Analysis of ryanodine receptor clusters in rat and human cardiac myocytes

Abstract

Single rat ventricular myocytes and human ventricle tissue sections were labeled with antibodies against the ryanodine receptor (RyR) and ␣-actinin to examine the 3D distribution of RyRs with confocal microscopy. Image contrast was maximized by refractive index matching and deconvolution. The RyR label formed discrete puncta representing clusters of RyRs or ''couplons'' around the edges of the myofilaments with a nearest-neighbor spacing of 0.66 ؎ 0.06 m in rat and 0.78 ؎ 0.07 m in human. Each bundle of myofibrils was served by approximately six couplons, which supplied a cross-sectional area of Ϸ0.6 m 2 in rat and Ϸ0.8 m 2 in human. Although the couplons were in reasonable registration with zlines, there were discontinuities in the longitudinal position of sarcomeres so that dislocations in the order of RyR clusters occurred. There was Ϸ53% longitudinal registration of RyR clusters, suggesting a nonrandom placement of couplons around the sarcomere. These data can explain the spherical propagation of Ca 2؉ waves and provide quantitative 3D data sets needed for accurate modeling of cardiac Ca 2؉ -induced Ca 2؉ release. By quantifying labeling intensity in rat ventricular myocytes, a lower limit of 78 RyRs per cluster (on average) was obtained. By modeling the couplon as a disk wrapping around a t-tubule and fitting cluster images, 95% of couplons contained between 120 and 260 RyRs (assuming that RyRs are tight packed with a spacing of 29 nm). Assuming similar labeling efficiency in human, from the fluorescence intensity alone we estimate that human ventricular myocytes contain Ϸ30% fewer RyRs per couplon than rat.calcium-induced calcium release ͉ excitation-contraction coupling ͉ sarcoplasmic reticulum I n cardiac ventricular muscle, excitation-contraction (EC) coupling arises from Ca 2ϩ release via clusters of ryanodine receptors (RyRs) in regions of close apposition between the sarcoplasmic reticulum (SR) and surface membranes in functional units called couplons (1, 2). Current work directed at understanding cardiac EC coupling is hindered by uncertainty in the size and 3D distribution of the couplons. Previous detailed analysis from electron micrographs has shown that typically 30-270 RyRs (depending on species) may be present in a couplon (1), but the thin sectioning associated with EM limits analysis of the spatial relationship between nearby and more distant couplons. Such knowledge is important, not only to make sense of the structures that underlie Ca 2ϩ sparks (3, 4) but also for detailed mathematical modeling of cardiac Ca 2ϩ metabolism.In this study, we have used immunocytochemistry combined with 3D imaging and analysis to both reveal the 3D organization of RyR clusters and estimate the numbers of RyRs within the couplon. Our analyses generally support some detailed quantitative measurements from EM (1), but also provide insight into organization in 3D at spatial scales that would be extremely laborious (if not impossible) to achieve by using conventional thin sectioning. In addition, the antibody labe...

Optical single-channel resolution imaging of the ryanodine receptor distribution in rat cardiac myocytes

“…Our data reveal that RyR cluster sizes vary widely from single RyRs to Ͼ100 RyRs, which seems at variance with the substantially larger estimates for the size of ''couplons'' within myocytes (17,27), although the variability in junction size and feet (i.e., RyR) content has been noted in ref. 28.…”

“…While this could reflect a genuine difference between peripheral and deeper RyR cluster size, it is possible that assumptions necessary for the estimation of cluster sizes from previous data led to an overestimate. For example, in both thin sectioning (17) and quantitative fluorescence imaging (27), the assumption of circularity and complete filling of the junction by close-packed RyRs has been used. Our data suggests that these key assumptions may be incorrect, and this conclusion is supported by some recent EM tomography (24,29).…”

Imaging Ca 2+ Nanosparks in Heart With a New Targeted Biosensor

“…This notion was confirmed by colocalization of GCaMP6f-J and GCaMP6f-T (latter data not shown) with type 2 RyR immunofluorescence ( Figure 1C) and the pattern of labeling was similar to that reported in highresolution studies of RyR distribution. 20 The t-tubular structure of the myocytes was well maintained with GCaMP6f-J and Di-4 fluorescent signals (a surface membrane marker) strongly colocalizing ( Figure 1D) after spectral unmixing (Online Figure I) again supporting the idea that the construct correctly trafficked to the dyadic junctions.…”

Size Matters: Ryanodine Receptor Cluster Size Affects Arrhythmogenic Sarcoplasmic Reticulum Calcium Release

“…First we assume that our mean cluster ΔF/F 0 of 1.39 (Figure 1D) corresponds to the average (or most frequent) RyR cluster size estimated from several prior studies that range mostly between 50 and 150 RyR/cluster, which in some high‐resolution cases were clusters of clusters or superclusters 2, 3, 4, 5, 6, 27. The mean ΔF/F 0 of 1.39 is indicated by a vertical broken line in Figure 1E.…”

“…In this model each RyR is formulated in a 4‐state Markovian model and regulated simultaneously by [Ca 2+ ] in both the dyadic cleft ([Ca 2+ ] cleft ) and SR luminal ([Ca 2+ ] SR ). Instead of the fixed number of the RyR cluster size in the original model, we investigated the effect of RyR cluster size ranging from 1 to 250, as indicated by experimental data 27. Increases of [Ca 2+ ] i of 200 nmol/L (Δ[Ca 2+ ], versus baseline) were used for Ca 2+ spark detection threshold.…”