No description available.

Neutrophils are thought to orchestrate myocardial remodeling during the early progression to cardiac failure through the release of reactive oxygen species, antimicrobial peptides, and proteases. Although neutrophil activation may be beneficial at early stages of disease, excessive neutrophil infiltration detrimentally leads to cardiomyocyte death and tissue damage. The neutrophil-derived serine protease cathepsin G (CG) has been shown to induce neonatal rat cardiomyocyte detachment and apoptosis by anoikis1. However the role of inflammatory serine proteases in cardiac remodeling and cardiac regeneration in-vivo is still unknown. We showed that cardiac injection of neutrophil derived protease led to early cardiac dilatation and dysfunction characterized by an increase in matrix metalloprotease (MMP) activation and extracellular matrix degradation along with an increase in myocyte death by apoptosis. To assess the role of these serine proteases, we used mice lacking dipeptidyl peptidase I (DPPI), an enzyme involved in major inflammatory protease activation. DPPI deficient mice demonstrated a more robust functional recovery after ischemia reperfusion (IR) and myocardial infarction (MI) injury, as well as significantly reduced myocyte apoptosis, cardiac dilatation, infarct size and mortality rate. Meanwhile, our data showed increased groups of cardiac stem cells and proliferating cardiac cells in the MI 7-days DPPI knockout mice. We also found enhanced DPPI expression in response to pathological stress stimuli in mice. These findings reveal an unrecognized role of DPPI as a key mediator of post-ischemia cardiac injury and show that inflammatory derived proteases may contribute to the pathological cardiac remodeling and cardiac regeneration, and may be considered as novel target for future therapies. / Physiology

Acute myocardial infarction (AMI) is a leading cause of morbidity and mortality in the world (4). Restoration of coronary flow to the ischemic myocardium by interventions such as angioplasty, thrombolytic treatment or coronary bypass surgery is the current standard therapy for AMI (5). However, reperfusion of the ischemic myocardium may result in paradoxical cardiomyocyte dysfunction and worsen tissue damage, in a process known as “reperfusion injury” (6). Ischemic reperfusion (IR) injury may intensify pathological processes that contribute to the generation of oxyradicals, disturbances in cation homeostasis, and depletion of cellular energy stores, which may elicit arrhythmias, contractile dysfunction, and ultrastructural damage of the myocardium. These changes can lead to heart failure and ultimately sudden death. The exact mechanisms of IR injury are not fully known (7). Molecular, cellular, and tissue alterations such as cell death, inflammation, neurohumoral activation, and oxidat / Bioengineering

Since integrity and functions of vascular endothelial cells are greatly affected by shear stress, a variety of in vitro systems to subject endothelial cells under precisely controlled fluid conditions has been developed. Complicated designs of the conventional flow devices, however, have impeded such implementation. In the present study, we designed and developed a novel parallel-plate flow chamber (PPFC). It consists of multiple layers of different materials to adjust the required geometries of the chamber and provide a wide span of biomedical research applications. Because the chamber stacks separate layers to constitute the flow channel, different pieces can be easily removed or replaced. Moreover, the multilayer design only requires 2D cutting, which is easier and faster to manufacture. It is also capable of accepting up to four glass slides facing each other so that the flow within the channel is exclusively formed by endothelial cells. Furthermore, it minimizes the pressure loss across the chamber while maximizing the effective area of endothelial cells up to 96 cm2. Results from mathematical analysis and dye injection experiments showed that a uniform magnitude of shear stress is applied throughout the entire surface of endothelial cells. In addition, the morphological changes and attenuated gene expression of pro-inflammatory mediators were observed in endothelial cells exposed to the physiologically relevant shear stress. These findings indicate that our newly designed PPFC can provide a better in vitro system for versatile applications of biomedical research. The reperfusion of blood flow occurred in a number of conditions such as stroke and organ transplantation immensely augments tissue injury and can cause more severe damage than prolonged ischemia. The injuries caused by cessation and reperfusion of blood flow are closely related to the inflammatory reactions involving in endothelium-leukocyte cascade responding to a shear stress exerted by the flow. Shear stress is also known to play an important role in human chronic diseases including atherosclerosis, neurological disorders, and cancer metastasis. Therefore, it is important to investigate the transmission of mechanical stimuli such as shear stress to various complex endothelial cell signaling pathways which process as a whole is often referred as mechanotransduction. Shear stress-mediated signaling pathways have been known to trigger endothelial cell responses and contribute to the pathophysiology of human vascular diseases. The present study was designed to apply the novel PPFC to biomedical research, especially ischemia/reperfusion injury. The changes in mRNA and protein expression of inflammatory mediators in endothelial cells were analyzed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. RBE4 and HMEC-1 cells were either maintained in continuous laminar flow condition (Normal Flow) or subjected to 1 h of flow cessation followed by reperfusion of flow (Ischemia/Reperfusion) for 24 h. Ischemia/Reperfusion significantly up-regulated expression of inflammatory mediators, such as IL-6, MCP-1, ICAM-1, VCAM-1, and E-selectin, in microvascular endothelial cells. Furthermore, antioxidant pyrrolidine dithiocarbamate (PDTC) significantly attenuated ischemia/reperfusion-induced overexpression of pro-inflammatory mediators. These data indicates that our newly designed PPFC provide a better in vitro system for versatile applications of biomedical research. / Master of Science

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Mitochondrial structure and function are inextricably linked ("structure-function"), with decrements in structure-function evident across diseases. Barriers to new therapies include a complete understanding of the underlying molecular culprits, as well as effective mitochondria-targeted therapies that mitigate injury. In these works, we investigate the role of cristae-shaping factors like cardiolipin in health and disease. In a series of studies, we tested the effects of the cell-permeable tetrapeptides, elamipretide and a postulated peptide, (arginine-tyrosine-lysine-phenylalanine; "RYKF"), on the recovery of mitochondrial structure-function after injury. Elamipretide is a clinical-stage compound currently under investigation for genetic and age-related mitochondrial diseases, yet the mechanism of action is not completely understood. We used a combination of physiological models, mitochondrial imaging, and biomimetic membrane studies to test the hypothesis that elamipretide and RYKF-cardiolipin interactions improved mitochondrial structure-function. Post-ischemic treatment with elamipretide sustained mitochondrial function across electron transport chain complexes. Endogenous RYKF expression similarly improved mitochondrial respiration after peroxide and hypoxia nutrient deprivation injuries. Using two parallel electron microscopy paradigms, we show elamipretide and RYKF treatment led to maintenance of mitochondrial ultrastructure and notably, improved cristae interconnectedness. Finally, we utilized a novel biomimetic membrane system to model the pathological mitochondrial membrane and found that elamipretide and RYKF both improved biophysical pressure-area relationships through a mechanism that appears to involve aggregating cardiolipin. Our data indicate that targeting pathophysiological mitochondrial membranes with cationic, lipophilic peptides can improve bioenergetics by sustaining cristae networks and support interdependent relationships between mitochondrial structure and function. / Doctor of Philosophy / Mitochondria, the powerhouses of the cell, form energy networks that produce over 90% of the body’s energy. Mitochondrial dysfunction is implicated across diseases, yet no FDA-approved treatments exist that improve mitochondrial energy production. In this study, we tested the effects of elamipretide, a peptide that localizes to mitochondria. Although elamipretide is currently in clinical trials for several diseases characterized by energetic deficiencies, its mechanism of action is not fully understood. Since mitochondrial structure and function are directly linked, we modeled heart attacks in cultured cells and rat hearts to test the hypothesis that elamipretide and a postulated analog, RYKF, glue damaged mitochondrial membranes back together to preserve structure and function during disease. In hearts subjected to a heart attack, elamipretide significantly protected mitochondrial energy production. Similarly, RYKF protected mitochondrial function in muscle cells exposed to peroxide stress. In damaged hearts imaged with electron microscopy, elamipretide and RYKF treatment significantly improved mitochondrial structure and notably, improved the interconnectedness of mitochondrial energy networks. Furthermore, elamipretide and RYKF improved the integrity of diseased mitochondrial membranes. Together, these data support our hypothesis that elamipretide and RYKF act as mitochondrial adhesion molecules to protect mitochondrial structure and sustain energy production during disease.

Dysfunction within complex I (CI) of the mitochondrial electron transport system has been implicated in a number of disease states ranging from cardiovascular diseases to neuro-ophthalmic indications. Herein, we provide three novel approaches to model and study the impacts of injury on the function of CI. Cardiovascular ischemia/reperfusion (I/R) injury has long been recognized as a leading contributor to CI dysfunction. Aside from the physical injury that occurs in the tissue during the ischemic period, the production of high levels of reactive oxygen species (ROS) upon reperfusion, led by reverse electron transport (RET) from CI, causes significant damage to the cell. With over 700,000 people in the US set to experience an ischemic cardiac event annually, the need for a pharmacological intervention is paramount. Unfortunately, current pharmacological approaches to treat I/R related injury are limited and the ones that have shown efficacy have often done so with mixed results. Among the current approaches to treat I/R injury antioxidants have shown some promise to help preserve mitochondrial function and assuage tissue death. The studies described herein have provided new, more physiologically matched, methods for assessing the impact of potential therapeutic interventions in I/R injury. With these methods we evaluated the efficacy of the coenzyme-Q derivative idebenone, a proposed antioxidant. Surprisingly, in both chemically induced models of I/R and I/R in the intact heart, we see no antioxidant-based mechanism for rescue. The mechanistic insight we gained from these models of I/R injury directed us to further examine CI dysfunction in greater detail. Through the use of two cutting edge genetic engineering approaches, CRISPR/Cas9 and Artificial Site-specific RNA Endonucleases (ASRE), we have been able to directly edit the mitochondria to accurately model CI dysfunction in disease. The use of these genetic engineering technologies have provided first in class methods for modeling three unique mitochondrial diseases. The culmination of these projects has provided tremendous insight into the role of CI in disease and have taken a significant step towards elucidating potential therapeutic avenues for targeting decrements in mitochondrial function. / Doctor of Philosophy / Within the mitochondria, “the powerhouse of the cell,” exists a series of five enzyme complexes that produce 90% of the energy for our cells need to function. The largest of these enzymes, complex I (CI), plays an important role in ensuring proper mitochondrial function. Injury to CI contributes to a number of diseases, but surprisingly few options exist to treat complex I. One of the most prevalent forms of CI dysfunction can be seen in ischemia/ reperfusion injury, a form of which is most commonly recognized as a heart attack. Surprisingly, the American Heart Association reports that in the next year over 700,000 people in the US will suffer from an ischemic event. With such a profound impact on the population, the need for new therapeutic developments is extremely high. Some current therapeutic approaches have been shown to be effective at treating cardiac dysfunction, but few address the dysfunction that occurs in the mitochondria. Here we test both a method for modeling these ischemia/reperfusion-based injuries and a potential therapeutic for treating these injuries within the context of CI dysfunction. We further evaluate CI dysfunction by using both established genetic engineering approaches as well as a completely new method to model CI disease. Through the use of two cutting edge genetic engineering approaches, we have been able to directly edit components of the mitochondria to accurately model CI dysfunction in disease. The use of these genetic engineering technologies have provided a first-in-class method for modeling three unique mitochondrial diseases. The culmination of these projects has provided tremendous insight into the role of CI in disease and have taken a significant step towards elucidating potential therapeutic avenues for targeting decrements in mitochondrial function.

Thesis (MSc)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: The ultimate consequence of the interruption of blood flow to the myocardium is necrosis. In view of the prevalence of coronary artery disease in the general population, and the deleterious effects of myocardial ischaemia on myocardial tissue, it is important to develop new strategies to protect the myocardium against ischaemia. Necrosis of myocardial tissue has for a long time been considered to be the main component of the damage incurred by myocardial infarction. Recently the importance of the contribution of apoptotic cell death in the context of myocardial ischaemia/reperfusion injury has become apparent. There is a general agreement that early reperfusion is necessary to salvage myocardial tissue from cell death. Preconditioning is the phenomenon whereby brief episodes of ischaemia and reperfusion protect the heart against a subsequent longer period of ischaemia. This endogenous mechanism is the strongest form of protection against myocardial infarction that has yet been described. Apart from ischaemie preconditioning (IPC), protection can also be elicited with pharmacologic agents, such as activation of the beta-adrenergic receptor with isoproterenol. Ischaemie preconditioning protects the myocardium against necrosis, arrhythmias and apoptosis, and increases functional recovery upon reperfusion. Betaadrenergic receptor stimulated preconditioning (PPC) has been shown to improve post-ischaemie functional recovery, but it is not known whether it also protects against myocardial infarction and apoptosis. The signaling pathways involved in preconditioning have been extensively studied. A distinction is usually made between factors that act as triggers, or as mediators of protection. Triggers activate cellular responses before the onset of sustained ischaemia, and its involvement is demonstrated by showing that inhibitors of the trigger bracketing the preconditioning protocol can block its protective effect, or that transient administration with washout before sustained ischaemia can activate a protective effect. A mediator operates during sustained ischaemia, and its involvement is demonstrated by showing that infusion of an inhibitor of its action immediately prior to sustained ischaemia (without washout) can block its protective effect. Another approach to demonstrate a mediator role is to attempt to activate signal transduction pathways during sustained ischaemia. As it is not possible to infuse substances during ischaemia, activators are infused immediately prior to ischaemia without washout of the agent and subsequently its effect on protection is observed. It is clear that the evolutionary conserved stress activated pathways are involved in preconditioning. There are three pathways i.e., the extracellular receptor activated pathways (ERK), c-jun terminal activated kinases (JNK) and p38 mitogen-activated protein kinases (MAPK). The precise role of the p38 MAPK pathway has not been elucidated. Experimental evidence has suggested a role for the activation of p38 MAPK as a trigger, as well as a mediator of the protective effect of preconditioning. There is however also strong evidence that the attenuation of p38 MAPK activation during sustained ischaemia, rather than its activation, is responsible for the protection that is observed. Furthermore, the role of p38 MAPK has only been investigated in relation to its protection against necrosis, but not apoptosis. AIMS: The aim of this study was to: (I) Establish a model of preconditioning in neonatal cardiomyocyte cell culture. The reason was that such a model could potentially enable one to rapidly elucidate the signal transduction pathways in an environment without the influence of non-cardiac cells. (II) Investigate whether IPC and ~PC protect against necrosis and apoptosis. (III) Elucidate the role of the stress-activated kinase, p38 MAPK, in preconditioning. METHODS: 1. Neonatal rat cardiomyocyte cell culture model A viability assay with 3-[4,5- Dimethylthaizol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) was first developed using different concentrations - a concentration of 0.25% was found to be optimal to determine viability. Neonatal cardiomyocyte cell cultures were subjected to sustained simulated "ischaemia" by using either 5 mM KCN plus deoxyglucose (DOG) for 5 min or potassium cyanide (KCN) for 45 min. Some cell cultures were preconditioned with either chemical ischaemia (5 mM KCN for 5 min) or isoproterenol (10-7 M) for 5 min and 60 min reoxygenation before being exposed to sustained simulated ischaemia. 2. Isolated adult rat cardiomyocyte model Isolated cardiac myocytes were exposed to 2 hours of hypoxia, which was induced by pelletting the cells by centrifugation, and covering them with a thin layer of mineral oil. Some groups were preconditioned with either hypoxia for 10 min at 37° C or isoproterenol (10-7 M) for 5 min, followed by reoxygenation for 20 minutes. The trypan blue exclusion method and MTT method developed in the neonatal cardiomyocytes were used to assess viability. 3. Isolated perfused rat heart model 3.1 Infarct size was determined in a model of regional ischaemia by using tetrazolium staining and determining the area of necrosis (exclusion of tetrazolium) as a percentage of area at risk. These hearts were subjected to 35 min global ischaemia and 30 min reperfusion. Some groups were preconditioned by three cycles of 5 min global ischaemia or addition of isoproterenol (10-7 M) for 5 min, followed by 5 min reperfusion before the onset of sustained regional ischaemia. 3.2 p38 MAPK activation and markers of apoptosis: p38 MAPK activation was determined using antibodies against dual phosphorylated p38 MAPK (i.e. activated p38 MAPK). Apoptosis was measured by using antibodies against activated caspase-3, and against a fragment of PARP (PARP cleavage). For these experiments isolated rat hearts were exposed to global ischaemia for 25 min followed by 30 min reperfusion. Some groups were preconditioned with three cycles of 5 min global ischaemia. A global ischaemia model was used in order to have sufficient tissue available for the Western blot determinations. This necessitated a shorter period of sustained ischaemia, as the globally ischaemie heart does not recover sufficiently after a longer period of ischaemia such as is necessary in regional ischaemia experiments. 3.3 The role of p38 MAPK in ischaemie preconditioning was investigated by administration of SB 203580 (1IJM),a selective inhibitor of p38 MAPK, either bracketing the preconditioning (i.e. to determine its role as a trigger) or for 10 min immediately prior to sustained ischaemia (i.e. to determine its role as a mediator). The second approach was to use anisomycin, an activator of p38 MAPK, as a trigger (infusion for 10 min followed by wash out) or as a mediator (10 min immediately prior to sustained ischaemia) in the same model as used for determination of p38 MAPK activity. The infusion of anisomycin for 10 min has been shown to elicit activation of p38 MAPK to a similar extent as has been observed with an ischaemie preconditioning protocol. The endpoints used were infarct size and markers of apoptosis. RESULTS: 1. Neonatal rat cardiomyocyte cell culture model It was not possible to establish a model of preconditioning of neonatal cardiomyocytes that was consistently successful. It was therefore decided to abandon the attempts and to use a different cell model. 2. Isolated adult rat cardiomyocyte model Isolated adult cardiomyocytes were preconditioned successfully, but produced too little material to perform simultaneous determinations of cell viability and Western blots (p38 MAPK activation and markers of apoptosis). It was therefore decided to use the isolated perfused adult rat heart. 3. Isolated perfused adult rat heart model 3.1 Both IPC and PPCprotect against infarction and apoptosis: Using two models of preconditioning i.e., IPC and PPC, the protective effects of preconditioning were demonstrated convincingly against infarction (necrosis). IPC and PPC both caused a significant reduction in infarct size (12.2±1.4 and 15.2±2.6%) versus Non-PC hearts (29.6±2.9%) (p < 0.001). Both forms of preconditioning also protected against apoptosis, by significantly reducing the markers of apoptosis, caspase-3 activation and PARP cleavage. The protection afforded by both forms of preconditioning was accompanied by a marked decrease in activation of p38 MAPK upon reperfusion. The relationship between p38 MAPK and the protection that was elicited by preconditioning was then investigated, namely whether p38 MAPK acted as a trigger, or as a mediator of protection. To investigate the role of p38 MAPK as a mediator or a trigger in preconditioning, use was made of (i) a specific inhibitor of p38 MAPK activation i.e., SB 203580 and (ii) a known activator of p38 MAPK i.e., anisomycin. 3.2 p38 MAPK as a trigger of protection: Administration of SB 203580 during the IPC protocol and washed out before sustained ischaemia did not abolish the protective effect of ischaemie preconditioning, and resulted in a small, but significant increase in caspase-3 activation and PARP cleavage. On the other hand, activation of p38 MAPK with anisomycin for 10 min followed by washout also resulted in a significant reduction in necrosis (infarct size 14.9±2.2 versus 29.6±2.9% in Non-PC hearts) (p < 0.001) and both markers of apoptosis. The latter results suggested that p38 MAPK was a trigger of preconditioning. If this was the case, why didn't SB 203580 abolish the protection of IPC? The most likely explanation was that multiple protective mechanisms were activated during a multi-cycle protocol of ischaemic preconditioning, of which activation of p38 MAPK was only one. Inhibition of p38 MAPK with SB 203580 would therefore not be expected to block the activation of those mechanisms that were independent of p38 MAPK, but were still capable of protecting against necrosis or apoptosis. It is very interesting that a small increase in apoptosis was observed when SB 203580 was used in this situation, as it may indicate that the protection against apoptosis was more dependent on the activation of p38 MAPK than the protection against necrosis, as no effect was seen on infarct size. Another explanation could be that infarct size determination was not sensitive enough to detect such small effects. 3.3 p38 MAPK as a mediator of protection: Inhibition of p38 MAPK activation with SB 203580 administered 10 min before sustained ischaemia caused a significant decrease in infarct size compared to Non-PC hearts (12.6±1.9 vs 29.6±2.9%) (p < 0.001) equivalent to that of hearts preconditioned with ischaemia. This was accompanied by a similar pattern of protection against apoptosis, with significantly reduced activation of caspase-3 activation and PARP cleavage. These results strongly supported a role for the attenuation of p38 MAPK activation as a mediator of preconditioning against ischaemia/reperfusion-mediated necrosis and apoptosis. However, the results of the experiments with anisomycin were at first glance not compatible with such a conclusion. The administration of the activator of p38 MAPK, anisomycin, for 10 min immediately prior to sustained ischaemia resulted in significant protection against necrosis (infarct size 16.6±2.4% vs 29.6±2.9% in Non-PC hearts) (p < 0.01) and reduced caspase-3 activation and PARP cleavage indicating less apoptosis. The reason for these findings were probably that this method of administration of anisomycin did in fact not activate p38 MAPK during sustained ischaemia, but actually served as a trigger to protect against ischaemia - similarly as if it had been infused with washout of the drug. Support for this notion was found in the fact that p38 MAPK activation was decreased upon reperfusion. These results suggested that the logistical problem of not being able to infuse a drug into the myocardium during ischaemia could not be overcome by immediate prior infusion, and that the administration of anisomycin in this way had activated downstream effectors of the p38 MAPK signal transduction pathway. An important contender for such an effector would be heat shock protein 27 (HSP27), which has been shown to play an important role in protection against apoptosis, and stabilisation of actin, and thus the cytoskeleton. Another possibility was that anisomycin had activated the JNK stress activated kinases. The elucidation of a role of this signal transduction pathway would necessitate the use of anisomycin in the presence of an agent such as curcumin, an inhibitor of JNK. Final conclusion: The work in this thesis showed that the stress activated kinase, p38 MAPK, was involved in the protective effect of ischaemie preconditioning. The results suggested a role for the activation of p38 MAPK as a trigger of protection, and the attenuation of p38 MAPK as a mediator of protection, which was observed in the reduction of both necrosis (infarct size) and apoptosis as determined with caspase- 3 activation and PARP cleavage. / AFRIKAANSE OPSOMMING: Die afsluiting van bloedvloei na die miokardium gee aanleiding tot nekrose. In die lig van die voorkoms van koronêre bloedvatsiekte onder die algemene populasie, en die nadelige effekte van miokardiale isgemie op miokardiale weefsel, is dit belangrik om nuwe strategieë te ontwikkel wat die miokardium teen isgemie beskerm. Nekrose van miokardiale weefsel word tradisioneel as die belangrikste komponent van die skade aangerig deur miokardiale infarksie beskou. Die belang van apoptotiese seldood in die konteks van miokardiale isgemie/herperfusie (I/R) het onlangs na vore getree. Dit word algeneem aanvaar dat vroeë vroegtydige herperfusie noodsaaklik is om miokardiale weefsel te beskerm teen seldood. Prekondisionering is 'n verskynsel waartydens kort episodes van IIR die hart teen 'n daaropvolgende langer periode van isgemie beskerm. Hierdie endogene meganisme is die kragtigste vorm van beskerming teen miokardiale infarksie tot dusver beskryf. Afgesien van isgemiese prekondisionering (IPC), kan beskerming ook deur farmakologiese middels, soos byvoorbeeld die aktivering van die beta-adrenerge reseptore met isoproterenol, ontlok word. IPC beskerm die miokardium teen nekrose, arritmieë en apoptose, en verhoog funksionele herstel na herperfusie. Daar is reeds aangetoon dat betaadrenerge prekonsionering (~PC) post-isgemiese funksionele herstel verbeter, maar dit is nog onbekend of beskerming ook teen miokardiale infarksie en apoptose verleen word. Die seintransduksie paaie betrokke tydens prekondisionering is reeds in detail bestudeer. Daar word gewoonlik tussen faktore wat optree as snellers, of as mediators van beskerming, onderskei. Snellers aktiveer sellulêre response voor die aanvang van volgehoue isgemie, en hul betrokkenheid word aangetoon deurdat inhibisie van snellers tydens die prekondisionering protokol, beskerming ophef. Snellers se effekete kan ook ontlok word deur hulle tydelike toe te dien en dan net voor volgehoue isgemie weer uit te was. Mediators oefen hulle effek tydens volgehoue isgemie uit, en hulle betrokkenheid word gedemonstreer deurdat toediening van inhibitors net voor volgehoue isgemie (sonder uitwas) hulle beskermende effekte ophef. Mediators se rol kan ook aangetoon word deur te poog om seintransduksie paaie tydens volgehoue isgemie te aktiveer. Aangesien dit ontmoontlik is om middels tydens isgemie te infuseer, word aktiveerders onmiddelik voor die aanvang van isgemie toegedien sonder om hulle uit te was, sodat hulle effekte op beskerming vervolgens bestudeer kan word. Dit is duidelik dat die evolusionêr-behoue stres geaktiveerde paaie tydens prekondisionering betrokke is. Daar is drie paaie nl. die ekstrasellulêre reseptor geaktiveerde pad (ERK), c-jun terminaal geaktiveerde kinases (JNK) en p38 mitogeen geaktiveerde proteïen kinases (MAPK). Die spesifieke rol van die p38 MAPK pad is nog nie ontrafel nie. Eksperimentele bewyse stel 'n rol vir die aktivering van p38 MAPK as 'n sneller, sowel as 'n mediator van die beskermende effek van prekondisionering, voor. Daar is egter ook sterk bewyse dat 'n afname in p38 MAPK aktivering tydens volgehoue isgemie, eerder as sy aktivering, verantwoordelik is vir die waargenome beskermende effek. Verder is die rol van p38 MAPK slegs in die konteks van beskerming teen nekrose, maar nie teen apoptose nie, bestudeer. DOELWITTE: Die doelwit van hierdie studie was: (I) Die vestiging van 'n prekondisionering model in neonatale kardiomiosiet in selkultuur. Hierdie model sou potensieel 'n spoedige ontrafeling van die seintransduksie paaie sonder die invloed van nie-kardiale selle bewerkstellig. (II Om ondersoek in te stelof IPC en PPCteen nekrose en apoptose beskerm. (III) Die ontrafeling van die rol van die stres geaktiveerde kinase, p38 MAPK, tydens prekondisionering. METODES: 1. Neonatale rot kardiomiosiet weefselkultuur model 'n Lewensvatbaarheids essai is ontwikkel deur van verskillende konsentrasies van 3-[4,5-dimetielthiazol-2-yl]-2,5-difeniel-tetrazolium bromied (MTT) gebruik te maak - 'n konsentrasie van 0.25% was optimaalom lewensvatbaarheid te bepaal. Neonatale kardiomiosiet weefselkulture is onderwerp aan volgehoue gesimuleerde "isgemie" deur gebruik te maak van 5 mM KCN plus deoksiglukose (DOG) vir 5 minute of 45 min KCN. Sommige weefselkulture is geprekondisioneer deur middel van chemiese isgemie (5 mM KCN vir 5 min) of van isoproterenol (10-7 M) vir 5 minute en 60 minute reoksigenasie alvorens dit bloot gestel is aan volgehoue gesimuleerde isgemie. 2. Geïsoleerde volwasse rot kardiomiosiet model Geïsoleerde kardiomiosiete is aan twee uur hipoksie blootgestel deur selle in 'n pellet te sentrifugeer en met 'n dun lagie mineraalolie te bedek. Sommige groepe is geprekondisioneer deur middel van 10 minute hipoksie by 37°C, of toediening van isoproterenol (10-7 M) vir 5 minute gevolg deur 20 minute reoksigenasie. Die tripaanblou uitsluitings metode en MTT metode soos ontwikkel in die neonatale kardiomiosiet model is gebruik om lewensvatbaarheid te bepaal. 3. Geïsoleerde geperfuseerde volwasse rot hart model 3.1 Infarkgrootte is bepaal met 'n model van streeks isgemie deur van tetrazolium kleuring gebruik te maak, waarna die area van nekrose (uitsluiting van tetrazolium) as 'n presentasie van die risiko area bepaal is. Hierdie harte was onderwerp aan 35 minute globale isgemie en 30 minute herperfusie. Sommige groepe is geprekondisioneer met 3 siklusse van 5 minute globale isgemie, of die toevoeging van isoproterenol (10-7 M) vir 5 minute, gevolg deur 5 minute herperfusie voor die aanvang van volgehoue streeks isgemie. 3.2 p38 MAPK aktivering en merkers van apoptose: p38 MAPK aktivering is bepaal deur gebruik te maak van anti-liggame teen tweeledige gefosforileerde p38 MAPK (d.w.s. geaktiveerde p38 MAPK). Apoptose is bepaal deur gebruik te maak van anti-liggame teen geaktiveerde kaspase-3, en teen 'n fragment van PARP (PARP kliewing). Tydens hierdie eksperimente is geïsoleerde rotharte bloot gestel aan 25 minute globale isgemie gevolg deur 30 minute herperfusie. Sommige groepe is geprekondisioneer met drie siklusse van 5 minute globale isgemie. Om voldoende weefsel vir Westerse klad tegnieke te verkry, is gebruik gemaak van 'n globale isgemie model. As gevolg hiervan was 'n kort periode van volgehoue isgemie genoodsaak, aangesien die globale isgemiese hart nie voldoende herstel na 'n langer periode van isgemie nie, soos wat benodig word in streeks isgemiese eksperimente. 3.3 Die rol van p38 MAPK tydens IPC is bepaal deur die toediening van 'n 1IJM konsentrasie van SB 203580, 'n selektiewe inhibitor van p38 MAPK, hetsy tydens prekondisionering (d.w.s. om die rol as 'n sneller te bepaal), óf vir 10 minute direk voor die aanvang van volgehoue isgemie (d.w.s. om dus sy rol as mediator te bepaal). Die tweede benadering was om anisomisien, 'n aktiveerder van p38 MAPK, as sneller (toediening vir 10 minute gevolg deur uitwassing) of as mediator (10 minute direk voor aanvang van volgehoue isgemie) in dieselfde model as in die geval van p38 MAPK aktiviteit bepaling, te gebuik. Die toediening van anisomisien vir 10 minute het aangetoon dat dit p38 MAPK aktivering kan ontlok tot dieselfde maate as die IPC protokol. Die eindpunte was infarkgrootte en merkers van apoptose. RESULTATE: 1. Neonatale rot kardiomiosiet weefselkultuur model Dit was nie moontlik om 'n suksesvolle model met konsekwente resultate vir die prekondisionering van neonatale kardiomiosiete te vestig nie. Daar is dus besluit om af te sien van hierdie pogings en eerder 'n alternatiewe selmodel te gebruik. 2. Geïsoleerde volwasse rot kardiomiosiet model Geïsoleerde volwasse kardiomiosiete is suksesvol geprekondisioneer, maar het te min materiaalopgelewer vir die gelyktydige bepaling van sellewensvatbaarheid, p38 MAPK aktivering en merkers vir apoptose. Daar is dus besluit om die geïsoleerde geperfuseerde volwasse rothart te gebruik. 3. Geïsoleerde geperfuseerde volwasse rothart model 3.1 Beide IPC en PPCbeskerm teen infarksie en apoptose: Deur gebruik te maak van twee prekondisionering modelle d.w.s. IPC en PPC, is die beskermende effekte van prekondisionering teen infraksie (nekrose) oortuigend gedemonstreer. Beide IPC en PPC het In betekenisvolle afname in infarkgrootle veroorsaak (12.2 ± 1.4 en 15.2 ± 2.6% respektiewelik), vs Nie-PC harte (29.6 ± 2.9%)(p < 0.001). Beide vorme van prekondisionering het ook teen apoptose beskerm deur die apoptose merkers, kaspase-3 aktivering en PARP kliewing te verlaag. Die beskerming verkry deur beide vorms van prekondisionering is geassosieer met In merkbare afname in die aktivering van p38 MAPK na herperfusie. Die verband tussen p38 MAPK en die beskerming ontlok deur prekondisionering is gevolglik ondersoek, naamlik of p38 MAPK optree as 'n sneller of as 'n mediator van beskerming. Om die rol van p38 MAPK as 'n mediator of sneller tydens prekondisionering te ondersoek is daar gebruik gemaak van (I) 'n spesifieke inhibitor van p38 MAPK aktivering nl. SB 203580 en (II) 'n bekende aktiveerder van p38 MAPK nl. anisomisien. 3.2 p38 MAPK as 'n sneller vir beskerming: Toediening van SB 203580 tydens die IPC protokol en uitwassing daarvan voor die aanvang van volgehoue isgemie het nie die beskermende effek van IPC opgehef nie, en het gelei tot 'n klein maar betekenisvolle verhoging in kaspase-3 aktivering en PARP kliewing. Andersins het die aktivering van p38 MAPK met anisomisien vir 10 minute gevolg deur In uitwas ook tot In betekenisvolle afname in nekrose (infarkgrootte 14.9 ± 2.2 vs 29.6 ± 2.9% in Nie-PC harte) (p < 0.001) in beide merkers van apoptose gelei. Laasgenoemde resultate dui daarop dat p38 MAPK inderdaad 'n mediator van prekondisionering is. Indien dit die geval is, waarom het SB 203580 nie die beskermende effek van IPC opgehef nie? Die mees waarskynlike verklaring is dat veelvuldige beskermingsmeganismes tydens 'n multi-siklus protokol van IPC geaktiveer word, waarvan p38 MAPK aktivering slegs een is. Dit is dus onwaarskynlik dat die inhibisie van p38 MAPK met SB 203580 die aktivering van daardie meganismes onafhanklik van p38 MAPK sal blokkeer en steeds in staat sal wees tot beskerming teen nekrose en apoptose. Dit is interessant dat In klein verhoging in apoptose waargeneem is toe SB 203580 gebruik is onder hierdie toestande, aangesien dit daarop kan dui dat die beskerming teen apoptose meer afhanklik was van die aktivering van p38 MAPK as die beskerming teen nekrose, siende dat geen effek op infarkgrootte waargeneem is nie. 'n Verdere verklaring kan wees dat die bepaling van infarkgrootte nie sensitief genoeg is om sulke klein effekte waar te neem nie. 3.3 p38 MAPK as 'n mediator vir beskerming: Inhibisie van p38 MAPK aktivering deur SB 203580 toediening 10 minute voor volgehoue isgemie het 'n betekenisvolle verlaging in infarkgrootte in vergelyking met Nie-PC harte veroorsaak (12.6 ± 1.9 vs 29.6 ± 2.9%) (p < 0.001) soortgelyk aan dié van harte geprekondisioneer met isgemie. Dit is geassosieer met In soortgelyke patroon van beskerming teen apoptose, met betekenisvolle verlaagde kaspase-3 aktivering en PARP kliewing. Hierdie resultate ondersteun die rol van die afname van p38 MAPK aktivering as 'n mediator van prekondisionering teen I/R-gemedieerde nekrose en apoptose. Die resultate van die anisomisien eksperimente was met die eerste oogopslag nie in oorstemming met hierdie gevolgtrekking nie. Die toedienning van die p38 MAPK aktiveerder, anisomisien, vir 10 minute voor volgehoue isgemie het tot 'n betekenisvolle beskerming teen nekrose aanleiding gegee (infarkgrootte 16.6 ± 2.4 vs 29.6 ± 2.9% in Nie-PC harte) (p < 0.01) en verlaagde kaspase-3 aktivering en PARP kliewing wat dui op verlaagde apoptose. Die rede vir hierdie bevindings is moontlik dat die metode van anisomisien toediening nie p38 MAPK geaktiveer het tydens volgehoue isgemie nie, maar eintlik gedien het as 'n sneller vir beskerming teen isgemie - amper asof dit toegedien sou word sonder om uitgewas te word. Ondersteuning vir hierdie aanname word gevind in die feit dat p38 MAPK aktivering verlaag is na herperfusie. Hierdie resultate stel voor dat die logistiese probleem dat In middel nie tydens isgemie toegedien kan word nie, nie oorkom kan word deur onmiddelike voortydige infusie nie, en dat die toediening van anisomisien op hierdie manier gelei het tot die aktivering van stroom-af effektors van die p38 MAPK seintransduksie pad. 'n Belangrike kandidaat vir so 'n effektor is "heat shock protein 27" (HSP27), wat reeds aangetoon is om 'n belangrike rol in die beskerming teen apoptose en destabilisering, en dus die sitoskelet, te speel. 'n Ander moontlikheid is dat anisomisien die JNK stres geaktiveerde kinases geaktiveer het. Die ontrafeling van die rol van hierdie seintransduksie pad noodsaak die gebruik van anisomisien in die teenwoordigheid van 'n agent soos curcumin, 'n JNK inhibitor. Finale gevolgtrekking: Die werk soos vervat in hierdie tesis toon aan dat die stres geaktiveerde kinase, p38 MAPK, betrokke is in die beskermings effek van isgemiese prekondisionering. Die resultate dui op 'n rol vir die aktivering van p38 MAPK as 'n sneller vir beskerming, en die afname in p38 MAPK as 'n mediator vir beskerming, soos waargeneem in die vermindering van veranderlikes van beide nekrose (infarkgrootte) en apoptose soos bepaal deur kaspase-3 aktivering en PARP kliewing.

Cette étude a été conçue afin d’évaluer l’effet d’un pré-traitement à long terme au célécoxib sur la taille d’infarctus suite à un infarctus du myocarde. Sachant que le célécoxib est un anti-inflammatoire et que des dommages myocardiques peuvent découler des processus inflammatoires, l’inhibition de l’inflammation devrait hypothétiquement réduire la taille d’un éventuel infarctus. Pour ce faire, un traitement au célécoxib (3 mg/kg/jour i.p.) ou au véhicule (DMSO 50% ; EtOH 15% ; eau distillée) a été administré chroniquement pendant 28 jours à des rats mâles Sprague-Dawley (n=18 par groupe) par pompes osmotiques ALZET. Après avoir été anesthésiés, les animaux ont été sujets à l’occlusion de l’artère coronaire gauche descendante, suivie d’une période de reperfusion de 24 heures. Les résultats démontrent que la taille de l’infarctus des animaux traités au célécoxib est significativement réduite comparativement à celle du groupe témoin (37,5±2,5% versus 48,0±2,6% de la zone à risque, p < 0,05). Par la suite, l’accumulation de neutrophiles indique une hausse de ces leucocytes pour la zone ischémique, sans toutefois discriminer entre les groupes traité et non-traité, qui contenaient aussi les couches sub-endocardique et sous-épicardique. Cependant, aucune différence significative est notée entre les groupes traité et témoin au niveau de l’expression de la prostaglandine E2 plasmatique et du facteur de nécrose tumorale alpha. D’un autre côté, l’apoptose, déterminée par le ratio de Bax/Bcl2 et par un essai TUNEL est significativement réduite pour la couche sub-endocardique de la zone à risque des animaux traités au célécoxib. Enfin, l’agrégation plaquettaire, induite à l’adénosine diphosphate et analysée dans le sang complet, suggère que le célécoxib diminue l’agrégation plaquettaire. Cette étude indique alors qu’un pré-traitement au célécoxib peut réduire la taille d’infarctus par un mécanisme impliquant l’apoptose. / This study was designed to evaluate the effect of long-term pre-treatment with celecoxib, a cyclooxygenase-2 inhibitor, on myocardial infarct size. Since celecoxib is an anti-inflammatory and that myocardial damages can be present in the occurrence of inflammatory processes, inhibition of inflammation should hypothetically reduce the size of an eventual infarct. Celecoxib (3 mg/kg/day i.p.) or vehicle (DMSO 50%; EtOH 15%; distilled water) was administered chronically to male Sprague-Dawley rats (n=18 per group) through ALZET osmotic pumps for 28 days. Under anaesthesia, the animals were then subjected to left anterior descending coronary artery occlusion for 40 minutes, followed by 24-hour reperfusion. The results show that myocardial infarct size in celecoxib-treated rats was significantly reduced compared to the control group (37.5±2.5% versus 48.0±2.6% of the area at risk, p < 0.05). Accumulation of neutrophils, estimated by myeloperoxidase levels, indicated an increase in the ischemic area without any significant difference between groups. No significant difference was observed between the treated and vehicle groups in terms of plasma prostaglandin E2 and tumour necrosis factor-alpha. Apoptosis, evaluated by Bax/Bcl-2 and terminal dUTP nick-end labelled-positive cells, was significantly decreased in the subendocardial layer of the ischemic area in celecoxib-treated rats. Adenosine diphosphate-induced platelet aggregation in whole blood suggested that celecoxib diminished platelet aggregation. This study indicates that pre-treatment with celecoxib can reduce infarct size by a mechanism which may involve apoptosis.

Le récepteur éboueur CD36 facilite l’internalisation des acides gras libres non estérifiés (AGNE) au niveau des tissus cardiaque et périphériques. Lors d’une ischémie-reperfusion du myocarde (MI/R), les dommages produits sont en partie liés à l’internalisation des AGNE et à la production d’espèces réactives de l’oxygène, contrairement à ce qui est observé chez des souris déficientes en CD36 (CD36-/-). Nous avons émis l’hypothèse selon laquelle le CP-3(iv), un ligand synthétique du récepteur CD36, exercerait un effet cardioprotecteur en réduisant la taille de la zone myocardique infarcie lors d’une ischémie transitoire du myocarde. Nos objectifs étaient 1) de déterminer l’effet cardioprotecteur du CP-3(iv) et 2) de définir son mécanisme. Pour cela, des études in vivo et ex vivo ont été faites. Des souris de type sauvage ont été traitées avec le CP-3(iv) (289 nmol/kg) par voie sous-cutanée pendant 14 jours avant d’être soumises à 30 minutes d’ischémie suivant la ligature de l’artère coronaire gauche descendante et de sa reperfusion pendant une période de 6 ou 48 heures. De plus, des coeurs isolés de souris ont été perfusés 30 minutes, suivi de 40 minutes à faible débit (10%) et de 30 minutes de reperfusion pendant laquelle le coeur est perfusé avec le CP-3(iv) à une concentration de 10-6 M. Nos travaux ont montré que l’effet cardioprotecteur d’un traitement préventif par le CP-3(iv) permet de diminuer la taille de l’infarctus et préserve l’hémodynamie cardiaque de façon dépendante du CD36 puisque cet effet est non visible chez les souris CD36-/-. De plus, le CP-3(iv) exerce non seulement un effet systémique, mais aussi un effet cardioprotecteur direct sur le coeur isolé. / The scavenger receptor CD36 facilitates the internalization of non-esterified fatty acids (NEFA) on cardiac and peripheral tissues. During myocardial ischemia and reperfusion (MI/R), the damage induced is in part related to the internalization of NEFA and the production of reactive oxygen species, in opposition to what is observed in CD36-deficient mice (CD36-/-). We hypothesized that CP-3(iv), a synthetic ligand of the CD36 receptor, provides a cardioprotective effect by reducing the infarct area during a transient myocardial ischemia. Our objectives were 1) to determine the cardioprotective effect of CP-3(iv) and 2) to define its mechanism. For this, in vivo and ex vivo studies have been done. Wild-type mice were treated with CP-3(iv) (289 nmol/kg) subcutaneously during 14 days before being submitted to 30 minutes of ischemia following left anterior descending coronary artery ligature and reperfusion for a period of 6 to 48 hours. In addition, isolated mouse hearts were perfused 30 minutes, followed by 40 minutes with low flow (10%) and 30 minutes of reperfusion during which the heart is perfused with CP-3(iv) at a concentration of 10-6 M. Our work has shown that the cardioprotective effect of preventive treatment with CP-3(iv) reduces the infarct size and preserves cardiac hemodynamics in a CD36-dependent manner because this effect is not visible in CD36-/- mice. In addition, CP-3(iv) not only exerts a systemic effect, but also a direct cardioprotective effect on the isolated heart.