Doctoral Degrees (Physiological Sciences)
Permanent URI for this collection
Browse
Browsing Doctoral Degrees (Physiological Sciences) by Author "Mapanga, Rudo Fiona"
Now showing 1 - 1 of 1
Results Per Page
Sort Options
- ItemNovel therapeutic agents that blunt hyperglycemia-induced cardiac contractile dysfunction(Stellenbosch : Stellenbosch University, 2013-03) Mapanga, Rudo Fiona; Essop, M. Faadiel; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Introduction Diabetes constitutes a major health challenge. Since cardiovascular complications are common in diabetic patients this will further increase the overall burden of disease. Furthermore, stress-induced hyperglycemia in non-diabetic patients with acute myocardial infarction is associated with higher inhospital mortality. Hyperglycemia-induced oxidative stress results in DNA damage and subsequent activation of poly-ADP-ribose polymerase (PARP) as a restorative mechanism. However, PARP attenuates glyceraldehyde–3-phosphate dehydrogenase (GAPDH) activity, thereby diverting upstream glycolytic metabolites into damaging non-oxidative glucose pathways (NOGP). For example, hyperglycemia-induced stimulation of four NOGP, i.e. the polyol pathway, hexosamine biosynthetic pathway (HBP), advanced glycation end products (AGE), and PKC activation elicit cardiovascular complications. The current thesis examined the regulation of NOGP in the setting of ischemia and reperfusion under hyperglycemic conditions. Here we hypothesized that administration of two unique therapeutic interventions, i.e. oleanolic acid (OA; clove extract) and benfotiamine (BFT; vitamin B1 derivative), can blunt oxidative stress and NOGP-induced cardiac dysfunction under hyperglycemic conditions following ischemia and reperfusion. Our choice for these agents was based on the principle that OA possesses antioxidant properties; and BFT stimulates transketolase (pentose phosphate pathway [PPP] enzyme) thereby shunting flux away from the NOGP pathways. Additionally, hyperglycemia-induced oxidative stress can also result in dysregulation of the ubiquitin-proteasome system (UPS) that removes misfolded proteins. There are conflicting data whether increased/decreased UPS is detrimental with hyperglycemia and/or in response to ischemia and reperfusion. In light of this, we also hypothesized that BFT and OA act as novel cardio-protective agents by diminishing myocardial UPS activity in response to ischemia and reperfusion under acute hyperglycemic conditions. Materials and Methods For the first part of the study, we employed several experimental systems: 1) H9c2 cardiac myoblasts were exposed to 33 mM glucose for 48 hr vs. controls (5 mM glucose); and subsequently treated with two OA doses (20 and 50 μM) for 6 and 24 hr, respectively; 2) 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 min reperfusion ± OA treatment; 3) Infarct size was determined using Evans Blue dye and 1% 2,3,5-triphenyl tetrazolium chloride (TTC) staining with 20 min regional ischemia and 2 hr reperfusion 4) In vivo coronary ligations were performed on streptozotocin-diabetic rats ± 0.45 mg/kg OA administration within the first two minutes of reperfusion; and 5) Effects of long-term OA treatment (2 weeks) on heart function were assessed in streptozotocin (STZ)-diabetic rats. Here, STZ was dissolved in citrate buffer (p.H 6.3) and diabetes was induced by administering 60 mg/kg i.p Tissues were collected at the end of the global ischemia experiments and analyzed for oxidative stress, apoptosis, UPS activity and HBP activation. For the second part of the study we employed several experimental systems: 1) Isolated rat hearts were perfused ex vivo with Krebs-Henseleit buffer containing 33 mM glucose vs. controls (11 mM glucose) for 90 min, followed by 30 min global ischemia and 60 min reperfusion ± 25, 50 and 100 μM BFT treatment, respectively, added during the first 20 min of reperfusion; 2) Infarct size determination as in #3 above but with 30 min regional ischemia and 2 hr reperfusion ± 100 μM BFT treatment; and 3) In vivo coronary ligations performed on streptozotocin-diabetic rats ± 0.50 mg/kg BFT treatment within the first two min of reperfusion. In parallel experiments, NOGP inhibitors were added during the first 20 min of reperfusion. The following inhibitors were individually employed: AGE pathway (100 μM aminoguanidine); PKC (5 μM chelerythrine chloride); HBP (40 μM 6-diazo-5-oxo-L-norleucine); and polyol pathway (1 μM zopolrestat); Infarct size determination as in #2) with 30 min regional ischemia and 120 min reperfusion ± similar treatments. Results Our data show decreased cardiac contractile function in response to ischemia and reperfusion under hyperglycemic conditions. This was linked to increased PARP and attenuated GAPDH activities, together with higher activation of the NOGP. Moreover, we found elevated myocardial oxidative stress, UPS and cell death under these conditions. OA treatment resulted in cardio-protection, i.e. for ex vivo and in vivo rat hearts exposed to ischemia and reperfusion under hyperglycemic conditions. In parallel, OA decreased oxidative stress, apoptosis, HBP flux and UPS activity following ischemia and reperfusion. Long-term OA treatment also improved heart function in streptozotocin-diabetic rats. Our data also reveal that acute BFT treatment significantly decreased myocardial oxidative stress and apoptosis, and provided cardio-protection in response to ischemia and reperfusion under hyperglycemic conditions. In parallel, BFT blunted hyperglycemia-induced activation of four NOGP in the rat heart. Acute administration of each of the NOGP inhibitors decreased PARP and enhanced GAPDH activities, while diminishing oxidative stress and myocardial apoptosis. Moreover, each of the NOGP inhibitors (individually) employed blunted activation of the other three pathways here examined. Hearts treated with NOGP inhibitors also displayed improved functional recovery and smaller infarct sizes following ischemia and reperfusion. Interestingly, NOGP inhibitors resulted in the same degree of change (for all above-mentioned parameters evaluated) when compared to each other. Conclusions This study shows that acute and chronic hyperglycemia trigger myocardial oxidative stress that eventually results in NOGP activation and contractile dysfunction following ischemia and reperfusion. Moreover, our findings establish - for the first time as far as we are aware - that there is a convergence of downstream NOGP effects in our model, i.e. increased myocardial oxidative stress, further NOGP pathway activation, apoptosis, and impaired contractile function. Thus a vicious metabolic cycle is established whereby hyperglycemia-induced NOGP further fuels its own activation by generating even more oxidative stress, thereby exacerbating damaging effects on the heart under these conditions. We also found that both OA and BFT treatment blunted high glucose-induced detrimental effects and provided robust cardio-protection in response to ischemia and reperfusion under hyperglycemic conditions (acute and chronic). These findings suggest that the UPS may be a unique therapeutic target to treat ischemic heart disease in individuals that present with stress-induced, acute hyperglycemia. Moreover, BFT exhibited its cardio-protective effects by NOGP inhibition after ischemia and reperfusion under acute and chronic high glucose conditions. A similar effect was observed at baseline although the underlying mechanisms driving this process still need to be elucidated. In summary, the findings of this thesis are highly promising since it may eventually result in novel, cost-effective therapeutic interventions to treat acute hyperglycemia (in non-diabetic patients) and diabetic patients with associated cardiovascular complications.