Postconditioning the isolated perfused rat heart : the role of kinases and phosphatases

Van Vuuren, Derick (Stellenbosch : Stellenbosch University, 2008-03)

Thesis (MScMed)--Stellenbosch University, 2008.

Thesis

ENGLISH ABSTRACT: It has recently been observed that the application of multiple short cycles of reperfusion and ischaemia, at the onset of reperfusion, elicits cardioprotection against injury due to prior sustained ischaemia. This phenomenon has been termed “postconditioning” (postC) and is of special interest due to its clinical applicability. Although much work has been done to delineate the mechanism of protection, there is still controversy regarding the precise algorithm of postC, the importance of the reperfusion injury salvage kinases (RISK), as well as uncertainty about the possible role of p38 MAPK and the protein phosphatases in postC cardioprotection. The aims of this study were therefore: I. To develop and characterise a cardioprotective postC protocol in the ex vivo rat heart, using both the retrogradely perfused and working heart models. II. To characterise the profiles of PKB/Akt, ERK p42/p44 and p38 MAPK associated with the postC intervention. III. To investigate the possible role of the serine/threonine protein phosphatases type 1 and type 2A (PP1 and PP2A) in the mechanism of postC. Hearts from male Wistar rats were perfused in both the retrograde Langendorff (at a perfusion pressure of 100 cmH2O and diastolic pressure set between 1 and 10 mmHg) and working heart models (preload: 15 cmH20 and afterload: 100 cmH20). Several different postC protocols were tested for their cardioprotective effect, as analysed by infarct size (IFS; determined by triphenyltetrazolium chloride (TTC) staining) and functional recovery. Experimental parameters tested were the number of cycles (3,4 or 6), the duration of the cycles (10, 15, 20 or 30 seconds), the method of application (regional or global) and temperature during the intervention (36.5 or 37 °C). Different sustained ischaemic insults were also utilised: 35 minutes regional (RI) or 20, 25, 30 and 35 minutes global ischaemia (GI). Hearts treated with a cardioprotective postC intervention or standard reperfusion after sustained ischaemia, were freeze-clamped at 10 and 30 minutes reperfusion in both perfusion models. Tissue samples were then analyzed using Western blotting, probing for total and phosphorylated PKB/Akt, ERK p42/p44 and p38 MAPK. The contribution of PKB/Akt and ERK p42/p44 activation to cardioprotection was also investigated by administration of inhibitors (A6730 and PD098059 respectively) in the final 5 minutes of ischaemia and the first 10 minutes of reperfusion, in the presence and absence of the postC intervention. The effect of these inhibitors were analyzed in terms of IFS and kinase profiles. The possible role of the phosphatases in postC was investigated by observing the effect of cantharidin (a PP1 and PP2A inhibitor) treatment directly before sustained ischaemia (PreCanth) or in reperfusion (PostCanth), in the presence and absence of postC, on IFS and kinase profiles. A postC protocol of 6x10 seconds global reperfusion / ischaemia, at 37°C, was found to give the best and most consistent reduction in infarct size in both the Langendorff (IFS in NonPostC: 47.99±3.31% vs postC: 27.81±2.49%; p<0.0001) and working heart (IFS in NonPostC: 35.81±3.67% vs postC: 17.74±2.73%, p<0.001) models. It could however only improve functional recovery in the Langendorff model (after 30 minutes GI: rate pressure product (RPP) recovery: NonPostC = 12.27±2.63% vs postC = 24.61±2.53%, p<0.05; and after 35 minutes GI: left ventricular developed pressure (LVDP) recovery: NonPostC = 28.40±7.02% vs postC = 48.49±3.14%, p<0.05). This protection was associated with increased PKB/Akt (NonPostC: 0.88±0.26 AU (arbitrary unit) vs postC: 1.65±0.06 AU; p<0.05) and ERK p42 (NonPostC: 2.03±0.2 AU vs postC: 3.13±0.19 AU; p<0.05) phosphorylation. Inhibition of PKB/Akt activation with A6730 (2.5 μM) abrogated the infarct sparing effect of postC. Administration of cantharidin, either before of after ischaemia, in the absence of postC, conferred an infarct sparing effect (IFS in PreCanth: 15.42±1.80%, PostCanth: 21.60±2.79%; p<0.05) associated with an increase in the phosphorylation of MAPK p38 (administration before ischaemia: NonCanth: 1.52±0.26 AU vs PreCanth: 2.49±0.17 AU, p<0.05; and administration after ischaemia: NonCanth: 5.64±1.17 AU vs PostCanth: 10.69±1.29 AU, p<0.05) and ERK p42 (when administered in reperfusion; NonCanth: 2.24±0.21 AU vs PostCanth: 3.34±0.37 AU; p<0.05). Cantharidin treatment combined with the postC intervention did not elicit an additive infarct sparing effect (postC: 17.74±2.72%, PreCanth-postC: 13.30±3.46% and PostCanth-postC: 15.39±2.67%). In conclusion: a postC protocol of 6x10 seconds global ischaemia / reperfusion, at 37°C, confers the best infarct sparing effect in both the Langendorff and working rat heart models. This protection is associated with ERK p42 and PKB/Akt phosphorylation, although only PKB/Akt is necessary for cardioprotection. We could not find evidence for PP1 and PP2A involvement in postC, although inhibition of these phosphatases per se does elicit an infarct sparing effect. The latter observation suggests that phosphatase activation during ischaemia / reperfusion is potentially harmful.

AFRIKAANSE OPSOMMING: Dit is onlangs waargeneem dat toediening van meervoudige siklusse herperfusie / iskemie, met die aanvang van herperfusie, die hart teen iskemie / herperfusie beskadiging beskerm. Hierdie verskynsel, bekend as postkondisionering (postC), geniet tans baie aandag vanweë die kliniese toepaslikheid van die ingreep. Ten spyte van intensiewe navorsing om die betrokke meganisme van beskerming vas te stel, is daar steeds kontroversie oor die presiese algoritme van die ingreep, asook die betrokkenheid van die sogenaamde iskemie herperfusie oorlewings kinases (RISK). Daar bestaan ook onsekerheid oor die rol van die stres-kinase, p38 MAPK, asook die proteïen fosfatases in die meganisme van beskerming teen iskemiese besering. Hierdie studie het dus drie doelstellings gehad: I. Ontwikkeling van ‘n postC protokol wat beskerming ontlok in die rothart ex vivo, deur gebruik te maak van beide die retrograad geperfuseerde ballon model, asook die werkhart model. II. Analiese van die profiele van die kinases PKB/Akt, ERK p42/p44 en p38 MAPK tydens herperfusie van postC en kontrole (NonPostC) harte. III. Ondersoek na die moontlike rol van die serien / treonien proteïen fosfatases tipe 1 en tipe 2A (PP1 en PP2A) in die meganisme van postC beskerming. Harte van manlike Wistar rotte is geperfuseer in beide die retrograad geperfuseerde ballon (d.i. die Langendorff) model (teen ‘n konstante perfusie druk van 100 cmH20 en ‘n diastoliese druk gestel tussen 1 en 10 mmHg), asook die werkhart model (teen ‘n voorbelading van 15 cmH20 en ‘n nabelading van 100 cmH20). Verskeie moontlike postC protokolle is getoets vir hul vermoë om kardiobeskerming te ontlok, in terme van funksionele herstel en infarktgrootte (IFS), soos bepaal deur trifenieltetrazolium chloried (TTC) kleuring. Die eksperimentele veranderlikes tydens die postC protokol wat ondersoek is, sluit in: die aantal siklusse (3, 4 of 6), die duur van die siklusse (10, 15, 20 of 30 sekondes), die wyse van postC toediening (streeks of globaal) en laastens die temperatuur tydens die ingreep (36.5 of 37 °C). Daar is ook gebruik gemaak van verskillende periodes iskemie: 35 minute streeks iskemie (RI), asook 20, 25, 30 en 35 minute globale iskemie (GI). Na 10 of 30 minute herperfusie is harte wat blootgestel is aan ‘n kardiobeskermende postC ingreep of gewone standaard herperfusie na iskemie, in beide perfusie modelle, gevriesklamp. Die weefsel proteïen-inhoud is verder geanaliseer deur van die Western blot tegniek gebruik te maak vir bepaling van die totale en fosforileerde vlakke van PKB/Akt, ERK p42/p44 en p38 MAPK. Die funksionele belang van PKB/Akt en ERK p42/p44 is verder ondersoek deur die effek van ‘n geskikte inhibitor (onderskeidelik A6730 en PD098059, toegedien tydens die laaste 5 minute van iskemie en die eerste 10 minute van herperfusie), in die teenwoordigheid en afwesigheid van die postC ingreep, op infarktgrootte en kinase aktiwiteit te monitor. Die moontlike rol van proteïen fosfatases in postC is ondersoek deur die effek van cantharidin (‘n PP1 en PP2A inhibitor) op infarktgrootte en kinase profiele te ondersoek. Cantharidin is óf onmiddelik voor iskemie óf tydens herperfusie toegedien, in die aan – en afwesigheid van die postC ingreep. Daar is bevind dat ‘n postC protokol van 6x10 sekondes globale iskemie / herperfusie, teen 37°C, die mees effektiewe en konstante verlaging in infarktgrootte teweeg gebring het in beide die ballon model (IFS in NonPostC: 47.99±3.31% vs postC: 27.81±2.49%; p<0.0001), asook die werkhart (IFS in NonPostC: 35.81±3.67% vs postC: 17.74±2.73%, p<0.001). Funksionele herstel kon egter slegs ontlok word in die ballon model (na 30 minute GI: tempo druk produk (RPP) herstel: NonPostC = 12.27±2.63% vs postC = 24.61±2.53%, p<0.05; en na 35 minute GI: linker ventrikulêre ontwikkelde druk (LVDP) herstel: NonPostC = 28.40±7.02% vs postC = 48.49±3.14%, p<0.05). Die infarkt-besparende effek van postC was geassosieer met ‘n toename in die fosforilasie van beide PKB/Akt (NonPostC: 0.88±0.26 AU (arbitrêre eenhede) vs postC: 1.65±0.06 AU; p<0.05) en ERK p42 (NonPostC: 2.03±0.2 AU vs postC: 3.13±0.19 AU; p<0.05). Inhibisie van PKB/Akt met A6730 (2.5 μM) het die infarkt-besparende effek van postC opgehef. Inhibisie van PP1 en PP2A opsigself, deur toediening van cantharidin óf voor óf na iskemie (in die afwesigheid van postC), het ‘n infarkt-besparende effek ontlok (IFS in PreCanth: 15.42±1.80%, PostCanth: 21.60±2.79%; p<0.05). Hierdie kardiobeskerming was geassosieer met ‘n toename in die fosforilasie van beide p38 MAPK (met toediening voor iskemie: NonCanth: 1.52±0.26 AU vs PreCanth: 2.49±0.17 AU, p<0.05; en toediening na iskemie: NonCanth: 5.64±1.17 AU vs PostCanth: 10.69±1.29 AU, p<0.05), asook ERK p42, indien cantharidin toegedien is tydens herperfusie (NonCanth: 2.24±0.21 AU vs PostCanth: 3.34±0.37 AU; p<0.05). Kombinasie van cantharidin behandeling met postC toediening kon egter nie ‘n kumulatiewe infarkt-besparende effek uitlok nie (postC: 17.74±2.72%, PreCanth-postC: 13.30±3.46% en PostCanth-postC: 15.39±2.67%). In samevatting: ‘n PostC protokol van 6x10 sekondes globale iskemie / herperfusie, teen 37°C, ontlok die mees effektiewe infarkt-besparende effek in beide die ballon, sowel as die werkhart modelle. Alhoewel hierdie beskerming geassosieer is met ‘n toename in die fosforilasie van beide PKB/Akt en ERK p42/p44 tydens herperfusie, is dit slegs PKB/Akt wat van funksionele belang is in die meganisme van kardiobeskerming. Ons kon geen bewyse vind vir die betrokkenheid van PP1 en PP2A in postC beskerming nie, alhoewel inhibisie van hierdie fosfatases opsigself infarkt-besparend is. Laasgenoemde waarneming toon dat fosfatase aktivering tydens iskemie / herperfusie skadelike gevolge mag hê.

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