Novel therapeutic agents that blunt hyperglycemia-induced cardiac contractile dysfunction

Mapanga, Rudo Fiona (2013-03)

Thesis (PhD)--Stellenbosch University, 2013.

Thesis

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.

AFRIKAANSE OPSOMMING: Inleiding Diabetes skep ‘n groot gesondheidsuitdaging. Omrede kardiovaskulêre komplikaseis algemeen onder diabetiese pasiënte is, sal dit oorkoepelend die las van hierdie siekte verder laat toeneem. Verder word stresgeïnduseerde hiperglukemie in nie-diabetiese pasiënte met akute miokardiale infarksie geassosieër met ‘n hoër binne-hospitaalmortaliteit. Hiperglukemies-geïnduseerde oksidatiewe stres veroorsaak DNA skade, en gevolglike aktivering van poli-ADF-ribose polimerase (PARP), as ‘n herstelmeganisme. Nietemin, PARP verminder gliseraldehied–3-fosfaatdehidrogenase (GAPDH) aktiwiteit om sodoende die opstroom glikolitiese metaboliete te herlei na skadelike nie-oksidatiewe glukose weë (NOGW). Byvoorbeeld, hiperglukemie-geïnduseerde stimulasie van vier NOGW, i.e. die poliolweg, heksosamienbiosintetiese weg, (HBW), gevorderde glukasie eindprodukte (GGE), en PKC aktivering, lei tot kardiovaskulêre komplikasies. Die huidige tesis ondersoek die regulering van NOGW in ‘n isgemiese-reperfussie onder hiperglukemiese toestande. Ons hipotetiseer dat die toediening van twee unieke terapeutises intervensies, i.e. oleanoliese suur (OS, naaltjie ekstrak), en benfotiamien (BFT, vitamien B1 derivaat) oksidatiewe stress kan versag, en NOGW geinduseerde kardiale disfunksie onder hiperglukemiese toestande na ischemie en reperfussie. Ons keuse vir hierdie middels is gebaseer op die beginsel dat OS antioksidanteienskappe bevat, en dat BFT transketolase (pentosefosfaat weg (PFW) ensiem) stimuleer en sodoende die fluks weg van die NOGW weg veroorsaak. Addisioneel kan hiperglukemiegeïnduseerde oksidatiewe stres ook tot wanregulering van die ubikwitien-proteosoomsisteem (UPS) wat wangevoude protïene verwyder, aanleiding gee. Daar bestaan kontrasterende data oor ‘n verhoogde/verlaagde UPS, tesame met hiperglukemie en/of in reaksie tot isgemie-reperfussie. In die lig hiervan, hipotetiseer ons dat BFT en OS as ‘n nuwe kardiobeskermingsmiddel kan optree deur miokardiale oksidatiewe stres en UPS aktiwiteit in reaksie op isgemie-reperfussie tydens akute hiperglukemiese toestande kan verlaag. Materiale en Metodes Vir die eerste deel van die studie het ons van verskeie eksperimentele sisteme gebruik gemaak: 1) H9c2 kardiale mioblaste is aan 33 mM glukose vir 48 uur vs. kontrole (5 mM glukose) blootgestel; en gevolglik met twee OS dosisse (20 en 50 μM) vir 6 en 24 hr, onderskeidelik behandel; 2) 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 ± OS behandeling; 3) 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 4) In vivo koronêre liggasies is op streptozotosien-diabetiese rotte uitgevoer ± 0.45 mg/kg OS toedienning binne die eerste twee minute van reperfussie; en 5) effekte van langtermyn OS behandeling (2 weke) op hartfunskie is in hierdie streptozotosien-diabetiese rotte ondersoek. Hier is STZ opgelos in ‘n sitraatbuffer (pH 6.3), en diabetes is geinduseer deur 60mg/kg i.p. toe te dien. Weefsels is aan die einde van die globale isgemie eksperimente versamel, en vir oksidatewe stres, apoptose, UPS aktiwiteit en HBW aktivering, ontleed. Vir die tweede deel van die studie het ons van verskeie eksperimentele sisteme gebruik gemaak: 1) geïsoleerde rotharte is ex vivo met Krebs-Henseleit buffer, wat 33 mM glukose vs. kontrole (11 mM glukose) bevat, vir 90 min geperfuseer. Daarna is dit gevolg met 30 min globale isgemie en 60 min reperfussie ± 25, 50 en 100 μM BFT behandeling onderskeidelik, gevolg, bykomend, gedurende die eerste 20 min reperfussie; 2) Infarkgrootte is bepaal soos in #3 hierbo, maar met 30 minute regionale ischemie en 2 uur reperfussie ± 100 μM BFT behandeling; en 3) In vivo koronêre liggasies is op streptozotosien-diabetiese rotte uitgevoer ± 0.50 mg/kg BFT behandeling binne die eerste twee minute van reperfussie. Met parallele eksperimente is NOGW inhibeerders bygevoeg binne die eerste 20 min van reperfussie. Die volgende inhibeerders is individueel ontplooi: GGE weg (100 μM aminoguanidien); PKC (5 μM chelleritrienchloried); HBW (40 μM 6-diazo-5-oxo-L-nor-leusien); en poliolweg (1 μM zopolrestaat); 2) Infarkgrootte is bepaal soos in #2) met die uitsondering van 30 min regionale isgemie en 120 min reperfussie ± identiese behandelings. Resultate Ons data toon aan dat kardiale kontraktiele funksie, in reaksie op isgemie-reperfussie onder hiperglukemiese toestande, verlaag. Dit is verwant aan verhoogde PARP en verminderde GAPDH aktiwiteit, tesame met ‘n hoër aktivering van die NOGW. Verder het ons bevind dat verhoogde miokardiale oksidatiewe stres, UPS en seldood onder die toestande voorkom. OS behandeling lei tot kardiale beskerming, i.e. vir ex vivo en in vivo rotharte wat aan isgemie-reperfussie onder hiperglukemiese toestande blootgestel is. Parallel hiermee het OS oksidatiewe stres, apoptose, HBW invloed, en UPS aktiwiteit na isgemie-reperfussie, verlaag. Langtermyn OS behandeling het ook hartfunksie in streptozotosien-diabetiese rotte verbeter. Ons data vertoon verder dat akute BFT behandeling, miokardiale oksidatiewe stres en apoptose, betekenisvol verlaag het in reaksie op isgemie-reperfussie onder hiperglukemiese toestande. Parallel hiermee het BFT hiperglukemiegeïnduseerde aktivering van vier NOGWë in die rothart, verminder. Akute toediening van die elk van die NOGW inhibeerders het PARP verlaag, en GAPDH aktiwiteite verhoog, terwyl oksidatiewe stres, en miokardiale apoptose verminder. Verder het elk van die NOGW inhibeerders wat (individueel) toegedien is, aktivering van die ander drie weë, hier ondersoek, verlaag. Die harte wat met NOGW inhibeerders behandel is het ook ‘n verbeterde herstel en kleiner infarkgrootte na isgemie-reperfussie getoon. Interessant is hoe die NOGW inhibeerders tot dieselfde graad verandering (vir al die bogemelde parameters wat geevalueer is) indien dit vergelyk word teen mekaar, gelei het. Gevolgtrekking Hierdie studie het bevind dat akute en chroniese hiperglukemie, miokardiale oksidatiewe stres ontlok, en dat dit geleidelik tot NOGW aktivering en kontraktiele wanfunksionering na isgemie-reperfussie lei. Verder het ons bevindinge vir die eerste keer, volgens ons wete, bewys dat daar ‘n ineenloping is van afstroom NOGW effekte in ons model, i.e. verhoogde miokardiale oksidatiewe stres, verdere NOGW weg aktivering, apoptose, en ingeperkte kontraktiele funksie. Dus, ‘n gebrekkige metaboliese siklus word verkry waarby hiperglukemies-geïnduseerde NOGW verder sy eie aktivering aanvuur deur meer oksidatiewe stres, en sodoende die skadelike effekte op die hart onder hierdie toestande verder versleg. Ons het verder bevind dat beide OS en BFT behandeling, hoë glukose-geïnduseerde skadelike effekte onderdruk, en kragtige kardiale-beskerming in reaksie op isgemie-reperfussie onder hiperglukemiese toestande (akuut en chronies), teweeg bring. Hierdie bevindinge dui moontlik daarop dat die UPS ‘n unieke terapeutiese teiken kan wees vir die behandeling van isgemiese hartsiekte in individue wat presenteer met stres-geïnduseerde, akute hiperglukemie. BFT het ook sy kardiale beskermende effekte getoon deur NOGW inhibering na isgemie-geïnduseerde reperfussie onder aktute en chroniese hoë glukose toestande. ‘n Soorgelyke effek is tydens die basislyn waargeneem, alhoewel die onderliggende meganisme wat hierdie proses dryf verder ondersoek moet word. Opsommend is ons bevindinge baie belowend omrede dit daartoe kan aanleiding gee tot ‘n nuwe, meer koste-effektiewe terapeutiese intervensie vir die behandeling van akute hiperglukemie (in niediabetiese pasiënte) en diabetiese pasiënte met geassosieërde kardiovaskulêre komplikasies.

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