Analysis of Ca_v1.2 and Ryanodine Receptor Clusters in Rat Ventricular Myocytes

Abstract

We analyzed the distribution of ryanodine receptor (RyR) and Ca_v1.2 clusters in adult rat ventricular myocytes using three-dimensional object-based colocalization metrics. We found that ∼75% of the Ca_v1.2 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, Ca_v1.2 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 Ca_v1.2. Extradyadic clusters of RyR contained ∼30 tetramers, whereas the extradyadic Ca_v1.2 clusters contained, on average, only four channels. Between 80% and 85% of both RyR and Ca_v1.2 molecules are within couplons. RyR clusters were in the closest proximity, with a median nearest-neighbor distance of 552 nm; comparable values for Ca_v1.2 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 Ca_v1.2 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.

Introduction

Normal excitation-contraction coupling (ECC) in the ventricle is dependent on correct positioning of two key proteins: the voltage-gated L-type Ca²⁺ channel (Ca_v1.2) situated in the surface and tubule membranes, and the type 2 ryanodine receptors (RyR) in the membrane of the junctional sarcoplasmic reticulum (jSR). Juxtaposition of these membranes across a narrow 15 nm gap brings the proteins into close apposition, where influx of Ca²⁺ through Ca_v1.2 initiates a much larger Ca²⁺ release from the SR through RyR via Ca²⁺ induced Ca²⁺ release. This distinct structural arrangement has been referred to as a calcium release unit, an excitation-contraction coupling unit, or a couplon (1). The physical arrangement of the proteins and the couplons is thought to be key in understanding how Ca²⁺ release is regulated and controlled.

Multiple publications have documented the distribution of RyR clusters in rodents (both rats and mice) (2–5). A notable omission from these studies is data regarding the distribution of Ca_v1.2, which would enable us to differentiate between clusters that are in couplons and those that are not (6). This, in combination with previously published data for whole-cell numbers of RyR and Ca_v1.2, would allow us to estimate the size and density of clusters, and the relative numbers of molecules in each cluster, and to determine the relevant intercluster distances. Super-resolution techniques (2) have revealed that each RyR cluster consists of smaller, irregularly shaped arrays. Diffraction-limited optical systems, such as those used here, see these arrays as a single cluster, and the distances we report are between clusters rather than within them; similarly, the sizes are for the whole cluster, not the arrays within them.

In this study, we obtained measurements in adult rat ventricular myocytes using immunocytochemistry and three-dimensional fluorescence microscopy, taking advantage of well-characterized antibodies for RyR and Ca_v1.2 (7). We used two imaging modalities to highlight different aspects of the data: horizontally oriented myocytes on a wide-field microscope, and vertically oriented myocytes on a confocal microscope. We used a new algorithm for determining colocalization by measuring clusters rather than individual voxels, as such a measure more likely represents the underlying functional relationships (8). In addition, we modified our display algorithms to show these clusters and highlight their relationship to one another.

Materials and Methods

All chemicals were purchased from Sigma-Aldrich (Oakville, Ontario, Canada) unless otherwise stated. The animals were handled in accordance with the guidelines of the Canadian Council on Animal Care.

Immunofluorescent labeling and data acquisition

Ventricular myocytes were isolated from the hearts of adult male Wistar rats weighing between 200 and 250 g. Dissociation of myocytes, fixation, permeabilization, and labeling were performed as described previously (9). We used an anti-RyR2 monoclonal antibody (Affinity Bioreagents, Golden, CO) and an affinity-purified rabbit polyclonal antibody against the pore-forming subunit of the voltage-gated calcium channel, Ca_v1.2 (a gift from Dr. W. Catterall, Seattle, WA) (7). The secondary antibodies were affinity-purified and highly cross-adsorbed to minimize cross-reaction, and were goat anti-mouse conjugated to Alexa 594 and goat anti-rabbit conjugated to Alexa 488 (Invitrogen, Burlington, Ontario, Canada). Wide-field microscopic images were acquired with an inverted Zeiss Axio Observer microscope equipped with a Plan Apo 63/1.4 oil immersion objective and EXFO Xcite illumination (Mississauga, Ontario, Canada). All filters were from Semrock (Rochester, NY; exciter FF01-494/20-25, dichroic FF506-Di01-25x36, emitter FF01-536/40-25 for Alexa 488; and exciter FF01-575/25-25, dichroic FF593-Di02-25/36, emitter FF01-624/40-25 for Alexa 594). Sixteen-bit images were collected on a thermoelectrically cooled CCD camera with an SITe 1502AB chip; voxel dimensions were 95 nm × 95 nm × 250 nm. For cross-sectional images, the labeled cells were embedded in 2% low-melting-point agarose to which 2 mM Trolox was added to inhibit photobleaching. The agarose was allowed to cool and solidify into a cylindrical shape, and was then sliced into thin disks that were placed on a coverslip (3). Images from the cross-sections were acquired with an Olympus FV 1000 Fluoview microscope equipped with a 60/1.2 water immersion objective and a 12-bit photomultiplier tube. The voxel dimensions were 51 nm × 51 nm × 170 nm.

Data analysis

Wide-field images were background subtracted and flat-field corrected, and both wide-field and confocal images were deconvolved using empirically measured point spread functions (PSFs) acquired from subresolution beads. For the confocal microscope, the full width at half-maximum (FWHM) values of the deconvolved PSFs were 250 nm in XY and 800 nm in Z for Alexa 488, and 280 nm in XY and 900 nm in Z for Alexa 594. For the wide-field microscope, the FWHM values of the deconvolved PSFs were 255 nm in XY and 790 nm in Z for Alexa 488, and 275 nm in XY and 860 nm in Z for Alexa 594. The procedures have been described in detail elsewhere (9).

We calculated colocalization between images using three metrics. The first metric, voxel colocalization, measures the percentage of voxels labeled with fluorophore A that also contain fluorophore B and vice versa. The second and third metrics are measures of object colocalization, which allows us to determine the relationship between the clusters that make up the dyad. Specifically, if the two components of the dyad overlap each other's brightest points, they are considered to be colocalized even if the number of voxels in one component is smaller than the other. From this information we derive two metrics: the percentage of clusters that are colocalized, and the percentage of voxels that are within the colocalized clusters. Nearest-neighbor distances (NNDs) were calculated between the centers of these clusters. These metrics and the methodology used to calculate them are described in greater detail in Fletcher et al. (8).

We determined the volume of the cell sections by identifying the coordinates of the cell surface using the algorithm developed by Lifshitz et al. (10), and described in detail by Scriven et al. (9). We then directly determined the volume according to the number of voxels inside the section.

The standard error of a ratio of means was calculated according to Cochran (11).

Results

In Fig. 1 A we show a stereo pair from the wide-field microscope, with RyR in red, Ca_v1.2 in green, and the colocalized voxels white. In these images, and in images acquired from 25 other cells isolated from six rats, we found that 40% ± 2.5% of the voxels labeled for RyR were also positive for Ca_v1.2, and 70% ± 2.8% the voxels labeled for Ca_v1.2 contained RyR (Table 1). To more easily visualize protein distribution around the Z disks, two complete disks were extracted from the image and rotated 90° about the X axis (a YZ orientation; Fig. 1 B). Two of the clusters (indicated by the white box) are magnified and displayed a single X plane at a time, from one side of the clusters to the other (Fig. 1 C, i–vi). The upper and larger of the two clusters contains more voxels labeled for RyR than for Ca_v1.2, and the former surround the latter. The lower spot is smaller in volume, covering only three planes, and contains no Ca_v1.2. We refer to the latter as extradyadic RyR.

Figure 1.

(A) Stereo pair, 6° separation, of a ventricular cell labeled for RyR (red) and Ca_v1.2 (green); colocalized voxels are in white. Scale bars are 5 μm in each dimension. (B) Indicated Z disks isolated and rotated 90° about the X axis; 12 planes deep in Y. (C) Magnified images of the clusters highlighted in B are displayed one plane at a time through the width of the clusters (i–vi).

Table 1.

Colocalization metrics

		Voxels		Clusters		Voxels within colocalized clusters
Myocyte orientation	Protein; wavelength	Cav1.2 with RyR	RyR with Cav1.2	Cav1.2 with RyR	RyR with Cav1.2	Cav1.2 with RyR	RyR with Cav1.2
Horizontal	Cav1.2; 488/RYR; 594 [25]	74.4 ± 2.8	41.9 ± 2.5	70.9 ± 3.3	62.6 ± 1.4	81.1 ± 2.1	74.0 ± 2.8
Vertical	Cav1.2; 488/RYR; 594 [13]	78.8 ± 2.5	39.5 ± 2.8	78.5 ± 2.8	69.3 ± 3.4	85.8 ± 1.8	77.4 ± 2.1
	Cav1.2; 594/RYR; 488 [12]	69.6 ± 1.9	44.5 ± 3.3	73.8 ± 3.5	64.6 ± 3.1	84.3 ± 1.8	75.9 ± 2.7

Rows indicate whether the data were acquired from the wide-field microscope with cells in a horizontal orientation or from the confocal with cells viewed end-on. Wavelengths of the secondary antibodies are listed after the semicolon, and numbers of observations are in brackets. Columns list the mean percentage ± SE colocalization between the listed proteins using three techniques (voxels, clusters, and voxels within the clusters).

Additional details, not readily visible in the stereo pair, are provided by the single XY planes displayed in Fig. 2 A i. The single arrow points to a longitudinal tubule traversing an entire sarcomere, and the double arrow points to the more frequent but smaller longitudinal tubules that extend various fractions of a sarcomere. To more clearly demonstrate the relationship between the relative sizes of the RyR and Ca_v1.2 clusters, all of the colocalized voxels have been removed (Fig. 2 A ii) to reveal empty rings of RyR (large arrow), clusters of extradyadic RyR (double arrow), and clusters of extradyadic Ca_v1.2 (small arrows). Fig. 2 A iii displays the same XY plane obtained with an algorithm that highlights the functional relationship between the clusters; all voxels within clusters that are colocalized are white. The use of this paradigm to calculate colocalization indicates that there are significantly more clusters of RyR colocalized with Ca_v1.2 than voxels (p < 0.01; Table 1), but little change in the values for Ca_v1.2 (p > 0.05). These data support the visual impression in Figs. 1 and 2 that the colocalized clusters of Ca_v1.2 are smaller than those of RyR. This is reinforced by calculating colocalization as the percentage of voxels within colocalized clusters (Table 1), which shows significant increases in colocalization of RyR with Ca_v1.2 compared with either the number of voxels or clusters colocalized. Qualitatively and quantitatively similar results were obtained from deconvolved confocal images acquired from cells viewed end-on (Fig. 2 B, Table 1). The consistency of the colocalization and the low variance of these values indicate that both the anti-RyR and anti-Ca_v1.2 antibodies are specific.

Figure 2.

Single image planes taken from three cells labeled for RyR and Ca_v1.2. (A) Wide-field microscope (RyR, red; Ca_v1.2, green). Couplons traversing an entire sarcomere (arrow) and part of a sarcomere (double arrow) are indicated. (B and C) Confocal microscope, cells viewed end-on. In B, RyR is red and Ca_v1.2 is green; the colors are reversed in C. Colocalized voxels are always white. Three display paradigms are used: (i) voxel colocalization, (ii) colocalized voxels are removed from the image, and (iii) cluster colocalization. Single arrows point to rings of RyR, which are magnified in the insets; double arrows point to extradyadic clusters of RyR. Scale bar: 5 μm.

To test whether the Ca_v1.2 clusters were actually smaller, or only appeared so because they were labeled with the shorter-wavelength fluorophore, we repeated the experiments but reversed the secondary antibody labeling. The results, shown in Fig. 2 C and Table 1, were unchanged.

Measurements of the cluster sizes are listed in Table 2, and the number of clusters examined and their density are shown in Table 3. Cluster sizes are expressed as ratios of means measured at the same wavelength; this allows direct comparison of the volumes without the confounding variable of wavelength. Four ratios were determined. The first two are RyR and Ca_v1.2 cluster sizes within a couplon compared with their extradyadic counterparts, (RyR^c/RyR^e) and (Ca_v1.2^c/Ca_v1.2^e). Neither the wavelength nor the cell orientation produced significant differences in the values (p > 0.05). The last two are the ratio of RyR to Ca_v1.2 within a couplon and in the extradyadic sites (RyR^c/Ca_v1.2^c and RyR^e/Ca_v1.2^e). These ratios were calculated using data acquired from cells in which RyR was labeled with Alexa 594 and Ca_v1.2 with Alexa 488, in combination with data acquired from experiments in which the labeling was reversed. This enabled us to determine the ratios of RyR and Ca_v1.2 clusters as though they were labeled with the same fluorophore, which is an experiment that we cannot perform directly. The analysis indicates that RyR clusters are ∼1.5 times larger than Ca_v1.2 clusters whether or not they are within a couplon. There are no significant differences in the ratios produced by the method of acquisition or the wavelength at which they were measured (p > 0.05), but they are significantly smaller than the RyR^c/ RyR^e and Ca_v1.2^c/ Ca_v1.2^e ratios (p < 0.01). Cluster densities were determined from the total volume of cell sections examined (63.2 pl).

Table 2.

Relative sizes of the ryanodine receptor and Ca_v1.2 clusters

	Confocal (vertical)		Wide-field (horizontal)
Wavelength	488	594	488	594
RyRc/RyRe	2.70 ± 0.26	2.45 ± 0.23		2.58 ± 0.15
Cav1.2c/Cav1.2e	2.25 ± 0.16	2.80 ± 0.31	2.74 ± 0.22
RyRc/Cav1.2c	1.58 ± 0.16	1.37 ± 0.17
RyRe/Cav1.2e	1.32 ± 0.09	1.57 ± 0.11

Data are the ratio of the mean volume of the indicated clusters (c, couplon; e, extradyadic). Vertical myocytes were viewed end-on with the confocal microscope; horizontal myocytes were lying flat and recorded with the wide-field microscope.

Table 3.

Cluster density

Clusters	All RyR	All Cav1.2	Couplons	Extradyadic RyR	Extradyadic Cav1.2
Numbers of clusters examined	56,969	52,571	37,851	19,118	14,720
Cluster density (per pl)	901.4	831.8	598.9	302.5	232.9

Total volume examined: 63.2 pl. Data were accumulated from all 50 cells.

Median NNDs are compiled in Table 4. The results of Wilcoxon rank sum signed tests indicate that they are all significantly different from each other (p < 0.01) with the exception of those with broad distributions (extradyadic RyR to extradyadic Ca_v1.2, extradyadic Ca_v1.2 to extradyadic Ca_v1.2, and extradyadic Ca_v1.2 to couplons). Distances from horizontally oriented myocytes are significantly larger than those from vertically oriented myocytes, but with the same exceptions. The histograms obtained from the vertically oriented myocytes are displayed in Fig. 3.

Table 4.

NNDs between indicated clusters

Clusters	All RyR	All Cav1.2	Couplons	Extradyadic RyR to extradyadic Cav1.2	Extradyadic RyR to extradyadic RyR	Extradyadic Cav1.2 to extradyadic Cav1.2	Extradyadic RyR to Couplon	Extradyadic Cav1.2 to Couplon
Horizontal	656 ± 18	691 ± 19	815 ± 21	890 ± 102	820 ± 31	863 ± 105	835 ± 33	1020 ± 87
Vertical	552 ± 14	618 ± 15	733 ± 18	775 ± 90	624 ± 26	1003 ± 53	674 ± 25	1088 ± 64

Table lists the median ± SE NNDs (in nm) between the indicated clusters.

Figure 3.

Histograms showing the distribution of the NNDs between the indicated clusters of molecules. Data are from the 25 cells imaged on the confocal microscope. (A) RyR. (B) Ca_v1.2. (C) Couplons. (D) Extradyadic RyR and Ca_v1.2. (E) Extradyadic RyR. (F) Extradyadic Ca_v1.2. (G) Extradyadic RyR to couplon. (H) Extradyadic Cav1.2 to couplon. A total of 42,259 clusters of RyR and 26,587 clusters of Ca_v1.2 were analyzed. Bin size: 50 nm.

Discussion

In this study we used different techniques to analyze the distributions of Ca_v1.2 and RyR clusters in adult rat ventricular myocytes. Wide-field microscopy has a higher signal/noise ratio, the CCD camera has a larger bit depth, and both the numerical aperture of the objective and the refractive index of the mounting and immersion media are greater, all of which increase the resolving power (12). We also used cells embedded in agarose and examined them end-on to view the Z disk in an orientation more nearly perpendicular to the microscope's optical axis, and therefore at greater resolution. This system uses a PMT with a lower bit depth, and a lower NA water immersion objective for use with the water-based agarose mounting medium, both of which lower the resolving power. Despite their differences, the results from both systems were comparable. This result disagrees with a recent report suggesting that colocalization analyses derived from myocytes lying flat on the coverslip are flawed, but the reasons for these differences are not clear (13).

The percentages of colocalized voxels (Table 1) are comparable to those we previously reported (6,14) and have been reproduced by others (15,16). There was a significant drop in colocalization of Ca_v1.2 with RyR and an increase in colocalization of RyR with Ca_v1.2 when we reversed the labels. These changes are consistent with the change in wavelength: for any given protein, the longer the wavelength of the indicator, the larger the number of labeled voxels. Although the changes reached statistical significance, they were small and the trends in the data were unaltered. Of importance, there were always more voxels labeled for RyR than for Ca_v1.2, which agrees with binding studies demonstrating more sites for ryanodine than for calcium channels (17).

We recently developed techniques in which we measure colocalization between objects consisting of clusters of voxels rather than the voxels themselves (8). In couplons, where the Cav1.2 clusters are 1.5 times smaller than the clusters of RyR, the use of object colocalization allows us to regard the whole couplon, including the outer ring of RyR (Fig. 2, A ii, B ii, C ii, insets), as a single colocalized object, which dramatically reduces the number of noncolocalized RyR voxels (Table 1). Assessing colocalization in this way provides greater insight because these clusters likely represent the primary functional units of the myocyte. The cluster size ratio RyR:Ca_v1.2 of 1.5 showed remarkably little variation with microscopic technique or recording wavelength (Table 2), implying that their relative sizes are tightly controlled. During development of the myocyte, RyR clusters are positioned over the Z-lines in corbular SR before the arrival of t-tubules and Ca_v1.2 (18), and it is tempting to speculate that the size of the RyR cluster determines that of the Ca_v1.2. Further, the extradyadic RyR clusters are consistently smaller than those in the couplons, and it may be that the RyR cluster must attain a certain size before association with Ca_v1.2 can take place. The spatial relationship between the colocalized clusters, with the smaller Ca_v1.2 cluster positioned near the center of the RyR cluster (Fig. 2, A ii, B ii, and C ii), was unexpected. If this were an artifact of wavelength, then reversing the wavelengths should have reversed the effect, but it did not (Fig.  2, C ii), implying that the relative position of the molecules is not determined by chance. Modeling has demonstrated that the relative position of the molecules impacts the gain of ECC (19,20), so grouping Ca²⁺ channels together near the center of the RyR cluster should increase the probability that the released Ca²⁺ ions will bind with a RyR, ensuring that the couplon is activated. Finally, these results disagree with freeze-fracture analyses suggesting that the molecules occupy roughly the same area in their respective membranes and that Ca_v1.2 is randomly positioned relative to RyR (18). This could be the result of some Ca_v1.2 molecules being inaccessible to the antibody, though these would all have to be at the periphery of the couplon. It is also possible that some of the intramembranous particles revealed by freeze-fracture are not Ca_v1.2, or there could be a combination of these effects.

Approximately 35% of RyR clusters are without an adjacent Ca_v1.2, and thus are what we call extradyadic clusters (6,9,14). The nature of these RyRs is elusive. On the basis of results from electron microscopy of papillary muscle, extradyadic clusters were originally thought to be in corbular sarcoplasmic reticulum (cSR), a sac of SR with readily identifiable RyR, on or near the Z-line, but without an adjacent t-tubule (21,22). Yet, despite an extensive electron microscopic analysis of ventricular muscle, we found RyR only in jSR, not in cSR, and no RyR in unexpected locations (14). This suggests that papillary and ventricular muscles have significant ultrastructural differences, and that the extradyadic clusters of RyR are in jSR. Because Ca_v1.2 cannot be detected in standard transmission electron micrographs, the extradyadic RyR may be indistinguishable from clusters that are opposite Ca_v1.2.

Roughly 25% of the Ca_v1.2 clusters are also outside the couplons, and these appear to be both on or near the Z-line and to be well off of it (Fig. 2 A ii). These could be clusters that are embedded in a surface or tubular membrane, or are trafficking intracellularly. Some of the extradyadic clusters of RyR and Ca_v1.2 could also be nascent couplons in the process of being assembled, or older couplons that are being degraded.

We previously estimated that the volume of ventricular cells isolated from animals of the same sex, weight, and species is 37.6 pl (23). Given the recorded cluster densities (Table 3), we estimate that an average cell would contain (rounded to the nearest decade) ∼22,520 couplons and 11,370 extradyadic clusters of RyR. From Table 2 we know that the ratio of RyR cluster sizes, couplon to extradyadic, is 2.5, and given estimates of ∼2,000,000 for the total number of RyR homotetramers in a rat ventricular myocyte (17,24), we solve for the numbers of RyR per couplon (R_c) as follows:

$${22520\text{R}}_{\text{c}}{+11370\text{R}}_{\text{c}}/2\text{.}5=2000000$$

giving ∼74 RyR in a couplon. This is similar to the values obtained by others using comparable optical techniques (74–285 RyR/couplon (3,5)), but smaller than that estimated from electron microscopy (1). Each extradyadic cluster would have ∼30 tetramers. Assuming that the ratio of RyR:Ca_v1.2 molecules in the rat is 7.3 (17), comparable calculations indicate that each couplon would contain ∼10 individual Ca_v1.2, which is very close to the three to 11 predicted for rabbit couplons (25). The extradyadic clusters would only have approximately four channels each.

Our calculations assume that the relative sizes of the clusters are proportional to the numbers of molecules they contain, which is true if the packing densities of both RyR and Ca_v1.2, though different, are constant. Given that the packing may not be complete, however, these values represent an upper limit.

With the exception of measurements involving extradyadic Ca_v1.2, the NNDs recorded from horizontally oriented myocytes are significantly larger than those obtained from vertically positioned myocytes, and this is the only metric in which the two yielded significantly different results. NND measurements from the former are likely compromised by the Z-disks being more nearly parallel to the microscope's optical axis, although the RyR NNDs we recorded from horizontally positioned myocytes are virtually identical to those previously obtained from vertically oriented myocytes (5). Regardless of the orientation, the trends in the data are the same: RyR clusters are the closest together, followed by clusters of Ca_v1.2 and couplons. Extradyadic RyRs are significantly closer to couplons than the couplons are to each other, and they are also significantly closer to each other than are the couplons. These results imply that extradyadic RyRs are preferentially distributed near couplons. No such preferences are seen in the distribution of extradyadic Ca_v1.2, as the NNDs show broad, flat distributions relative to all of the clusters, including themselves.

Ca²⁺ sparks can be initiated by the opening of a single Ca_v1.2, although the probability is significantly less than one (26). Calculations based on data obtained from rabbit myocytes suggest that 10 Ca_v1.2 channels raise the probability of activation of a couplon to near unity (25). This is identical to the 10 Ca_v1.2/couplon we calculated using a different approach. Inoue and Bridge's (25) calculation was based on the open probability of Ca_v1.2 being 0.15, a value that is the subject of ongoing debate (27). The function of extradyadic RyRs is unclear. If they are in jSR with morphological characteristics indistinguishable from those of a couplon (9), they could have a dramatic impact on excitation-contraction coupling. We know from studies of Ca²⁺ waves that the nanospaces between the RyR and the surface membrane are accessible to Ca²⁺ diffusing from the myoplasm (28), but their sensitivity to myoplasmic Ca²⁺ will depend on the molecules to which they are adjacent. We have provided evidence that the extra-dyadic RyR are adjacent to caveolin-3 and the signaling molecules it anchors (9). Further structural definition will have to await results from super-resolution immunofluorescence microscopy and electron tomography.