Effects of dietary red palm oil supplementation on myocardial infarct size in normal and hypercholesterolaemic rats: the role of matrix metalloproteinase 2, glutathione peroxidase transcription and extracellular signal regulated kinase phosphorylation

Abstract

Cardiovascular disease remains one of the leading causes of death globally. Many pharmacotherapeutic strategies are constantly being developed in order to effectively reduce the prevalence, as well as the morbidity and mortality associated with cardiovascular disease. In recent years, studies in our laboratory were able to demonstrate that dietary red palm oil (RPO) supplementation could offer protection against myocardial ischaemia/reperfusion injury. Several possible mechanisms for this protection were proposed, including: 1) upregulation of nitric oxide (NO), 2) cyclic guanylyl-mono-phosphate (cGMP) signaling, 3) scavenging of harmful free radicals associated with ischaemia/reperfusion injury and 4) the modulation of mitogen activated protein kinase (MAPK) signaling pathways which inhibit myocyte apoptosis. These findings created opportunities for further investigations to be conducted in order to elucidate RPO mediated pathways involved in cardiac protection. Matrix metalloproteinase 2 (MMP2) is an endogenous protease which is normally associated with the digestion of gelatin. This protease has recently become the focus of many cardiovascular studies, as it was found to be a mediator of ischaemia/reperfusion injury. Activation of MMP2 by free radicals leads to induction of myocyte death through degradation of intracellular targets. Research has shown that dietary RPO supplementation is able to increase activity of glutathione peroxidase (GPX) which is an endogenous antioxidant expressed in most mammalian tissue, including the heart. Increased antioxidant activity in the heart may lead to a reduction in ischaemia/reperfusion injury. However, the mechanism by which RPO supplementation was able to increase GPX activity is not known. The aims of the study were: 1) To investigate whether dietary RPO supplementation can reduce myocardial infarct size of rats fed a standard rat chow diet and rats fed a cholesterol supplemented diet 2) To investigate the whether inhibition of MMP2 plays a role in RPO mediated protection against ischaemia/reperfusion injury 3) To determine whether dietary RPO supplementation regulates GPX activity through gene transcription. Three study designs are described in this thesis where use was made of a male Wistar rat model being fed RPO supplemented diets for a 5 to 9 week period. Study design 1 compared rats placed on RPO supplemented diets with sunflower oil (SFO) fed rats and standard rat chow (SRC) fed rats while Study design 2 compared rats fed cholesterol supplemented diets with rats placed on a cholesterol+RPO supplemented diet. Study design 3 makes use of similar groups as Study design 1. After the supplementation period, rats were sacrificed and the excised hearts perfused on either a Langendorff perfusion apparatus or a working heart perfusion apparatus. Myocardial infarct size and aortic output recovery were measured in order to determine whether RPO does offer protection against ischaemia/reperfusion injury. MMP2 activity was measured in coronary effluent samples of the hearts, in order to determine its level of activity in RPO supplemented hearts. GPX1, -3 and -4 gene transcription were also measured by quantitative real time polymerase chain reaction (qrtPCR), in hearts of RPO supplemented rats, and compared to controls after supplementation. Other assays performed include ELISA (enzyme linked immunosorbent assay) for the determination of serum cholesterol, serum triglyceride and myocardial 3-nitrotyrosine levels as well as western blots to determine protein kinase B (also known as Akt (PKB/Akt)) and extracellular signal-regulated kinases (ERK) in myocardial tissue. Our results showed that dietary RPO supplementation was able to reduce myocardial infarct size in SRC fed rats (9.17 ± 1.03% in the RPO group, versus 30.20 ± 3.97% in the SRC group), as well as rats fed a cholesterol supplemented diet 26,87 ± 2,96% in the HCRPO group, versus 37.16 ± 3.58% in rats fed a cholesterol supplemented diet). Dietary RPO supplementation was also able to increase aortic output recovery in SRC fed rats (47.16 ± 5.46 % versus 13.44 ± 6.34 %). The decreased infarct size was associated with decreased MMP2 activity during reperfusion in the SRC fed rats (1389.27 ± 124.34 arbitrary units versus 1724.42 ± 69.77 arbitrary units; 72kDa isoform: 2635.03 ± 163.02 arbitrary units versus 3201.63 ± 104.97 arbitrary units). Dietary SFO supplementation reduced MMP activity at the same time point, with no reduction in infarct size. Dietary supplementation of RPO and cholesterol to rats, led to decreased MMP2 activity before ischaemia (228.43 ± 28.06 arbitrary units versus 450.83 ± 33.62 arbitrary units). The MMP2 activity was significantly increased in the cholesterol+RPO supplemented group when compared to the cholesterol fed rats after ischaemia (2107.06 ± 50.99 arbitrary units versus 1821.90 ± 56.92 arbitrary units). RPO supplementation did not show any significant differences in GPX transcription. We therefore conclude that dietary RPO supplementation reduced myocardial infarct size in SRC fed rats and rats fed a cholesterol supplemented diet. Inhibition of MMP2 activity was also shown not to be a major pathway of protection involved in RPO mediated protection against ischaemia/reperfusion injury. Furthermore, our results are in agreement with previous studies which show that dietary RPO supplementation is able to improve aortic output recovery. RPO supplementation does however, not increase GPX activity through upregulation of GPX gene transcription.