UBC Theses and Dissertations
Dynamic distribution of ryanodine receptors in cardiac muscles Asghari, Parisa
The focus of this thesis is to address the location and distribution of the type 2 Ryanodine Receptor (RyR2) in mammalian cardiac myocytes with respect to their function. These integral membrane proteins function as Ca²⁺-activated Ca²⁺ ion channels as well as a scaffold for a large number of signaling molecules that modulate the release of Ca²⁺ through the channel. The relative position of the RyR2 tetramers is therefore a critical determinant of their function. To study this question, I have used a combination of immunofluorescence microscopy, transmission electron microscopy, and tomography to map the position of the tetramers in whole cells and in cell sections and have used tissue obtained from both rat and human hearts. Biochemical and physiological techniques were used to correlate structure with function. I have found that RyR2s are located only in three regions: in couplons on the surface, transverse tubules and on most of the axial tubules. In all regions, most but not all of the RyR2s colocalize with the voltage-gated Ca²⁺ channel (Cav1.2), suggesting that they play a role in excitation-contraction coupling. Some RyR2 are colocalized with cavelin-3 and not with Cav1.2 and hypothesized that these ‘extra-couplonic’ RyR2 might be regulated by the multitude of signaling molecules associated with caveolin-3 to modulate Ca²⁺ release. Dual-tilt electron tomography produced en face views of both rat and human dyads, enabling a direct examination of RyR2 arrangement. Both species showed that tetramer packing was non-uniform containing a mix of checkerboard and side-by-side arrangements as well as isolated tetramers. Finally, I showed that the tetramers’ arrangement depended on the Mg²⁺ concentration and on their phosphorylation status; in low Mg²⁺ and after phosphorylation RyR2s were positioned in largely checkerboard arrangements while in response to high Mg²⁺ the tetramers were positioned largely side by side. These tetramer arrangements: side by side, mixed and checkerboard were associated with progressively increasing spark frequencies. The correlation between tetramer arrangement and spark frequency suggests that tetramer rearrangement may be another mechanism whereby physiological processes operate and provides potential new mechanisms by which the activity of RYR2, the dyad and cardiac contractility may be regulated.
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