Abstract:
The ventricular cardiac myocyte is the most prominent cell of the heart by volume and weight and is responsible for the contractive force necessary to pump blood around the body. Operation of the ventricular myocyte centre occurs through the coupling of electrical activity and the subsequent mechanical contraction, a process known as excitation contraction coupling (EC Coupling). EC coupling centre around the precise handling of both extracellular and internal calcium ion stores in a process called calcium induced calcium release (CICR). This process is dependent on the precise nanometer scale arrangement of the Ryanodine receptor (RyR) protein within the SR membrane which act as the primary calcium release channel for the SR calcium stores. This thesis aims to investigate the ryanodine receptor and associated structures using super resolution imaging modality to investigate nanoscale changes in structural distribution. Section one focuses on the imaging of the Z disc – the region of localisation for RyR release sites. Results show thicknesses of z disc were measured at 100 nm with transverse arrangement showing all fibril cores being within a unified 500 nm distance band. The second section demonstrates the distribution of RyR proteins in transverse rat cardiac myocyte tissue sections. Results demonstrated a large variation in cluster sizes with approximate exponential distribution. Mean cluster size was 63 RyR with mean edge to edge separation of 130 nm in general agreement with previous confocal data. Colocalisation of RyR with the T-tubule – SR crosslinking protein JPH showed a high level of colocalisation compared with an idealised colocalisation simulation of RyR vs RyR (61% vs 78%). Data is consolidated with diffraction limited data highlighting the detection falloff at smaller cluster sizes. The final section investigated differences in human RyR distribution for normal, idiopathic dilated cardiomyopathy, and rat cells. Results showed little change in cluster size distribution between all three cases, however a change in nearest adjacent neighbour distances were observed. A reduction in colocalisation between JPH and RYR is further seen in humans as compared with rats. These results highlight the subtle structural features seen in the arrangement of RyR clusters, and how super resolution imaging provides greater clarity and additional details on top of the previous attempts at analysis with conventional microscopes.