Thessis (PhD)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: Although there is evidence for a protective role of long-chain polyunsaturated fatty acids (PUFAs) in cardiovascular disease, their mechanism of action as well as their participation in intracellular signalling processes remain to be elucidated. Therefore the aims of this study were twofold: (i) to characterize the roles of the mitogen-activated protein kinases (MAPKs) and protein kinase B (PKB/Akt) in ischaemia/reperfusion-induced apoptosis of neonatal cardiomyocytes and (ii) to establish whether long-chain PUFAs protect the heart via manipulation of these kinases. Rat neonatal ventricular myocytes exposed to simulated ischaemia and reperfusion (Sl/R) were used to characterize the role(s) of extracellular signalregulated kinase (ERK), p38 and c-Jun NH2-terminal protein kinase (JNK), as well as PKB/Akt in apoptosis. The effects of an omega-3 fatty acid (eicosapentaenoic acid - EPA) and an omega-6 fatty acid (arachidonic acid - ARA) on the response of neonatal rat cardiomyocytes to Sl/R with regard to the above parameters were determined. Exposure of the myocytes to SI (energy depletion induced by KCN and 2- deoxy-D-glucose) reduced cell viability, as measured by the 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, and stimulated apoptosis (increased caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage). However, morphological evidence of increased apoptosis (Hoechst 33342 staining) occurred only after reperfusion. A rapid activation of p38 and PKB/Akt Ser473 occurred during SI, while significant activation of ERK and JNK was observed during reperfusion only. Myocytes pre-treated with SB203580, a p38-inhibitor, displayed a significant increase in cell viability and attenuation of apoptosis during Sl/R, while SP600125, a specific JNK inhibitor, significantly increased both caspase-3 activation and the apoptotic index. However, PD98059, an ERK inhibitor, was without effect. Wortmannin, a PI3-kinase inhibitor, reduced PKB/Akt Thr308 but not Ser473 phosphorylation during Sl/R and caused a significant increase in Although there is evidence for a protective role of long-chain polyunsaturated fatty acids (PUFAs) in cardiovascular disease, their mechanism of action as well as their participation in intracellular signalling processes remain to be elucidated. Therefore the aims of this study were twofold: (i) to characterize the roles of the mitogen-activated protein kinases (MAPKs) and protein kinase B (PKB/Akt) in ischaemia/reperfusion-induced apoptosis of neonatal cardiomyocytes and (ii) to establish whether long-chain PUFAs protect the heart via manipulation of these kinases. Rat neonatal ventricular myocytes exposed to simulated ischaemia and reperfusion (Sl/R) were used to characterize the role(s) of extracellular signalregulated kinase (ERK), p38 and c-Jun NH2-terminal protein kinase (JNK), as well as PKB/Akt in apoptosis. The effects of an omega-3 fatty acid (eicosapentaenoic acid - EPA) and an omega-6 fatty acid (arachidonic acid - ARA) on the response of neonatal rat cardiomyocytes to Sl/R with regard to the above parameters were determined. Exposure of the myocytes to SI (energy depletion induced by KCN and 2- deoxy-D-glucose) reduced cell viability, as measured by the 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, and stimulated apoptosis (increased caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage). However, morphological evidence of increased apoptosis (Hoechst 33342 staining) occurred only after reperfusion. A rapid activation of p38 and PKB/Akt Ser473 occurred during SI, while significant activation of ERK and JNK was observed during reperfusion only. Myocytes pre-treated with SB203580, a p38-inhibitor, displayed a significant increase in cell viability and attenuation of apoptosis during Sl/R, while SP600125, a specific JNK inhibitor, significantly increased both caspase-3 activation and the apoptotic index. However, PD98059, an ERK inhibitor, was without effect. Wortmannin, a PI3-kinase inhibitor, reduced PKB/Akt Thr308 but not Ser473 phosphorylation during Sl/R and caused a significant increase in Although there is evidence for a protective role of long-chain polyunsaturated fatty acids (PUFAs) in cardiovascular disease, their mechanism of action as well as their participation in intracellular signalling processes remain to be elucidated. Therefore the aims of this study were twofold: (i) to characterize the roles of the mitogen-activated protein kinases (MAPKs) and protein kinase B (PKB/Akt) in ischaemia/reperfusion-induced apoptosis of neonatal cardiomyocytes and (ii) to establish whether long-chain PUFAs protect the heart via manipulation of these kinases. Rat neonatal ventricular myocytes exposed to simulated ischaemia and reperfusion (Sl/R) were used to characterize the role(s) of extracellular signalregulated kinase (ERK), p38 and c-Jun NH2-terminal protein kinase (JNK), as well as PKB/Akt in apoptosis. The effects of an omega-3 fatty acid (eicosapentaenoic acid - EPA) and an omega-6 fatty acid (arachidonic acid - ARA) on the response of neonatal rat cardiomyocytes to Sl/R with regard to the above parameters were determined. Exposure of the myocytes to SI (energy depletion induced by KCN and 2- deoxy-D-glucose) reduced cell viability, as measured by the 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, and stimulated apoptosis (increased caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage). However, morphological evidence of increased apoptosis (Hoechst 33342 staining) occurred only after reperfusion. A rapid activation of p38 and PKB/Akt Ser473 occurred during SI, while significant activation of ERK and JNK was observed during reperfusion only. Myocytes pre-treated with SB203580, a p38-inhibitor, displayed a significant increase in cell viability and attenuation of apoptosis during Sl/R, while SP600125, a specific JNK inhibitor, significantly increased both caspase-3 activation and the apoptotic index. However, PD98059, an ERK inhibitor, was without effect. Wortmannin, a PI3-kinase inhibitor, reduced PKB/Akt Thr308 but not Ser473 phosphorylation during Sl/R and caused a significant increase in PARP cleavage during reperfusion, but had no effect on caspase-3 activation or the apoptotic index. EPA and ARA (20 jiM, present before and after SI) significantly reduced caspase-3 activation, PARP-cleavage and the apoptotic index during reperfusion. This was associated with increased ERK- and decreased p38 phosphorylation. Vanadate (a tyrosine phosphatase inhibitor), but not okadaic acid (a serine-threonine phosphatase inhibitor), significantly reduced ARAinduced inhibition of p38 phosphorylation, suggesting involvement of tyrosine phosphatases during Sl/R. MKP-1, a dual-specificity phosphatase, was targeted and a significant induction of MKP-1 by ARA and EPA was observed. An in vitro dephosphorylation assay confirmed that this phosphatase might be responsible for the inhibition of p38 activation. It was also demonstrated that the protective actions of ARA are PI3-K dependent. The results suggest that p38 has a pro-apoptotic role while JNK phosphorylation is protective and that these kinases act via caspase-3 to prevent or promote cell survival in response to SI/R-induced injury. It was demonstrated for the first time that EPA and ARA protect neonatal cardiac myocytes from ischaemia/reperfusion-induced apoptosis through induction of a dual-specific phosphatase, MKP-1, causing dephosphorylation of the proapoptotic kinase, p38. These beneficial effects of ARA and EPA were also reflected by improvement in functional recovery during ischaemia/reperfusion of the isolated perfused rat heart model. / AFRIKAANSE OPSOMMING: Dit word algemeen aanvaar dat lang-ketting poli-onversadigde vetsure teen kardiovaskulere siektes beskerm, maar hul meganisme van aksie sowel as hul invloed op intrasellulere seinoordragpaaie is egter onbekend. Die doelwitte van hierdie studie is dus tweevoudig: (i) om die belang van mitogeen-geaktiveerde proteien kinases (MAPKs) en protein kinase B (PKB/Akt) in isgemie/herperfusie-geinduseerde apoptose vas te stel en (ii) om te bepaal of lang-ketting poli-onversadigde vetsure die hart, deur manipulering van hierdie kinases, beskerm. Rot neonatale ventrikulere miosiete, blootgestel aan gesimuleerde isgemie en herperfusie (Sl/H), is gebruik om die aktivering van ekstrasellulere seingereguleerde kinase (ERK), p38, c-Jun NH2-terminale protein kinase (JNK) asook PKB/Akt tydens apoptose, te karakteriseer. Die effek van ‘n omega-3 vetsuur (eikosapentaenoSsuur - EPA) en ‘n omega-6 vetsuur (aragidoonsuur - ARA) op die respons van bogenoemde kinases in neonatale kardiomiosiete tydens Sl/H, is ondersoek. Blootstelling van miosiete aan SI (energie-uitputting gemduseer deur kaliumsianied en 2-deoksi-D-glukose) het ‘n afname in die vermoe van die sel om te oorleef, soos gemeet deur die MTT (3-[4,5-dimetieltiazol-2-yl]-2,5- difeniel tetrazolium bromied) bepaling, tot gevolg gehad. ‘n Toename in apoptose (kaspase-3 aktivering en poli(ADP-ribose) polimerase (PARP) kliewing) is ook waargeneem. Morfologiese bewyse van apoptose (Hoechst 33342 kleuring) was egter eers tydens herperfusie sigbaar. SI is gekenmerk deur vinnige aktivering van p38 en PKB/Akt Ser473, terwyl ERK en JNK fosforilering slegs tydens herperfusie waargeneem is. Vooraf-behandeling met SB203580, ‘n p38 inhibitor, het ‘n beduidende toename in sellewensvatbaarheid asook ‘n afname in die apoptotiese indeks tydens Sl/H teweeggebring, terwyl SP600125, ‘n spesifieke JNK inhibitor, apoptose bevorder het. PD98059, ‘n ERK inhibitor, het geen invloed op apoptose tydens Sl/H gehad nie. Wortmannin, ‘n PI3-kinase inhibitor, het Thr308 (nie Ser473) fosforilering onderdruk, gepaargaande met ‘n toename in PARP kliewing, maar dit het geen invloed op kaspase-3 aktivering of die apoptotiese indeks gehad nie. EPA en ARA (20 (iM, teenwoordig voor en na SI) het kaspase-3 aktivering en PARP kliewing asook die apoptotiese indeks tydens herperfusie beduidend verminder. Beide vetsure het ook ‘n beduidende toename in ERK en afname in p38 fosforilering veroorsaak. Vanadaat (‘n serien-threonien fosfatase inhibitor), maar nie “okadaic” suur (‘n tirosien fosfatase inhibitor), kon die ARA-gemduseerde inhibisie van p38 ophef nie. Induksie van MKP-1, ‘n tweeledige-spesifieke fosfatase, is beduidend deur ARA en EPA tydens herperfusie verhoog. 'n In vitro defosforileringbepaling het bevestig dat hierdie fosfatase wel betrokke by die inhibisie van p38 kan wees. Daarbenewens is gevind dat die beskermende aksie van ARA PI3-K afhanklik is. Hierdie resultate wys dat fosforilering van p38 pro-apoptoties is, terwyl JNK beskermend is en dat hierdie kinases via kaspase-3 seldood of oorlewing tydens SI/H-geinduseerde beskadiging bemiddel. In hierdie model is daar vir die eerste keer getoon dat EPA en ARA neonatale kardiale miosiete teen isgemie/herperfusie-geinduseerde apoptose beskerm deur induksie van MKP- 1, wat defosforilering van die pro-apoptotiese kinase, p38 teweegbring. Hierdie voordelige effekte van EPA en ARA is ook sigbaar in die funksionele herstel tydens isgemie/herperfusie van die geisoleerde rothart model.

published_or_final_version / Medicine / Master / Doctor of Medicine

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Physiology / Master / Master of Philosophy

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Medicine / Master / Master of Philosophy

published_or_final_version / Nursing Studies / Master / Master of Philosophy

Current dietary recommendations have placed increasing emphasis on dietary fat as an important element to decrease risk of cardiovascular disease (CVD). Although total fat and the fatty acid composition of diets influence the risk of CVD, the optimal amounts of different fatty acids are not well defined, especially if n-6 and n-3 fatty acids are considered. Despite the fact that postmenopausal women are at increased risk of CVD, few studies have investigated the influence of dietary fatty acids on this risk. Therefore, this study was designed to determine the effect of supplementation with different fatty acids on risk factors of CVD in postmenopausal women. Sixteen healthy, postmenopausal women were randomly assigned in a three-period crossover trial to treatments of 15 g/d supplements of oleic acid-rich sunflower oil (TS), linoleic acid-rich safflower oil (SO), and eicosapentaenoic acid- and docosahexaenoic acid-rich fish oil (FO). Each treatment period lasted 5 weeks followed by a 7-week washout interval. When the women were supplemented with FO compared to supplementation with either TS or SO, the concentration of high density lipoprotein cholesterol tended to increase (p=0.07 and 0.05, respectively) as did the size of the low density lipoprotein (LDL) particle (P=0.03 in both instances) while the concentration of triacylglycerol (p=0.0001 and 0.02, respectively) and apolipoprotein B (apo B) (P=0.005 and P=0.01, respectively) decreased. The concentration , i.e., total cholesterol, cholesterol ester, free cholesterol, phospholipids, α- and γ-tocopherol, of the two LDL subfractions was not influenced by any of the oil supplements but was greater in the large (L) subfraction than the small (S). When the oxidation of the two subfractions was measured by monitoring the formation of conjugated dienes, the lag time was shorter in both fractions after supplementation with FO compared to supplementation with SO (P=0.0001) or TS (P=0.0001) but the effect was greater in the L subfraction. The rate of formation of conjugated dienes, which was slower after FO supplementation than supplementation with either TS (P=0.02) or SO (P=0.001), was faster in the L compared to the S subfraction. When oxidation was measured by monitoring the increase in negative charge on apo B over 23 hr, only the 1 hr time point differed. The increase was greater in the FO-supplemented group than either the TS- or SO-supplemented groups (P=0.001 in both instances). The change was greater in S LDL (P=0.007). These findings demonstrate a greater potential antiatherogenic property of dietary n-3-rich oil than n-6- or n-9-rich ones as indicated by changes to plasma lipids, lipoproteins, apo B, and particle size but the influence of the oxidative susceptibility of L and S subfractions is less conclusive. / Graduation date: 2000

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