Thesis (MScMedSc)--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Obesity is a major risk factor for ischaemic heart disease. Obesity-induced metabolic abnormalities have been associated with increased oxidative stress which may play an important role in the increased susceptibility to myocardial dysfunction and ischaemiareperfusion (I/R) injury seen in obesity. The pineal gland hormone, melatonin, has powerful antioxidant properties. Previous studies have shown that short-term or acute melatonin administration protects the normal healthy heart of lean animals against I/R damage. However, the effects of melatonin on the heart in obesity remain unknown. Moreover, the myocardial signalling mechanisms associated with the cardioprotective effects of melatonin have not been established. Using a rat model of diet induced obesity, we set out to: 1) investigate the effects of chronic melatonin administration on the development of diet-induced systemic alterations including biometric and metabolic parameters and oxidative stress, 2) determine whether chronic melatonin treatment protects the myocardium against ischaemia-reperfusion injury, and 3) determine whether melatonin treatment confers cardioprotection by altering the reperfusion injury salvage kinase (RISK) pathway signalling and the pro-apoptotic p38 MAPK, AMPK and GLUT-4 expression. Male rats weighing 200±20g were randomly allocated to four groups: 1) C, control rats receiving a standard commercial rat chow and drinking water without melatonin; 2) CM, control rats receiving melatonin (4mg/kg/day) in drinking water; 3) D, diet-induced obesity rats, receiving a high calorie diet and drinking water without melatonin; 4) DM, diet-induced obesity rats, receiving melatonin in drinking water. After 16 weeks of treatment and feeding, rats were weighed and blood and myocardial tissue collected to document biochemical and molecular biological changes. Hearts were perfused on the isolated working rat heart perfusion apparatus for the evaluation of myocardial function and infarct size. The Reperfusion Injury Salvage Kinases (RISK) pathway (PKB/Akt (Ser-473), ERK p42/ p44) and p38 MAPK (mitogenactivated protein kinase) were investigated in pre-and post-ischaemic hearts using Western blotting techniques. Post-ischaemic activation of AMPK (5’AMP-activated protein kinase) (Thr- 172) and GLUT-4 (glucose transporter) expression were also investigated. Serum and baseline myocardial glutathione (GSH) content were measured. In addition, serum lipid peroxidation products: thiobarbituric reactive substances (TBARS), conjugated dienes (CD) and lipid hydroperoxide (LOOH), were also determined. The high-calorie diet caused increases in body weight, visceral adiposity, heart weight, serum insulin, leptin, blood triglycerides, and low HDL-cholesterol levels. Blood glucose levels were similar for both diet fed rats and controls. Myocardial glutathione, serum glutathione, total cholesterol, TBARS, LOOH, CD as well as total cholesterol (TC) levels were not affected by the high calorie diet. Chronic melatonin treatment reduced body weight gain, visceral adiposity, heart weight, blood triglycerides, serum insulin, HOMA index, serum leptin (DM vs D, p<0.01), and increased blood HDL-C in diet treated rats while there was no effect on these parameters in control rats, despite the reduction in body weight, heart weight and visceral adiposity. Melatonin treatment had no effect on myocardial or serum GSH and LOOH in either control or diet animals. It however reduced TBARS and CD in the diet and control groups, respectively. At baseline, chronic melatonin treatment caused a significant increase in phospho-PKB/total PKB ratio and a concomitant reduction in phospho-p38 MAPK/total p38 MAPK ratio of control hearts while there were no such effects on diet-induced-obesity hearts. Infarct size was significantly reduced by melatonin in both diet and control groups (DM: 16.6±2.0%; D: 38.4±2.6% (p < 0.001), and CM: 12.8±1.5%; C: 30.4±1.0%, p<0.001). After coronary artery occlusion and 30 minutes of reperfusion, melatonin increased percentage recovery of aortic output (DM: 28.5±6.5%; D: 6.2±6.2%, p<0.01), cardiac output (DM: 44.4±5.2%; D: 26.6±5.1%, p < 0.01) and total work (DM: 34.5±5.6%; D: 20.4±7.9%, p<0.05) of diet-induced obesity hearts, while having no effect on control hearts. During reperfusion, hearts from melatonin treated rats had increased activation of PKB/Akt (p<0.01), ERK42/44 (p<0.05), and reduced p38 MAPK activation (p<0.05). There was no difference in post-ischaemic activation of AMPK (Thr-172) and GLUT-4 expression in either control or diet fed rats. We successfully demonstrated that chronic melatonin treatment prevented the development of diet-induced metabolic abnormalities and improved ex vivo myocardial function. Melatonin protected the heart against ischaemia-reperfusion injury that was exacerbated in obesity. This was achieved by activation of the RISK pathway. The antioxidant properties of melatonin were involved in these cardioprotective effects. / AFRIKAANSE OPSOMMING: Vetsug of obesiteit is een van die hoof risikofaktore vir iskemiese hartsiekte. Obesiteitgeinduseerde metaboliese abnormaliteite gaan met verhoogde oksidatiewe stres gepaard wat op sy beurt ‘n belangrike rol mag speel in die miokardiale wanfunksie en verhoogde vatbaarheid vir iskemie-herperfusie (I/H) beskadiging, kenmerkend van vetsug. Melatonien, die hormoon afgeskei deur die pineaalklier, is ‘n kragtige anti-oksidant. Vorige studies het getoon dat kort-termyn of akute toediening van melatonien die normale hart van gesonde diere teen I/H beskadiging deur middel van sy anti-oksidant aksies beskerm. Die effek van melatonien op die hart in obesiteit is egter nog onbekend. Hierbenewens is die miokardiale seintransduksie meganismes geassosieer met die beskermende effekte van die hormoon nog nie ontrafel nie. ‘n Model van dieet-geinduseerde obesiteit in rotte is gebruik om die volgende te bepaal: (i) die effek van kroniese melatonientoediening op die ontwikkeling van dieet-geinduseerde sistemiese veranderinge soos biometriese en metaboliese parameters en oksidatiewe stres (ii) die effek van kroniese melatonienbehandeling op die respons van die hart op I/H beskadiging en (iii) die rol van herperfusie beskadiging op die aktivering van PKB/Akt en ERK42/44 (die sg RISK seintransduksiepad), die pro-apoptotiese p38MAPK, AMPK sowel as die uitdrukking van GLUT-4. Manlike Wistar rotte (200±20g) is ewekansig in vier groepe verdeel: (i) C, kontrole rotte wat ‘n standaard rotdieet en drinkwater sonder melatonien ontvang (ii) CM, kontrole rotte wat melatonien (4mg/kg/dag) ontvang (iii) D, dieet-geϊnduseerde vet rotte wat ‘n hoë kalorie dieet en drinkwater sonder melatonien ontvang (iv) DM, dieet-geϊnduseerde vet rotte wat melatonien (4mg/kg/dag) in die drinkwater ontvang. Na 16 weke van behandeling, is die rotte geweeg, bloed en hartweefsel gekollekteer vir biochemiese en molekulêre biologie bepalings. Harte is geperfuseer volgens die werkhartmodel, blootgestel aan iskemie/herperfusie vir evaluering van funksionele herstel en infarktgrootte. Uitdrukking en aktivering van PKB/Akt (Ser-473), ERKp42/p44 en p38MAPK van pre-en postiskemiese hartweefsel is met behulp van Western blot bepaal. Postiskemiese aktivering van AMPK (5’AMP-aktiveerde proteϊen kinase) (Thr-172) en GLUT-4 (glukose transporter) is op soortgelyke wyse bepaal. Serum en basislyn hartweefsel glutatioon (GSH) inhoud asook tiobarbituursuur reaktiewe substans (TBARS), gekonjugeerde diene (CD) en lipiedhidroperoksied (LOOH) konsentrasies is bepaal. Resultate Die hoë kalorie diet het ‘n toename in liggaamsgewig, visserale vet, hartgewig, serum insulien, leptien, plasma trigliseried en lae HDL-cholesterol vlakke teweegebring. Bloed glukosevlakke was egter dieselfde in die vet en kontrole rotte. Miokardiale glutatioon, serum glutatioon, totale cholesterol, TBARS, LOOH, CD is nie deur die dieet beinvloed nie. Chroniese melatonien behandeling het die liggaamsgewig, visserale vet, hartgewig, plasma trigliseried, serum insulien en leptien, HOMA indeks verlaag (DM vs D, p<0.05) en die HDL-cholesterol verhoog in die dieetrotte, terwyl dit geen effek op hierdie parameters in kontrole rotte gehad het nie (uitgesonderd ‘n afname in liggaamsgewig, hartgewig en visserale vet). Melatonien behandeling het geen effek op hart of serum GSH en LOOH in kontrole en vet rotte gehad nie. Dit het egter die TBARS en CD in beide vet en kontrole rotte verlaag. Chroniese melatonien toediening het ‘n beduidende toename in basislyn fosfo PKB//totale PKB ratio en ‘n afname in fosfo p38MAPK/totale p38MAPK ratio teweegebring in harte van kontrole rotte, maar soortgelyke effekte is nie in die harte van die vet rotte waargeneem nie. Infarktgrootte is beduidend deur melatonienbehandeling verlaag in beide dieet en kontrole groepe (DM: 16.6± 5.2%, D: 38.4 ±2.6% (p<0.001); CM: 12.8± 1.5%; C 30.4±1.0 (p<0.001). Na koronere arterie afbinding en 30 min van herperfusie, het melatonien die persentasie herstel van aorta omset (DM: 28.5± 6.5%; D: 6.2± 6.2%, p<0.01), kardiale omset ( DM: 44.4± 5.2%D: 26.6±5.1%, p<0.01) en totale werk (DM: 34.5 5.6%; D 20.4± 7.9%, p<0.05) in die harte van dieetrotte verbeter, terwyl dit sonder effek was in kontrole harte. Tydens herperfusie het harte van melatonienbehandelde rotte verhoogde aktivering van PKB/Akt (p<0.01) en ERKp42/p44 (p<0.05) getoon, terwyl aktivering van p38MAPK verlaag is (p<0.05). Geen verskil in postiskemiese aktivering van AMPK en GLUT-4 uitdrukking is in beide kontrole en dieetrotte waargeneem nie. Ons het daarin geslaag om aan te toon dat chroniese melatonienbehandeling die ontwikkeling van dieet-geϊnduseerde metaboliese abnormaliteite beduidend kan voorkom en ex vivo miokardiale funksie verbeter. Melatonien het ook die hart teen iskemie/herperfusie beskadiging beskerm in beide kontrole en dieetrotte. Bogenoemde veranderinge het met aktivering van PKB/Akt en ERKp42/p44 gepaard gegaan. Die anti-oksidant effekte van melatonien was heelwaarskynlik hierby betrokke.