Biomedical Engineering - Theses
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ItemRole of ultrastructural alterations in diabetic cardiomyopathy( 2019)Cardiomyocytes inside the heart are densely packed with parallel columns of myofibrils and mitochondria. Growing evidence indicates a strong correlation between alterations in this sub-cellular ultrastructure and the alterations in energy metabolism during various pathological conditions of heart. The central hypothesis explored in this thesis is that changes in cardiac sub-cellular architecture in pathological conditions can also affect cardiac bioenergetics by interfering with various mechanisms of intracellular energy transport. Type 1 diabetic cardiomyopathy is an ideal candidate for a model disease state to understand this hypothesized interplay between ultrastructure and metabolism. It exhibits many common conditions which accompany heart failure, such as increased mitochondrial reactive oxygen species production and decreased reserve of creatine phosphate. In a preliminary study, 2D electron microscope images collected from control and streptozotocin induced type I diabetic rat hearts were analysed. It was found that diabetic cardiomyopathy leads to an increased mitochondrial fission and formation of large mitochondrial clusters. Further analysis showed that effective surface-to-volume ratio of mitochondrial clusters increases by 22.5% in diabetic cells. Subsequently, a compartmental model of cardiac energy transfer was developed. This simple model predicted that this increase in the surface-to-volume ratio can have a moderate compensatory effect by elevating the availability of adenosine triphosphate (ATP) in the cytosol when ATP synthesis within the mitochondria is compromised. Next, 3D electron microscope images from control animals were investigated. The analysis revealed that cardiac mitochondria are arranged non-uniformly in parallel columns of varying sizes. Following this, the compartmental model was extended to a reaction diffusion based 2D finite element model incorporating a realistic description of the observed sub-cellular ultrastructure. The new model predicted that rapid diffusion of creatine and creatine phosphate acts to maintain homogenous ATP distribution and uniform force dynamics in the control cardiomyocytes, despite the heterogeneous mitochondrial organization. Subsequently, 3D electron microscope images of cardiomyocytes from streptozotocin (STZ) induced type I diabetic rats were compared with controls. The analysis revealed that mitochondrial distribution along the transverse sections was significantly more heterogeneous in type I diabetes compared to control cells. Moreover, mitochondrial area fraction in the studied type I diabetic cells was higher than the control cells. Finally, 2D models of cardiac energy metabolism were created based on the electron microscope images collected from the control and diabetics cells. The results indicated that an increased fraction of mitochondria in diabetic cells can compensate for the reduced ATP synthesising capacity of diabetic mitochondria. The models also predicted that lower activity of mitochondrial enzymes in type I diabetes, coupled with the observed non-uniform mitochondrial distribution, can lead to large spatial variation in concentration of ATP and adenosine diphosphate (ADP). The heterogeneous metabolic landscape in the diabetic cell cross sections was also reflected in large spatial gradients of myofibrillar ATP consumption rate. This finding is important since ATP consumption rate correlates with the speed of muscle shortening. Different parts of a diabetic cell might contract at different rates, which can decrease the energy efficiency of the cell and also damage the cell structure. Thus, this thesis, combining image analysis with computational modelling, provides new insights into how the ultrastructure regulates the metabolism of the cardiomyocytes in disease and health.
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ItemDecoding calcium signalling crosstalk in cardiac hypertrophy( 2017)The concentration of calcium ions (Ca2+) within a cell is important for governing many different processes across a range of cell types. In heart muscle cells, proper calcium handling is critical for maintaining the rhythmic cycle of contraction and relaxation. Chronic stresses can drive changes in calcium signalling which trigger heart cells to grow in size. This process is called hypertrophy and is a common cause of heart failure. It was unknown how a cell could distinguish calcium signals leading to cell growth from those leading to contraction. The central hypothesis explored in this thesis is that calcium can simultaneously yet specifically effect two distinct responses in a cardiomyocyte by altering the shape of the cellular calcium transient, but how this might be achieved without disrupting contractile function was unknown. New line-scan data shows that IP3R-mediated Ca2+ release widens the cellular transient following RyR-mediated Ca2+-induced Ca2+-release events. The data supports the hypothesis that RyR and IP3R systems interact by inducing a global yet transient elevation in Ca2+. A mathematical model of the whole-cell adult rat ventricular myocyte Ca2+ transient was developed by combining existing models of RyR and IP3R and fitting to the line-scan data. The model includes the two major compartments that interpret the calcium signalling and provide spatial separation -- the cytosol which achieves the calcium-dependent contractile response, and the nucleus which achieves calcium-dependent gene expression. The compartmental model was found to reproduce the observed shape of the Ca2+ transient, but only if IP3Rs exhibit a refractory response. This difference in time-course kinetics could underlie the signal separation between cell contraction and cell growth. Advanced immunofluorescence imaging and statistical methods were used to map the spatial positioning of RyRs and IP3Rs in adult rat heart cells. A statistical tool was developed to simulate physiologically-realistic protein distributions on images of the cell architecture. A spatial model of the Ca2+ transient based on realistic RyR distributions showed that both cellular architecture and the distance between RyR clusters could affect local signalling events. For the first time, super-resolution data was used to establish the relationship between RyRs and IP3Rs. Data analysis indicated that cardiomyocyte-specific IP3R cluster distribution reduces the effective spatial distance between RyR clusters which may promote Ca2+ signal propagation or which may enhance Ca2+ signal strength and longevity. This research combining mathematical modelling and advanced imaging has shown that RyR and IP3R proteins co-exist within the same areas of the heart cell and can modify the normal contractile signal without disrupting it. The modification of the Ca2+ signal may subsequently be interpreted by the cell as a signal to alter calcium-dependent gene expression. This finding is important because it reveals how the cell growth signal might be encoded in the heart cell, and this encoding mechanism may be extensible to many other signaling processes in different cells.