David R.L. Scriven

Distribution of Proteins Implicated in Excitation-Contraction Coupling in Rat Ventricular Myocytes

We have examined the distribution of ryanodine receptors, L-type Ca(2+) channels, calsequestrin, Na(+)/Ca(2+) exchangers, and voltage-gated Na(+) channels in adult rat ventricular myocytes. Enzymatically dissociated cells were fixed and dual-labeled with specific antibodies using standard immunocytochemistry protocols. Images were deconvolved to reverse the optical distortion produced by wide-field microscopes equipped with high numerical aperture objectives. Every image showed a well-ordered array of fluorescent spots, indicating that all of the proteins examined were distributed in discrete clusters throughout the cell. Mathematical analysis of the images revealed that dyads contained only ryanodine receptors, L-type Ca(2+) channels, and calsequestrin, and excluded Na(+)/Ca(2+) exchangers and voltage-gated Na(+) channels. The Na(+)/Ca(2+) exchanger and voltage-gated Na(+) channels were distributed largely within the t-tubules, on both transverse and axial elements, but were not co-localized. The t-tubule can therefore be subdivided into at least three structural domains; one of coupling (dyads), one containing the Na(+)/Ca(2+) exchanger, and one containing voltage-gated Na(+) channels. We conclude that if either the slip mode conductance of the Na(+) channel or the reverse mode of the Na(+)/Ca(2+) exchanger are to contribute to the contractile force, the fuzzy space must extend outside of the dyad.

Axial tubules of rat ventricular myocytes form multiple junctions with the sarcoplasmic reticulum.

Ryanodine receptors (RyRs) are located primarily on the junctional sarcoplasmic reticulum (SR), adjacent to the transverse tubules and on the cell surface near the Z-lines, but some RyRs are on junctional SR adjacent to axial tubules. Neither the size of the axial junctions nor the numbers of RyRs that they contain have been determined. RyRs may also be located on the corbular SR and on the free or network SR. Because determining and quantifying the distribution of RyRs is critical for both understanding and modeling calcium dynamics, we investigated the distribution of RyRs in healthy adult rat ventricular myocytes, using electron microscopy, electron tomography, and immunofluorescence. We found RyRs in only three regions: in couplons on the surface and on transverse tubules, both of which are near the Z-line, and in junctions on most of the axial tubules--axial junctions. The axial junctions averaged 510 nm in length, but they occasionally spanned an entire sarcomere. Numerical analysis showed that they contain as much as 19% of a cells RyRs. Tomographic analysis confirmed the axial junctions architecture, which is indistinguishable from junctions on transverse tubules or on the surface, and revealed a complexly structured tubule whose lumen was only 26 nm at its narrowest point. RyRs on axial junctions colocalize with Ca(v)1.2, suggesting that they play a role in excitation-contraction coupling.

The molecular architecture of calcium microdomains in rat cardiomyocytes.

Abstract: We have used standard indirect immunofluorescence techniques in combination with wide‐field microscopy and image deconvolution to assess the distribution of proteins implicated in excitation‐contraction coupling and Ca2+ homeostasis in adult rat cardiomyocytes. We begin by discussing our earlier results and summarizing what is known about the molecular architecture of this species to provide a rationale for the work presented here. The previous results showed that the dyads contain Ca2+ channels and ryanodine receptors, but few Na+ channels or Na+/Ca2+ exchangers. The latter proteins were not colocalized elsewhere on the membrane, and we have now found that they appear to be minimally associated with caveolin‐3. None of the molecules examined are distributed uniformly in the membranes in which they are located but are organized into discrete clusters attached to the underlying cytoskeleton, an arrangement that, at the level of light microscopy, does not appear to be affected by the enzymatic dissociation used to study single cells. Analysis of how the clusters are organized and distributed throughout the volume of the cell suggests that there may be differences in excitation‐contraction coupling between the cell surface and the interior.

Microarchitecture of the Dyad

This review highlights recent and ongoing discoveries that are transforming the previously held view of dyad structure and function. New data show that dyads vary greatly in both structure and in their associated molecules. Dyads can contain varying numbers of type 2 ryanodine receptor (RYR2) clusters that range in size from one to hundreds of tetramers and they can adopt numerous orientations other than the expected checkerboard. The association of Ca(v)1.2 with RYR2, which defines the couplon, is not absolute, leading to a number of scenarios such as dyads without couplons and those in which only a fraction of the clusters are in couplons. Different dyads also vary in the transporters and exchangers with which they are associated producing functional differences that amplify their structural diversity. The essential role of proteins, such as junctophilin-2, calsequestrin, triadin, and junctin that maintain both the functional and structural integrity of the dyad have recently been elucidated giving a new mechanistic understanding of heart diseases, such as arrhythmias, hypertension, failure, and sudden cardiac death.

Image acquisition for colocalization using optical microscopy.

Colocalization, in which images of two or more fluorescent markers are overlaid, and coincidence between the probes is measured or displayed, is a common analytical tool in cell biology. Interpreting the images and the meaning of this identified coincidence is difficult in the absence of basic information about the acquisition parameters. In this commentary, we highlight important factors in the acquisition of images used to demonstrate colocalization, and we discuss the minimum information that authors should include in a manuscript so that a reader can interpret both the fluorescent images and any observed colocalization.

Analysis of Cav1.2 and Ryanodine Receptor Clusters in Rat Ventricular Myocytes

We analyzed the distribution of ryanodine receptor (RyR) and Cav1.2 clusters in adult rat ventricular myocytes using three-dimensional object-based colocalization metrics. We found that ∼75% of the Cav1.2 clusters and 65% of the RyR clusters were within couplons, and both were roughly two and a half times larger than their extradyadic counterparts. Within a couplon, Cav1.2 was concentrated near the center of the underlying RyR cluster and accounted for ∼67% of its size. These data, together with previous findings from binding studies, enable us to estimate that a couplon contains 74 RyR tetramers and 10 copies of the α-subunit of Cav1.2. Extradyadic clusters of RyR contained ∼30 tetramers, whereas the extradyadic Cav1.2 clusters contained, on average, only four channels. Between 80% and 85% of both RyR and Cav1.2 molecules are within couplons. RyR clusters were in the closest proximity, with a median nearest-neighbor distance of 552 nm; comparable values for Cav1.2 clusters and couplons were 619 nm and 735 nm, respectively. Extradyadic RyR clusters were significantly closer together (624 nm) and closer to the couplons (674 nm) than the couplons were to each other. In contrast, the extradyadic clusters of Cav1.2 showed no preferential localization and were broadly distributed. These results provide a wealth of morphometric data that are essential for understanding intracellular Ca2+ regulation and modeling Ca2+ dynamics.

Multi-image colocalization and its statistical significance.

Accurately localizing molecules within the cell is one of main tasks of modern biology, and colocalization analysis is one of its principal and most often used tools. Despite this popularity, interpretation is often uncertain because colocalization between two or more images is rarely analyzed to determine whether the observed values could have occurred by chance. To address this, we have developed a robust methodology, based on Monte Carlo randomization, to measure the statistical significance of a colocalization. The method works with voxel-based, intensity-based, object-based, and nearest-neighbor metrics. We extend all of these to measure colocalization in images with three colors. We also introduce three new metrics; blob colocalization, where the blob consists of a local maximum surrounded by a three-dimensional group of voxels; cluster diameter, to measure the clustering of fluorophores in three or more images; and the intercluster distance to measure the distance between these clusters. The robustness of these metrics was tested by varying the image thresholds over a broad range, which produced no change in the statistical significance of the colocalizations. A comparison of blob colocalization with voxel and Manders colocalization metrics shows that the different measures produce consistent results with similar values for significance and nonsignificance. Using our methodology, we are able to determine not only whether the labeled molecules colocalize with a probability greater than chance, but also whether they are sequestrated into different compartments. The program, written in C++, is freely available as source, as well as in a Linux version.

Nonuniform and Variable Arrangements of Ryanodine Receptors Within Mammalian Ventricular Couplons

Rationale: Single-tilt tomograms of the dyads in rat ventricular myocytes indicated that type 2 ryanodine receptors (RYR2s) were not positioned in a well-ordered array. Furthermore, the orientation and packing strategy of purified type 1 ryanodine receptors in lipid bilayers is determined by the free Mg2+ concentration. These observations led us to test the hypothesis that RYR2s within the mammalian dyad have multiple and complex arrangements. Objectives: To determine the arrangement of RYR2 tetramers in the dyads of mammalian cardiomyocytes and the effects of physiologically and pathologically relevant factors on this arrangement. Methods and Results: We used dual-tilt electron tomography to produce en-face views of dyads, enabling a direct examination of RYR2 distribution and arrangement. Rat hearts fixed in situ; isolated rat cardiomyocytes permeabilized, incubated with 1 mmol/L Mg2+, and then fixed; and sections of human ventricle, all showed that the tetramer packing within a dyad was nonuniform containing a mix of checkerboard and side-by-side arrangements, as well as isolated tetramers. Both phosphorylation and 0.1 mmol/L Mg2+ moved the tetramers into a predominantly checkerboard configuration, whereas the 4 mmol/L Mg2+ induced a dense side-by-side arrangement. These changes occurred within 10 minutes of application of the stimuli. Conclusions: The arrangement of RYR2 tetramers within the mammalian dyad is neither uniform nor static. We hypothesize that this is characteristic of the dyad in vivo and may provide a mechanism for modulating the open probabilities of the individual tetramers.

Couplons in rat atria form distinct subgroups defined by their molecular partners.

Standard local control theory, which describes Ca2+ release during excitation–contraction coupling (ECC), assumes that all ryanodine receptor 2 (RyR2) complexes are equivalent. Findings from our laboratory have called this assumption into question. Specifically, we have shown that the RyR2 complexes in ventricular myocytes are different, depending on their location within the cell. This has led us to hypothesize that similar differences occur within the rat atrial cell. To test this hypothesis, we have triple-labelled enzymatically isolated fixed myocytes to examine the distribution and colocalization of RyR2, calsequestrin (Casq), voltage-gated Ca2+ channels (Cav1.2), the sodium–calcium exchanger (Ncx) and caveolin-3 (Cav3). A number of different surface RyR2 populations were identified, and one of these groups, in which RyR2, Cav1.2 and Ncx colocalized, might provide the structural basis for ‘eager’ sites of Ca2+ release in atria. A small percentage of the dyads containing RyR2 and Cav1.2 were colocalized with Cav3, and therefore could be influenced by the signalling molecules it anchors. The majority of the RyR2 clusters were tightly linked to Cav1.2, and, whereas some were coupled to both Ca 1.2 and Ncx, none were with Ncx alone. This suggests that Cav1.2-mediated Ca2+ -induced Ca2+ release is the primary method of ECC. The two molecules studied that were found in the interior of atrial cells, RyR2 and Casq, showed significantly less colocalization and a reduced nearest-neighbour distance in the interior, compared with the surface of the cell. These differences might result in a higher excitability for RyR2 in the interior of the cells, facilitating the spread of excitation from the periphery to the centre. We also present morphometric data for all of the molecules studied, as well as for those colocalizations found to be significant.

Characterisation of glucose transporters in the intact coronary artery endothelium in rats: GLUT-2 upregulated by long-term hyperglycaemia

Aims/hypothesisWe have examined the effects of streptozotocin-induced type 1 diabetes on the expression and subcellular distribution of the classic sugar transporters (GLUT-1 to 5 and sodium-dependent glucose transporter-1 [SGLT-1]) in the endothelial cells of an en face preparation of septal coronary artery from Wistar rats.MethodsThe presence of the GLUT isoforms and SGLT-1 in the endothelial cell layer was determined by immunohistochemistry using wide-field fluorescence microscopy coupled to deconvolution, and was quantified by digital image analysis.ResultsWe found that all of the transporters were expressed within these cells and that all except SGLT-1 were preferentially located on the abluminal side. The heaviest labelling was adjacent to the cell-to-cell junctions where the luminal and abluminal membranes are in close proximity, which may reflect a spatial organisation specialised for vectorial glucose transport across the thinnest part of the cytoplasm. Long-term hyperglycaemia, induced by streptozotocin, significantly downregulated GLUT-1, 3, 4 and 5 and dramatically upregulated GLUT-2, leaving SGLT-1 unchanged.Conclusions/interpretationWe conclude that the high susceptibility of endothelial cells to glucose toxicity may be the result of the subcellular organisation of their GLUTs and the increased expression of GLUT-2.