Effect of different substrates on functional performance and kinase activation during reperfusion after ischaemia in hearts from obese insulin resistant rats

Fan, WenJun (2017-03)

Thesis (PhD)--Stellenbosch University, 2017

Thesis

ENGLISH ABSTRACT : Obesity is an important risk factor for the development of insulin resistance, the metabolic syndrome and diabetes and has also been implicated as one of the major risk factors for coronary heart disease. Ischaemic heart disease impacts on both cardiac metabolism and function. During early reperfusion after ischaemia, several protein kinases are specifically activated, including PI3K/PKB, MAPKs (ERK, JNK and p38 MAPK), and the tyrosine kinases. Activation of PKB and ERK, the so-called reperfusion injury salvage kinase (RISK) pathway, is associated with a reduction in infarct size and/or improvement in functional recovery. PKB is an enzyme central also to insulin signalling and glucose uptake. Activation of the JNK signaling pathway has been suggested to be a prerequisite for PKB activation; however, its role in ischaemia/reperfusion (I/R) injury remains controversial. We hypothesize that obesity-induced insulin resistance will affect infarct size, functional recovery and interactions between PKB, JNK, ERK, p38MAPK and PTEN activation during reperfusion after exposure to ischaemia. The aim of the study was therefore to assess the effects of hyperphagia-induced obesity and insulin resistance in rats on the response of the heart to I/R injury, with particular attention to the intracellular signalling pathways during early reperfusion. To further elucidate the role of JNK, we used SP600125, a specific inhibitor of JNK. Methods: Insulin resistance was induced by feeding rats a high caloric diet for 16 weeks (DIO). Hearts from DIO and age-matched controls (C) were perfused in the working mode (preload 15cm H2O; afterload 100cm H2O) and subjected to (i) 15 min global ischaemia followed by different reperfusion times for evaluation of functional recovery and freeze-clamping of tissues for Western blot or (ii) 35 min regional ischaemia followed by 2 hours reperfusion for infarct size determination (IS), using tetrazolium staining. Substrates were glucose (G) (10mM), glucose (10mM) plus BSA (3%) (G+B), and glucose (10mM) plus fatty acid (1.2mM palmitate / 3% BSA) (G+FA). The JNK inhibitor, SP600125, was administered either before ischaemia or during reperfusion after ischaemia. Infarct size, functional recovery as well as expression and activation of PKB, ERK, JNK, p38MAPK and PTEN were used as endpoints. Results: (I) In the presence of glucose alone as substrate, the hearts from DIO rats exhibited an improved tolerance to ischaemia/reperfusion (I/R) injury as reflected by an increase in functional recovery (after exposure to 15 min global ischaemia) as well as a reduction in infarct size (after 35 min regional ischaemia) compared with the age-matched controls. This was associated with early activation of PKB and JNKp54/p46 at 10 min reperfusion, with down regulation of activation of these kinases after 30 min reperfusion. (II) Contrary to expectations, the combination of a high concentration of fatty acids and glucose as substrates (G+FA) afforded significantly more protection against I/R injury in hearts from both DIO and control rats, when compared with the respective groups perfused with glucose alone as substrate. This improved protection in both groups was associated with increased activation of the PKB pathway. Interestingly, perfusion with glucose and a high concentration of fatty acid maintained PKB activation throughout the reperfusion phase, in contrast to the transient activation seen with glucose alone as substrate. (III) SP600125 (10 uM), administrated either before ischaemia or during early reperfusion after ischaemia, almost completely inhibited the JNK pathway and exacerbated myocardial I/R injury, particularly in hearts from DIO rats. Conclusion: Our study demonstrates, in contrast to several other studies, that dietary-induced obesity and high perfusate fatty acid concentrations, increase the tolerance of the ex vivo myocardium to I/R injury. It was also found that, contrary to expectations, a high concentration of circulating fatty acid was not detrimental to hearts of normal rats during I/R, indicating the beneficial actions of fatty acids on the outcome of I/R injury. This protection was shown to be associated with activation of PKB and JNK during early reperfusion. Administration of the selective JNK inhibitor, SP600125, before or after myocardial ischaemia indicates that JNK and its downstream signalling pathways are critical in mediating protection against I/R in our study. SP-induced effects were also associated with lower activation of PKB. Our results suggest that the cross-talk between the JNK and PKB pathways in the post-ischaemic myocardium may be a major contributing factor to the outcome of I/R injury The data presented here, although seemingly dichotomous, actually solidify the hypothesis that JNK signalling specifically and simultaneously modulates pro- and antiapoptotic effector mechanisms within cardiomyocytes. They also reflect an extraordinary complexity of the heart‘s metabolic, functional, and structural changes in obesity. In addition, the results obtained showed that moderate hyperphagia-induced obesity does not have a harmful effect on the ischaemic-reperfused heart and in fact, reduced the sensitivity of the heart to I/R damage. This was further substantiated by the beneficial effects of fatty acids in the perfusate. Taken together, our results are potentially of clinical significance, and confirm the importance of events during early reperfusion as possible therapeutic targets.

AFRIKAANSE OPSOMMING : Vetsug is 'n belangrike risikofaktor in die ontwikkeling van insulienweerstandigheid, die metaboliese sindroom en diabetes en word beskou as een van die hoof risikofaktore van koronêre hartvatsiektes. Iskemiese hartsiekte op sy beurt, affekteer beide miokardiale metabolisme en funksie. In die vroeë fase van herperfusie na miokardiale iskemie word verskeie proteïen kinases soos byvoorbeeld PI3K/PKB, die MAPKs (ERK, JNK en p38 MAPK), asook tirosien kinases, geaktiveer. Aktivering van PKB en ERK, die sogenaamde herperfusie-besering herwinningspad (RISK), word met ‗n vermindering van infarktgrootte en/of ‗n verbeterde funksionele herstel, geassosieer. PKB staan ook sentraal aan insulienseintransduksie en glukose opname. Aktivering van die JNK seintransduksiepad is voorgestel om ‗n voorvereiste vir die aktivering van PKB te wees maar die rol van hierdie pad in iskemie/herperfusie (I/H) besering, is tans kontroversieël. Ons hipotese is dat vetsug-geïnduseerde insulienweerstandigheid miokardiale infarktgrootte, funksionele herstel asook die interaksie tussen PKB, JNK, ERK, p38MAPK en PTEN aktivering gedurende herperfusie na iskemie, sal beïnvloed. Die doel van hierdie studie was dus om die effek van hiperfagie-geïnduseerde vetsug en insulienweerstandigheid in rotte op die respons van die hart op I/H besering te bepaal met besondere aandag aan die intrasellulêre seintransduksiepaaie tydens vroeë herperfusie. Om die rol van JNK uit te lig en te evalueer, is van ‗n spesifieke inhibitor van JNK, SP600125, gebruik gemaak. Metodes: Insulienweerstandigheid is ontlok deur rotte vir 16 weke ‗n hoë-kalorie dieet te voer (DIO). Harte van die DIO en ouderdomsgepaarde diere (C) is volgens die werkhartmetode geperfuseer (voorbelading 15cm H2O; nabelading 100cm H2O) en blootgestel aan (i) 15min globale iskemie gevolg deur verskillende herperfusietye vir die evaluering van funksionele herstel asook vriesklamping van weefsel vir Western klad analises of (ii) 35min streeksiskemie gevolg deur 2 uur herperfusie vir die bepaling van infarktgrootte (IS) met behulp van tetrazolium kleuring. Substrate gebruik: glukose (G) (10mM), glukose (10mM) plus BSA (3%) (G+B) en glukose (10mM) plus vetsure (1.2mM palmitaat/3% BSA) (G+FA). Die JNK inhibitor, SP600125, is of voor iskemie of gedurende herperfusie na iskemie toegedien. Infarktgrootte, funksionele herstel asook uitdrukking en aktivering van PKB, ERK, JNK, p38MAPK and PTEN is as eindpunte gebruik. Resultate: (I) In die teenwoordigheid van slegs glukose as substraat kon die harte van DIO rotte I/H besering beter as die ouderdomsgepaarde kontroles weerstaan, aangedui deur ‗n verbeterde funksionele herstel (na blootstelling aan 15min globale isgemie) sowel as kleiner infarktgrootte (na 35min streeksiskemie). Dit is gekenmerk deur vroeë aktivering van PKB en JNKp54/p46 na 10min herperfusie asook afregulering van die aktivering van hierdie kinases na 30min herperfusie. (II) In teenstelling met wat verwag is, het die kombinasie van ‗n hoë konsentrasie versure met glukose as substrate (G+FA) beduidende verhoogde beskerming teen I/H besering verleen in harte van beide DIO en kontrole rotte, in vergelyking met die ooreenstemmende groepe wat slegs met glukose as substraat geperfuseer is. In beide groepe is hierdie verbeterde beskerming met verhoogde aktivering van die PKB pad geassosieer. Dit is ook interessant dat perfusie met glukose en ‗n hoë konsentrasie vetsure, die aktivering van PKB tydens die hele herperfusiefase kon onderhou, in teenstelling met die verbygaande aktivering waargeneem met glukose alleen as substraat. (III) Toediening van SP600125 (10uM) voor iskemie of gedurende die vroeë fase van herperfusie na iskemie, kon die JNK pad feitlik heeltemal onderdruk en het I/H besering, veral in die harte van DIO rotte, vererger. Gevolgtrekking: Hierdie studie, in teenstelling met verskeie ander studies, toon aan dat dieet-geïnduseerde vetsug asook hoë konsentrasies vetsure in die perfusaat, die weerstandigheid van die ex vivo miokardium teen I/H besering, kan verhoog. Dit is ook gevind dat, in teenstelling met wat verwag is, ‗n hoë sirkulerende vetsuurkonsentrasie nie nadelig vir harte van normale rotte, blootgestel aan I/H, is nie, inderdaad ‗n voordelige effek van vetsure op die uitkoms van I/H besering aantoon. Hierdie beskerming het gepaard gegaan met die aktivering van beide PKB en JNK gedurende vroeë herperfusie. Toediening van die selektiewe JNK inhibitor SP600125 voor of na miokardiale iskemie, het aangetoon dat, in ons studie, JNK en sy geassosieerde seinstransduksiepaaie krities belangrik as bemiddelaar van I/H besering is. Hierdie effekte het gepaard gegaan met laer aktivering van PKB. Ons resultate dui dus daarop dat ‗n interaksie tussen die JNK en PKB seintransduksiepaaie in die post-iskemiese miokardium, ‗n belangrike bydraende faktor in die uitkoms van I/H besering mag wees. Alhoewel die data wat hier aangebied word, teenstrydig mag voorkom, ondersteun dit juis die hipotese dat JNK seintransduksie spesifiek en tergelykertyd pro- en anti-apoptotiese meganismes in kardiomiosiese mag moduleer. Dit reflekteer ook die uitsonderlike kompleksiteit van die hart se metaboliese, funksionele en strukturele veranderinge in vetsug. Die resultate dui ook daarop dat matige hiperfagie-geïnduseerde vetsug nie nadelige effekte op die iskemies/herperfuseerde hart het nie maar eintlik die sensitiwiteit van die hart teenoor I/H beskadiging verminder. Hierdie aanname is verder onderskryf deur die voordelige effekte wat met vetsure in die perfusaat waargemee, is. Wanneer saamgevat, het die resultate van hierdie studie potensiëel klinies belangrike implikasies en bevestig die belangrikheid van gebeurtenisse tydens vroeë herperfusie as moontlike terapeutiese teikens.

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