Proteasome Inhibitors : a novel therapy that blunt hyperglycemia-induced cardiac contractile dysfunction

Adams, Buin (2015-04)

Thesis (MSc)--Stellenbosch University, 2015.

Thesis

ENGLISH ABSTRACT: Diabetes is considered a major threat to human health in both developed and developing nations. Cardiovascular disease which is common in diabetic patients has increased the overall disease affliction. Moreover, stress-induced hyperglycemia has led to increased mortality and morbidity in patients with an acute myocardial infarction (MI), whether the patient has diabetes or not. In addition, acute MI might stem from stress-induced hyperglycemia capability to increase inflammation and oxidative stress resulting in a worse functional cardiac outcome. Hyperglycemia-induced oxidative stress can similarly result in the formation of miss folded or damaged proteins that may be eliminated by the ubiquitin-proteasome system (UPS). Futhermore, hyperglycemia-induced oxidative stress can also result in dysregulation of the UPS that removes these misfolded proteins. Additionally, an increasing body of evidence implicates UPS dysfunction in cardiac diseases and hyperglycemia which has been associated with increased inflammation and blunted cardiac function in response to ischemia-reperfusion. Literature however is blurred whether a reduction or a rise in the UPS is damaging with hyperglycemia and in response to ischemia-reperfusion. In light of this, we hypothesized that UPS inhibitors such as Z-Leu-Leu-Leu-al (MG-132) and lactacystin, protects the rat heart against ischemia-reperfusion under hyperglycemic perfusion conditions. Isolated rat hearts were perfused ex vivo with Krebs-Henseleit buffer containing 33 mM glucose vs. controls (11 mM glucose) for 60 min, followed by 20 min global ischemia and 60 minutes reperfusion ± PI treatment (MG-132 and lactacystin), anti-inflammatory (Ibuprofen) and anti-oxidant (NAC). Infarct size was determined using Evans Blue dye and 1% 2,3,5-triphenyl tetrazolium chloride (TTC) staining with 20 minutes regional ischemia and 2 hours reperfusion ± PI’s treatments. Tissues were collected at the end of the global ischemia experiments and analyzed for UPS activity, oxidative stress, apoptosis and inflammation. Our data expressed a reduced cardiac contractile function in response to ischemia and reperfusion under hyperglycemic conditions as well as an increase in UPS activity. PI treatment resulted in cardio-protection for ex vivo rat heart model exposed to ischemia and reperfusion under hyperglycemic conditions as well as ibuprofen and NAC. In parallel lactacystin treatment significantly decreased myocardial oxidative stress, apoptosis, and inflammation which provided cardio-protection in response to ischemia and reperfusion under hyperglycemic conditions This study shows that acute hyperglycemia elicits myocardial oxidative stress, apoptosis and inflammation that in time results in an increase in contractile dysfunction following ischemia and reperfusion. However, we found that PI treatment with both MG-132 and lactacystin blunted high glucose-induced damaging effects which resulted in a robust cardio-protection in response to ischemia and reperfusion under hyperglycemic conditions, by reducing oxidative stress, decreasing apoptosis and limiting inflammation. A parallel outcome was observed at baseline although the underlying mechanisms driving this process still need to be clarified. Our findings indicate that the UPS may be a unique therapeutic target to treat ischemic heart disease in diabetic patients, and non-diabetic individuals that present with stress-induced hyperglycemia. In summary, this thesis established that PIs act as a novel cardio-protective intervention to treat acute hyperglycemia with associated cardiovascular complications.

AFRIKAANSE OPSOMMING: Diabeties word beskou as ‘n baie groot problem vir menslieke gesondhied vir biede die ontwikkel en onontwikkelende lande. Kardiovaskulêre siekte wat normaal met diabetiese pasiente geassoseerd word veroorsaak ‘n toeneemende druk, wat hierdie siekte laat toeneem. Verder meer vergroot stresgeïnduseerde hiperglukemie die mortaliteit van pasiente met of sonder diabeties wat akute miokardiale infarksie onder lede het. Akute miokardiale infarksie kan ook ontstaan van stresgeïnduseerde hiperglukemie se bekwaamheid om meer inflamasie en oksidante stress te veroorsaak wat in ‘n meer swakker funksionele kardiale toestand. Hiperglukemiegeïnduseerde oksidatiewe stres ook tot wanregulering van die ubikwitien-proteosoomsisteem (UPS) wat wangevoude protïene verwyder, aanleiding gee. Kontrasterende data bestaan van verhoogde/verlaagde UPS aktiwietiet, sowel as met hiperglukemie en/of in reaksie tot isgemie-reperfussie. As gewolg hiervan,, hipotetiseer ons dat Z-Leu-Leu-Leu-al (MG-132) and lactacystin as ‘n nuwe kardiobeskermingsmiddel kan optree deur miokardiale oksidatiewe stress, inflamasie en UPS aktiwiteit te verlaag in reaksie op isgemie-reperfussie tydens akute hiperglukemiese toestande kan verlaag. Geïsoleerde rotharte is ex vivo met Krebs-Henseleit buffer, wat, 33 mM glukose vs. kontrole (11 mM glukose) bevat, vir 60 min geperfuseer, daarna is dit deur 20 min globale isgemie gevolg en 60 min reperfussie ± PI behandeling (MG-132 and lactacystin), antiflammatoriese behandeling (Ibuprofen) en antioxidant behandeling (NAC). Infarkgrootte is bepaal deur Evans bou kleursel en 1% 2. 3-5 tripfeniel tetrazoloimcholierd (TTC) kleuring met 20 minute regionale ischemie, en 2 uur reprefussie ± PI’s behandeling. Weefsels is aan die einde van die globale isgemie eksperimente versamel, en vir oksidatewe stres, apoptose en inflammasie ontleed. Ons data toon aan dat kardiale kontraktiele funksie in reaksie op isgemie-reperfussie onder hiperglukemiese toestande verlaag het asook ‘n toename in UPS aktiwitiet veroorsaak. PI behandeling het gelei tot kardiale beskerming vir ex vivo rotharte wat aan isgemie-reperfussie onder hiperglukemiese toestande blootgestel was sowel as ibuprofen en NAC. Parallel hiermee het lactacystin oksidatiewe stres, apoptose, inflmasie, en UPS aktiwiteit na isgemie-reperfussie, verlaag in reaksie isgemie-reperfussie onder hiperglukemiese toestande. Hierdie studie het bevind dat akute hiperglukemie, miokardiale oksidatiewe stres lei tot oksidante stress, apoptose, en inflamasie na kontraktiele wanfunksionering na isgemie-reperfussie lei. Ons het bevind dat beide MG-132 en lactacystin behandeling, hoë glukose-geïnduseerde skadelike effekte onderdruk, en kardiale-beskerming in reaksie op isgemie-reperfussie onder hiperglukemiese toestande ondervind was deur oksidante stress, apoptose, en inflamasie te verlaag. ‘n Soorgelyke effek is tydens die basislyn waargeneem, alhoewel die onderliggende meganisme wat hierdie proses meer ondersoek instel. Ons bevinding dei dat die UPS ‘n nuwe behandeling teiken kan word in sgemie-geïnduseerde reperfussie onder aktute en chroniese hoë glukose toestande. In opsomming, het die tesis belowend bevindinge gevind wat ‘n nuwe terapeutiese intervensie vir die behandeling van akute hiperglukemie met geassosieërde kardiovaskulêre komplikasies gebruik kan word.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/97119
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