Browsing by Author "Lumkwana, Dumisile"
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- ItemIdentifying appropriate attachment factors for isolated adult rat cardiomyocyte culture and experimentation(Stellenbosch : Stellenbosch University, 2014-04) Lumkwana, Dumisile; Lopes, John; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences, Division of Medical Physiology.ENGLISH ABSTRACT: Introduction: Primary culture of isolated adult rat cardiomyocytes (ARCMs) is an important model for cardiovascular research, but successful maintenance of these cells in culture for their use in experiments remains challenging (Xu et al, 2009; Louch et al, 2011). Most studies are done on acutely isolated cardiomyocytes immediately after isolation, which is due to low survival of these cells in culture. Obstacles in culture are due to the type of medium and attachment factors (tissue culture adhesives) used to culture and grow these cells. Although we previously identified an optimum medium and adhesive for culture, an adhesive that permits cells to remain attached to the culture surface until after an ischemia/reperfusion insult was elusive. Aims: We therefore aimed to identify the best attachment factor and concentration that will allow adult rat cardiomyocytes to remain attached to the culture surfaces after ischemia/reperfusion experiments. Methods: Cardiomyocytes were isolated from adult Wistar rat hearts and cultured overnight on different concentrations (25 -200 μg/ml) of collagen 1, collagen 4, extracellular matrix (ECM), laminin/entactin (L/E) and laminin. Following overnight cultures, experiments were done in PBS and in PBS versus MMXCB to compare ARCM attachment and viability. Cardiomyocytes cultured on ECM, L/E and L (25−200μg/ml) were subjected to 1 hour of simulated ischemia using MMXCB that contained 3mM SDT and 10mM 2DG, followed by 15 minutes reperfusion. Cell viability was determined by staining cells with JC-1 and images of cells in a field view of 1.17μm/mm2 were captured using fluorescence microscopy. The cells were analysed according to morphology and fluorescence intensity. Results: Total and rod-shaped ARCMs attachment was improved when MMXCB was used as an experimental buffer instead of PBS. Regardless of the buffer used, morphological viability was poor on substrates of Col 1 and Col 4. In contrast to collagens, ARCMs attached efficiently and morphological viability was high on substrates of ECM, L/E and L in MMXCB, but this was greatly reduced in PBS. Mitochondrial viability was high in MMXCB compared to PBS on Col 1 and Col 4 at 75−175μg/ml and on ECM, L/E and L at all concentrations, except at 50 and 150μg/ml ECM, 175μg/ml L/E and 25μg/ml L. When cardiomyocytes cultured on ECM, L/E and L were subjected to simulated ischemia, total ARCMs, rod-shaped and R/G fluorescence (mitochondrial viability) was reduced at all concentrations compared to the control group. Hypercontracted cells were higher in the ischemic treated cells compared to the controls on ECM at 75−150μg/ml and 200μg/ml, L/E at 50,100μg/ml and 175μg/ml and on L at 125μg/ml. Total numbers of ARCMs attached on ECM, L/E and L in the ischemic group consisted of similar numbers of non-viable hypercontracted and viable rod-shaped cells. Conclusion: Cardiomyocytes should be cultured on ECM or L/E or L at concentrations from 25−200μg/ml in MMXCB. PBS is harmful to cultured ARCMs and should thus not be used as an experimental buffer. Ischemia/reperfusion can be simulated on ARCMs cultured on ECM, L/E or L from 25−200μg/ml, provided that a modified culture buffer is used as experimental buffer.
- ItemInvestigating the role of rilmenidine and spermidine on autophagic flux and cell death in a model of Alzheimer's disease(Stellenbosch : Stellenbosch University, 2020-04) Lumkwana, Dumisile; Loos, Ben; Kinnear, Craig; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Introduction - Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by multiple cognitive deficits. The neuropathology of AD is underpinned by two molecular hallmarks; intracellular protein aggregates known as neurofibrillary tangles (NFTs), composed of hyper-phosphorylated Tau and extracellular amyloid beta (Ab) plaques, composed of Ab peptides derived from the amyloid precursor protein (APP). Both of these occur as a result of an imbalance in proteostasis, leading to neuronal toxicity. Although, we have advanced our understanding of the molecular machinery that regulates the rate of protein degradation through autophagy at basal levels and increasingly so the many aspects of its dysfunction in AD, the deviation of autophagic activity from basal levels and its change during disease pathogenesis in neuronal tissue remains largely unclear. Over the recent years, substantial progress has been made in modulating autophagy using pharmacological agents in vitro and in vivo and mounting evidence points towards autophagy modulation using pharmacological agents as one of the major therapeutic strategies for neurodegenerative diseases. Although spermidine and rilmenidine both enhance autophagy, the relationship between autophagy activity, the extent of protein clearance and cell death onset remains unclear. Moreover, the impact of their concentration on autophagic flux and subsequent protein clearance as well as neuronal toxicity is unclear. Therefore, this study aimed to unravel the impact of using a high and low concentration of spermidine and rilmenidine on autophagic flux, neuronal toxicity and protein clearance using distinct neuronal injury model systems. Methods - Murine hypothalamus-derived GT1-7 neuronal cells and the mouse neuroblastoma (N2a) cell line stably expressing Swedish double mutation APP695 (Swe) associated with AD pathology were used. GT1-7 cells transfected with mRFP-GFP-LC3 and GFP-LC3-RFP-LC3DG were treated with a low and high concentration of spermidine and rilmenidine in the absence and presence of saturating concentrations of bafilomycin, after which the autophagic flux profile was characterized, assessing cellular viability, autophagosome pool, autolysosome pool, autophagosome flux, transition time, p62 puncta and autophagic vacuoles. Cellular viability assays, western blotting, fluorescence microscopy, transmission electron microscopy and correlative light and electron microscopy linked to quantitative morphometric analysis was performed. In addition, potential protective effects of a low and high concentration of spermidine and rilmenidine were assessed in a paraquat (PQ)-induced neuronal toxicity model and in an APP over-expression model. Cellular viability, ROS damage, cell death onset and autophagic activity were assessed in the PQ-induced toxicity model, while in the APP model, cellular viability and protein clusters were assessed and quantified using cellular viability assays and single molecule imaging, i.e. direct Stochastic Optical Reconstruction Microscopy (d-STORM). Moreover, a Correlative Light and Electron Microscopy (CLEM) method was developed and implemented to morphometrically assess the effects of a low and high concentration of spermidine on the localization of autophagosomes in 3 dimensions. Finally, potential protective effects of spermidine at two distinct concentrations were assessed using transgenic mice expressing GFP-LC3, treated with PQ to induce neuronal toxicity associated with neurodegeneration. Results - Our results indicate a concentration-dependent effect of spermidine and rilmenidine on autophagic flux with the detected change in flux depending on the specificity and sensitivity of the method employed. In addition, in the in vitro model of PQ-induced toxicity, our results revealed that spermidine at a low concentration and not a high concentration protected against cell toxicity, ROS damage, cell death as well as microtubule destabilization. To our surprise, both concentrations of rilmenidine failed to protect against ROS damage and cell death and also failed to robustly upregulate autophagy in the same model. Moreover, in the in vitro model of neuronal toxicity induced by APP over-expression, our results showed that both concentrations of spermidine and rilmenidine protected against cytotoxicity. Here, spermidine at a low concentration effectively cleared APP clusters and reduced their size, especially after 48 h of APP over-expression, while rilmenidine, reduced the number and size of APP clusters in a concentration-dependent manner at the same time point. Taken together, the in vitro results reveal a concentration-dependent effect, that is cell type and injury specific, impacting autophagy and cell death control. Moreover, we have successfully implemented a 3D CLEM protocol and revealed that spermidine, in combination with BafA1 decreases autophagosome volume while increasing their surface area in a concentration-dependent manner. Lastly, in vivo, our results reveal that PQ-induced toxicity impacts the brain regions differentially, with the hippocampus being highly susceptible to PQ-induced injury followed by the cortex. Moreover, our results show that both dosages of spermidine robustly protected against oxidative stress, neuronal damage, microtubule destabilization, and upregulated autophagy, in the hippocampus and cortex. However, the low dose of spermidine resulted in more enhanced protection, although, autophagy was here regionspecifically upregulated in a manner dependent on the dose utilized, with the higher dose of spermidine increasing LC3-II in the hippocampus, and the lower dose increasing LC3-II in the cortex. Conclusion - Our results indicate the critical importance of using multiple tools to assess autophagy and show that a concentration-dependent effect of the two selected drugs on autophagic flux exists. In addition, we provide evidence of the distinct, context dependent protective roles of spermidine and rilmenidine in an in vitro model of APP over-expression as well as the protective roles of spermidine using in vitro and in vivo models of PQ-induced neuronal toxicity. These results suggest that firstly administration of spermidine may represent a favourable therapeutic strategy for the treatment of AD and secondly, concentration / flux screening may be more critical for optimal autophagy control than previously thought. Future studies, using an in vivo model over-expressing APP are warranted to further verify the protective effects of spermidine, to foster clinical translation and therapeutic intervention in neurodegenerative disorders.