Dissertations / Theses on the topic 'Postconditioning'

Ischaemia and reperfusion in the heart leads to necrotic cell death (infarction) and contractile dysfunction (stunning). Ischaemic preconditioning (non-lethal periods of ischaemia prior to a longer ischaemic episode) and postconditioning (modified reperfusion) both reduce infarct size and are thought to act via activation of reperfusion injury salvage kinase (RISK) pathways to prevent the opening of the mitochondrial permeability transition pore (MPTP) at reperfusion. Catecholamines are released centrally and locally during myocardial ischaemia and activate adrenoceptors. This investigation was concerned with the roles of p-adrenoceptor activation in preconditioning and postconditioning. Studies utilising isolated paced atrial and ventricular tissues demonstrated that preconditioning against stunning could be achieved by ischaemic preconditioning, or pre-treatment of tissues with isoprenaline, a p-adrenoceptor agonist. Both forms of protection were blocked by propranolol, a-adrenoceptor antagonist. Postconditioning was not protective in this model. A Langendorff model of regional ischaemia was used to determine effects of -adrenoceptor agonists and antagonists on infarct size. In this model, ischaemic postconditioning was blocked by timolol, a non-selective p-adrenoceptor antagonist. Formoterol, a-adrenoceptor agonist, given at reperfusion, mimicked postconditioning. The non-selective adrenoceptor agonist, adrenaline, when applied at reperfusion, worsened reperfusion contracture but had no effect on infarct size. The application of a selective-adrenoceptor antagonist (CGP-20712A) at reperfusion led to a reduction of infarct size whereas p2 (ICI-118,551) and p3 (SR-59230A) antagonists had the opposite effect. If the results are replicable in man in vivo they would be of clinical relevance because a commonly used class of drugs (p-adrenoceptor antagonists) have the potential to abrogate the protection of postconditioning. Another widely available class of drugs (P-adrenoceptor agonists) can have cardioprotective effects at reperfusion.

Background- Coronary heart disease is the leading cause of death in the Western world and targeting those strategies which limit the damage sustained as a result of a lethal myocardial infarction has been a major goal for many years. Two such strategies, ischaemic preconditioning and ischaemic postconditioning are the most powerful endogenous cardio-protective phenomena known to man. The mechanisms involved in the newly described phenomenon of ischaemic postconditioning are not fully known. Additionally, the vast majority of studies investigating ischaemic preconditioning have been performed in normal hearts, leading to the suggestion that preconditioning is a "healthy heart" phenomenon. However, conflicting evidence exists as to whether the diabetic myocardium can be protected by ischaemic preconditioning (IPC). This thesis examines the mechanisms involved in these strategies of cardioprotection: (1) ischaemic postconditioning in normal hearts, and (2) ischaemic pre- and postconditioning in type II diabetic hearts. Methods and Results- Using a Langendorff isolated rat heart model we demonstrated that ischaemic postconditioning significantly reduced myocardial infarct size in normal rat hearts, and that this effect was comparable to that of ischaemic preconditioning. Western blot analysis demonstrated for the first time that postconditioning-induced protection is mediated via the PI3K-Akt pro- survival signalling cascade and its downstream targets, namely, eNOS and p70S6K. However, we found that type II diabetic rat hearts could not be protected using the same postconditioning protocol as a result of insufficient Akt phosphorylation. Conversely, the type II diabetic myocardium can be protected by ischaemic preconditioning but the threshold required to achieve this protection is elevated compared to non-diabetic hearts. This elevation in threshold is required to achieve sufficient phosphorylation of Akt, to execute the protective signal induced by ischaemic preconditioning. Conclusion- Our study demonstrates that in normal hearts, ischaemic postconditioning is a powerful strategy for myocardial protection and is mediated via the PI3K-Akt pro-survival signalling cascade. However, presumably due to differences in cellular signalling physiology, ischaemic postconditioning does not have a similar effect in type II diabetic hearts, and the beneficial effect of ischaemic preconditioning is only seen if the preconditioning stimulus is increased sufficiently to achieve enough Akt phosphorylation to mediate the protective signal. This suggests that the human diabetic population may be more resistant to the protective effects of IPC, but that provided the preconditioning stimulus is sufficient, the diabetic myocardium can be protected.

Thesis (MScMed)--Stellenbosch University, 2008.
ENGLISH ABSTRACT: It has recently been observed that the application of multiple short cycles of reperfusion and ischaemia, at the onset of reperfusion, elicits cardioprotection against injury due to prior sustained ischaemia. This phenomenon has been termed “postconditioning” (postC) and is of special interest due to its clinical applicability. Although much work has been done to delineate the mechanism of protection, there is still controversy regarding the precise algorithm of postC, the importance of the reperfusion injury salvage kinases (RISK), as well as uncertainty about the possible role of p38 MAPK and the protein phosphatases in postC cardioprotection. The aims of this study were therefore: I. To develop and characterise a cardioprotective postC protocol in the ex vivo rat heart, using both the retrogradely perfused and working heart models. II. To characterise the profiles of PKB/Akt, ERK p42/p44 and p38 MAPK associated with the postC intervention. III. To investigate the possible role of the serine/threonine protein phosphatases type 1 and type 2A (PP1 and PP2A) in the mechanism of postC. Hearts from male Wistar rats were perfused in both the retrograde Langendorff (at a perfusion pressure of 100 cmH2O and diastolic pressure set between 1 and 10 mmHg) and working heart models (preload: 15 cmH20 and afterload: 100 cmH20). Several different postC protocols were tested for their cardioprotective effect, as analysed by infarct size (IFS; determined by triphenyltetrazolium chloride (TTC) staining) and functional recovery. Experimental parameters tested were the number of cycles (3,4 or 6), the duration of the cycles (10, 15, 20 or 30 seconds), the method of application (regional or global) and temperature during the intervention (36.5 or 37 °C). Different sustained ischaemic insults were also utilised: 35 minutes regional (RI) or 20, 25, 30 and 35 minutes global ischaemia (GI). Hearts treated with a cardioprotective postC intervention or standard reperfusion after sustained ischaemia, were freeze-clamped at 10 and 30 minutes reperfusion in both perfusion models. Tissue samples were then analyzed using Western blotting, probing for total and phosphorylated PKB/Akt, ERK p42/p44 and p38 MAPK. The contribution of PKB/Akt and ERK p42/p44 activation to cardioprotection was also investigated by administration of inhibitors (A6730 and PD098059 respectively) in the final 5 minutes of ischaemia and the first 10 minutes of reperfusion, in the presence and absence of the postC intervention. The effect of these inhibitors were analyzed in terms of IFS and kinase profiles. The possible role of the phosphatases in postC was investigated by observing the effect of cantharidin (a PP1 and PP2A inhibitor) treatment directly before sustained ischaemia (PreCanth) or in reperfusion (PostCanth), in the presence and absence of postC, on IFS and kinase profiles. A postC protocol of 6x10 seconds global reperfusion / ischaemia, at 37°C, was found to give the best and most consistent reduction in infarct size in both the Langendorff (IFS in NonPostC: 47.99±3.31% vs postC: 27.81±2.49%; p<0.0001) and working heart (IFS in NonPostC: 35.81±3.67% vs postC: 17.74±2.73%, p<0.001) models. It could however only improve functional recovery in the Langendorff model (after 30 minutes GI: rate pressure product (RPP) recovery: NonPostC = 12.27±2.63% vs postC = 24.61±2.53%, p<0.05; and after 35 minutes GI: left ventricular developed pressure (LVDP) recovery: NonPostC = 28.40±7.02% vs postC = 48.49±3.14%, p<0.05). This protection was associated with increased PKB/Akt (NonPostC: 0.88±0.26 AU (arbitrary unit) vs postC: 1.65±0.06 AU; p<0.05) and ERK p42 (NonPostC: 2.03±0.2 AU vs postC: 3.13±0.19 AU; p<0.05) phosphorylation. Inhibition of PKB/Akt activation with A6730 (2.5 μM) abrogated the infarct sparing effect of postC. Administration of cantharidin, either before of after ischaemia, in the absence of postC, conferred an infarct sparing effect (IFS in PreCanth: 15.42±1.80%, PostCanth: 21.60±2.79%; p<0.05) associated with an increase in the phosphorylation of MAPK p38 (administration before ischaemia: NonCanth: 1.52±0.26 AU vs PreCanth: 2.49±0.17 AU, p<0.05; and administration after ischaemia: NonCanth: 5.64±1.17 AU vs PostCanth: 10.69±1.29 AU, p<0.05) and ERK p42 (when administered in reperfusion; NonCanth: 2.24±0.21 AU vs PostCanth: 3.34±0.37 AU; p<0.05). Cantharidin treatment combined with the postC intervention did not elicit an additive infarct sparing effect (postC: 17.74±2.72%, PreCanth-postC: 13.30±3.46% and PostCanth-postC: 15.39±2.67%). In conclusion: a postC protocol of 6x10 seconds global ischaemia / reperfusion, at 37°C, confers the best infarct sparing effect in both the Langendorff and working rat heart models. This protection is associated with ERK p42 and PKB/Akt phosphorylation, although only PKB/Akt is necessary for cardioprotection. We could not find evidence for PP1 and PP2A involvement in postC, although inhibition of these phosphatases per se does elicit an infarct sparing effect. The latter observation suggests that phosphatase activation during ischaemia / reperfusion is potentially harmful.
AFRIKAANSE OPSOMMING: Dit is onlangs waargeneem dat toediening van meervoudige siklusse herperfusie / iskemie, met die aanvang van herperfusie, die hart teen iskemie / herperfusie beskadiging beskerm. Hierdie verskynsel, bekend as postkondisionering (postC), geniet tans baie aandag vanweë die kliniese toepaslikheid van die ingreep. Ten spyte van intensiewe navorsing om die betrokke meganisme van beskerming vas te stel, is daar steeds kontroversie oor die presiese algoritme van die ingreep, asook die betrokkenheid van die sogenaamde iskemie herperfusie oorlewings kinases (RISK). Daar bestaan ook onsekerheid oor die rol van die stres-kinase, p38 MAPK, asook die proteïen fosfatases in die meganisme van beskerming teen iskemiese besering. Hierdie studie het dus drie doelstellings gehad: I. Ontwikkeling van ‘n postC protokol wat beskerming ontlok in die rothart ex vivo, deur gebruik te maak van beide die retrograad geperfuseerde ballon model, asook die werkhart model. II. Analiese van die profiele van die kinases PKB/Akt, ERK p42/p44 en p38 MAPK tydens herperfusie van postC en kontrole (NonPostC) harte. III. Ondersoek na die moontlike rol van die serien / treonien proteïen fosfatases tipe 1 en tipe 2A (PP1 en PP2A) in die meganisme van postC beskerming. Harte van manlike Wistar rotte is geperfuseer in beide die retrograad geperfuseerde ballon (d.i. die Langendorff) model (teen ‘n konstante perfusie druk van 100 cmH20 en ‘n diastoliese druk gestel tussen 1 en 10 mmHg), asook die werkhart model (teen ‘n voorbelading van 15 cmH20 en ‘n nabelading van 100 cmH20). Verskeie moontlike postC protokolle is getoets vir hul vermoë om kardiobeskerming te ontlok, in terme van funksionele herstel en infarktgrootte (IFS), soos bepaal deur trifenieltetrazolium chloried (TTC) kleuring. Die eksperimentele veranderlikes tydens die postC protokol wat ondersoek is, sluit in: die aantal siklusse (3, 4 of 6), die duur van die siklusse (10, 15, 20 of 30 sekondes), die wyse van postC toediening (streeks of globaal) en laastens die temperatuur tydens die ingreep (36.5 of 37 °C). Daar is ook gebruik gemaak van verskillende periodes iskemie: 35 minute streeks iskemie (RI), asook 20, 25, 30 en 35 minute globale iskemie (GI). Na 10 of 30 minute herperfusie is harte wat blootgestel is aan ‘n kardiobeskermende postC ingreep of gewone standaard herperfusie na iskemie, in beide perfusie modelle, gevriesklamp. Die weefsel proteïen-inhoud is verder geanaliseer deur van die Western blot tegniek gebruik te maak vir bepaling van die totale en fosforileerde vlakke van PKB/Akt, ERK p42/p44 en p38 MAPK. Die funksionele belang van PKB/Akt en ERK p42/p44 is verder ondersoek deur die effek van ‘n geskikte inhibitor (onderskeidelik A6730 en PD098059, toegedien tydens die laaste 5 minute van iskemie en die eerste 10 minute van herperfusie), in die teenwoordigheid en afwesigheid van die postC ingreep, op infarktgrootte en kinase aktiwiteit te monitor. Die moontlike rol van proteïen fosfatases in postC is ondersoek deur die effek van cantharidin (‘n PP1 en PP2A inhibitor) op infarktgrootte en kinase profiele te ondersoek. Cantharidin is óf onmiddelik voor iskemie óf tydens herperfusie toegedien, in die aan – en afwesigheid van die postC ingreep. Daar is bevind dat ‘n postC protokol van 6x10 sekondes globale iskemie / herperfusie, teen 37°C, die mees effektiewe en konstante verlaging in infarktgrootte teweeg gebring het in beide die ballon model (IFS in NonPostC: 47.99±3.31% vs postC: 27.81±2.49%; p<0.0001), asook die werkhart (IFS in NonPostC: 35.81±3.67% vs postC: 17.74±2.73%, p<0.001). Funksionele herstel kon egter slegs ontlok word in die ballon model (na 30 minute GI: tempo druk produk (RPP) herstel: NonPostC = 12.27±2.63% vs postC = 24.61±2.53%, p<0.05; en na 35 minute GI: linker ventrikulêre ontwikkelde druk (LVDP) herstel: NonPostC = 28.40±7.02% vs postC = 48.49±3.14%, p<0.05). Die infarkt-besparende effek van postC was geassosieer met ‘n toename in die fosforilasie van beide PKB/Akt (NonPostC: 0.88±0.26 AU (arbitrêre eenhede) vs postC: 1.65±0.06 AU; p<0.05) en ERK p42 (NonPostC: 2.03±0.2 AU vs postC: 3.13±0.19 AU; p<0.05). Inhibisie van PKB/Akt met A6730 (2.5 μM) het die infarkt-besparende effek van postC opgehef. Inhibisie van PP1 en PP2A opsigself, deur toediening van cantharidin óf voor óf na iskemie (in die afwesigheid van postC), het ‘n infarkt-besparende effek ontlok (IFS in PreCanth: 15.42±1.80%, PostCanth: 21.60±2.79%; p<0.05). Hierdie kardiobeskerming was geassosieer met ‘n toename in die fosforilasie van beide p38 MAPK (met toediening voor iskemie: NonCanth: 1.52±0.26 AU vs PreCanth: 2.49±0.17 AU, p<0.05; en toediening na iskemie: NonCanth: 5.64±1.17 AU vs PostCanth: 10.69±1.29 AU, p<0.05), asook ERK p42, indien cantharidin toegedien is tydens herperfusie (NonCanth: 2.24±0.21 AU vs PostCanth: 3.34±0.37 AU; p<0.05). Kombinasie van cantharidin behandeling met postC toediening kon egter nie ‘n kumulatiewe infarkt-besparende effek uitlok nie (postC: 17.74±2.72%, PreCanth-postC: 13.30±3.46% en PostCanth-postC: 15.39±2.67%). In samevatting: ‘n PostC protokol van 6x10 sekondes globale iskemie / herperfusie, teen 37°C, ontlok die mees effektiewe infarkt-besparende effek in beide die ballon, sowel as die werkhart modelle. Alhoewel hierdie beskerming geassosieer is met ‘n toename in die fosforilasie van beide PKB/Akt en ERK p42/p44 tydens herperfusie, is dit slegs PKB/Akt wat van funksionele belang is in die meganisme van kardiobeskerming. Ons kon geen bewyse vind vir die betrokkenheid van PP1 en PP2A in postC beskerming nie, alhoewel inhibisie van hierdie fosfatases opsigself infarkt-besparend is. Laasgenoemde waarneming toon dat fosfatase aktivering tydens iskemie / herperfusie skadelike gevolge mag hê.

Includes bibliographical references (leaves 158-192).
Ischaemic postconditioning (IPostC) is a powerful protective mechanism that activates prosurvival intrinsic signalling cascades to limit reperfusion injury but the exact signalling pathway involved in this cardioprotective effect still remains unclear.

Thesis (M.A.) University of Missouri-Columbia, 2006.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (June 27, 2007) Includes bibliographical references.

Timely coronary artery reperfusion is essential to prevent myocyte death following myocardial infarction. The act of restoring blood flow however, paradoxically reduces the beneficial effects of reperfusion. This phenomenon, termed myocardial reperfusion injury, refers to the injury of cardiac myocytes that were viable immediately before reperfusion. Recent studies have shown that the timing and hemodynamic sequence of events which govern reperfusion can help to minimize the severity of reperfusion injury. The term postconditioning describes a modified form of reperfusion that involves a series of flow interruptions which confer significant cardioprotection to the heart. This thesis investigates ischemic postconditioning and endothelial nitric oxide synthase (eNOS) phosphorylation as cardioprotective therapies against reperfusion injury. In the first half of this thesis, we test the hypothesis that phosphorylation of eNOS serves as a cardioprotection nodal point for ischemic postconditioning. We show that phosphorylation of eNOS increases enzyme activity and that its product, nitric oxide, plays a critical role in cardioprotection. A number of cardiac dysfunctions arise after reperfusion and we address the effects of postconditioning on infarct size and myocardial blood flow. The second half of this thesis introduces the use of magnetic relaxometry sensors to detect cardiac biomarkers. The ability to non-invasively measure infarct size in small animals would be helpful in studying models of myocardial ischemia-reperfusion injury. We investigate the use of implantable biosensors in vivo and show that the cumulative detection of cardiac biomarkers correlates with infarct severity.
Engineering and Applied Sciences

L’infarctus du myocarde, problème majeur de santé publique, est caractérisé par une nécrose cardiomyocytaire. Des séries d’occlusions-reperfusions courtes, réalisées avant l’ischémie (Préconditionnement (PréC) ischémique) ou au moment de la reperfusion (Postconditionnement (PostC) ischémique), protègent le coeur contre des lésions d’ischémie-reperfusion (IR). Les mécanismes intracellulaires impliqués restent obscurs. Nous avons étudié la signalisation intracellulaire du PréC et du PostC, et la cardioprotection qui en découle, sur un modèle de coeur isolé perfusé de souris. Le PréC ischémique peut être mimé par une activation directe du canal potassique mitochondrial ATP dépendant (mitoKATP), entraînant la mise en place d’une boucle d’auto-amplification incluant l’activation d’Akt, l’inhibition de GSK-3β et l’ouverture du mitoKATP. Cette réponse est liée à la production modérée d’espèces dérivées de l’oxygène par le mitoKATP et aboutie à une cardioprotection. La voie de développement Wnt est capable de moduler le PréC via GSK-3β. La voie de survie mTOR, cible de GSK-3β est aussi impliquée et pourrait induire des modifications traductionnelles lors de la réponse adaptative à l’IR. Le PostC ischémique peut également être mimé par activation directe du mitoKATP lors de la reperfusion, engendrant une protection du coeur et la mise en place d’une boucle d’auto-amplification similaire à celle du PréC, comprenant Akt, GSK-3β et le mitoKATP. Le PostC est dépendant de GSK-3β, mais contrairement au PréC, il n’impliquerait pas les voies Wnt et mTOR. Cette étude est la première démontrant que le PréC implique les voies de survie mTOR et de développement Wnt avec un rôle central de GSK-3β
Myocardial infarction is a major problem of public health, whose prognosis is related to the extent of the infarcted territory. Transient episodes of ischemia/reperfusion before ischemia (ischemic PreConditioning (PreC)), or at the onset of reperfusion (ischemic PostConditioning (PostC)) confer myocardium resistance to lethal ischemia. However the exact mechanism of PreC and PostC remains obscure. Our objectives were to examine signaling events during PreC and PostC and their effects on cardioprotection in an isolated mouse heart model. We provide evidence that pharmacological PreC by direct activation of mitoKATP, like ischemic PreC, involve an amplification loop involving ROS production and resulting in a sustained down-regulation of GSK-3β via Akt activation and a constant opening of mitoKATP. The mTOR pathway is a target of this loop and participates to cardioprotection. Disruption of Wnt pathway by sFRP1 modulates this loop inducing GSK-3β activation. This is the first evidence that PreC involves both a pro-survival mTOR pathway and an embryonic developmental Wnt pathway targeting GSK-3β. During ischemic and pharmacological PostC, the same amplification loop is activated, including Akt, GSK-3β and the mitoKATP. Unlike PreC, PostC did not induce the mTOR survival pathway: neither phosphorylation of mTOR nor of its targets p70S6K and 4E-BP1 were observed. However, cardiac overexpression of a Wnt antagonist, impairing PreC through GSK3-β, was unable to abolish cardioprotection afforded by PostC. PostC signaling differs from the preC pathway. Despite these discrepancies, GSK-3β plays a key role in both types of cardioprotection

Les maladies cardiovasculaires sont une des principales causes de morbidité et de mortalité au monde. La plus courante est l’infarctus du myocarde définit pathologiquement par la mortalité cellulaire dû à une ischémie prolongée d’une partie du ventricule gauche. L'ischémie est caractérisée par un apport sanguin insuffisant causé par une obstruction d’une artère coronaire. La restauration, en clinique, du flux sanguin, appelée reperfusion, est considérée comme la méthode la plus efficace contre les dommages ischémiques. Paradoxalement, cette restauration du flux sanguin est associée à une exacerbation de la lésion tissulaire, entraînant alors des lésions d'ischémie-reperfusion (I/R). Dans le but de limiter ces lésions, le conditionnement ischémique myocardique est une avancée majeure dans le domaine de la cardioprotection. Ce protocole confère ses effets cardioprotecteurs via le recrutement de divers mécanismes endogènes suivant l’activation de deux voies intracellulaires : la voie RISK (Reperfusion Injury Salvage Kinase) et/ou la voie SAFE (Survivor Activator Factor Enhancer). Ces voies impliquent l'activation de différentes cascades de signalisation et de protéines kinases. En particulier, concernant la voie SAFE, le transducteur de signal et l'activateur de transcription-3 STAT3, a été identifié comme un acteur clé dans le postconditionnement ischémique (PostCI). Il est suggéré que les effets cardioprotecteurs attribués à STAT3 soient liés à ses effets en tant que facteur de transcription et en tant que régulateur de l’activité mitochondriale, mais tout n’est pas encore connu. En revanche, il est admis que STAT3 est activé par la phosphorylation ciblant les résidus tyrosine 705 et sérine 727. Dans nos travaux actuels, nous avions initialement pour objectif d’étudier les rôles cardioprotecteurs mitochondriaux de STAT3 après une I/R et un PostCI. Cependant, nous n'avons pas été en mesure de détecter STAT3 dans les mitochondries de cardiomyocytes adultes de souris, dans des conditions basales et de stress, en utilisant différentes approches. Fait intéressant, nous avons montré une localisation exclusive de STAT3 dans les myocytes cardiaques adultes, le long des tubules T, et nous avons mis en évidence les inconvénients des techniques précédemment utilisées.Outre les rôles putatifs de STAT3 dans les mitochondries, nous avons ciblé ses effets dans la signalisation et la génomique au cours de l'I/R et du PostCI. Nous avons tout d’abord cherché à déterminer, pendant l’I/R et le PostCI, la cinétique temporelle d’activation de STAT3 et des autres kinases de la voie RISK, notamment Akt et les MAPK ERK1 / 2, JNK et p38. En outre, nous avions pour objectif d’étudier les liens entre les voies SAFE et RISK en déchiffrant les liens entre STAT3 et les kinases RISK au cours du PostCI. Nous avons montré qu’après une ischémie et un temps court de reperfusion, STAT3 et ERK1/2 sont activés, et que l’utilisation d’un PostCI active d’autant plus STAT3 en induisant exclusivement la phosphorylation de sa tyrosine. Nous avons également montré que l’interconnexion entre les voies SAFE et RISK, dans le protocole PostCI utilisé, se fait par STAT3 et ERK1/2. À partir de ces résultats, nous nous sommes dirigés vers la génomique grâce à laquelle nous avons étudié l'activité de STAT3 au cours de l'IPoC. À cet égard, nous avons montré que STAT3 est impliqué dans la régulation de la réponse inflammatoire au cours de la PostCI. Dans l’ensemble, cette étude présente une approche globale des fonctions mitochondriales, de signalisation et génomiques de STAT3 dans le contexte de la protection cardiaque
Cardiovascular diseases are leading causes of morbidity and mortality worldwide. Among the mostly prevailing cardiovascular diseases is myocardial infarction, which is pathologically defined as myocardial death due to a prolonged ischemia. Ischemia is an insufficient supply of blood caused by a blockade in the coronary arteries. The early restoration of blood flow is considered the most effective method against the ischemic lesions. Paradoxically, this blood flow restoration is associated with an exacerbation of the tissue injury, leading to the ischemia-reperfusion (I/R) injury. To avoid this injury, the myocardial ischemic conditioning protocol has rejuvenated the field of cardioprotection. This protocol confers its cardioprotective effects via recruiting various endogenous mechanisms following the activation of two intracellular pathways: the reperfusion injury salvage kinase (RISK) or survivor activator factor enhancer (SAFE) pathways. These pathways involve the activation of different signaling cascades and protein kinases. Zooming in through the SAFE pathway, the signal transducer and activator of transcription-3, STAT3, has been identified as a prominent key player in ischemic postconditioning (IPoC). The cardioprotective effects attributed to STAT3 are suggested to be linked to its roles as a transcription factor and as a regulator of the mitochondrial activity, but these are not well studied and elaborated. STAT3 is activated by phosphorylation, which targets the tyrosine 705 and serine 727 residues. In our current work, we initially aimed to investigate the mitochondrial cardioprotective roles of STAT3 following I/R and IPoC. However, we were not able to detect STAT3 in the mitochondria of adult mouse cardiomyocytes under variousbasal and stress conditions using different approaches. Interestingly, we showed an exclusive STAT3 pattern in adult cardiac myocytes, along the T-tubules, and highlighted drawbacks of previously used techniques. Aside from the mitochondrial roles of STAT3, we targeted its signaling and genomic roles during I/R and IPoC. We first aimed to determine, during I/R and IPoC, the temporal kinetics of activation of STAT3 and the other kinases of the RISK pathway including Akt and the MAPKs ERK1/2, JNK and p38. In addition, we aimed to decipher the interlink between the SAFE and RISK pathways through deciphering the interlink between STAT3 and the RISK kinases following IPoC. We showed that a short reperfusion time activates STAT3 and ERK1/2 following ischemia, and that the application of IPoC further activates STAT3 through inducing its tyrosine phosphorylation. We also showed that the interlink between SAFE and RISK pathways, in the IPoC protocol we used, is through STAT3 and ERK1/2. From this signaling level, we moved toward the genomic level whereby we investigated the genomic activity of STAT3 during IPoC. In this regard, we have shown that STAT3 is involved in the regulation of the inflammatory response during IPoC. Overall, this study presents a global approach of STAT3’s mitochondrial, signaling and genomic functions in the context of cardiac protection

Thesis (MSc)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Context: Cardiovascular diseases are the leading cause of death globally. Myocardial infarction is responsible for the highest number of deaths due to cardiovascular disease. Objective: We have previously shown that acute benfotiamine administration at the onset of reperfusion is associated with decreased infarct size and preserved contractile function in response to ischemia-reperfusion. We aimed to evaluate the involvement of the phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) pro-survival signaling pathways in mediating these cardioprotective effects. Materials and Methods: Part One - Hearts were rapidly excised from Wistar rats and mounted on a Langendorff perfusion apparatus. After stabilization, hearts were subjected to 30 minutes of regional ischemia and 120 minutes of reperfusion. The control group received no treatment. Experimental groups were treated with 100 μM benfotiamine ± 0.1 μM Tyrphostin AG490 or Wortmannin (inhibitors of JAK2 and PI3K, respectively), dissolved in dimethyl sulfoxide. The vehicle control group received an equivalent dose of dimethyl sulfoxide. All treatments were administered for 20 minutes at the onset of reperfusion. Functional parameters were measured throughout, to test the effects of benfotiamine ± pro-survival pathway inhibitors on functional recovery. In addition, hearts were stained with Evans blue and triphenyltetrazolium chloride to assess the effects of benfotiamine ± pro-survival pathway inhibitors on infarct size. Part Two - Hearts that were perfused ± 30 minutes of global ischemia and ± 20 minutes of benfotiamine administration, were used to assess PI3K/Akt and JAK/STAT signaling in response to ischemia-reperfusion and benfotiamine treatment. As with previous experiments, benfotiamine was administered at a concentration of 100 μM, at the onset of reperfusion. Tissues were assessed by Western blot analysis. Results: 20 minutes of acute benfotiamine administration at the onset of reperfusion led to a decrease in infarct size (35.6 ± 2.4% vs. 55.7 ± 5.0% [p<0.05]). Inhibition of PI3K/Akt signaling by addition of Wortmannin abrogated this infarct-limiting effect (51.5 ± 1.3% vs. 35.6 ± 2.4% [p<0.05]). However, inhibition of JAK/STAT signaling had no effect. There were no significant differences in left ventricular developed pressure, coronary flow rate or heart rate during the experiments. In addition, 20 minutes of acute benfotiamine administration at the onset of reperfusion lead to an increase in phospho-FOXO/FOXO in the cytosolic fraction, but no significant change in phospho-STAT3/STAT3 in the nucleus. Conclusions: Our results suggest that acute benfotiamine administration at the onset of reperfusion may act to reduce infarct size via activation of PI3K/Akt pro-survival signaling.
AFRIKAANSE OPSOMMING: Konteks: Kardiovaskulêre siekte is die hoofoorsaak van sterftes wêreldwyd. Miokardiale infarksie is verantwoordelik vir die grootste aantal sterftes weens kardiovaskulêre siekte. Doel: Ons het voorheen getoon dat akute benfotiamientoediening met die aanvang van reperfusie geassosieer is met „n verkleining in die infarkgrootte, en dit het verder ook die kontraktiele funksie in reaksie op ischemie-reperfusie behou. Ons doel was om die betrokkenheid van die fosfatidielinositol 3-kinase/Akt (PI3K/Akt) en Janus kinase/seintransduseerde en aktiveerder van transkripsie (JAK/STAT) pro-oorlewings seinweg in die mediasie van hierdie kardiobeskermende effekte te evalueer. Materiale en Metodes: Deel een - Harte is vinnig vanuit Wistarrotte verwyder en op die Langendorff-perfusieapparaat gemonteer. Na stabilisering is die harte blootsgestel aan 30 minute regionale ischemie en 120 minute reperfusie. Die kontrole groep het geen behandeling ontvang nie. Eksperimentele groepe is met 100 μM benfotiamien ± 0.1 μM Tirfostien AG490 of Wortmannin (inhibeerders van JAK2 en PI3K, onderskeidelik) behandel, opgelos in dimetielsulfoksied. Die draer-kontrole groep het „n ekwivalente dosis van dimetielsulfoksied ontvang. Alle behandelings is toegedien vir 20 minute aan die begin van die reperfusie. Funksionele parameters is deurgaans gemeet om te toets vir die effekte van benfotiamien ± pro-oorlewingsweg inhibeerders op funksionele herstel. Verder is die harte met Evans-blou en trifenieltetrazoliumchloried gekleur om die effek van benfotiamien ± pro-oorlewingsweg inhibeerders op die infarkgrootte te bepaal. Deel twee - Harte is vir ± 30 minute perfuseer met globale ischemie en ± 20 minute met benfotiamientoediening. Dit was gebruik om PI3K/Akt en JAK/STAT seine as gevolg van ischemie-reperfusie en benfotiamienbehandeling te ondersoek. Soos met die vorige eksperimente, is benfotiamien toegedien by ‟n konsentrasie van 100 μM met die aanvang van reperfusie. Weefsel is ondersoek deur middel van Western blot analise. Resultate: 20 minute van akute benfotiamientoediening, met die aanvang van reperfusie, het tot „n verkleining in die infarkgrootte (35.6 ± 2.4% vs. 55.7 ± 5.0% [p<0.05]) gelei. Inhibering van die PI3K/Akt seinweg deur toediening van Wortmannin het die infark-beperkende effek opgehef (51.5 ± 1.3% vs. 35.6 ± 2.4% [p<0.05]). Inhibering van JAK/STAT seine het egter geen effek getoon nie. Daar was geen beduidende verskille in linkerventrikulêr-ontwikkelde druk, koronêre-vloeitempo of harttempo tydens die eksperimente nie. Verder, 20 minute van akute benfotiamientoediening met die aanvang van reperfusie het „n toename in fosfo-FOXO/FOXO in die sitosoliese-fraksie veroorsaak, maar geen beduidende verandering in fosfo-STAT3/STAT3 is in die nukleus waargeneem nie. Gevolgtrekkings: Ons resultate suggereer dat akute benfotiamientoediening met die aanvang van reperfusie moontlik die infarkgrootte via aktivering van die PI3K/Akt pro-oorlewingsein kan verklein.

L'infarctus du myocarde (IDM) est consécutif à une occlusion coronaire provoquant une ischémie prolongée. La taille de l'infarctus est le déterminant majeur de la récupération du myocarde et de la survie du patient. La reperfusion coronaire la plus précoce (par thrombolyse ou angioplastie) est reconnue comme étant le seul traitement recommandé. Cependant, malgré les effets bénéfiques, la reperfusion s'accompagne d'effets délétères appelés « lésions de reperfusion ». Récemment, l'efficacité du postconditionnement ischémique (PostC) a été démontrée en clinique. Il permet, lorsqu'il est utilisé au cours de l'angioplastie primaire ou de sauvetage, de réduire significativement la taille de l'infarctus. L'application du protocole de PostC reste cependant limitée aux patients admis dans les centres d'angioplastie, ce qui exclut tous les patients thrombolysés. Afin d'améliorer la cardioprotection, il est nécessaire de déterminer la fenêtre thérapeutique temporelle optimale d'administration du PostC. De plus, il est nécessaire de développer des stratégies thérapeutiques nouvelles, adjuvants de la reperfusion, qui pourront être administrées dès que le diagnostic d'IDM est posé. L'objectif de notre travail a donc été d'étudier l'influence du délai d'application du PostC sur l'efficacité de cardioprotection dans un modèle murin d'ischémie-reperfusion et de rechercher des substances pharmacologiques cardioprotectrices. Nos travaux révèlent que l'efficacité du PostC est maintenue s'il est appliqué les 30 premières minutes après le début de la reperfusion. D'autre part, nous avons évalué le potentiel thérapeutique de peptides inhibiteurs ciblant l'apoptose responsable de la mort cellulaire au cours de l'ischémie-reperfusion. L'utilisation de peptides Tat-BH4 et Pip2b-BH4 pour le traitement des lésions d'ischémie-reperfusion myocardique a fait l'objet d'une étude preuve-de-concept in vivo. Les constructions peptidiques inhibant l'interaction FAS-DAXX, injectées en intraveineuse au moment de la reperfusion, induisent une diminution de 50% de la taille d'infarctus et de l'apoptose. Ils ont fait l'objet d'un dépôt de brevet car ils constituent des outils pharmacologiques à haut potentiel thérapeutique
Myocardial infarction (MI) results from a coronary occlusion leading to severe ischemia. Infarct size is a major determinant of myocardial salvage and mortality. Prompt revascularization (either by thrombolysis or primary angioplasty) is recommended for AMI patients but leads to deleterious effects called "reperfusion injury". Recently, ischemic postconditioning (PostC) efficiency has been reported in patients. However, its application during primary angioplasty is limited to patients admitted in angioplasty centers thereby excluding thrombolysed patients. In order to improve cardioprotection, it is necessary to define the time window for optimal cardioprotection and to develop new pharmacological strategies for AMI patients. Our work, using an in vivo mouse model of ischemia-reperfusion, shows that PostC efficiency is maintained if applied the first 30 minutes after the onset of reperfusion. Furthermore, we evaluated the therapeutic potential of peptide inhibitors targeting apoptosis, which is responsible for cell death during ischemia-reperfusion. As a proof-of-concept study, the cardioprotective effects of Tat-BH4 and Pip2b-BH4 peptidic constructs have been revealed in vitro and in vivo. The peptidic constructs targeting FAS-DAXX interaction, injected intravenously as a single bolus at the time of reperfusion, reduced by 50% both infarct size and apoptosis. These pharmacological tools have been patented due to their high therapeutic potential

Orientador: Noma Sueli Pinheiro Modolo
Resumo: Introdução: a lesão por isquemia-reperfusão (LIR) é uma importante causa de lesãorenal aguda experimentada na prática clínica. A restauração da perfusão aos tecidosapós um período de isquemia inicia uma cascata de inflamação associada ao acúmulode íons, formação de espécies reativas de oxigênio (ERO), disfunção endotelial eativação imune. O condicionamento isquêmico é a aplicação de breves ciclos deinterrupção seguidas de restauração do fluxo sanguíneo, tendo o objetivo de adaptaros tecidos à isquemia. Pode ser aplicado antes do estímulo principal, como précondicionamento(PCI), ou depois, sendo denominado pós-condicionamento (PCoI).Metodologia: estudo experimental realizado com 40 ratos wistar, divididos em cincogrupos para análise comparativa: Sham (S): laparotomia; Controle (C): laparotomia e30 min de isquemia; Pré-condicionamento (PRE): laparotomia, PCI e 30 min deisquemia; Pré e Pós-condicionamento (PRE/POS): laparotomia, PCI, 30 min deisquemia e PCoI; Pós-condicionamento (POS): laparotomia, 30 min de isquemia ePCoI. A comparação entre os grupos foi realizada pela análise bioquímica sérica decreatinina, ureia, lipocalina associada à gelatinase de neutrófilos (NGAL) e histolologia.Resultados: apenas o grupo Sham apresentou valores estatisticamente menores dosmarcadores de lesão renal e menor incidência de lesão tubular renal à histologia(SAbstract: Background: Ischemia-reperfusion injury (IRI) is an unavoidable aspect of transplantation, as well as an important cause of acute kidney injury in clinical practice. Restoration of the blood supply after an ischemic period activates an inflammatory cascade associated with multiple processes, including ion accumulation, free reactive oxygen species (ROS) formation, endothelial dysfunction, and immune activation. Ischemic “conditioning” refers to the application of a brief series of ischemic periods followed by reperfusion in the setting of major ischemia. In ischemic preconditioning (IPC), the conditioning stimulus is applied before the major ischemic event, whereas in ischemic postconditioning (IPoC), it is applied after the event. Methods: Forty Wistar rats were randomized into five groups: Sham (S): laparotomy; Control (C): laparotomy and 30 min ischemia; Preconditioning (PRE): laparotomy, IPC, and 30 min ischemia; Preconditioning and Postconditioning (PRE/POST): laparotomy, IPC, 30 min ischemia, and IPoC; Postconditioning (POST): laparotomy, 30 min ischemia, and IPoC. Serum analyses of creatinine and neutrophil gelatinaseassociated lipocalin (NGAL) were performed, and renal histology was also examined. Results: Severe tubular injury and increases in creatinine were observed in all groups except the S group, and no significant differences were detected between the other groups (SDoutor

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Introdução: a lesão por isquemia-reperfusão (LIR) é uma importante causa de lesão renal aguda experimentada na prática clínica. A restauração da perfusão aos tecidos após um período de isquemia inicia uma cascata de inflamação associada ao acúmulo de íons, formação de espécies reativas de oxigênio (ERO), disfunção endotelial e ativação imune. O condicionamento isquêmico é a aplicação de breves ciclos de interrupção seguidas de restauração do fluxo sanguíneo, tendo o objetivo de adaptar os tecidos à isquemia. Pode ser aplicado antes do estímulo principal, como précondicionamento (PCI), ou depois, sendo denominado pós-condicionamento (PCoI). Metodologia: estudo experimental realizado com 40 ratos wistar, divididos em cinco grupos para análise comparativa: Sham (S): laparotomia; Controle (C): laparotomia e 30 min de isquemia; Pré-condicionamento (PRE): laparotomia, PCI e 30 min de isquemia; Pré e Pós-condicionamento (PRE/POS): laparotomia, PCI, 30 min de isquemia e PCoI; Pós-condicionamento (POS): laparotomia, 30 min de isquemia e PCoI. A comparação entre os grupos foi realizada pela análise bioquímica sérica de creatinina, ureia, lipocalina associada à gelatinase de neutrófilos (NGAL) e histolologia. Resultados: apenas o grupo Sham apresentou valores estatisticamente menores dos marcadores de lesão renal e menor incidência de lesão tubular renal à histologia (SBackground: Ischemia-reperfusion injury (IRI) is an unavoidable aspect of transplantation, as well as an important cause of acute kidney injury in clinical practice. Restoration of the blood supply after an ischemic period activates an inflammatory cascade associated with multiple processes, including ion accumulation, free reactive oxygen species (ROS) formation, endothelial dysfunction, and immune activation. Ischemic “conditioning” refers to the application of a brief series of ischemic periods followed by reperfusion in the setting of major ischemia. In ischemic preconditioning (IPC), the conditioning stimulus is applied before the major ischemic event, whereas in ischemic postconditioning (IPoC), it is applied after the event. Methods: Forty Wistar rats were randomized into five groups: Sham (S): laparotomy; Control (C): laparotomy and 30 min ischemia; Preconditioning (PRE): laparotomy, IPC, and 30 min ischemia; Preconditioning and Postconditioning (PRE/POST): laparotomy, IPC, 30 min ischemia, and IPoC; Postconditioning (POST): laparotomy, 30 min ischemia, and IPoC. Serum analyses of creatinine and neutrophil gelatinaseassociated lipocalin (NGAL) were performed, and renal histology was also examined. Results: Severe tubular injury and increases in creatinine were observed in all groups except the S group, and no significant differences were detected between the other groups (S