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.

L’objectif central de cette thèse de Doctorat était d’investiguer les dysfonctions mitochondriales qui surviennent précocement au cours de la phase compensée du remodelage ventriculaire pathologique et qui pourraient jouer un rôle causal dans la progression vers l’insuffisance cardiaque. Nos travaux antérieurs, réalisés à l’aide d’un modèle de surcharge volumique chronique induite par une fistule aorto-cavale (ACF) chez le Rat WKHA, ont montré qu’au cours du remodelage ventriculaire, les mitochondries développaient une vulnérabilité à l’ouverture du pore de perméabilité transitionnelle (PTP : un élément clé de la signalisation de la mort cellulaire) [1]. Ceci était observable au stade compensé du remodelage en absence des dysfonctions mitochondriales majeures typiquement observées dans le cœur insuffisant. Ces résultats nous ont amenés à suggérer que la vulnérabilité à l’ouverture du PTP pourrait constituer un mécanisme précoce favorisant la progression de la cardiopathie. Dans l’étude 1 de cette thèse, nous avons tenté de tester cette hypothèse en induisant une ACF chez deux souches de rats affichant de très nettes différences au niveau de la propension à développer l’insuffisance cardiaque : les souches WKHA et Sprague Dawley (SD). Nos études in vitro sur organelles isolées et in situ sur l’organe entier ont permis de confirmer que, dans le cœur ACF, les mitochondries développent une vulnérabilité à l’ouverture du PTP et à l’activation de la voie mitochondriale de la mort cellulaire lorsqu’exposées à des stress pertinents à la pathologie (surcharge calcique, ischémie-reperfusion [I-R]). Cependant, bien que comparativement aux animaux WKHA, les animaux SD démontraient un remodelage ventriculaire plus rapide et prononcé et une progression précoce vers l’insuffisance cardiaque, aucune différence n’était observable entre les deux groupes au niveau des dysfonctions mitochondriales, suggérant quelles ne sont pas à l’origine de la progression plus rapide de la pathologie chez la souche SD, à tout le moins en réponse à la surcharge volumique. Nous avons par la suite déterminé, à l’aide des mêmes approches expérimentales, si cette vulnérabilité mitochondriale était observable dans une cardiopathie d’étiologie différente, plus spécifiquement celle qui est associée à la dystrophie musculaire de Duchenne (DMD), une maladie génétique causée par une mutation de la protéine dystrophine. Nos études menées (études 2-4) sur de jeunes souris mdx (le modèle murin de la DMD) exemptes de tout signe clinique de cardiopathie n’ont révélé aucune différence au niveau des fonctions mitochondriales de base. Cependant, tout comme dans le modèle d’ACF, les mitochondries dans le cœur de souris mdx étaient significativement plus vulnérables à l’ouverture du PTP lorsque soumises à une I-R (étude 2). Par ailleurs, nous avons démontré que l’administration aiguë de sildénafil aux souris mdx induisait une abolition de l’ouverture du PTP et de ses conséquences signalétiques, une diminution marquée du dommage tissulaire et une meilleure récupération fonctionnelle à la suite de l’I-R (étude 3). Nous avons ensuite testé chez la souris mdx l’administration aiguë de SS31, un peptide anti-oxydant ciblé aux mitochondries, cependant aucun effet protecteur n’a été observé, suggérant que le tamponnement des radicaux libres est d’une utilité limitée si les perturbations de l’homéostasie calcique typiques à cette pathologie ne sont pas traitées simultanément (étude 4). Globalement, les travaux effectués au cours de cette thèse démontrent que la vulnérabilité à l’ouverture du PTP constitue une dysfonction précoce et commune qui survient au cours de remodelages ventriculaires pathologiques d’étiologies différentes. Par ailleurs, ces travaux suggèrent des stratégies d’intervention pharmacologiques ciblant ce processus, dont l’efficacité pour la prévention de l’insuffisance cardiaque demande à être établie. / The central objective of this doctoral thesis was to investigate the mitochondrial dysfunction that occurs early during the compensated phase of pathological ventricular remodeling and which may play a causal role in the progression to heart failure. Our previous work using a model of chronic volume overload induced by aorto-caval fistula (ACF) in rats WKHA showed that during the ventricular remodeling, mitochondria developed a vulnerability to permeability transition pore opening (PTP: a key component of cell death signaling) [1]. This was observed at the stage of compensated remodeling in the absence of major mitochondrial dysfunction typically observed in the failing heart. These results led us to suggest that the vulnerability to PTP opening could be a mechanism facilitating the progression of the cardiomyopathy. In our first study of this thesis we have attempted to test this hypothesis by inducing ACF in two strains of rats displaying sharp differences in the propensity to develop heart failure: WKHA strains and Sprague Dawley (SD). Our studies in vitro on isolated organelles and in situ on the whole organ have confirmed that, in the ACF heart, mitochondria develop a vulnerability to PTP opening and activation of mitochondrial cell death when exposed to stresses relevant to the pathology (calcium overload, ischemia-reperfusion [I-R]). However, SD animals compared to WKHA showed a more rapid and pronounced ventricular remodeling and early progression to heart failure, no difference was found between the two groups in terms of mitochondrial dysfunction, suggesting that this is not behind the more rapid progression of the disease in the SD strain, at least in response to volume overload. We subsequently determined, using the same experimental approaches, if this vulnerability was observed in mitochondria of heart disease from other etiology more specifically that associated with Duchenne muscular dystrophy (DMD), a genetic disease caused by a mutation of the protein dystrophin. Our studies (studies 2-4) on young mdx mice (the mouse model of DMD) free of clinical signs of heart disease showed no difference in basal mitochondrial functions. However, as in the model of ACF, the mitochondria of mdx mice heart were significantly more vulnerable to PTP opening when subjected to I-R (study 2). Furthermore, we demonstrated that acute administration of sildenafil to mdx mice abolished the PTP opening and its signaling consequences, markedly reduced of tissue damage and improved functional recovery following I-R (Study 3). We then tested in mdx mice acute administration of SS31, an antioxidant peptide that targets and accumulates in mitochondria. However, no protective effect was observed, suggesting that the buffering of free radicals have a limited utility if the typical perturbations of the calcium homeostasis in this disease are not treated simultaneously (Study 4). Overall, the work done during this thesis show that the vulnerability to PTP opening is a common and early dysfunction that occurs during pathological ventricular remodeling of different etiologies. Moreover, these studies suggest pharmacological intervention strategies targeting this process, whose effectiveness in preventing heart failure needs to be established.

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...

Cardiovascular diseases represent the most important health risk factors because they are responsible for more than 50% of total mortality. Among them, the ischemic heart disease is leading cause of mortality. From the whole spectrum of different cardioprotective phenomena we have selected: 1) adaptation to chronic normobaric hypoxia (CNH) as the traditional experimental model in our laboratory area and 2) protective effect of exercise which in recent years represents promising and clinically relevant protective mechanism. The whole thesis is based on two studies. Aim of the first study was to characterize the expression of the main pro-inflammatory cytokine, TNF-α, in hearts of rats adapted to CNH. Chronic TNF-α inhibition by infliximab was used for discovering of certain role of TNF-α in CNH. We showed that increased myocardial level of TNF-α during adaptation to CNH was contributed via its receptor TNFR2 and nuclear factor κB-dependent activation of protective redox signalling with increased antioxidant defence. This adaptive pathway participates on the infarct size-limiting effect of CNH. Aim of the second study was find out whether exercise training and CNH could play synergy in cardiac protection in rats model. We reported that CNH and exercise reduced infarct size but their combination...

As conexinas são as principais proteínas componentes das junções do tipo GAP no fígado. Existem poucos estudos sobre o papel das junções GAP nas lesões hepáticas, principalmente nas células não-parenquimatosas. O processo de isquemia e de reperfusão (IR) é um fenômeno complexo que está muito presente na prática clínica e envolve inúmeras vias metabólicas, e acredita-se que a comunicação celular parece ser uma via importante de interação entre células hepáticas, que respondem às alterações da homeostase e insultas. O objetivo da pesquisa foi comprovar a participação da conexina Cx43 na lesão de IR hepática, em um modelo experimental em roedores. A utilização de animais deficientes para Cx43 (\"Knockout\" heterozigotos Cx43+/-) permitiu verificar a evolução da lesão de IR, na deficiência desta conexina. A lesão de IR foi induzida pelo clampeamento do pedículo hepático durante uma hora. Realizou-se então a comparação dos níveis de enzimas hepáticas entre os animais \"Knockout\" e o grupo controle. Com isso se comprovou que a presença da Cx43 melhora o prognóstico em lesões hepáticas. Atualmente, com medicações capazes de bloquear ou aumentar a presença da Cx43 e, tendo em vista os altos índices de doenças hepáticas no Brasil e no mundo, esta é uma importante contribuição nesta área do conhecimento / Connexins are the main protein components of the gap junctions in the liver. There are few studies on the role of gap junctions on liver injury, and they are mainly on non-parenchymal cells. The process of ischemia and reperfusion (IR) is a complex phenomenon that involves many metabolic processes, and we believe that cellular communication is an important interaction mechanism between hepatic cells that respond to alteration of homeostasis and insults. The aim of the project was to prove that connexin Cx43 plays an important role in lesions in hepatic IR in experimental rodent models. The use of Cx43 deficient models (knockout heterozygous Cx43+/-) allowed us to verify the evolution of the liver injury from IR in the deficiency of this connexin. The IR lesion was induced by clamping the hepatic pedicle for one hour. We then carried out a comparison of the levels of hepatic enzymes between the knockout mice and the control group. This demonstrated that the presence of Cx43 altered the prognosis for liver injury. Given that there are medications capable of blocking or increasing the presence of Cx43 selectively or not, and bearing in mind the high rates of hepatic pathologies in Brazil and the rest of the world, this article contributes to understanding of the participation of connexin 43 in liver injury during ischemia and reperfusion

Atualmente, define-se como translocação bacteriana o deslocamento de bactérias e/ou seus produtos, como as endotoxinas, da luz do TGI para sítios estéreis. A ocorrência de translocação bacteriana tem sido sugerida em diversos estudos experimentais e clínicos. Apesar de todos estes estudos sustentarem a hipótese da ocorrência de translocação bacteriana, eles não demonstram que a bactéria detectada no sangue e em sítios estéreis tem efetivamente origem no TGI do animal ou paciente. Portanto, o objetivo da primeira fase deste trabalho, consistiu no desenvolvimento de um modelo experimental que comprovasse que bactérias isoladas em sítios estéreis são realmente de origem intestinal e que pudesse posteriormente viabilizar o estudo da translocação bacteriana. Para isto, realizou-se a colonização de ratos através da inoculação, via gavagem, de solução de Enterococcus faecalis resistente a vancomicina (ERV) e E. coli produtora de Beta-lactamase de espectro estendido (ESBL). O perfil de resistência destas cepas foi utilizado como marcador. Posteriormente, este estudo avaliou a translocação bacteriana em ratos submetidos a isquemia-reperfusão hepática com e sem estase venosa intestinal, utilizando o modelo de colonização. Quarenta e seis animais foram divididos nos seguintes grupos: Grupo I (n=15) ratos submetidos a isquemia hepática e estase intestinal por 30 minutos, e 1h de reperfusão; Grupo II (n=15) ratos submetidos a 30 minutos de isquemia hepática parcial sem estase intestinal, e 1h de reperfusão;Grupo III (n=8) ratos controle que apenas sofreraam manipulação cirúrgica e Grupo IV (n=8) ratos controle não cirúrgico. Os grupos foram analisados em relação: a ocorrência de translocação bacteriana; proporção de animais com crescimento da cepa pré-definida de ERV e E. coli ESBL, por órgão ou tecido; Concentração de LPS no sangue portal e sistêmico. Os resultados obtidos evidenciaram presença marcante de crescimento microbiológico positivo para cepas inoculadas na maioria dos órgãos ou tecidos analisados nos diferentes grupos. Desta forma, conseguimos comprovar que a bactéria detectada em sítios estéreis tem efetivamente origem no TGI daquele animal. A translocação de bactérias, para os órgãos sólidos, ocorreu com maior freqüência nos ratos submetidos a isquemia e reperfusão com estase intestinal. A translocação bacteriana para o pulmão ocorreu com maior freqüência nos grupos cirúrgicos, inclusive controle, do que no controle não cirúrgico. A translocação de endotoxinas, medida pela concentração sangüínea sistêmica, ocorreu com maior intensidade nos ratos submetidos a isquemia e reperfusão hepática com estase intestinal. Não houve diferenças entre os grupos quanto a proporção de animais com cultura positiva no sangue sistêmico e porta e concentração de endotoxinas no sangue porta. / Bacterial translocation is defined as the passage of viable bacteria and/or their products, such as endotoxins, from inside the gastrointestinal tract (GIT) to normally sterile sites. Experimental studies demonstrate that increase of the permeability of the intestinal mucosa and other conditions such as intestinal bacterial overgrowth or host immune deficiency may be associated with this phenomenon. The occurrence of bacterial translocation has been suggested in some experimental and clinical studies. Although all these studies sustained the occurrence of the bacterial translocation but they did not demonstrate that it has effectively origin in the TGI of the animal or patient. The first objective of this study was to develop a GIT colonization experimental model in rats with resistant Enterococcus faecalis (E.faecalis) and E.coli, to be used in further studies of bacterial translocation intending, The resistance profile of these strains is used as a marker. Afterwards, this study evaluated the bacterial translocation in rats submitted to hepatic ischemic-reperfusion with or without intestinal vein stasis. Forty six animals were used as follows: Group I (n=15) mice submitted to ischemic hepatica and intestinal stasis for 30 minutes, and 1h of reperfusion; Group II (n=15) mice submitted to partial ischemic hepatica for 30 minutes without intestinal stasis, and 1h of reperfusion; Group III (n=8) control of mice which only suffered surgical manipulation and Group IV (n=8) Group of mice without surgical control. The groups were analyzed concerning: the occurrence of bacterial translocation; proportion of animals with predefined ERV and E.Coli ESBL growth increase per organ or tissue; LPS concentration in the portal and systemic blood. The results demonstrated remarkable appearance of positive microbiological growth for inoculated stains mostly in the analyzed tissues within the different groups. By this way, we were able to prove that the bacteria present in sterile sites have effectively their origin in the TGI of that animal. The bacteria translocation in solid organs occurred frequently in group I submitted to the ischemia-reperfusion with intestinal stasis. The bacterial translocation in lung occurred more frequently in the surgical groups including chirurgical control. The endotoxin in systemic blood concentration occurred more intensively in group I submitted to the ischemia-reperfusion with intestinal stasis.

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