Analysis of ryanodine receptor clusters in rat and human cardiac myocytes
2007
Soeller, Christian | Crossman, David | Gilbert, Ray | Cannell, Mark B.
Single rat ventricular myocytes and human ventricle tissue sections were labeled with antibodies against the ryanodine receptor (RyR) and α-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 μm in rat and 0.78 ± 0.07 μm in human. Each bundle of myofibrils was served by approximately six couplons, which supplied a cross-sectional area of [almost equal to]0.6 μm² in rat and [almost equal to]0.8 μm² in human. Although the couplons were in reasonable registration with z-lines, there were discontinuities in the longitudinal position of sarcomeres so that dislocations in the order of RyR clusters occurred. There was [almost equal to]53% longitudinal registration of RyR clusters, suggesting a nonrandom placement of couplons around the sarcomere. These data can explain the spherical propagation of Ca²⁺ waves and provide quantitative 3D data sets needed for accurate modeling of cardiac Ca²⁺-induced Ca²⁺ 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 [almost equal to]30% fewer RyRs per couplon than rat.
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