Correlative super-resolution analysis of cardiac calcium sparks and their molecular origins in health and disease

Figure 1.. An experimental framework for spatially correlating Ca2+ sparks to the underlying RyR2 channel organization. (a) A TIRF micrograph of a spontaneous Ca2+ spark (white arrowhead) in the sub-surface regions of a rat right ventricular myocyte loaded with Fluo-4 AM and immersed in 5 mM [Ca2+]o. (b) A split view of a similar, fixed rat right ventricular myocyte immuno-stained for RyR2 imaged with standard TIRF microscopy (upper-left) and DNA-PAINT (lower-right), demonstrating the level of resolution improvement in the latter. (c) Magnified view of the region indicated by the box in panel (b) illustrating a single RyR2 cluster. The individual punctate densities of labelling (arrowheads) represented individual RyR2 channels; (d) A schematic illustration of a ventricular cardiomyocyte where the SR forms junctions with both the surface plasmalemma and the t-tubular invaginations. The magnified view illustrates the predominantly sub-surface RyR2 clusters observed with the thin TIRF field (depth indicated by asterisk). (e) Experimental pipeline developed to allow correlative Ca2+ spark TIRF imaging and DNA-PAINT of acutely isolated cardiomyocytes. (f) The image alignment involves the…

Figure 2.. Local interrogation of RyR2 organization in Ctrl and MCT- RV cardiomyocytes. DNA-PAINT super-resolution maps of RyR2 labelling in (a) Ctrl and (b) MCT-RV rat cardiomyocytes. The arrowheads indicate the dissipated morphology of RyR2 clusters in the latter. (c,d) show two-dimensional super-resolution maps of RyR2, colour-coded for local, average Ca2+ spark mass in Ctrl-RV and MCT-RV myocytes, respectively. The insets show the original DNA-PAINT images in the corresponding region (larger versions of the insets shown in electronic supplementary material figure S2); the colour scale represents the average spark mass (in arbitrary units), estimated by xySpark. (e,f) Scattergrams of the Ca2+ spark mass, plotted on log10 scales against the RyR2 locally-counted within each ‘spark footprint’ in Ctrl-RV myocytes (n = 1050 sparks; 15 cells; 6 animals) and MCT-RV myocytes (n = 1529 sparks; 15 cells; 6 animals), the latter featuring sparks with smaller spark mass in regions with larger RyR2 counts. The line fits are y = 0.272x2 – 0.3 and y = 0.022x2 – 0.2 respectively. (g) Overlaid frequency histograms of the number of RyR2 clusters consisting of ≥5 RyR2 within the footprint of sparks recorded…

Figure 3.. Spatial encoding of the spontaneous Ca2+ spark patterns in the RyR2 organization. (a) An overlay of an RyR2 super-resolution image (cyan) of a Ctrl-RV myocyte and a two-dimensional map of the centroids of all the spontaneous Ca2+ sparks (red circles) recorded within a 15 second time window. (b,c) Overlays of the DNA-PAINT super-resolution images of near-surface RyR2 labelling (cyan) and the recurring spark sites (magenta) in Ctrl-RV and MCT-RV myocytes respectively. (d,e) Magnified view of the windows indicated by dashed boxes in panels (b,c) respectively, illustrating that the recurring spark sites typically bridge or tessellate with local groups of RyR2 cluster rather than overlie them. (f) Overlaid percentage histograms of the RyR2 count underneath the spark footprint in Ctrl-RV (blue) and MCT-RV (red) as well as simulations where the detected spark positions were randomized (cyan), and where RyR2 positions were randomized. (g) The equivalent overlays of the percentage histograms of the mean neighbour distance between RyR2 puncta underneath each spark, and (h) the average distance to each of the three nearest neighbours to each RyR2 underneath spark. Scale bars: (a–c) 500 nm;…