Browsing by Author "Smith, Angelique"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    The role of Phosphodiesterase 4 in insulin- and phytocannabinoids-induced cardio-protection, and B-adrenergic cardiac damage.
    (Stellenbosch : Stellenbosch University, 2015-03) Smith, Angelique; Lopes, John; Webster, Ingrid; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.
    ENGLISH ABSTRACT: Introduction: Cardiovascular disease (CVD) is one of the main non-communicable diseases globally contributing to deaths each year. Ischemic heart disease leading to myocardial infarction (or heart attack as it is commonly known) is one of the leading causes of death in the United States (Raven et al. 2008) and in South Africa (Stats SA, 2013). Much research is and has been done to understand the underlying factors that lead to this disease, but this study evaluates the role of phosphodiesterase 4 (PDE 4) in mediating damage or protection during an ischemic event. PDEs function in a coordinated manner under the instruction of various stimuli, mostly known to be from G-protein coupled receptors (GPCRs), in order to promote stimulus-specific cellular physiological effects. PDE4 was considered in this thesis because PDE4 exerts a dominant role in global cellular cAMP hydrolysis in rat cardiomyocytes (Johnson et al 2012) and is responsible for mediating 1- and 2-AR specific effects in normoxic cardiomyocytes (Mika et al 2014, Gregg et al 2010). This is through cAMP compartmentation (Nikolaev et al 2006), which might also be true for ischemia/reperfusion, but has never been evaluated. Thus the PDEs expressed in cardiomyocytes might be responsible for the sensitization of cardiomyocytes to ischemic damage. Aim: The aim was to find an alternative way to intervene during acute myocardial ischemia/infarction, through the GPCR pathways by evaluating the role of phosphodiesterase 4 (PDE 4) in mediating damage or protection during an ischemic event. Methods: Cardiomyocytes were isolated from adult male Wistar rat hearts and cultured overnight on 100ug/ml laminin / entactin (L / E). Following overnight cultures at 5%CO2 and 37°C, experiments were done in modified medium X culture buffer (MMXCB) with different treatment conditions of rolipram (a PDE4 inhibitor), dobutamine (b1AR-agonist), formoterol (b2AR-agonist), isoproterenol (b1- and b2AR-agonist), HU210 (synthetic cannabinoid) and THC (delta-9 tetrahydrocannabinol) to compare adult rat cardiomyocytes (ARCM) attachment, contracture and viability. Cardiomyocytes cultured on L/E were subjected to 20 minutes of simulated ischemia [using MMXCB that contained 3mM 2-deoxyglucose (2DG) and 10mM Sodium dithionite (SDT)] with the different treatment drugs administered during ischemia, followed by 15 minutes reperfusion. Cell viability was determined by staining cells with JC-1 and images of cells in a field view that was captured using fluorescence microscopy. The cells were analysed according to cell length, morphology and fluorescence intensity with the help of ImageJ Fiji (open source software at http://fiji.sc/Fiji). Results: A Significant difference in the cell length parameter was observed between the normoxic untreated groups compared to the ischemic untreated groups and this indicated that the ischemic conditions were sufficient to result in hypercontracture induced by ischemia/reperfusion. Regardless of eliciting a proper ischemic insult, no treatments administered during ischemia could alter hyper contracture in the cardiomyocytes. Mitochondrial viability, measured in red over green fluorescence intensity did not always portray a significant difference between the normoxic untreated and ischemic untreated groups. The mitochondrial viability did however increase significantly when 10nM HU210 was administered during ischemia compared to ischemia untreated group. 10uM THC increase the viability when compared to ischemia untreated. With the following experiments the results could not be obtained. The rod-shaped ARCMs attachment as measured by percentage cell viability according to morphology, did show a significant decrease after ischemic stimulation, but no differences could be monitored when the different treatment conditions was compared as the percentage viability decreased greatly to levels of 20% and less. Conclusion: This study suggests that B-AR pathways did not have an effect on cardiomyocyte hyper contracture during an severe ischemia insult, nor insulin. It did show that the cannabinoid-receptors increased mitochondrial viability during ischemia and does indeed play a role during ischemia/reperfusion. However, the exact mechanisms for these effects seen are unknown and need further investigation. PDE4 inhibition with and without different agonists also failed to alter hypercontracture. This lack of change in cardiomyocyte survival parameters in response to the different protective and damaging drugs is considered to be a result of the harsh ischemic conditions used. It is recommended to instead incorporate hypoxia with these treatments to evaluate the effects of PDE4 in the presence and absence of b-AR, cannabinoid and insulin signalling.
  • Thumbnail Image
    Item
    Sanguinarine Non- Versus Re-Circulation During Isolated Heart Perfusion - A Jekyll and Hyde Effect?
    (Springer, 2014) Webster, Ingrid; Smith, Angelique; Huisamen, Barbara; Lochner, Amanda; Biomedical Sciences: Medical Physiology