Relationship of Ryanodine Receptors to the Sarcolemma in Rabbit Ventricular Myocytes

Abstract

To investigate the spatial relationship between the sarcolemma and ryanodine receptors (RyRs) we dual-labeled cells using Alexa fluor dyes and simultaneously imaged them with confocal microscopy. We deconvolved the images and subjected them to digital processing. We obtained three-dimensional reconstructions from cells in two configurations: lying flat and standing on end. In the flat configuration, RyR clusters appeared to be arranged in sheets near Z-disks. The distance between sheets was ∼2 μm. Although some clusters are closely associated with detectable sarcolemma, the majority of them are not (>70%). With cells standing on end in agar we obtained XY scans orthogonal to the cell long axis. This improved the separation of RyR clusters and the classification based on their relationship to the sarcolemma. These data confirmed our initial finding obtained on cells lying flat, that most of RyRs are non-junctional (see figure). It is unclear whether they contribute to the Ca transient. If they do, the transient would reflect the combined activity of locally controlled couplons with the activation of non-junctional RyRs, which are not locally controlled.View Large Image | View Hi-Res Image | Download PowerPoint Slide