Protein phosphorylation in Ischaemia and reperfusion of the heart : a focus on protein phosphatase 2A

White, Charlize (2020-03)

Thesis (PhD)--Stellenbosch University, 2020.

Thesis

ENGLISH ABSTRACT: Understanding the mechanism and signalling pathways involved in myocardial ischaemia/reperfusion (I/R) injury is a prerequisite for elucidating any novel therapeutic intervention to reduce I/R injury. Historically, protein kinases have been the focus of research on I/R injury while information regarding phosphatases in I/R injury is lacking. Clarifying the role of phosphatases in the cellular response to I/R injury is critical to enhance existing cardioprotective interventions or identify new therapeutic targets. This study aimed to investigate the effects of protein phosphatase 2A (PP2A, a major phosphatase in the heart) inhibition on the outcomes of I/R and to characterize the phosphoproteome of myocardial tissue exposed to these interventions. For this study, tissue was generated using the isolated working mouse heart model. Administration of a PP2A inhibitor, okadaic acid (OA; 50 nM), prior to 20 minutes global ischaemia (GI) reduced infarct size (28 ± 19%) when compared to control (56 ± 23%; n=9; p < 0.0106), but did not exert a major effect on functional recovery. Phosphoproteomic analysis conducted on hearts exposed to OA prior to 20 minutes GI followed by 10 minutes reperfusion revealed that 898 proteins and 184 phosphorylated proteins were influenced by PP2A inhibition. Pathway analysis indicated that the major pathways affected by PP2A inhibition were mitochondrial function and oxidative phosphorylation, epithelial adherens signalling and remodelling, and cytoskeletal signalling. Two proteins from the data set, striatin and heat shock protein (HSP) 90α, were selected for confirmation and further investigation. Mitochondrial respiration was also investigated. In line with the phosphoproteomic results, OA administration increased the amount of phosphorylated HSP90α during reperfusion (1.83 ± 0.83 Arbitrary Units (AU) vs 0.69 ± 0.09 AU; n = 2-4; p = 0.0480) and increased the amount of striatin during reperfusion (1.93 ± 0.84 AU vs 0.80 ± 0.06 AU; n = 2-4; p = 0.0436). Striatin, but not HSP90α coimmunoprecipitated with PP2A, indicating that the increased phosphorylation of HSP90α was an indirect effect of PP2A inhibition. As striatin recruits regulatory proteins into large signalling complexes that mostly include PP2A, co-immunoprecipitation may indicate the presence of these complexes in the heart. OA was also administered directly to isolated mitochondria, where oxidative phosphorylation analysis was performed polarographically (using a Clark-type electrode). No differences in oxidative phosphorylation or respiratory control ratio were observed. Mitochondria were separated into the outer mitochondrial membrane (OMM) and mitoplast (inner mitochondrial membrane and matrix) using digitonin. Western blot analysis indicated that that catalytic and scaffolding subunits of PP2A are probably localized to the OMM, while striatin is located within the mitoplast, a novel finding. Finally, following an incidental observation, the inhibitory effects of sodium orthovanadate (SOV) on PP2A was investigated. This study is the first to demonstrate that SOV is a weak, competitive inhibitor of PP2A (IC50 ≈ 2.2 mM). This study is, to our knowledge, the first to use a phosphoproteomic approach to investigate the effects of phosphatase inhibition during myocardial I/R. Our data indicate that PP2A is associated with numerous proteins and processes during I/R, including oxidative phosphorylation and cytoskeletal dynamics.

AFRIKAANSE OPSOMMING: Begrip van die meganisme en seintransduksie-paaie wat betrokke is in miokardiale iskemie/herperfusie (I/H) besering is ‘n voorvereiste daarvoor om nuwe terapeutiese ingrepe te ontwikkel om I/H besering te verminder. Histories was meeste van die navorsings-fokus op die rol van kinases in I/H besering, terwyl inligting aangaande fosfatases in hierdie konteks steeds beperk is. Duideliker insig in die rol van fosfatases in die sellulêre reaksie op I/H besering is krities om bestaande kardiobeskermende intervensies te versterk of nuwe ingrepe te identifiseer. Hierdie studie het ten doel gehad om die effekte van proteïen fosfatase 2A (PP2A, ‘n beduidende fosfatase in die hart) inhibisie op die gevolge van I/H te ondersoek, asook om die fosfoproteoom van miokardiale weefsel wat aan hierdie intervensies blootgestel is te ondersoek. Die geïsoleerde werkende muishart model is gebruik om weefsel voor te berei. Toediening van ‘n inhibitor van PP2A, okadaïensuur (OA; 50 nM) onmiddelik voor 20 minute globale iskemie (GI) het infarktgrootte verminder in vergelyking met kontrole (28 ± 19% vs 56 ± 23%; n=9; p < 0.0106), maar het nie ‘n beduidende effek op funksionele herstel gehad nie. Fosfoproteomiese analiese het 898 proteïene en 184 gefosforileerde proteïene geïdentifiseer wat beïnvloed is deur die toediening van OA direk voor 20 minute GI, gevolg deur 10 minute herperfusie. Analise van sellulêre paaie het getoon dat die belangrikste paaie wat beïnvloed word deur PP2A inhibisie betrekking het op mitochondriale funksie en oksidatiewe fosforilasie, epiteel adherens seintransduksie en hermodellering, en sitoskelet seintransduksie. Twee proteïene vanuit die datastel, striatien en hitteskokproteïen (HSP) 90α, is gekies vir bevestiging en verdere ondersoek. Mitochondriale respirasie is ook ondersoek. In lyn met die fosfoproteomika resultate het toediening van OA die hoeveelheid gefosforileerde HSP90α tydens herperfusie verhoog (1.83 ± 0.83 Arbitrêre Eenhede (AE) vs 0.69 ± 0.09 AE; n = 2 4; p = 0.0480), asook die totale hoeveelheid striatien tydens herperfusie verhoog (1.93 ± 0.84 AE vs 0.80 ± 0.06 AE; n = 2-4; p = 0.0436). Striatien, maar nie HSP90α nie, is geko-immunopresipiteer met PP2A wat daarop dui dat die toename in fosforilasie van HSP90α die gevolg is van ‘n indirekte effek van PP2A inhibisie. Striatien werf regulatoriese proteïene na groot seintransduksie komplekse wat meestal PP2A bevat. Ko-immunopresipitasie van striatien dui dus op die moontlike teenwoordigheid van hierdie komplekse in die hart. OA is direk toegedien aan geïsoleerde mitochondria waarop polarografiese analiese van oksidatiewe fosforilasie, met behulp van ‘n Clark-tipe elektrode, gedoen is. Geen verskille in oksidatiewe fosforilasie of respiratoriese beheer-ratio is waargeneem nie. Mitochondria is verder verdeel in die mitochondriale buitemembraan (MBM) en mitoplast (die mitochondriale binnemembraan en matriks) deur van digitonien gebruik te maak. Western-klad analiese het getoon dat die katalitiese en steier-subeenhede van PP2A waarskynlik in die MBM voorkom, terwyl striatien in ‘n nuutvinding in die mitoplast geïdentifiseer is. Laastens, na aanleiding van ‘n toevallige waarneming, is die inhibitoriese effek van natriumorthovanadaat (NOV) op PP2A ondersoek. Hierdie is die eerste studie wat demonstreer dat NOV ‘n swak, kompeterende inhibitor van PP2A is (IC50 ≈ 2.2 mM). Na ons kennis, is hierdie studie die eerste om van ‘n fosfoproteomiese benadering gebruik te maak om die effekte van fosfatase inhibisie tydens miokardiale I/H te ondersoek. Ons data dui daarop dat PP2A verwant is aan talryke proteïene en prosesse tydens I/H, insluitend oksidatiewe fosforilasie en sitoskelet dinamika.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/107785
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