Figure 4: Calcium imaging in tissue explants of TN-XXL mice.

(a,b) Calcium imaging in mesenteric arteries. (a) Fluorescence image of a mesenteric blood vessel in vivo showing TN-XXL expressed in endothelia (arrows) and SMC (arrowhead). (b) Ex vivo calcium imaging of seven endothelial cells in a mesenteric artery explant. The graph below shows the calcium responses upon application of 3 μM acetylcholine (Scale bars a,b, 25 μm). (c,d) Immunocytochemistry and calcium imaging of the SAN region of the mouse heart. (c) Immunocytochemistry of SAN explants. The left image illustrates cpCitrine fluorescence, the image in the middle highlights the staining with anti-HCN4 antibodies, a marker of the SAN (red). Right merged image shows co-localization (Scale bar, 500 μm). (d) Calcium imaging of cells in SAN explants. 30 Hz high-speed imaging of three cells (blue, red and black) from three different spontaneously beating SAN explants recorded at 30 °C (Scale bars, 25 μm). (e,f) Wide-field calcium imaging of whole embryonic hearts of TN-XXL transgenic mice. (e) Fluorescence image of a day 8.5 embryonic heart (left). Dotted circles refer to the regions of interest (ROIs) plotted in (f) (A, atria; V, ventricles). The phase plot in the right picture illustrates the progression of the calcium signal over one heartbeat cycle. The colour code indicates the time at which the maximum ΔR/R was observed at a given pixel (Scale bar, 150 μm). (f) Calcium transients of spontaneous beatings recorded from the heart shown in (e). Shown are intensities of CFP (blue) and cpCitrine (orange) emission channels (ROI 1) and the corresponding ratio (black). The lower graph shows a blow-up of four beating cycles for the indicated ROIs.