Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2006. / Introduction: Obesity, which is implicated in the development of the metabolic syndrome (MS) is reaching epidemic proportions worldwide. MS significantly increases the risk of developing cardiovascular disease, which includes coronary artery disease. The current absence of animal models of diet induced obesity and the MS makes the investigation of the cardiovascular consequences of MS virtually impossible. As a result the effects of the MS on cardiac function, morphology and susceptibility to ischaemia are not well understood. Aims: We set out to: 1) develop and characterize a rodent model of dietinduced obesity and the MS, 2) investigate the susceptibility of hearts from these animals to ischaemia/reperfusion induced injury and, 3) determine whether angiotensin II (Ang II) and endothelin-1 (ET-1) plays a role in cardiac remodelling and/or the severity of ischaemia and reperfusion injury in this model. Methods: Male Wistar rats were fed a standard rat chow diet or cafeteria diet (CD) for 16 weeks. After the feeding period rats were sacrificed and blood and myocardial tissue samples were collected to document biochemical changes in these animals. Hearts were perfused on the isolated working rat heart perfusion apparatus to assess myocardial mechanical function before and after ischaemia. In a separate series of experiments, hearts underwent coronary artery ligation to determine the incidence and duration of ventricular arrhythmias during ischaemia and reperfusion, using electrocardiography. To assess a possible link between myocardial remodelling and ischaemia/reperfusion injury and myocardial Ang II and ET-1 content, we also measured these peptides under basal conditions and during ischaemia. Two-dimensional targeted Mmode echocardiography was used to assess in vivo myocardial mechanical function in control and obese rats. Results: After 16 weeks on the CD, obese rats satisfied the World Health Organization (WHO) criteria for the MS by having visceral obesity, insulin resistance, dyslipidaemia and an elevated systolic blood pressure, compared to control rats. Circulating Ang II levels, but not ET-1 levels, were elevated in CD fed rats. Obese rats had cardiac hypertrophy and ex vivo basal myocardial mechanical function was depressed in the CD fed rat hearts compared to control rat hearts. CD fed rat hearts had poorer aortic output (AO) recoveries compared to hearts from control rats. These hearts also had a higher incidence and duration of reperfusion arrhythmias. No such functional differences were seen in the in vivo experiments. No differences in basal or ischaemic myocardial Ang II and ET-1 levels were seen in either group. Conclusion: We have developed and characterized a model of diet-induced obesity and the MS. Obesity is associated with cardiac hypertrophy and an increased myocardial susceptibility to ischaemia and reperfusion injury in our model. The hearts from obese rats were also more prone to reperfusion ventricular arrhythmias. As myocardial function was only poorer in the ex vivo obese animal experiments, our data suggests that the obesity associated changes in function observed in the ex vivo studies may be related to the absence of circulating substrates or factors, which are essential for their normal mechanical function.

Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Both, the cytokine Tumor Necrosis Factor-α (TNF-α) and the enzyme cytosolic phospholipase A2 (cPLA2) are crucial driving forces in mediating the cellular inflammatory response and are involved in ischaemic injury. During an ischaemic insult, TNF-α is endogenously generated. Apart from the recognized effects of TNF- α, such as the induction of apoptosis, proliferation and differentiation, if present in low dosages, it also mediates cytoprotective effects. Upon activation, cPLA2 contributes to the ischaemic challenge with the generation of mediators of cellular injury and apoptosis. Upon stimulation, this calcium dependent enzyme translocates to the phospholipid compartment of the cell membrane and induces the hydrolysis of sn-2 ester bonds in phospholipids. It governs the release of free fatty acids and lysophospholipids and generates role players of inflammation. We suggest a role for cPLA2 in the TNF-α mediated cytoprotection, with a distinct phosphorylation and translocation pattern. Aims The involvement of cPLA2 in TNF-α mediated cytoprotection in the C2C12 murine skeletal muscle cell line in tolerance to ischaemia was examined. To investigate the nature of the cPLA2 phosphorylation pattern, the mitogen activated protein kinases (MAPKs) p38 and extracellular regulated kinase (ERK) as contributors to cPLA2 phosphorylation and activation, were examined at appropriate time points. To dissect out the cPLA2 interplay and dependencies with these MAPKs within the pathway context, the selective cPLA2 inhibitor arachidonyl trifluoromethyl ketone (AACOCF3) was employed and its effect on cell viability was examined. Fluorescence microscopy was used to substantiate cPLA2 activation, by assessing its cellular distribution, translocation and cell organelle target preference, using co-localization and z-stack techniques. In addition, the induction of the apoptotic pathway through analysis of caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavage was examined. The role of caspase-3 in cPLA2 turnover was addressed employing the caspase inhibitor, Z-DEVD-FMK. Methods Cells were grown in Dulbecco’s Modified Eagles Medium (DMEM) with 10% fetal bovine serum (FBS), and incubated under 5% CO2 conditions, until 50%-70% confluent. Using DMEM supplemented with 1% horse serum, cell differentiation into myotubes was induced. Differentiated cells were preconditioned for 30 min classically, with 0.5 ng/ml TNF-α or the cPLA2 selective inhibitor AACOCF3 (10 μM) respectively. Followed by a 60 min washout period the cells were subjected to 8 hrs simulated ischaemia. Cellular viability; and cPLA2 phosphorylation- and translocation events were assessed using Western blots and advanced immunocytochemistry and imaging techniques. Results Preconditioning with TNF-α, ischaemic preconditioning; and the use of the cPLA2 inhibitor AACOCF3, attenuated the decrease is cell viability brough about by ischaemia. Western blot analysis indicates the induction of the apoptotic pathway with caspase-3 and PARP cleavage. A significantly reduced translocation of pcPLA2 to the nuclear region in the TNF-α preconditioned group compared to the ischaemic group, as reflected by reduced mean nuclear fluorescence intensity, was observed. A z-stack analysis confirmed that the nuclear and endonuclear region was the target organelle for cPLA2. 3-dimensional co-localazation analysis of pcPLA2 with the nuclear marker nucleoporin p62 mirrored these results. Discussion and conclusion Our results provide evidence that there is a role for cPLA2 in TNF-α mediated cytoprotection. Although we do not observe a differential activation pattern in terms of cPLA2 phosphorylation at various time points within the ischaemic event, and no differential inactivation of cPLA2 via caspase-mediated cPLA2 cleavage, we describe a differential cPLA2 translocation pattern, similar to that in IPC. Through inhibition of cPLA2 translocation, a functional cPLA2 inhibition might be achieved. This would imply inhibition of the inflammatory pathway and a subsequent reduction in the generation of inflammatory mediators. In addition we describe an effect of TNF-α preconditioning on the efficacy of the caspase inhibitor Z-DEVD-FMK. Our results shed light on the survival mechanisms employed by the ischaemically challenged cell in a setting of TNF-α mediated cytoprotection. This might lead to novel approaches in the context of inflammation treatment, through agents that control differential cPLA2 trafficking within the cell. / AFRIKAANSE OPSOMMING: Beide, die sitokien “Tumor Necrosis Factor-α (TNF-α)” en die ensiem, sitosoliese fosfolipase A2 (cPLA2) is uiters belangrike bemiddelaars van die sellulêre inflammatoriese respons en is verder ook betrokke by isgemiese selskade. TNF-α word endogeen gegenereer tydens ‘n isgemiese intervensie. Afgesien van ‘n verskeidenheid effekte, soos die inisiëring van apoptose, sel-proliferasie en - differensiasie, bemiddel dit ook selbeskermende meganismes indien dit in lae konsentrasies in die sel teenwoordig is. Na aktivering dra cPLA2 by tot die isgemiese intervensie deur die vorming van bemiddelaars van selskade en apoptose. Hierdie kalsium-afhanklike ensiem translokeer na die fosfolipied membraankomponent na stimulering en induseer die hidrolise van die sn-2 esterbinding in die fosfolipied. Die vrystelling van vry vetsure en lisofosfolipiede word sodoende bewerkstellig wat verder gemetaboliseer kan word tot inflammatoriese bemiddelaars. Ons stel voor dat cPLA2 ‘n rol in TNF-α bemiddelde selbeskerming speel en dat dit gepaardgaan met kenmerkende fosforilerings- en translokeringspatrone. Doelwitte Die rol van cPLA2 tydens TNF-α bemiddelde selbeskerming is in ‘n C2C12 skeletspiersellyn na blootstelling aan isgemie ondersoek. Die rol van die MAPKs, p38 en ERK, is ondersoek om vas te stel of hulle betrokke is by die aktivering van cPLA2. Die selektiewe cPLA2 inhibitor, AACOCF3, is gebruik om te bepaal of die fosforilering van MAPKs ook cPLA2-afhanklik is. Die sellulêre cPLA2 verspreiding, translokering en teiken selorganelle is ook ondersoek met behulp van fluoresensie mikroskopie deur gebruik te maak van ko-lokalisering en z-plaat tegnieke. Verder, is die indusering van die apoptotiese paaie ondersoek deur tegnieke wat kaspase- en PARP kliewing meet. Die kaspase inhibitor, Z-DEVD-FMK, is gebruik om vas te stel of kaspase-3 ‘n rol speel in cPLA2 kliewing in ons selmodel. Metodes Selle is gekweek in Dulbecco’s gemodifiseerde Eagles Medium (DMEM) waarby 10% fetale kalf serum (FBS) gevoeg is, en wat geïnkubeer is in 5% CO2 totdat dit 50%-70% konfluent was. Die selle is verder gedifferensieer in miobuise deur gebruik te maak van DMEM waarby 1% perdeserum gevoeg is. Gedifferensieerde selle is vir 30 min klassiek geprekondisioneer asook respektiewelik met 0.5 ng/ml TNF-α en die cPLA2 selektiewe inhibitor, AACOCF3 (10 μM). Na ‘n 60 minute uitwas periode is die selle blootgestel aan 8 h gesimuleerde isgemie. Sellulêre lewensvatbaarheid, cPLA2 fosforilering- and translokering is ondersoek deur onderskeidelik gebruik te maak van die “Western” klad metode en gesofistikeerde immunositochemiese beeld tegnieke. Resultate Prekondisionering met TNF-α, isgemiese prekondisionering asook inhibisie van as cPLA2 met die inhibitor, AACOCF3, het ‘n beduidende toename in sellewensvatbaarheid tot gevolg gehad. Daar is ook dmv die “Western” klad tegniek bewys dat apoptose geïduseer word deur middel van kaspase-3- en PARP kliewing. Daar is insiggewend minder translokasie van cPLA2 na die nukluêre fraksie in die isgemiese groep in vergelyking met die TNF-α geprekondisioneerde groep waargeneem (die gemiddelde nukluêre fluoreserende intensiteit is bepaal om voorafgaande feit te staaf). Die cPLA2 teiken organel is geverifieer as die nukleus en die endonukluêre gebied deur middel van z-plaat analises. Drie-dimensionele kolokaliserings analises van pcPLA2 met die nukluêre merker, nucleoporin p62 het hierdie resultate bevestig. Bespreking en Gevolgtrekking Ons resultate verskaf bewyse vir ‘n rol vir cPLA2 in TNF-α bemiddelde selbeskerming. Alhoewel daar nie ‘n differensiële aktiveringspatroon in terme van cPLA2 fosforilering tydens verskeie tydspunte in die isgemiese intervensie waargeneem is nie, en ook geen kaspase-3 bemiddelde kliewing van cPLA2 nie, word ‘n differensiële translokeringspatroon soorgelyk aan die isgemiese prekondisioneringsgroep, waargeneem. Funsksionele cPLA2 inhibisie kan dus moontlik bewerkstellig word deur inhibisie van cPLA2 translokasie. Die inflammatoriese respons kan dus moontlik so inhibieer word en die vorming van minder inflammatoriese bemiddelaars tot gevolg hê. Verder het TNF-α prekondisionering ook ‘n effek op die effektiwiteit van die kaspase-inhibitor, ZDEVD- FMK. Ons resultate werp ook lig op die meganismes wat deur selle onder isgemiese toestande uitgeoefen word tydens TNF-α bemiddelde selbeskerming. Hierdie resultate mag lei tot nuwe benaderings in die konteks van behandeling teen inflammasie deur gebruik te maak van middels wat cPLA2 translokering in die sel beheer.

Thesis MSc (Physiological Sciences))--University of Stellenbosch, 2010. / 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.

Thesis (PhD)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: The reperfusion injury after myocardial ischemia is relevant in the clinical setting, after cardiopulmonary bypass for cardiac surgery, after PTCA and stenting and after cardiopulmonary resuscitation. The components of the reperfusion injury considered in this study were myocardial stunning and reperfusion arrhythmias. Calcium antagonists have been shown to be beneficial in attenuating the myocardial reperfusion injury in the in vitro and in vivo laboratory setting (Lamping, Gross 1985, Przyklenk and Kloner 1988, Taylor 1990, Ehring 1992, Gross and Piper 1992). However systemic administration of a dose of calcium antagonist, large enough to attenuate the myocardial reperfusion injury in the clinical setting, would inevitably lead to unwanted systemic side effects of the drug. The aim of this study was to investigate the hypothesis that an adequate dose of verapamil administered timeously, directly into the ischemic myocardium, would attenuate the reperfusion injury, either when administered from the onset of ischemia, or from 3 minutes before reperfusion. The anesthetized open chest porcine model of myocardial ischemia (15 min total LAD occlusion) and reperfusion was employed in this study. A low dose of verapamil (0.5 mg/8mt or 0.0625mg/mt), a high dose of verapamil (2mg/8m or O.25mg/ml), or vehicle (saline) (8ml) was infused over 8 minutes, directly into the LAD coronary artery supplying the ischemic segment. The infusion was started either at the onset of ischemia, or from 3 minutes before reperfusion. The time taken for the various parameters to return to pre ischemic values was compared between the different groups. The results showed that the high dose of verapamil (2mg) attenuated the reperfusion injury both when administered from the onset of ischemia, and when administered from 3 minutes before reperfusion, compared to either the low dose of verapamil, or the saline infusions. The high dose of verapamil groups had a faster recovery of both systolic contractile function and diastolic function and a lower incidence of ventricular fibrillation on reperfusion. There were no systemic effects of verapamil infusion in any of the groups. The clinical setting of cardiac surgery expressly lends itself to the clinical application of this finding. There is direct access to the coronary arteries both before ischemia and before reperfusion. A small dose of calcium channel blocking drug, with no systemic effect can be administered into the aortic root at the onset of ischemia, just prior to cardioplegia (when the heart is still warm), and after rewarming a few minutes prior to removal of the aortic cross clamp. / AFRIKAANSE OPSOMMING: Die reperfusie besering na miokardiale isgemie is klinies relevant na kardiopulmonêre omleiding vir hart chirurgie, na kardiologiese PTKA en stut prosedures en na kardiopulmonale ressussitasie. Die komponente van die reperfusie besering wat in hierdie studie oorweeg is, is miokardiale tydelike omkeerbare onderdrukking (stunning) en reperfusie arritmieë. Kalsium antagoniste is gewys om effektief te wees in beperking van die reperfusie besering in beide in vitro en in vivo laboratorium eksperimente (Lamping, Gross 1985, Przyklenk en Kloner 1988, Taylor 1990, Ehring 1992, Gross en Piper 1992). Sistemiese toediening van 'n dosis kalsium kanaal blokker, voldoende om die miokardiale reperfusie besering in die pasiënt te beperk, lei egter tot ongewenste sistemiese newe effekte van die middel. Die doel van die studie was om die hipotese te ondersoek dat 'n voldoende dosis verapamil, wat betyds direk toegedien is aan die isgemiese miokardium, die reperfusie besering sal beperk, ongeag of dit toegedien is vanaf die begin van isgemie, of van 3 minute voor reperfusie. Die vark model van miokardiale isgemie en reperfusie is aangewend in die studie. Die varke was tydens die eksperiment onder narkose, met die borskas oop, en 15 minute totale LAD okklusie is toegepas. 'n Lae dosis verapamil (0.5mg/8ml of 0.0625 mg/mt), of hoë dosis veraparnil (2mg/8mt of 0.25mg/mt), of saline (8mt) is oor 8 minute toegedien direk in die LAD arterie wat die isgemiese segment voorsien. Die infuus is begin direk na die aanvang van isgemie, of 3 minute voor die aanvang van reperfusie. Die tyd geneem vir terugvoer van parameters na pre isgemiese waardes is tussen die groepe vergelyk. Die resultate toon dat die hoë dosis veraparnil die reperfusie besering beperk in vergelyking met die lae dosis veraparnil of saline infusies, ongeag of dit van die begin van isgemie, of van 3 minute voor reperfusie toegedien word. Die groepe wat die hoë dosis veraparnil ontvang het, het vinniger herstel van sistoliese en diastoliese funksie getoon en het'n laer insidensie van reperfusie disritmieë, gewys. Geen sistemiese effekte van veraparnail infuus is waargeneem nie. Die kliniese toepassing van hierdie bevinding is by uitstek geskik vir toepassing tydens kardiopulmonale omleiding by kardiale chirurgie. Daar is direkte toegang tot koronêre arteries voor isgemie en voor reperfusie. 'n Klein dosis kalsium antagonis, met weglaatbare sistemiese effekte, kan toegedien word in die aorta wortel met die aanvang van isgemie, net voor kardioplegie toediening (hart steeds warm), en na verwarming, 'n paar minute voor verwydering van die aorta kruis klem.

Phosphodiesterase Type 5 (PDE5) inhibitors are cardioprotective against ischemia/reperfusion (I/R) injury. However, it remains uncertain if I/R affects PDE5. We hypothesized that generation of reactive oxygen species (ROS) during I/R leads to upregulation of PDE5, which contributes to pathological changes following acute myocardial infarction (AMI). Adult male ICR mice were subjected to 30 minutes of in vivo or ex vivo I/R. To examine the role of ROS, a subset of hearts were perfused with 100 µM hydrogen peroxide (H2O2). Expression and activity of PDE5, pPDE5, and cGMP-dependent protein kinase (PKG) were measured by Western blots and spectrophotometric assay. The results show that ischemia and I/R significantly increased expression of PDE5. H2O2 had no effect on PDE5 expression and activity but significantly increased PKG activity. We conclude that acute cardiac ischemia or I/R upregulate PDE5 independent of oxidant stress in the heart.

Diplomová práce Abstract - Iveta Brabcová Abstract Ischemia-reperfusion heart injury is one of the most significant diseases affecting mankind and therefore current research pays more attention to its prevention and knowledge of the possible mechanisms which protect the heart. Adaptation to hypoxia has been known for several decades as a cardioprotective intervention but the main issues of protective mechanisms which are induced by the adaptation are still not completely understood. An important role of mitochondria as the main producers of energy and reactive oxygen species which can play a signalizing role in these mechanisms is confirmed in many studies. For this reason a special conplastic strain SHR/OlaIpcv-mtBN/Crl was created. This strain carries the nuclear genome of spontaneously hypertensive rat (SHR) and the mitochondrial genome of normotensive, highly resistant strain Brown Norway (BN). The aim of this study was to compare the expression of selected gene transcripts in the area of energy metabolism, of genes which are related to mitochondrial biogenesis and signaling and antioxidant systems. Comparing the expression was analyzed between strains and after chronic hypoxia adaptation, which cause cardioprotective phenotype in both of these strains. Our results showed a different expression HIF-1α...

Implication du PTP dans la mort cellulaire β pancréatique L'hyperglycémie, l'hyperfructosémie et l'ischémie-reperfusion sont délétères pour la viabilité cellulaire β pancréatique, jouant un rôle majeur dans la perte de la masse cellulaire β. Le pore de transition de perméabilité mitochondriale (PTP) est un canal mitochondrial impliqué dans le déclenchement de la mort cellulaire. Des données récentes montrent l'implication du PTP et du stress oxydant dans la toxicité induite par l'ischémie-reperfusion sur cardiomyocytes et également dans la glucotoxicité induite sur cellules endothéliales. La première partie de notre étude a visé à étudier l'implication de l'ouverture du PTP dans la mort cellulaire des cellules INS-1 et des îlots pancréatiques humains soumis à de fortes concentrations de glucose et de fructose. Nous démontrons que l'incubation des cellules INS-1 et des îlots pancréatiques humains en présence de 30 mM de glucose ou 2,5 mM de fructose déclenche une ouverture du PTP et induit la mort cellulaire. La metformine et la Cyclosporine A (CsA) préviennent l'ouverture du PTP et la mort cellulaire induite par le glucose et le fructose. La deuxième partie de notre travail montre que l'exposition des INS-1 à une heure de carence en substrat concomitante d'une hypoxie, suivie d'une restauration des conditions basales conduit à l'ouverture du PTP et à une majoration drastique de la mort cellulaire. Ces deux évènements sont totalement prévenus par l'incubation préalable par la CsA et la metformine mais aussi par la N-Acétyl-Cystéine (NAC) ou par l'exposition à une anoxie, soulignant ainsi le rôle fondamental du stress oxydant dans le déclenchement de l'ouverture du PTP et de la mort cellulaire. Nous montrons qu'au cours de l'ischémie-reperfusion simulée, la production de superoxide est bi-phasique : nous décrivons un premier pic de production au cours de la carence en substrat, lié à un flux reverse d'électrons au sein du complexe I de la chaîne respiratoire. Ce premier pic est suivi d'un deuxième pic de production après la restauration du niveau de substrats et d'O2, lié à l'ouverture du PTP. La NAC, l'anoxie ou la metformine préviennent les deux pics de production de superoxide tandis que la CsA prévient seulement le second pic. Enfin, nous montrons que l'hypoxie seule n'induit ni stress oxydant, ni ouverture du PTP ni mortalité cellulaire. L'ensemble de notre travail démontre le rôle central du PTP dans la gluco-fructotoxicité et dans la toxicité induite par l'ischémie-reperfusion sur la cellule β pancréatique. Ainsi, prévenir l'ouverture du PTP peut-être une approche intéressante pour préserver la viabilité cellulaire β. / PTP involvement in β pancreatic cell death Hyperglycemia, hyperfructosemia and ischemia-reperfusion play a major role in the progression of β cell loss in diabetes mellitus. The permeability transition pore (PTP) is a mitochondrial channel involved in cell death. PTP opening and oxidative stress have been shown to be involved in ischemia-reperfusion injury on cardiomyocytes and in hyperglycemia-induced cell death in endothelial cells. In the first part of this work, we have examined the involvement of PTP opening in INS-1 cells and human pancreatic islets cell death induced by high levels of glucose or fructose. We first reported that Metformin and Cyclosporin A (CsA) prevented Ca2+-induced PTP opening in permeabilized and intact INS-1 cells. We then shown that incubation of INS-1 cells and human islets in the presence of 30 mM glucose or 2.5 mM fructose induced PTP opening and led to cell death. Because both Metformin and CsA prevented glucose and fructose induced PTP opening, and hampered glucose and fructose induced cell death, we conclude that PTP opening is involved in high glucose and high fructose induced INS-1 and human islets cell death. We therefore suggest that preventing PTP opening might be a new approach to preserve β cell viability. In the second part of the work, we demonstrate that the incubation of INS-1 cells in the absence of energy substrates in hypoxic condition for 1 hour followed by incubation in normal condition led to PTP opening and to a dramatic increase in cell death. Both events were totally prevented when PTP opening was inhibited by either Cyclosporin A (CsA) or Metformin or when the cells were incubated in the presence of the antioxidant N-acetyl-cystein (NAC), in anoxia, highlighting the implication of oxidative stress is the commitment of PTP opening. Superoxide production increased during the removal of energy substrates, due to reverse electron flux through complex I and again increased when normal energy substrate and O2 were restored, due to PTP opening. NAC, anoxia or Metformin prevented the two phases of oxidative stress, while CsA prevented only the second one. Hypoxia alone did not induce oxidative stress, PTP opening or cell death. Our work demonstrates the implication of PTP opening in ischemia-reperfusion injury and gluco- fructotoxicty in β pancreatic cells. We therefore suggest that preventing PTP opening might be a new approach to preserve β cell viability.

La progression de l'incidence de l'insuffisance rénale chronique dans le monde nécessite d'améliorer les mesures visant à ralentir sa progression et son évolution vers l'insuffisance rénale terminale. Le régime pauvre en protéines ou la prévention des épisodes d'ischémie- reperfusion appartiennent à ces mesures de néphroprotection mais leur bénéfice n'est qu'incomplètement compris. Le rein étant un des organes les plus vascularisé, rapporté à son poids, l'évaluation concomitante de la perfusion rénale ([15O]H2O), du métabolisme oxydatif ([11C]acetate) puis du contenu tissulaire en oxygène (BOLD-IRM) est fondamentale dans la mesure où ces trois mesures ne sont pas corrélées de manière linéaire. La TEP-IRM, appareil hybride combinant les fonctionnalités de la TEP et de l'IRM, permet une évaluation concomitante de ces trois paramètres de manière non invasive et sur rein isolé ce qui permet de l'utiliser en recherche clinique pour l'évaluation de l'effet de différentes interventions. Les régimes pauvres en protéines ont démontré qu'ils permettaient un ralentissement du déclin de la fonction rénale mais le mécanisme à l'origine de cet effet bénéfique n'est pas connu. Nous avons démontré que le contenu en AGE (Advanced Glycation End Products ou protéines modifiées) d'une charge protéique était responsable de la mobilisation de la réserve fonctionnelle rénale avec une augmentation de la perfusion et du métabolisme oxydatif plus importants après une charge protéique riche en AGE par rapport à une charge protéique pauvre en AGE. Nos résultats permettent de s'interroger sur l'intérêt d'une alimentation pauvre en AGE pour les insuffisants rénaux chroniques au-delà d'une restriction protéique pure. La prévention des épisodes d'ischémie- reperfusion est également particulièrement importante dans le cadre de la néphroprotection alors que la revascularisation d'une sténose de l'artère rénale s'accompagne obligatoirement de lésions de reperfusion. L'utilisation de la ciclosporine avant la dilatation d'une sténose de l'artère rénale dans le cadre d'un pré conditionnement a permis de réduire les lésions rénales chez la souris mais ce bénéfice clinique n'a pas été démontré chez l'homme. L'étude CICLOSAAR qui a pour but d'évaluer le bénéfice d'un pré conditionnement par la ciclosporine A avant dilatation d'une sténose de l'artère rénale sur des paramètres fonctionnels rénaux (perfusion, métabolisme oxydatif et contenu tissulaire en oxygène) est en cours. Dans un second temps et afin d'améliorer l'applicabilité clinique de ces techniques d'imagerie fonctionnelle, nous avons démontré que l'acétate, qui était jusque-là utilisé comme un marqueur de métabolisme oxydatif, pouvait également être utilisé comme un marqueur de perfusion rénale grâce à la modélisation de son uptake par le rein. Au total, la TEP- IRM est un outil fondamental pour l'évaluation des paramètres fonctionnels rénaux de manière non invasive chez l'humain. La mise en évidence de l'impact du contenu en AGE sur la perfusion et le métabolisme rénal devrait permettre d'affiner nos conseils diététiques aux insuffisants rénaux pour prévenir la progression de la maladie vers l'insuffisance rénale terminale. L'étude CicloSAAR actuellement en cours devrait permettre d'améliorer les mesures mises en oeuvre pour protéger le rein des lésions de reperfusion après dilatation d'une sténose de l'artère rénale. Enfin, nos résultats sur la forte corrélation entre les données de perfusion en acétate et en eau devraient permettre d'utiliser ce traceur pour obtenir la perfusion et le métabolisme oxydatif à l'issue d'une seule injection de radio-traceur et renforcer l'applicabilité clinique de ces techniques / The increase in the incidence of chronic renal failure worldwide requires improvements in measures to slow its progression to end-stage renal failure. The low-protein diet or the prevention of episodes of ischemia-reperfusion belong to these measures of nephroprotection but their benefit is only incompletely understood. The kidney is one of the most vascularized organs, compared to its weight, the concomitant evaluation of renal perfusion ([15O] H2O), oxidative metabolism ([11C] acetate) and tissue content in oxygen (BOLD-IRM) is fundamental in that these three measures are not linearly correlated. PETMRI, a hybrid device combining the functionalities of PET and MRI, allows a concomitant evaluation of these three parameters in a non-invasive and isolated kidney way which allows to use it in clinical research for the evaluation of the effect of different interventions. Low-protein diets have been shown to slow down the decline in renal function, but the mechanism behind this beneficial effect is not known. We have demonstrated that the AGE (Advanced Glycation End Products) content of a protein load is responsible for the mobilization of the renal functional reserve with an increase in perfusion and oxidative metabolism after a high- AGE high- protein compared to a low- AGE high- protein load. Our results make it possible to question the interest of a low-AGE diet for chronic renal insufficiency beyond a pure protein restriction. The prevention of episodes of ischemia reperfusion is also particularly important in the context of nephroprotection, whereas the revascularization of a stenosis of the renal artery is necessarily accompanied by reperfusion injury. Use of ciclosporin prior to dilation of renal artery stenosis in pre-conditioning reduced renal damage in mice, but this clinical benefit has not been demonstrated in humans. The CICLOSAAR study, which aims to evaluate the benefit of pre-conditioning with ciclosporin before dilation of renal artery stenosis with renal functional parameters (perfusion, oxidative metabolism and tissue oxygen content) is in progress. In a second step and to improve the clinical applicability of these functional imaging techniques, we have shown that acetate, which was previously used as a marker of oxidative metabolism, could also be used as a marker of renal perfusion through the modeling of its uptake by the kidney. In total, PET-MRI is a fundamental tool for the evaluation of renal functional parameters in a non-invasive way in humans. Demonstrating the impact of the AGE content of a protein load on perfusion and renal metabolism should help refine our dietary advice to patients with chronic kidney disease to prevent progression of the disease to end-stage renal failure. The current CicloSAAR study is expected to improve the measures implemented to protect the kidney from reperfusion injury after dilatation of renal artery stenosis. Finally, our results on the strong correlation between acetate and water perfusion data should allow this tracer to be used to obtain perfusion and oxidative metabolism after a single radio tracer injection and reinforce clinical applicability of these techniques

INTRODUÇÃO: As técnicas de preservação pulmonar visam a melhorar a qualidade do enxerto e aumentar sua tolerância ao período de isquemia fria. A técnica mais usada atualmente consiste na perfusão da artéria pulmonar com Perfadex. O alto custo associado à importação dessa solução e as dificuldades logísticas dos portos e aeroportos brasileiros com relação a materiais médicohospitalares têm causado problemas para os centros de transplante pulmonar brasileiros. Daí a necessidade de uma solução de preservação pulmonar produzida no Brasil. O objetivo desse estudo é comparar a solução Perfadex com a solução de fabricação nacional LPD-G, quanto ao grau de lesão de isquemia-reperfusão, em um modelo de perfusão pulmonar ex vivo (PPEV). MÉTODOS: Foram usados doadores em morte cerebral, cujos pulmões foram recusados. Cada caso era incluído aleatoriamente em um dos grupos: Grupo 1, a preservação pulmonar era realizada com Perfadex, e Grupo 2, era usado o LPD-G, solução fabricada no Brasil com composição idêntica a do Perfadex. Após a captação, os pulmões eram armazenados a 4 °C por 10 horas. A reperfusão ocorria em um sistema de PPEV, no qual o bloco pulmonar era ventilado e perfundido por uma solução acelular a 37 °C por 60 minutos. A lesão de isquemia-reperfusão era medida através de parâmetros funcionais (gasometria, resistência vascular pulmonar, complacência pulmonar, relação peso úmido/peso seco) e histológicos. Foram feitas biópsias pulmonares em 3 tempos: antes da captação, após o período de isquemia fria e depois da reperfusão. Vários critérios foram usados (edema alveolar, edema intersticial, hemorragia etc.) para criar um Escore de Lesão Pulmonar (ELP). A contagem de células apoptóticas foi feita usando a metodologia TUNEL (TdT-mediated dUTP nick end labeling). RESULTADOS: Após a reperfusão, a capacidade de oxigenação média foi de 405,3 mmHg no Grupo 1 e 406,0 mmHg no Grupo 2 (p = 0,98). A mediana da resistência vascular pulmonar nos pulmões do Grupo 1 foi de 697,6 dina.s.cm-5, enquanto no Grupo 2, esse valor foi de 378,3 dina.s.cm-5 (p = 0,035). A complacência pulmonar média ao final da reperfusão foi de 46,8 cmH2O no Grupo 1 e de 49,3 ml/cmH2O no Grupo 2 (p = 0,816). A razão entre o peso úmido e o peso seco foi em média 2,06 e 2,02 nos Grupos 1 e 2, respectivamente (p = 0,87). Na biópsia realizada após reperfusão, o ELP médio foi de 4,37 e 4,37 nos Grupos 1 e 2, respectivamente (p = 1,0); a contagem de células apoptóticas foi de 118,75/mm2 e 137,50/mm2 nos Grupos 1 e 2, respectivamente (p = 0,71). CONCLUSÕES: A qualidade da preservação pulmonar obtida com a solução LPD-G nacional é semelhante a obtida com o Perfadex. A aplicação clínica da nova solução pode reduzir custos, facilitando a manutenção e a abertura de centros de transplante pulmonar / INTRODUCTION: Pulmonary preservation techniques aim at improving graft quality and increasing tolerance during reperfusion and cold ischemia times. Currently, the most used technique consists of pulmonary artery anterograde perfusion with Perfadex. The high cost associated with the importation of this solution and the logistical difficulties of our ports and airports regarding medical supplies have caused problems for lung transplant centers in Brazil. Therefore there is need for a preservation solution manufactured in Brazil. The aim of this study is to compare the pulmonary preservation solutions Perfadex and LPD-G manufactured in Brazil in an ex vivo lung perfusion (EVLP) model. METHODS: Donors with brain death, whose lungs had been declined by transplantation teams were used. Cases were randomized into two groups: in Group 1, Perfadex was used for pulmonary preservation. In Group 2, LPDnac, a solution manufactured in Brazil and whose compositon is identical to Perfadex, was used. After harvesting, lungs were stored at 4 °C for 10 hours. An EVLP system was used and the pulmonary block was ventilated and perfused by an acellular solution at 37 °C for 60 minutes. Ischemic-reperfusion injury was measured by functional (blood gas, pulmonary vascular resistance, lung compliance, wet/dry weight ratio) and histological parameters. Pulmonary biopsies were performed at three time points: before harvesting, 10 hours after cold ischemia and 60 minutes after reperfusion. Samples were prepared for light microscopy analysis. Several criteria were used (alveolar edema, interstitial edema, hemorrhage etc.) to create a lung injury score (LIS). Apoptotic cell count was carried out using the TUNEL methodology (TdT-mediated dUTP nick end labeling). RESULTS: After reperfusion, mean oxygenation capacity was 406.0 mmHg in Group 2 and 405.3 mmHg in Group 1 (p = 0.98). Mean pulmonary vascular resistance in Group 2 lungs was 378.3 dina.s.cm-5, whereas in Group 1 it was 697.6 dina.s.cm-5 (p = 0.035). Mean pulmonary compliance by the end of reperfusion was 49.3 ml/cmH2O in Group 2 and 46.8 cmH2O in Group 1 (p = 0.816). Mean wet/dry weight ratio was 2.02 and 2.06 in Groups 2 and 1, respectively (p = 0.87). Mean LIS for the biopsy performed after reperfusion was 4.37 and 4.37 in Groups 2 and 1, respectively (p= 1.0); apoptotic cell count was 137.50/mm2 and 118.75/mm2 in Groups 2 and 1, respectively (p = 0.71). CONCLUSION: The preservation solution manufactured in Brazil proved to be as good as Perfadex. The clinical application for the new solution may reduce costs, favoring the maintenance and opening of pulmonary transplantation centers

Introdução: O trauma é um fenômeno que cursa com lesão tecidual, sendo que o trauma cirúrgico (TC) apresenta a referida lesão como consequência de um ato cirúrgico. A isquemia seguida de reperfusão (IR) é um evento comum em várias condições patológicas, bem como em diversos procedimentos cirúrgicos, principalmente transplantes. É frequente o desenvolvimento de lesões teciduais locais e remotas após trauma e após a I/R, parte de um fenômeno conhecido como síndrome da resposta inflamatória sistêmica (SRIS), frequentemente seguida pela falência de múltiplos órgãos (FMO). Estudos provaram o envolvimento do neutrófilo em tais síndromes como resultado da ação de enzimas proteolíticas secretadas a partir de grânulos citoplasmáticos, radicais livres produzidos por explosão respiratória e citocinas liberadas após a infiltração nos tecidos. Nesse contexto, foi provado que o pré-condicionamento isquêmico (PCI), definido como curtos episódios de isquemia precedendo a IR, protege contra essas lesões, com menor ativação de neutrófilos. No entanto, o conhecimento a respeito dos mecanismos operantes nos neutrófilos após o trauma cirúrgico, a isquemia seguida de reperfusão ou o pré-condicionamento isquêmico, ainda são preliminares. Objetivo: Analisar com maior profundidade o impacto dessas condições (TC, IR e PCI) no proteoma e fosfoproteoma do neutrófilo. Métodos: Foi realizada a análise de parâmetros hematológicos juntamente com a análise proteômica e fosfoproteômica de neutrófilos de ratos submetidos a TC, IR e PCI, comparados ao grupo controle. A análise proteômica foi realizada em sistema de nLC-MS/MS orbitrap de alto desempenho, usando marcação com iTRAQ, enriquecimento de fosfopeptídios e pré-fracionamento por HILIC. A análise estatística baseada em clusters utilizando scripts em R mostrou proteínas com abundância relativa diferencial em todas as condições. Resultados: A avaliação dos parâmetros hematológicos antes e depois de TC, IR e IPC demonstrou alterações no número, forma e tamanho de linfócitos, hemácias, plaquetas e, principalmente, neutrófilos (granulócitos). Observou-se um claro aumento na contagem de neutrófilos após TC e IR, sendo que tal aumento foi prevenido pelo PCI. Um total de 393 proteínas foram determinadas como reguladas para abundância relativa entre o grupo controle e o grupo TC. A maioria das proteínas encontradas como reguladas em comum nos grupos TC e IR estão relacionadas à apoptose (caspase-3), motilidade celular (PAK2), transdução de sinal (IL-5, IL-6 e TNF) e degradação pelo sistema proteassoma no neutrófilo. Maior produção de espécies reativas de oxigênio e disfunção da migração direcional de neutrófilos (PKC-delta) com aumento do tempo de vida dos neutrófilos são eventos iniciais importantes que podem resultar em mais dano tecidual e em infecção. A análise proteômica de neutrófilos de ratos após PCI levou à identificação de 2437 grupos de proteínas atribuídos a 5 clusters diferentes, contendo proteínas de abundância relativa significativamente aumentada ou diminuída em IR e PCI. O estudo de vias desses clusters baseado no KEGG revelou aumento nas vias de fagocitose mediada por Fc-gama R, sinalização por quimiocinas, adesão focal e migração transendotelial, citoesqueleto de actina, metabolismo e diminuição nas vias ribossomais, de transporte de RNA, de processamento de proteínas. A regulação da fosforilação de proteínas após IR e PCI mostrou algumas vias como quimiocinas, Fc-gama, GPCR, migração celular e vias pró e antiapoptóticas, sendo que a via de splicing alternativo foi a que apresentou regulação mais evidente (p < 0.0001). A regulação da abundância, bem como da fosforilação, presença de motivos e de domínios levou à identificação de fosfatases, como Fgr, GRK2, PKC delta, ptpn6 e ptprc reguladas por IR, bem como stk38, pkn1, syk e inpp5d reguladas por PCI. A interação mais marcante entre proteínas foi demonstrada como sendo entre os receptores de Fgr e Ptp. Conclusão: Concluímos que as alterações causadas por TC, IR e PCI levaram a intenss alterações na abundância de algumas proteínas e em eventos de fosforilação em neutrófilos, levando ao efeito destrutivo observado após a IR e ao efeito protetor consequente ao PCI / Introduction: Trauma is a phenomenon that involves tissue injury, whereas the surgical trauma (ST) has such injury as a consequence of a surgery. Ischemia reperfusion is common event in many surgical procedures, especially in transplants, as well as in many pathological conditions. Local and remote tissue injuries usually develop after trauma and ischemic reperfusion, part of a phenomenon known as systemic inflammatory response syndrome, frequently followed by multiple organ failure (MOF). Studies have proven the involvement of the neutrophil in all these injuries as a result of proteolytic enzymes secreted from cytoplasmic granules, free radicals produced by respiratory burst, cytokines released after tissue infiltration. In that context, ischemic preconditioning (IPC), that are short episodes of ischemia before ischemia reperfusion, was proved to be protective against these injuries with less activation of neutrophils. However the knowledge about the underlying mechanism operating in the neutrophil after surgical trauma, ischemia reperfusion and preconditioning is preliminary. Objective: To deeply analyze the impact of these conditions (ST, IR and IPC) on the neutrophil proteome and phosphoproteome. Methodology: We did hematological analysis along proteomics and phospho proteomics through high throughput nLC-MS/MS analysis by orbitrap using iTRAQ labeling, phospho peptide enrichments, and HILIC pre-fractionation. Neutrophils from control, ST, IR and IPC conditions after extraction were processed for proteomic analysis. Statistical package using R based on cluster analysis led to the detection of differentially regulated proteins in all conditions. Results: The evaluation of the hematological parameters before and after ST, IR or IPC on blood cells stated alteration in size, number and shape of lymphocytes, RBCs, platelets and specially neutrophils (granulocytes). In the analysis, a clear increase in neutrophil count after ST and IR with such increase prevented by IPC. A total of 393 proteins were found differentially regulated between control and trauma groups. Most of the common proteins found regulated in trauma and IR seem to be related to apoptosis (caspase-3), cell motility (PAK2) and signal transduction in IL5, IL6 and TNF and proteasomal degradation in neutrophil. Higher oxygen species production and dysfunction of directional neutrophil migration (PKC delta) with increase in the life span of neutrophils are early important events that can finally result into more tissue damage and infection. The total proteomic analysis of rat neutrophils after IPC led to the identification of 2437 protein groups assigned to five different clusters with significantly up and downregulated proteins in IR and IPC. Cluster based KEGG pathways analysis revealed up-regulation of chemokine signaling, focal adhesion, leukocyte transendothelial migration, actin cytoskeleton, metabolism and Fc gamma R mediated phagocytosis, whereas downregulation in ribosome, spliceosome, RNA transport, protein processing in endoplasmic reticulum and proteasome, after intestinal ischemic preconditioning. The phosphoregulated proteins containing domains and motifs in the regulated peptides after IR and IPC led to the identification of some of important players such as chemokine, Fc gamma, GPCR, migration and pro/anti-apoptotic pathways. The phosphoproteins from alternative splicing was the pathway presenting the most remarkable regulation with a p-value of 0.0001. The regulation in expression as well as in phosphorylation, the presence of motifs and domains led to the identification of kinases and phosphatases including Fgr, GRK2, PKC delta, ptpn6 and ptprc in neutrophils after IR whereas stk38, pkn1, syk, and inpp5d in neutrophil due to IPC. The highest protein-protein interaction was shown by Fgr and Ptp receptors. Conclusion: We concluded that the changed stimulus produced after ST, IR and IPC led to the huge alteration in proteins expression and phosphorylation events in the neutrophil proteome as mentioned in our work, that leads to final destructive and protective phenotype of neutrophils respectively