The Significance of Mitophagy in Myocardial Ischaemia/ Reperfusion: the effect of melatonin

Dube, Kopano Rebaona (2018-03)

Thesis (MSc)--Stellenbosch University, 2018.

Thesis

Introduction: Myocardial ischaemia and concomitant cell damage are caused by a reduction in the blood supply to the heart. To date, the most effective strategy to salvage the myocardium is timely reperfusion which is unfortunately associated with further tissue damage. This phenomenon, termed ischaemia reperfusion injury, is associated with mitochondrial structural damage which could lead to death of cells previously damaged by ischaemia. Damaged and dysfunctional mitochondria play a key role in mediating tissue damage in this setting, thus the swift yet selective removal of these damaged organelles by mitochondrial autophagy – mitophagy could be of importance in cell survival and therefore is a potential therapeutic target. Studies have shown that upregulation of autophagy during ischaemia/reperfusion is cardioprotective, however, very little is known about the role of mitophagy in this setting. Subsequently, the aims of this study were to (i) characterise the effect of ischaemia/reperfusion on functional recovery during reperfusion and to correlate this with mitochondrial oxidative phosphorylation capacity, infarct size and mitophagy in the working heart model using male Wistar rats; (ii) evaluate the effect of mitophagy manipulation on cardioprotection using the parameters listed above. To achieve this, used was made of melatonin, the pineal hormone, which is well-known for its cardioprotective effects. Methods: Male Wistar rat hearts were perfused ex vivo in the working mode using Krebs-Henseleit buffer and glucose (10mM) as substrate. After a stabilization period of 30 min, hearts were subjected to 20min global ischaemia followed by 30min reperfusion during which time functional recovery was monitored. Mitochondria were isolated from hearts at different times during the perfusion protocol: after stabilization for 30min, after 20min global ischaemia and after 30min of reperfusion. The mitochondrial pellets were used for measurement of mitochondrial oxidative phosphorylation using an Oxygraph as well as for western blotting to evaluate a number of indicators of mitophagy. In addition, hearts were subjected to the perfusion protocol as described above and freeze-clamped at the same time intervals for subsequent Western blotting for mitophagy markers in the cytosolic fraction. In a separate series melatonin (0.3, 50M) was added to the perfusate for 10min before and 10 min after ischaemia and the same parameters evaluated as above. For evaluation of infarct size by the tetrazolium method, hearts were stabilized for 30min, followed by 35min of regional ischaemia and 60min reperfusion. Results: Exposure of hearts to either 35min regional ischaemia/ 60min reperfusion or 20min global ischaemia/ 30min reperfusion was associated with impaired recovery of myocardial function during reperfusion, characterized by significant reduction in several haemodynamic endpoints including coronary flow, aortic output and total work performed. Exposure to 20min global ischaemia per se had no effect on mitochondrial oxidative phosphorylation function, but a significant reduction in QO2 States 3 and 4 was observed after reperfusion, with glutamate/malate as substrates. Contrary to expectations, ischaemia/reperfusion did not upregulate mitophagy, as indicated by the reduced expression of PINK1, Parkin and TOM70 as well as markers of the alternative pathway (ULK1, DRP-1 and Rab9). Melatonin at both concentrations studied, significantly reduced the myocardial infarct size (p<0.0001), but did not improve mechanical recovery during reperfusion. In the global ischaemia model, melatonin increased mitochondrial oxphos during reperfusion only. While not having marked effects on the conventional PINK1/Parkin pathway, melatonin caused significant increases in the expression and phosphorylation of ULK1 and DRP-1, suggesting upregulation of the alternative pathway of mitophagy. Conclusion: In this experimental model ischaemia/ reperfusion reduced (i) contractile function and (ii) oxidative phosphorylation during reperfusion. It also subdued both the (iii) conventional and alternative mitophagy pathways suggesting that mitochondrial fission, which is a prerequisite for mitophagy, may be impaired under these conditions. These changes may contribute to the impaired functional recovery during reperfusion. Melatonin’s cardioprotective effects were associated with upregulation of a novel mitophagy signalling pathway, the significance of which in its cardioprotective actions needs to be further elucidated.

Miokardiale iskemie en selbeskadiging word deur ‘n vermindering in die bloedvoorsiening aan die hart teweeggebring. Tot op hede, is herperfusie van die iskemiese hart die mees effektiewe strategie om selbeskadiging te voorkom. Ongelukkig gaan herperfusie ook met verdere selbeskadiging gepaard, die sogenaamde verskynsel van herperfusie beskadiging. Herperfusie gaan gepaard met verdere beskadiging van selle wat reeds deur die voorafgaande periode van iskemie affekteer is. Beskadigde en disfunksionele mitochondria speel ‘n belangrike rol in hierdie scenario, gevolglik is die spoedige en selektiewe verwydering van hierdie organelle deur die proses van mitochondriale outofagie/mitofagie van kardinale belang vir oorlewing. Mitofagie mag dus ‘n potensiële terapeutiese teiken wees vir beskerming van die iskemies/herperfuseerde hart. Dit is voorheen getoon dat opregulering van outofagie tydens iskemie/herperfusie die hart teen beskadiging beskerm, maar min of geen inligting is beskikbaar aangaande die rol van mitofagie in hierdie verband nie. Die doelwitte van hierdie studie was dus (i) karakterisering van die effek van iskemie/herperfusie op funksionele herstel tydens herperfusie en die verband met mitochondriale oksidatiewe fosforilasie, infarkt grootte en mitofagie in ‘n model van die geïsoleerde, geperfuseerde rothart; (ii) evaluasie van mitofagie manipulasie op beskerming van die hart deur gebruik te maak van melatonien, ‘n hormoon afgeskei deur die pineaalklier. Metodes Harte van manlike Wistar rotte is ex vivo geperfuseer met Krebs-Henseleit buffer en glukose (10mM) as substraat, volgens die werkhart tegniek. Na ‘n stabilisasie periode van 30min, is harte onderwerp aan 20min globale iskemie gevolg deur 30min herperfusie waartydens funksionele herstel gemonitor is. Mitochondria is op verskillende tye tydens die perfusie protokol geïsoleer naamlik na stabilisasie, na 20min globale iskemie en na 30min herperfusie. Hierdie pellets is gebruik vir die meting van die oksidatiewe fosforilasie proses met behulp van ‘n Oksigraaf sowel as vir western blots vir evaluering van merkers van mitofagie. ‘n Addisionele series van harte is ook geperfuseer volgens bogenoemde protokol en die harte gevriesklamp vir bereiding van sitosoliese fraksies vir daaropvolgende Western blots. Melatonien (0.3, 50mM) is by die perfusaat gevoeg 10min voor en 10min na globale iskemie en dieselfde parameters evalueer soos bo gelys. Vir bepaling van infarkt grootte met die tetrazolium metode, is harte gestabilseer vir 30min, gevolg deur 35min streeks iskemie en 60 min herperfusie. Resultate Blootstelling van die geperfuseerde hart aan of 35min streeks iskemie/60min herperfusie of 20min globale iskemie/30 min herperfusie het funksionele herstel tydens herperfusie beduidend onderdruk, soos aangedui deur die verlaging in koronêre vloei, aorta en kardiale omset. Blootstelling van die hart aan 20min globale iskemie per se het geen effek op mitochondriale oksidatiewe funksie gehad nie, maar ‘n beduidende onderdrukking in QO2 staat 3 en staat 4 is na herperfusie waargeneem, met glutamaat/malaat as substrate. In teenstelling met verwagtinge, het iskemie/herperfusie nie mitofagie opgereguleer nie, maar onderdruk, soos getoon deur die verminderde uitdrukking van mitochondriale PINK1, Parkin en TOM70 sowel as merkers van die alternatiewe pad van mitofagie (ULK1, DRP-1 en Rab9). Beide hoë en lae melatonien konsentrasies het die infarkt grootte beduidend verlaag, maar nie funksionele herstel tydens herperfusie verbeter nie. Melatonien het mitochondriale oksidatiewe fosforilasie slegs tydens herperfusie na 20min globale iskemie verbeter. Melatonien het geen effek op die konvensionele pad van mitofagie gehad nie, maar het die uitdrukking en fosforilering van ULK1 en DRP-1 beduidend verhoog, wat dui op opregulering van die alternatiewe pad van mitofagie. Gevolgtrekking In die eksperimentele model wat gebruik is, het miokardiale iskemie/herperfusie die kontraktiele herstel en oksidatiewe fosforilasie proses onderdruk tydens herperfusie. Dit het ook beide die konvensionele en alternatiewe paaie van mitofagie onderdruk, wat moontlik dui op inhibisie van die mitochondriale fissie proses. Beide hierdie prosesse mag tot die onderdrukking in funksie bydra. Daarenteen was die beskerming deur melatonien verleen, geassosieer met opregulering van die alternatiewe mitofagie proses en dus moontlik mitochondriale fissie. Die belang van hierdie proses in die beskerming teen iskemiese beskadiging deur melatonien moet egter nog verder ondersoek word.

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