Novel therapeutic agents that blunt hyperglycemia-induced cardiac contractile dysfunction

[摘要] ENGLISH ABSTRACT: IntroductionDiabetes constitutes a major health challenge. Since cardiovascular complications are common indiabetic patients this will further increase the overall burden of disease. Furthermore, stress-inducedhyperglycemia in non-diabetic patients with acute myocardial infarction is associated with higher inhospitalmortality. Hyperglycemia-induced oxidative stress results in DNA damage and subsequentactivation of poly-ADP-ribose polymerase (PARP) as a restorative mechanism. However, PARPattenuates glyceraldehyde–3-phosphate dehydrogenase (GAPDH) activity, thereby diverting upstreamglycolytic metabolites into damaging non-oxidative glucose pathways (NOGP). For example,hyperglycemia-induced stimulation of four NOGP, i.e. the polyol pathway, hexosamine biosyntheticpathway (HBP), advanced glycation end products (AGE), and PKC activation elicit cardiovascularcomplications. The current thesis examined the regulation of NOGP in the setting of ischemia andreperfusion 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 andNOGP-induced cardiac dysfunction under hyperglycemic conditions following ischemia andreperfusion. Our choice for these agents was based on the principle that OA possesses antioxidantproperties; and BFT stimulates transketolase (pentose phosphate pathway [PPP] enzyme) therebyshunting flux away from the NOGP pathways. Additionally, hyperglycemia-induced oxidative stresscan also result in dysregulation of the ubiquitin-proteasome system (UPS) that removes misfoldedproteins. There are conflicting data whether increased/decreased UPS is detrimental withhyperglycemia and/or in response to ischemia and reperfusion. In light of this, we also hypothesizedthat BFT and OA act as novel cardio-protective agents by diminishing myocardial UPS activity inresponse to ischemia and reperfusion under acute hyperglycemic conditions.Materials and MethodsFor the first part of the study, we employed several experimental systems: 1) H9c2 cardiac myoblastswere exposed to 33 mM glucose for 48 hr vs. controls (5 mM glucose); and subsequently treated withtwo OA doses (20 and 50 μM) for 6 and 24 hr, respectively; 2) Isolated rat hearts were perfused exvivo 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 20min regional ischemia and 2 hr reperfusion 4) In vivo coronary ligations were performed onstreptozotocin-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 byadministering 60 mg/kg i.p Tissues were collected at the end of the global ischemia experiments andanalyzed for oxidative stress, apoptosis, UPS activity and HBP activation.For the second part of the study we employed several experimental systems: 1) Isolated rat heartswere perfused ex vivo with Krebs-Henseleit buffer containing 33 mM glucose vs. controls (11 mMglucose) for 90 min, followed by 30 min global ischemia and 60 min reperfusion ± 25, 50 and 100 μMBFT treatment, respectively, added during the first 20 min of reperfusion; 2) Infarct size determinationas 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 withinthe first two min of reperfusion. In parallel experiments, NOGP inhibitors were added during the first 20min of reperfusion. The following inhibitors were individually employed: AGE pathway (100 μMaminoguanidine); PKC (5 μM chelerythrine chloride); HBP (40 μM 6-diazo-5-oxo-L-norleucine); andpolyol pathway (1 μM zopolrestat); Infarct size determination as in #2) with 30 min regional ischemiaand 120 min reperfusion ± similar treatments.ResultsOur data show decreased cardiac contractile function in response to ischemia and reperfusion underhyperglycemic 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 vivoand in vivo rat hearts exposed to ischemia and reperfusion under hyperglycemic conditions. Inparallel, OA decreased oxidative stress, apoptosis, HBP flux and UPS activity following ischemia andreperfusion. Long-term OA treatment also improved heart function in streptozotocin-diabetic rats. Ourdata also reveal that acute BFT treatment significantly decreased myocardial oxidative stress andapoptosis, and provided cardio-protection in response to ischemia and reperfusion underhyperglycemic conditions. In parallel, BFT blunted hyperglycemia-induced activation of four NOGP inthe rat heart. Acute administration of each of the NOGP inhibitors decreased PARP and enhanced GAPDHactivities, while diminishing oxidative stress and myocardial apoptosis. Moreover, each of the NOGPinhibitors (individually) employed blunted activation of the other three pathways here examined. Heartstreated with NOGP inhibitors also displayed improved functional recovery and smaller infarct sizesfollowing ischemia and reperfusion. Interestingly, NOGP inhibitors resulted in the same degree ofchange (for all above-mentioned parameters evaluated) when compared to each other.ConclusionsThis study shows that acute and chronic hyperglycemia trigger myocardial oxidative stress thateventually 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 convergenceof downstream NOGP effects in our model, i.e. increased myocardial oxidative stress, further NOGPpathway activation, apoptosis, and impaired contractile function. Thus a vicious metabolic cycle isestablished whereby hyperglycemia-induced NOGP further fuels its own activation by generating evenmore oxidative stress, thereby exacerbating damaging effects on the heart under these conditions. Wealso found that both OA and BFT treatment blunted high glucose-induced detrimental effects andprovided robust cardio-protection in response to ischemia and reperfusion under hyperglycemicconditions (acute and chronic). These findings suggest that the UPS may be a unique therapeutictarget to treat ischemic heart disease in individuals that present with stress-induced, acutehyperglycemia. Moreover, BFT exhibited its cardio-protective effects by NOGP inhibition afterischemia and reperfusion under acute and chronic high glucose conditions. A similar effect wasobserved at baseline although the underlying mechanisms driving this process still need to beelucidated. In summary, the findings of this thesis are highly promising since it may eventually result innovel, cost-effective therapeutic interventions to treat acute hyperglycemia (in non-diabetic patients)and diabetic patients with associated cardiovascular complications.

[发布机构] Stellenbosch University