultate toon dat MKP-1 ‘n belangrike rol in seloorlewing speel tydens iskemie/herperfusiesskade in die hart, deur sy deelname in die regulering van autofagiese aktiwiteit as ‘n stres reaksie teen apoptotiese en nekrotiese seldood.
Description
Thesis MSc (Physiological Sciences))--University of Stellenbosch, 2010.
Keywords
Ischaemia, Autophagy, Apoptosis, Necrosis, MKP-1, Reperfusion injury, Dissertations -- Physiology (Human and animal), Theses -- Physiology (Human and animal), Dual specificity phosphatase 1, Map kinase phosphatase 1, Heart -- Diseases