Analysis of ryanodine receptor clusters in rat and human cardiac myocytes

Single rat ventricular myocytes and human ventricle tissue sections were labeled with antibodies against the ryanodine receptor (RyR) and a-actinin to examine the 3D distribution of RyRs with confocal microscopy. Image contrast was maximized by refractive index matching and cleconvolution. 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 mu m in rat and 0.78 +/- 0.07 mu m in human. Each bundle of myofibrils was served by approximately six couplons, which supplied a cross-sectional area of approximate to 0.6 mu m(2) in rat and approximate to 0.8 mu m(2) 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 approximate 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 approximate to 30% fewer RyRs per couplon than rat.