Store-Operated Calcium Entry Increases Nuclear Calcium in Adult Rat Atrial and Ventricular Cardiomyocytes

Figures

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  Figure 1. Experimental protocol used to elicit store-operated calcium entry in ventricular myocytes. (A) Experimental protocol showing the various solutions used to elicit store-operated calcium entry (SOCE). (B) Representative linescan images of cytoplasmic (black) and nuclear (red) Ca at 1 Hz stimulation (a,c–h) or without stimulation (b,i,j) from a WKY ventricular myocyte with corresponding normalized averaged fluorescence traces. Scan line was positioned perpendicular to the longitudinal axis of the cell traversing one nucleus, allowing for simultaneous recordings of cytosolic (black) and nuclear (red) Ca. Cells were first electrically stimulated at 1 Hz resulting in cytosolic and nuclear CaTs (a). After a resting period (b), the stimulation was turned on again (c) and NT was switched to a 0 Ca solution with 0.5 μM thapsigargin (TG), 5 μM verapamil, and 10 μM KB-R7943 resulting in elimination of CaTs (d–f). Twenty mM caffeine was applied twice (g,h) to empty intracellular Ca stores. After another short resting period (i), a 2 mM Ca (NT) solution was applied for 90 s to evoke store-operated calcium entry (j). The display range (after background correction) was 0–1200 for linescan images…

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  Figure 2. SR Ca load in 0 Ca/TG/verapamil/KB-R7943 solution determined by two caffeine bolus applications. (A) Representative linescan images of Fluo-4 fluorescence caffeine transients in a field-stimulated (1 Hz) ventricular myocyte from WKY during the first (left) and second (right) caffeine bolus application and corresponding normalized fluorescence traces (below), cytosolic (black) and nuclear (red) traces are shown. Same cell and images as in Figure 1. (B) Averaged values of cytosolic (grey) and nuclear (red) first caffeine amplitude (left) and second caffeine amplitude (right). Wilcoxon test; p-value as indicated; n = 39 cells from N = 16 rats. The display range (after background correction) was 0–1200 for both linescan images.

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  Figure 3. Robust SOCE in cytoplasm and nucleus of ventricular cardiomyocytes. (A) Representative normalized fluorescence traces of CaTs at 1 Hz stimulation (left) and SOCE evoked by re-addition of 2 mM Ca (right) of a ventricular myocyte. Cytosolic (black) and nuclear (red) traces are shown. (B) Averaged values of CaT amplitudes (ΔF/Frest). (C) Averaged values of SOCE amplitudes (ΔF/Frest). (D) SOCE amplitudes in the cytoplasm and nucleus normalized to the respective CaT in the very same cell. Cytosolic (Cyto, grey) and nuclear (Nuc, red) data is shown. (E) Linear correlation of nuclear and cytosolic SOCE amplitudes (Spearman correlation). (F) Average values of nuclear/cytosolic ratios of CaT amplitudes (black) or SOCE amplitudes (grey). Wilcoxon/Mann–Whitney test; p-values as indicated; **, p < 0.01; ***, p < 0.001; n = 39 cells from N = 16 rats.

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  Figure 4. SOCE in ventricular myocytes is reduced by pharmacological inhibitors of TRPC and Orai channels. (A) Representative normalized fluorescence traces of CaTs at 1 Hz stimulation (left) and normalized fluorescence traces of SOCE (right) of an untreated control myocyte in the presence of DMSO (a) and of ventricular myocytes measured in the presence of 1 μM S66 (b), 3 μM BTP-2 (c), 5 μM SKF96365 (d), or 1 μM S66 plus 5 μM SKF96365 (e). Cytosolic (black) and nuclear (red) traces are shown. (B) Averaged values of SOCE/CaT (%) for cytoplasm (left) and nucleus (right) of untreated control cells (ctl) and of cells treated with the various inhibitors, as indicated, depicted as scatter dot plots and box plots with medians. DMSO ctl: n = 35, N = 17; S66: n = 26, N = 4; BTP-2: n = 31, N = 5; SKF: n = 28, N = 5; S66/SKF: n = 20, N = 3; Kruskal–Wallis test with Dunn’s multiple comparisons test; p-values as indicated; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

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  Figure 5. SOCE persists after detubulation of ventricular myocytes, but CaT is decreased. (A) Representative 2D confocal image (left) of a ventricular myocyte stained with the cell membrane dye di-8-ANEPPS (10 μM). Normalized fluorescence traces of CaTs at 1 Hz stimulation (middle) and SOCE (right) of a different cell (same cell as shown in Figure 3A). Cytosolic (black) and nuclear (red) traces are shown. (B) Representative 2D confocal image (left) of a detubulated ventricular myocyte stained with di-8-ANEPPS. Normalized fluorescence traces of CaTs at 1 Hz stimulation (middle) and SOCE (right) of a different detubulated cell. The display range for the images in (A) and (B) was 250–3000. (C) Averaged values of CaT amplitudes in cytoplasm (grey) and nucleus (red), (D) SOCE amplitudes, and (E) SOCE amplitudes normalized to the respective CaT in the very same cell in control (ctl) or detubulated (det) ventricular myocytes. Wilcoxon/Mann–Whitney test; ctl: n = 39, N = 16 (same cells and data as in Figure 3); detubulated: n = 84, N = 12; p-values as indicated; **, p < 0.01; ***, p < 0.001.

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  Figure 6. Characteristics of SOCE in atrial cardiomyocytes. (A) Representative 2D confocal image (left) of an atrial myocyte; cell membrane was stained with di-8-ANEPPS (10 μM). Display range was 280–2300. Normalized fluorescence traces (of a different cell) of CaTs at 0.5 Hz stimulation (middle) and SOCE (right) with corresponding original linescan images shown above. Display range (after background correction) for both linescan images was 0–800. Cytosolic (black) and nuclear (red) traces are shown. (B) Averaged values of CaT amplitudes (ΔF/Frest), (C) SOCE amplitudes (ΔF/Frest), and (D) SOCE amplitudes normalized to the respective CaT amplitude in the very same cell. Data for cytosol (Cyto, grey) and nucleus (Nuc, red) is shown. (E) Linear correlation of nuclear and cytosolic SOCE amplitudes (Spearman correlation). (F) Average values of nuclear/cytosolic ratios of CaT amplitudes (black) or SOCE amplitudes (grey). Wilcoxon test; n = 21 cells from N = 5 rats; p-values as indicated; ***, p < 0.001.

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  Figure 7. SOCE in atrial myocytes is reduced by pharmacological inhibitors of TRPC and Orai channels. (A) Representative normalized fluorescence traces of CaTs at 0.5 Hz stimulation (left) and normalized fluorescence traces of SOCE (right) of an untreated control atrial myocyte in the presence of DMSO (a) and of atrial myocytes measured in the presence of 1 μM S66 (b), 3 μM BTP-2 (c), 5 μM SKF96365 (d), or 1 μM S66 plus 5 μM SKF96365 (e). Cytosolic (black) and nuclear (red) traces are shown. (B) Averaged values of SOCE/CaT (%) for cytoplasm (left) and nucleus (right) of untreated control cells (ctl) and of cells treated with the various inhibitors, as indicated, depicted as scatter dot plots and box plots with medians. Kruskal–Wallis test with Dunn’s multiple comparisons test; DMSO ctl: n = 41, N = 17; S66: n = 27, N = 5; BTP-2: n = 29, N = 4; SKF: n = 32, N = 4; S66/SKF: n = 28, N = 7; p-values as indicated; ***, p < 0.001.

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  Figure 8. Expression of TRPC and STIM proteins differs between atrial and ventricular tissue. (A) Representative immunoblots of TRPC1, 3, 6, and STIM1 with housekeeping protein GAPDH in ventricular (V) and atrial (A) tissue. (B) Averaged data of expression of TRPC1, 3, 6, and STIM1 in atrial and ventricular tissue from 8 WKY hearts. Protein expression was normalized to GAPDH expression and ventricular data served as reference (=100%). Student’s t-test; p-values as indicated; **, p < 0.01; ***, p < 0.001.