Research Article 1167

IntroductionExcitation–contraction coupling (ECC) in both atrial andventricular myocytes depends on Ca2+-induced Ca2+ release(CICR). During CICR, a small influx of Ca2+ through voltage-gated calcium channels (Cav1.2) initiates a much larger releaseof Ca2+ from type 2 ryanodine receptors (RyR2) located in theadjacent junctional sarcoplasmic reticulum (SR) (Fabiato, 1983).In ventricular cells, a well-developed transverse and axial tubularsystem (TATS) spreads depolarization into the myocyte, resultingin a nearly synchronous SR Ca2+ release throughout the entirecell (Wier et al., 1995). Most atrial cells, however, lack suchtubules and show a unique spatiotemporal pattern of Ca2+ releaseduring ECC, characterized by a subsarcolemmal ring of elevatedCa2+ concentration, which variably propagates into the centralbulk of the myocyte in direct proportion to cellular Ca2+ load andCa2+ influx (Berlin, 1995; Huser et al., 1996; Mackenzie et al.,2001). Mackenzie and colleagues (Mackenzie et al., 2001) alsoshowed that there are so-called ‘eager sites’ (those sites thatrespond very quickly to rises in Ca2+ concentration) along theperiphery of atrial myocytes; these are separated by one or more‘failure sites’, which exhibit weak regenerative Ca2+ responseswith approximately one-quarter the magnitude of the ‘eager’sites. Cav1.2 channels are primarily distributed along the peripheryof atrial myocytes and are highly colocalized with RyR2 (Carl etal., 1995). However, although this colocalization explains theresponsiveness of peripheral sites to electrical depolarization, itdoes not explain why the so-called eager and failure sites, both

classified as junctional RyR2, respond so differently to electricalstimulation.

One of the many proteins associated with RyR2 in amacromolecular complex is calsequestrin (Casq) (Bers, 2004).Multiple studies have shown that Casq acts as a luminal Ca2+

sensor (Beard et al., 2005) and a regulatory protein that alters theopen probability of RyR2 (Gyorke et al., 2004; Terentyev et al.,2007). This protein is distributed primarily along Z-lines and ishighly colocalized with RyR2 throughout the ventricular myocyte(Scriven et al., 2000); however, its localization in atrial myocytes,particularly with respect to RyR2, is unknown.

Caveolin-3 (Cav3), the dominant isoform of caveolin incardiomyocytes, is responsible for the formation of lipid rafts andcaveolae in the cell membrane, but it is also found outside of thesetwo structures (Patel et al., 2008). Rafts and/or caveolae have beenpostulated to play an important role in ECC; in the adult ratventricular myocyte, removing cholesterol and disrupting thecaveolae and/or rafts using methyl--cyclodextrin (MBC) reducedboth the amplitude of the [Ca2+]i transient and the contraction(Calaghan and White, 2006). The use of MBC in rat arterial smoothmuscle and neonatal cardiomyocytes, which lack a defined TATS,much like atrial myocytes, resulted in a reduction in the frequency,width and amplitude of Ca2+ sparks, without modulation of thecurrent through the Cav1.2 channels (Lohn et al., 2000). Their datasuggest that caveolin might have a role in regulating local SR Ca2+

release, and might indicate the presence of Cav1.2 channels nearCav3.

SummaryStandard local control theory, which describes Ca2+ release during excitation–contraction coupling (ECC), assumes that all ryanodinereceptor 2 (RyR2) complexes are equivalent. Findings from our laboratory have called this assumption into question. Specifically, wehave shown that the RyR2 complexes in ventricular myocytes are different, depending on their location within the cell. This has ledus to hypothesize that similar differences occur within the rat atrial cell. To test this hypothesis, we have triple-labelled enzymaticallyisolated 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+ releasein atria. A small percentage of the dyads containing RyR2 and Cav1.2 were colocalized with Cav3, and therefore could be influencedby the signalling molecules it anchors. The majority of the RyR2 clusters were tightly linked to Cav1.2, and, whereas some werecoupled to both Cav1.2 and Ncx, none were with Ncx alone. This suggests that Cav1.2-mediated Ca2+-induced Ca2+ release is theprimary method of ECC. The two molecules studied that were found in the interior of atrial cells, RyR2 and Casq, showed significantlyless colocalization and a reduced nearest-neighbour distance in the interior, compared with the surface of the cell. These differencesmight result in a higher excitability for RyR2 in the interior of the cells, facilitating the spread of excitation from the periphery tothe centre. We also present morphometric data for all of the molecules studied, as well as for those colocalizations found to besignificant.

Key words: Atrial myocyte, Excitation–contraction coupling, Ryanodine receptor, Cav1.2, Sodium–Calcium exchanger, Caveolin-3, Calsequestrin

Accepted 5 November 2010Journal of Cell Science 124, 1167-1174 © 2011. Published by The Company of Biologists Ltddoi:10.1242/jcs.080929

Couplons in rat atria form distinct subgroups definedby their molecular partnersMeredith N. Schulson, David R. L. Scriven, Patrick Fletcher and Edwin D. W. Moore*Department of Cellular and Physiological Sciences, University of British Columbia, Life Sciences Institute, 2350 Health Sciences Mall, Vancouver,BC V6T 1Z3, Canada*Author for correspondence (edmoore@interchange.ubc.ca)

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The type 1 sodium–calcium exchanger (Ncx) is known to be thesole member of the Ncx family of proteins present in thecardiomyocyte (Quednau et al., 1997) and has long been implicatedin the regulation of ECC. First viewed as a possible alternativemechanism by which to activate CICR, it is now largely agreedthat Ncx acts as a modulator of the process in cardiac myocytes(Bers, 2008). Structural data (Scriven et al., 2005) demonstratethat, whereas both couplons (Cav1.2 and RyR2) and Ncx moleculesare closely associated with Cav3 on the surface of rat ventricularmyocytes, they are only sparsely colocalized with one anotherwithin the cell interior. However, other studies have shown thatNcx is separate from Cav3, both in developing (Lin et al., 2009)and adult (Cavalli et al., 2007) ventricular cardiomyocytes. Owingto a lack of a well-developed TATS, Ncx is largely distributedalong the cell periphery in atria (Bootman et al., 2006) but mightcolocalize with proteins such as RyR2 and Cav1.2 channels there.If so, it could act as an important modulator of ECC.

ResultsControlsBefore beginning the triple-labelling experiments, a series ofcontrols was performed in order to ensure the validity of ourprotocols. First, rat atrial myocytes were labelled with antibodiesagainst RyR2, Cav1.2, Casq, Ncx or Cav3 individually. This wasdone in order to examine not only the distribution of each protein,but also to ensure that the labelling characteristics did not changewhen the cells were incubated with multiple antibodiessimultaneously. Deconvolved images from the surface and interior(left- and right-hand panels respectively, for each pair of images)of the labelled cells are shown in Fig. 1. At the surface, antibodiesagainst both RyR2 and Casq (Fig. 1A,B, left-hand panels) displayedhighly punctate labelling, spread fairly evenly across the membrane.In the interior of the myocyte (Fig. 1A,B, right-hand panels), thelabelling of these two proteins had a similar ladder-like pattern.Discrete clusters of both RyR2 and Casq lined up largely along the

Z-lines but also appeared in some longitudinal elements betweenZ-lines (arrows). We also confirmed the presence of a smallsubsarcolemmal gap in the RyR2 labelling (Carl et al., 1995;Mackenzie et al., 2001), and show that this phenomenon alsoexists for the Casq distribution in atrial cells (arrowheads).

Fig. 1C–E shows the distribution of Cav1.2, Ncx and Cav3proteins, respectively. At the surface (Fig. 1C, left-hand panel),clusters of Cav1.2 were spread evenly across the membrane. Ncxlabelling on the surface (Fig. 1D, left-hand panel), by contrast, wasdense but somewhat uneven, and appeared as even larger aggregatesthan those of Cav1.2. Note that the very bright staining at one sideof the membrane is an artefact created by imaging the sarcolemmain parallel with the optical axis of the microscope (edge effect). Anantibody directed against the Cav3 protein also collected in largeclusters on the sarcolemmal membrane (Fig. 1E, left-hand panel)that appeared to be oriented in longitudinal striations. Cav1.2, Ncxand Cav3 proteins were only expressed to a very small degree inthe interior of atrial myocytes (Fig. 1C–E, right-hand panels) andthe antibodies against these three proteins label only faintly in thisregion. This labelling probably represents either synthesis and/ordegradation of the proteins or the presence of rudimentary t-tubulesin some atrial cells.

The ability of the Zenon kits to eliminate cross-reaction betweenthe secondary antibodies against two monoclonal primaryantibodies was tested using primary antibodies against proteinsthat are known to localize to distinct regions of the cell (RyR2 andthe nuclear pore complex). We labelled myocytes simultaneouslywith these two antibodies using the manufacturer’s protocol andwere able to confirm that there was no cross-talk between the twolabels (supplementary material Fig. S1). The results of these controlexperiments confirmed that we could label with two monoclonalantibodies simultaneously without fear of cross-reaction.

The final step in confirming the validity of our triple-labellingexperiments was to compare the colocalization values betweenpairs of proteins that occurred in more than one experiment, to see

1168 Journal of Cell Science 124 (7)

Fig. 1. Single-labelling of atrial myocytes. An atrialmyocyte segment labelled with antibodies against RyR2(A), Casq (B), Cav1.2 (C), Ncx (D) or Cav3 (E). For eachantibody, the image shown is either a 1.5-mm-thick sectiontaken from the cell surface (left panels), or a single 250-nm plane taken from approximately the middle of the cell(right panels). For the interior segments of RyR2 (A) andCasq (B), there is a discrete subsarcolemmal ‘gap’ with nolabelling of either protein (upper inset, arrows), whereasthe lower inset shows that some longitudinal elements oflabelling are present (arrowheads). Scale bar: 5mm.

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whether they were the same as one another. We found no significantdifferences (see supplementary material Table S1 for a detailedanalysis), and, for this reason, pooled the results for the dualcolocalizations that were common to more than one experiment(Table 1). With the completion of these control experiments andcalculations, we were confident that the labelling characteristics ofeach protein involved in our triple-labelling experiments areindependent of one another and that our results are a true indicationof the structural organization of atrial myocytes and the associationof these proteins within the cells.

Triple-labelling experimentsThe first of three triple-labelling experiments reported hereexamines the potential association between RyR2, Ncx and Cav1.2in rat atria. Fig. 2 shows a section from a representative atrial cell,with RyR2 in blue, Ncx in green and Cav1.2 in red. Each image(A–C) is the same 1-mm-thick layer from the surface of themyocyte. Fig. 2A shows the entire field of view; this exhibited asignificant edge effect because of the abundance of Ncx labelling.In this region, the cell curvature caused an increase in apparentcolocalization owing to the reduced resolution along the z-axis(Scriven et al., 2008). To prevent the edge effect from causingerrors in our calculations, we used the cell segment shown in Fig.2B (outlined segment in Fig. 2A) to measure colocalization. Fig.2C shows the colocalization between the three molecules. Theprimary association appears to be between RyR2 and Cav1.2(magenta; Table 1). There were also a significant number of RyR2–Cav1.2–Ncx triplets (white; Table 2). Neither the Ncx–RyR2 (cyan)nor the Cav1.2–Ncx (yellow) represent significant colocalizations(Table 1). Ncx seems to be mainly organized into large clustersthat are either completely separate from the RyR2–Cav1.2 unit, anddo not associate with either protein (e.g. Fig. 2B), or that areintegral to the RyR2–Cav1.2 unit.

Fig. 3 shows triple labelling of RyR2 (blue), Casq (red) and Ncx(green) in a 1-mm-thick segment from the cell surface of arepresentative atrial myocyte. The full cell is shown in Fig. 3A, theoutlined segment shows the area that excluded the edges and wasused for the analysis. Fig. 3B is an expanded version of the boxedregion in Fig. 3A, whereas Fig. 3C shows the object colocalizationbetween the three molecules. The most dominant form ofassociation between these proteins was RyR2 with Casq alone(magenta), with about 50% of the voxels colocalized (Table 1).There were a significant number of RyR2–Casq–Ncx triplets (white;Table 2), as well as a few dual colocalizations [Ncx–RyR2 (cyan)and Ncx–Casq (yellow)] neither of which is significant (Table 1).It is worth noting that there was a large amount of Ncx that wasnot colocalized with either RyR2 or Casq (Fig. 3B).

RyR2 and Casq are the only molecules studied that have asubstantial presence in the interior of the atrial cell. A comparisonof the surface and interior colocalization of RyR2 and Casq (Table3), showed that there was significantly less colocalization in theinterior than at the surface, irrespective of whether one comparesobject colocalization or the number of voxels in the colocalizedobjects. In addition, the size of both the RyR2 and Casq objectsdiffers significantly between the interior and surface, with Casqsmaller in the interior, whereas the RyR2 was larger (Table 4).Furthermore, the median nearest-neighbour distances for both RyRand Casq were significantly less (P<0.05) in the interior of the cellthan they were on the surface (Table 5), although neither thecluster diameter nor the inter-cluster distance differed between theinterior and surface (Table 6).

In Fig. 4, we show an atrial myocyte labelled with antibodiesagainst RyR2 (blue), Cav1.2 (red) and Cav3 (green). Fig. 4A showsa 1-mm-thick layer, from the bottom cell surface, with a markededge effect. In Fig. 4B, we display the segment outlined in Fig. 4Athat excludes the edge regions. Object colocalization is shown in

1169Couplon subgroups in rat atria

Fig. 2. Triple-labelling of RyR2, Ncx and Cav1.2. An atrialmyocyte labelled with antibodies against RyR2 (blue), Ncx(green) and Cav1.2 (red). All images are 1-mm-thick slices fromthe cell surface. (A)View of the entire cell, showing the surfacedistribution and colocalization of RyR2, Ncx and Cav1.2.(B)Segment of the cell surface from the area shown in A.(C)The same segment, showing only the object colocalizationof RyR2–Cav1.2–Ncx (white), RyR2–Cav1.2 (magenta), Ncx–RyR2 (cyan) and Ncx–Cav1.2 (yellow). Scale bars: 5mm.

Table 1. Dual colocalizations

Molecule pairs Colocalization (%) Reverse pairs Colocalization (%) n

RyR2–Cav3 8.36±2.40 Cav3–RyR2 7.77±2.60 5RyR2–Ncx 10.02±1.29 Ncx–RyR2 8.17±1.19 17RyR2–Casq 61.31±3.08 Casq–RyR2 54.47±2.39 9RyR2–Cav1.2 52.90±7.77 Cav1.2–RyR2 59.25±7.52 13Cav3–Cav1.2 4.22±0.78 Cav1.2–Cav3 3.70±0.81 5Ncx–Casq 7.28±2.07 Casq–Ncx 8.96±1.94 9Ncx–Cav1.2 7.19±1.01 Cav1.2–Ncx 9.89±1.60 8

Colocalizations that occur at a significantly greater level than that expected by chance (P<0.01) are shown in bold. Values in italics represent colocalizationsthat occur at a significantly lesser level than that expected by chance (P>0.975), suggesting that they are in separate domains. Values are number of voxels in thecolocalizing objects divided by the total number of voxels for that molecule.

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Fig. 4C, with the triple colocalization RyR2–Cav1.2–Cav3 (white;Table 2) and the colocalization between RyR2 and Cav1.2 (magenta;Table 1) being highly significant. Colocalization of RyR2 withCav3 (cyan) was not significant, whereas the observed values forthe Cav3–Cav1.2 pair (yellow) were much less than those expectedby chance (Table 1), indicating that these two molecules areprobably in separate domains. The large colocalization betweenRyR2 and Cav1.2 was reflected in the large number of tightlycoupled magenta clusters with a few triplets (white) interspersed.

Our analysis technique (Fletcher et al., 2010) allowed us toderive three useful metrics for describing the positioning of themolecules within the atrial cell; these are the median nearest-neighbour distance of the individual molecules (Table 5); thecluster diameter, a measure of how closely associated thecolocalized molecules are (Table 6); and the inter-cluster distance,a measure of how separated the colocalizations are. The values inTable 6 include only those colocalizations that were statisticallysignificant (see Tables 1 and 2).

DiscussionA number of our results correspond well with earlier findings inrat ventricular myocytes (Scriven et al., 2000). The association ofRyR2 with Ncx in atrial cells was not significantly different fromthat expected by chance, and atrial RyR2 had large and significantcolocalizations with both Cav1.2 and Casq. The nearest-neighbourdistances for the surface RyR2 and Cav1.2 (Table 5), as well as theinter-cluster distance for the RyR2–Cav1.2 (Table 6), wereremarkably similar to those found in the ventricle (Scriven et al.,2010), with differences of 20 nm or less in their values. In addition,the nearest-neighbour distance for Cav3 indicated that its densitywas higher than that for either RyR2 or Cav1.2, which agrees withour earlier finding from the ventricle surface (Scriven et al., 2005).Ncx had a nearest-neighbour value (and thus density) similar tothat of Cav3, although we do not have values from the ventriclesurface that we can compare it with. Results that differ from thosein the ventricle involve Cav3: RyR2 and Cav3 alone were notsignificantly colocalized, unlike ventricular myocytes, wheresurface Cav3 seems to associate equally with RyR2 and Cav1.2,

presumably at the dyads (Scriven et al., 2005). In atrial cells, therewas a small but significant population of RyR2, Cav1.2 and Cav3triplets (Table 2).

The role of Ncx in ECC has been subject of an extended debate.Numerous models with various assumptions have producedconflicting results (Sher et al., 2008), and, although it is wellestablished that Ncx plays a major role in Ca2+ extrusion in thecardiomyocyte, its contribution to the Ca2+ influx, which couldtrigger CICR, is uncertain. Evidence from rabbit neonatalventricular cells, which do not have a well-developed TATS, as foratrial myocytes, shows that they have a heightened level of RyR2–Ncx colocalization (Dan et al., 2007) and that they rely on Ncx-mediated CICR (Huang et al., 2008) during their early development.Our results indicate that this mechanism is absent in the matureatria; as there was no significant colocalization between RyR2 andNcx, couplons consisting solely of these two molecules (andexcluding Cav1.2) are unlikely to exist and cannot contribute toCICR. As ~50% of the RyR2 colocalized only with Cav1.2 channels,it is probable that Cav1.2-mediated CICR is the primary method ofinitiating Ca2+ release in the atrial myocyte.

We found a statistically significant number (~13%) of RyR2–Cav1.2–Ncx triplets on the atrial surface, with a spacing ofapproximately twice that of the simple RyR2–Cav1.2 couplon(Table 6). This result raises questions as to the role of Ncx in thesetriplets and why only a limited number of dyads are involved. Bers(Bers, 2008) argues that, if the Ncx faces the restricted space of thedyadic cleft, the high Ca2+ concentrations that exist during theaction potential would limit the role of Ncx to Ca2+ extrusion.Simulations by Noble and colleagues (Sher et al., 2008) indicatethat, although Ncx primarily acts as a agent of Ca2+ extrusion anddoes not significantly contribute to Ca2+ influx, it might increasethe probability of Cav1.2 opening by reducing [Ca2+] in the dyadicspace, thus decreasing Ca2+-dependent inactivation. Furthermore,Noble’s group found that at least 10%, but no more than 18%, ofthe Ncx should be colocalized with RyR2 for their model toreproduce previously measured currents. Although the model wasdesigned for ventricular tissue, these findings suggest that theRyR2, Cav1.2 and Ncx triplets represent a subgroup of dyads with

1170 Journal of Cell Science 124 (7)

Fig. 3. Triple-labelling of RyR2, Ncxand Casq. An atrial myocyte segmentlabelled with antibodies against RyR2(blue), Ncx (green) and Casq (red). Allimages are 1-mm-thick slices from thecell surface. (A)View of the entire cell,showing surface distribution andcolocalization of RyR2, Ncx and Casq.(B)Segment of the cell surface from thearea shown in A. (C)The same segment,showing only the object colocalization ofRyR2–Ncx–Casq (white), RyR2–Casq(magenta), RyR2–Ncx (cyan) and Ncx–Casq (yellow). Scale bars: 5mm.

Table 2. Triple colocalizations

Molecule triplets Colocalization (%) Molecule triplets Colocalization (%) Molecule triplets Colocalization (%) n

RyR2–Cav3–Cav1.2 4.61±1.32 Cav3–Cav1.2–RyR2 3.54±1.24 Cav1.2–RyR2–Cav3 4.58±1.56 5RyR2–Ncx–Cav1.2 12.88±1.62 NCX–Cav1.2–RyR2 11.92±2.07 Cav1.2–RyR2–Ncx 15.26±2.19 8RyR2–Ncx–Casq 5.81±0.95 Ncx–Casq–RyR2 4.73±1.09 Casq–RyR2–Ncx 5.85±0.96 9

Colocalizations that occur at a significantly greater level than that expected by chance (P<0.01) are shown in bold. Each colocalization is calculated withrespect to the first molecule listed. Values are number of voxels in the colocalizing objects divided by the total number of voxels for that molecule.

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increased excitability. We hypothesize that these dyads form thestructural basis for the ‘eager sites’ of Ca2+ release (Mackenzie etal., 2001) at the surface of atrial myocytes.

Another small (~3%) but significant group of triplets is RyR2–Ncx–Casq, whose sparsity is reflected in their large interclusterdistances (Table 6). Although it is possible that these representRyR2–Ncx dyads modulated by Casq, this seems unlikely giventhe absence of this type of dyad without Casq. We believe that thisis a subgroup of the RyR2–Cav1.2–Ncx conglomeration, in whichthe Ncx is close enough (probably in the dyad) to colocalize withCasq. As Casq does not always colocalize with RyR2, we can splitthe RyR2–Cav1.2–Ncx group into two – one in which the RyR2excitability is modulated by Casq, and one in which it is not.Confirmation of this hypothesis would require quadruple labellingso that we can look at all four proteins simultaneously.

If we assume that Casq acts as both a luminal Ca2+ sensor for,and a Po inhibitor of, RyR2 channels when it is bound (Gyorke etal., 2004; Terentyev et al., 2007), the lower colocalization of RyR2and Casq in the interior of atrial myocytes means it is probable thatthey are more excitable than their surface counterparts. This wouldfacilitate the diffusion of Ca2+ waves from the periphery to thecentre, which is necessary in cells lacking a t-tubular structure. Inaddition, the nearest-neighbour distances for both RyR2 and Casqare significantly less in the interior than on the surface of the cell(Table 5), which would increase the likelihood of inward diffusionof the Ca2+ waves. Woo and colleagues (Woo et al., 2003a) noteda larger number of combined sparks (i.e. sparks that consisted oftwo to five single sparks) in the centre of atrial cells compared withthat evident in their periphery, which could support a role for Casqas a modulator of excitability. They also reported that spontaneousCa2+ sparks are more frequent in the cell periphery than in theinterior, but attributed this to an interaction between the 1c C-terminal tail of Cav1.2 and RyR2 (Woo et al., 2003b), which occursonly in the cell periphery, rather than to any effect of Casq. Asecond significant finding is that the sizes of the Casq clusters(both colocalized and not) are significantly smaller in the interiorof the cell compared with at the periphery (Table 4). Although it isdifficult to make an exact correspondence between voxel numbersand molecule density, it seems probable that there is much lessCasq within the cell than along its edge, which might representsmaller stores of Ca2+. By contrast, RyR2 clusters are larger in the

interior than in the exterior. It is not clear whether this is due to thelimited space available at the surface or to some functionaldifference.

An indication of the position of Ncx in relation to the RyR2–Cav1.2 is given by the cluster diameters of RyR2–Ncx–Cav1.2 andRyR2–Ncx–Casq, which were approximately twice those of RyR2–Cav1.2 and RyR2–Casq (Table 6). This suggests that although Ncxis close enough to the RyR2–Cav1.2 couplon to influence [Ca2+]within the dyadic space, it is further away from RyR than isCav1.2, and it is not in contact with these molecules. This meansthat it is unlikely that either fluorescence resonance energy transfer(FRET) or immunoprecipitation could be used to confirm thisresult. Furthermore, a recent paper (Nichols et al., 2010) has shownthat, despite their well-known close association in both theventricular and atrial cardiomyocyte, RyR2 and Cav1.2 do not co-immunoprecipitate, making it unlikely that RyR2 and Ncx woulddo so.

The two significant dual colocalizations, RyR–Cav1.2 and RyR–Casq (Table 1), as measured in different experiments, havecolocalization values that are not significantly different from eachother and sum to greater than 100% of the total RyR. In addition,they have identical cluster diameters and similar interclusterdistances (Table 6). In the ventricle, electron micrographs showthat Casq is associated with the couplon (Franzini-Armstrong etal., 1987), so it is probable that there is significant overlap betweenthe two groups, and that they form the triplet RyR–Cav1.2–Casq.If there is complete overlap between the two groups, and all of the

1171Couplon subgroups in rat atria

Fig. 4. Triple-labelling of RyR2, Cav3 and Cav1.2. An atrial myocytelabelled with antibodies against RyR2 (blue), Cav3 (green) and Cav1.2 (red).All images are 1-mm-thick slices from the cell surface. (A)View of the entirecell, showing surface distribution and colocalization of RyR2, Cav3 andCav1.2. (B)Segment of the cell surface from the area shown in A. (C)Thesame segment, showing only the object colocalization of RyR2–Cav3–Cav1.2(white), RyR2–Cav1.2 (magenta), Cav3–Cav1.2 (yellow) and RyR2–Cav3(cyan). Scale bars: 5mm.

Table 3. RyR–Casq colocalization

Interior colocalization (%) Surface colocalization (%)

Molecular pair Object Voxels in objects Object Voxels in objects

Casq with RyR2 16.35±1.82 37.56±2.41 33.28±1.54 60.39±2.29RyR2 with Casq 28.07±4.03 47.28±5.18 42.31±3.55 67.16±3.34

n9. All values listed for interior colocalization are significantly less (P<0.05) than their corresponding values at the surface. Values from the surface includeRyR–Casq that is colocalized with Ncx.

Table 4. RyR and Casq object sizes

Mean object size (voxels)

Molecule Interior Surface

Casq 8.08±1.02 13.91±0.73a

RyR2 25.19±2.19 18.91±0.93a

n9 cells. All objects, whether colocalized or not, are included in thecalculations.

aP<0.05 compared with that in the interior.

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RyR is in the triplet, summing the significant colocalizationsindicates that over 20% of the RyR would not be accounted for.There are a number of possibilities: not all of the RyR is in thetriplet, and the three associations RyR–Cav1.2, RyR–Casq andRyR–Cav1.2–Casq are all present; some of the RyR is by itself; orRyR is associated with a molecule that we have not tested for.These possibilities are not mutually exclusive. As shown above,‘naked’ RyR is definitely present in the interior of the atrial cell,so it is not far-fetched to suggest its existence on the atrial surface.

A surprising finding was that there was no significant pairingbetween Cav3 with either RyR2 or Cav1.2: the only significantassociation of Cav3 that we found was in the triplet RyR2–Cav1.2–Cav3, which we suspect is due to dyads being close to a group ofCav3 molecules. The proportion of total RyR2 colocalized withCav3 alone is not statistically significant, which suggests thatCav1.2 channels in the RyR2–Cav1.2–Cav3 group are immediateneighbours of Cav3, rather than occupying lipid rafts or caveolaeat these sites. Thus, a small proportion of the dyads are closeenough to be affected by signalling molecules associated withCav3, but the cluster diameters (Table 6) indicate that, althoughCav3 is close to the couplon, it is probably not in physical contactwith the sarcolemmal Cav1.2. The functional consequences of thisassociation are difficult to estimate; there are multiple signallingmolecules (e.g. -adrenergic and A1 receptors) associated withCav3. Cholesterol removal, which disrupts caveolae and lipid rafts,has been shown to cause a reduction in the frequency, width andamplitude of Ca2+ sparks (Lohn et al., 2000), although it is notclear whether this effect is due to a disruption of signalling betweenCav3-associated molecules and the dyad or to the deleteriouseffects of cholesterol removal from the membrane. If it is theformer, molecules associated with Cav3 might alter the openprobability of RyR2, resulting in a change in the sparkcharacteristics.

Because they were derived from different experiments, onecould theorise that the two triplets RyR2–Cav1.2–Ncx and RyR2–Cav1.2–Cav3 actually represent the quadruplet RyR2–Cav1.2–Cav3–Ncx. However, as the number of RyR2–Cav1.2–Cav3 tripletsis far fewer than those of RyR2–Cav1.2–Ncx (Table 2), and theirintercluster distances are very different (Table 6), it is unlikely thatthe groups match up. Even if significant numbers of the quadrupletdid exist, there would still be a large amount of unmatched RyR2–Cav1.2–Ncx.

Finally, isolated Cav1.2 was significantly less colocalized withCav3 than one would expect by chance, implying that these twomolecules are specifically directed to separate cellular domains.This differs from ventricular tissue, where Cav3 and Cav1.2 arecolocalized on the cell surface (Scriven et al., 2005; Balijepalli etal., 2006).

ConclusionsWe have provided evidence to support our hypothesis that atrialRyR2 complexes are heterogeneous and can be targeted to specificintracellular domains. In particular, we have identified a number ofsurface populations of RyR2, on the basis of its binding partners,that we believe are structurally and functionally distinct. These are:RyR with Cav1.2 and Casq (couplons modulated by Casq); RyR2with Cav1.2 and Cav3 (couplons modulated by moleculesassociated with Cav3). RyR2 with Cav1.2 and Ncx (the eagersites); and RyR2 with Cav1.2, Casq and Ncx (eager sites modulatedby Casq). In addition, the following might also be present: RyR2with Cav1.2, RyR2 with Casq, and RyR2 alone; however, theirrelative proportions are unclear. We also conclude that Cav1.2-mediated CICR is the dominant form in atrial myocytes and thatthere is no evidence to support a RyR2–Ncx-mediated CICR.Finally, we have found that the RyR and Casq within the cellinterior is different from that on the surface, and these differencesmight assist in the propagation of the Ca2+ wave into the cellinterior.

One of the central tenets of local control theory – that allcouplons are functionally equivalent – is challenged by thediscovery of the different RyR2 complexes shown here. Althougheach individual RyR2 complex might be independently controlled,their molecular constituents and binding partners are highly unlikelyto be identical.

Materials and MethodsAll chemicals were purchased from Sigma–Aldrich unless otherwise stated. Animalhandling was performed in accordance with the guidelines of the Canadian Councilon Animal Care.

Cell isolation and preparationAtrial myocytes were isolated from freshly excised hearts using the method ofRodrigues and Severson (Rodrigues and Severson, 1997). Briefly, adult male Wistarrats weighing 200–250 g were given 200 units of heparin (Organon Canada, Toronto,ON) intraperitoneally at 15–20 minutes prior to killing with sodium pentobarbital(20 mg per 100 g of body weight; MTC Pharmaceuticals, Cambridge, ON). Theheart was immediately excised and hung for retrograde Langendorff perfusion withwarm (37°C) Joklik MEM (M0518) supplemented with 23 mM NaHCO3, 1.2 mMMgSO4, and 1 mM DL-carnitine (final concentrations), and equilibrated with 95%O2 and 5% CO2. Perfusion was adjusted to give a flow of 7 ml/minute and maintainedfor 5 minutes to drain the heart of blood. It was then switched to a Joklik solutioncontaining 162 units/ml type II collagenase (Worthington Biochemical, Lakewood,NJ). Once the heart softened, the atria were separated from the ventricles, mincedand filtered through a Nitex nylon mesh (200 mm). Preparations contained ~30–50%rod-shaped quiescent cells, which were incubated for 20 minutes at 37°C in M-199(M4530) supplemented with 25 mM HEPES (pH 7.4), 1 mM DL-carnitine, 0.1 mMinsulin, 0.56 mM penicillin, 0.14 mM streptomycin sulphate, 2 mM EGTA and 0.01g/ml fatty-acid-free BSA (final concentrations). Cells were then fixed for 10 minutesin freshly made 2% paraformaldehyde. Fixation was quenched with 100 mM glycine(pH 7.4) for 10 minutes, after which cells were washed three times for 10 minuteseach in PBS (137 mM NaCl, 8 mM NaH2PO4, 2.7 mM KCl and 1.5 mM KH2PO4,pH 7.4). Cells were then permeabilized with 0.1% Triton X-100 for 10 minutesfollowed by three 10-minute washes in PBS.

ImmunolabellingPrimary antibodies were against: RyR2 (mouse monoclonal; MA3-916, AffinityBioReagents, Golden, CO); calsequestrin (rabbit polyclonal; PA1-913, Affinity

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Table 5. Median nearest-neighbour distances

Molecule Nearest-neighbour distance (nm)

RyR2 (surface) 566RyR2 (interior) 525a

Casq (surface) 464Casq (interior) 425a

Ncx 469Cav1.2 597Cav3 493

aInterior values are significantly less (P<0.05; Wilcoxon rank sum test)than those on the surface.

Table 6. Cluster diameter and inter-cluster distances

Cluster diameter Inter-clusterColocalization (nm) distance (nm)

RyR2–Cav3–Cav1.2 259 2194RyR2–Ncx–Cav1.2 250 1370RyR2–Ncx–Casq 212 2233RyR2–Cav1.2 115 748RyR2–Casq (surface) 115 697RyR2–Casq (interior) 115 853

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BioReagents); Cav3 (mouse monoclonal; 610420, BD Biosciences); Ncx (mousemonoclonal; R3F1, Swant, Bellinzona, Switzerland); and Cav1.2 [rabbit polyclonal;CNC1, a gift from William Catterall (Hell et al., 1993)]. Secondary antibodies wereaffinity purified and highly cross-adsorbed to minimize species cross-reactivity, andwere either goat anti-rabbit-IgG or goat anti-mouse-IgG conjugated to Alexa Fluor350, 488 or 594 (Invitrogen).

Incubations involving one monoclonal and one polyclonal primary antibody wereperformed sequentially overnight at 4°C. All labelling experiments involving twomonoclonal primary antibodies were performed using mouse IgG1 Zenon labellingkits (Invitrogen). Experiments involving one polyclonal and two monoclonalantibodies were performed in one of two ways. In the first method, cells wereincubated with one monoclonal antibody overnight at 4°C, and then with an anti-mouse-IgG secondary antibody for 1.5 hours at room temperature. The secondmonoclonal primary was conjugated to its fluorophore using a Zenon labelling kitand then added to cells for 1 hour at room temperature. The third primary antibody,a polyclonal, was added to cells overnight at 4°C and then an anti-rabbit-IgGsecondary antibody conjugated to a chosen fluorophore was added for 1.5 hours atroom temperature. In the second method, cells were incubated with the polyclonalantibody overnight at 4°C and then with an anti-rabbit-IgG secondary antibody for1.5 hours at room temperature. The myocytes were then incubated simultaneouslywith the two monoclonal antibodies that had been conjugated to their appropriatefluorophore using a mouse IgG1 Zenon labelling kit. For each triple labellingexperiment, an appropriate control experiment was performed in order to ensure nocross-reaction between the monoclonal antibodies.

Control experimentsWe used the Zenon kit (Invitrogen) to label monoclonal antibodies against the RyRand the nuclear pore complex (NPC). These two molecules are known to be inseparate domains. Supplementary material Fig. S1 shows the results from thisexperiment. RyR and its corresponding secondary antibody (Alexa-Fluor-594-conjugated; shown in red) and NPC (Alexa-Fluor-488-conjugated; shown in green)do indeed stay localized to separate regions of the cell – the Z lines (arrow) andnuclear pore region (arrowhead), respectively. There is negligible red labelling in thenuclear region and/or green labelling at the Z-lines, indicating no cross-reactionbetween the secondary antibodies. Colocalization analysis of these two proteinsconfirms what we see in Fig. 2 (0.32% RyR colocalizes with NPC; 6.88% NPCcolocalizes with RyR; whole-cell values). The higher percentage of NPC with RyRis a result of a much lower density of NPC as compared with RyR, and also ofcolocalization along the z-axis where RyR lies on top of, and underneath, the nuclearmembrane. We also performed this experiment by labelling RyR overnight at 4°C,then adding a goat Alexa-Fluor-594-conjugated anti-mouse IgG1 secondary antibodyfor 1.5 hours at room temperature. This was followed by incubation with a NPCantibody that had been conjugated to a goat Alexa-Fluor-488-conjugated anti-mouseIgG secondary antibody with the Zenon labelling kit. The results were identical tothe image in supplementary material Fig. S1 (data not shown), and thereforeconfirmed a second protocol that could be used to label cells with two monoclonalantibodies without cross-reaction.

Imaging, deconvolution and analysisImages were captured using a Zeiss AxioObserver inverted microscope with a63�/1.4 NA oil immersion objective and were then passed through a narrowbandpass filter (Semrock, Rochester, NY) specific for the chosen fluorophore.Images were captured on a thermoelectrically cooled charged-couple device (CCD)camera with an 80% quantum efficiency (SITe SI502AB chip). The z-position of thesample was controlled by a PZM2000 piezo stage (Applied Scientific Instrumentation,Eugene, OR). The pixel size of these images was 95 nm in the x- and y-planes, andimages were acquired at 250-nm intervals in the z-plane.

Images were deconvolved using the algorithm developed by Carrington andcolleagues (Carrington et al., 1995). All images were dark-current- and background-subtracted, and flat-field corrected, to allow for non-uniformity in illumination andcamera sensitivity across the field of view. After deconvolution, the images werealigned using fiduciary markers that emitted in all wavelengths. Finally, images werepeeled, one layer of voxels at a time, and numbered from 0 (surface) to 12 (centre)(Scriven et al., 2005). Because of some uncertainty in the exact position of thesurface, the –1 layer (immediately outside the surface) was also included.Colocalization and labelling density (number of lit voxels over the total number ofvoxels) were measured as a function of the distance into the cell; ‘surface’ and‘interior’ represented the values obtained from layers –1 to +2 and +4 to +10,respectively. Interior colocalization and density was only calculated for the RyR2–Casq pair, as no other proteins had significant labelling in the interior. All valueswere obtained from cell segments chosen to eliminate possible edge effects (Scrivenet al., 2008). Voxels or blobs encompassing all three antibodies (e.g. A with B andC) were counted as triply colocalized and separate from the dual colocalizations (e.g.A with B, B with C, and C with A) given that the triple grouping was thought to befunctionally different from the doublets (Fletcher et al., 2010).

Determining that colocalization has occurred between two or more proteins is notsufficient as one has also to determine whether the measured colocalization issignificant or if it could have occurred purely by chance. We have recently publishedtechniques to analyze and determine the statistical significance of images using three

fluorophores (Fletcher et al., 2010) using both voxel colocalization and an object-based ‘blob’ colocalization. Briefly, this latter approach splits the images into three-dimensional blobs, each centred on one of the local maxima within the image. Twoor more blobs are colocalized when the maxima of the blobs falls within the bodyof the other. Colocalization of blob objects gives a measure of how groups of voxels(and thus molecules) interact with one another and omits colocalizations that occurowing to peripheral overlap between the edges of the blobs. Significance wascalculated by generating a series of images in which the blobs were placed at randomthroughout the region of interest; the values generated were then compared with thatof the observed value. Values with P≤0.025 were regarded as being significantlycolocalized, P≥0.975 as being significantly separate and 0.025<P<0.975 as occurringby chance. A number of useful metrics can be derived from this approach: themedian nearest-neighbour distance is the median distance between blob centres fora particular molecule, the cluster diameter is the minimum-sized sphere that can bedrawn about the maxima of two or more colocalized blobs, and the interclusterdistance is the median distance between the centre of these spheres.

We thank William Catterall for the gift of the anti-Cav1.2 antibody(National Institutes of Health grant R01 HL085372) and Qixia Yu fortechnical assistance. This work was supported by grants from theCanadian Institutes of Health Research (MOP12875), the Heart andStroke Foundation of British Columbia and the Yukon, and the NaturalSciences and Engineering Research Council of Canada (to E.D.W.M.).

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/124/7/1167/DC1

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