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

Figures

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  Figure 1. Spatial organization and distribution of ryanodine receptor (RyR) clusters in permeabilized rat ventricular cardiac myocytes. A, Representative confocal F‐FKBP12.6 fluorescence image of a rat cardiac myocyte with a 2.0 zoom factor producing an x‐y pixel of 0.10×0.10 μm. Each yellow dot represents the local maximum of F‐FKBP12.6 fluorescence. Panel a is enlarged part of (A) (white box). B, Labeling of RyR clusters based on a threshold of 90th percentile of the intensity distribution after baseline correction. Different colors were randomly used so that individual clusters could be visibly distinguished. C, Fraction of RyR clusters as a function of the nearest RyR cluster in the transverse direction (red line) and longitudinal direction (green line, Gaussian fits) for the same cell shown in A. D, Fraction of RyR clusters as a function of average F‐FKBP12.6 relative fluorescence ΔF/F0 per cluster (n=10 cells, N=2 rats). Red labels are after translation of ΔF/F0 to estimated number of RyRs from E. E, Estimation of RyRs per cluster as a function of F‐FKBP12.6 relative fluorescence ΔF/F0. The curve is based on the fact that the mean number of RyRs per cluster (≈84 RyRs) should…

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  Figure 2. RyR cluster size and shape. A, Histogram of RyR cluster area for images like that in Figure 1B (n=4 cells, N=1 rat, Lorentzian fit). B, Plot of number of RyR tetramers vs physical area for each cluster (n=4 cells, N=1 rat, Quadratic polynomial fit). C, Major vs minor axis for each junctional cluster, with average ratio implied by regression line cluster (n=4 cells, N=1 rat, linear regression). D, The longer (major axis) was more often oriented along the transverse direction (along T‐tubules). RyR indicates ryanodine receptors.

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  Figure 3. Ca2+ spark peak detection. A, Representative confocal Fluo‐4 fluorescence frame extracted from 600 sequential frames. White outline indicates cell border, and yellow outlines indicate sparks detected for this frame (inset shows enlargement of region 1) with major and minor axes depicted. B, 3‐Dimensional surface plot of the same frame as in A with the signal over the spark threshold detection shown. The threshold is set at 1 standard deviation above the local mean intensity. C and D, Distribution of Ca2+ spark major axis and minor axis lengths (N=8 cells, n=1 rat, Lorentzian fit). E, Distribution of spark shape eccentricity (see also RyR cluster eccentricity in Figure S5, Gaussian fit). RyR indicates ryanodine receptors.

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  Figure 4. Ca2+ sparks spatial distribution vs RyR clusters. A, Representative Ca2+ spark frame with detected peak locations circled in white. B, Each white dot indicates the peak location of each Ca2+ spark in the whole series of 600 frames. Bb, Enlargement showing sparks typically located along Z‐lines (arrow 1), but also at hot spots (arrow 2) and some between Z‐lines (arrow 3). C through E, Quantitative results obtained for n=10 cells, N=2 rats showing the fraction of Ca2+ sparks vs distance to nearest RyR cluster (C) when corrected for larger area at larger distances (blue line: Gaussian fit) (D), and vs distance to Z‐line with cumulative Ca2+ spark probability superimposed (blue line, Lorentzian fit; red line, 1 phase association fit) (E). D, This panel shows the fraction of Ca2+ spark normalized by the area of the disk where the spark is detected. RyR indicates ryanodine receptors.

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  Figure 5. Ca2+ Spark frequency and RyR cluster locations. A, Representative confocal FKBP12.6 fluorescence image (top). Z‐lines are obtained by skeletonizing binary mask after thresholding (middle). Fluo‐4 image is average of all frames from time series (bottom). B, Superimposition of Z‐lines and average Fluo‐4 image with green dots representing a Ca2+ spark peaks. Bb, enlargement (white box) showing only Z‐lines and spark peak sites. C, Quantitative results (for n=10 cells, N=2 rats) of overall Ca2+ spark frequency as a function of FKBP12.6 fluorescence (ΔF/F0). D, Same data as in C, but normalized by fraction of RyR clusters (from Figure 1D). RyR indicates ryanodine receptors.

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  Figure 6. Ca2+ Spark frequency and amplitude as function of RyR cluster size. A and B, Ca2+ spark frequency (A) and amplitude (B) from experimental data with abscissa transformed to RyR numbers (based on Figure 1E). C and D, Simulations of RyR cluster size effect on Ca2+ spark frequency and amplitude at steady state with [Ca]SR=700, 770, and 650 μmol/L (with experimental data from (A) (blue). RyR indicates ryanodine receptors; SR, sarcoplasmic reticulum.