SR-Mitochondria Crosstalk Shapes Ca Signalling to Impact Pathophenotype in Disease Models Marked by Dysregulated Intracellular Ca Release

Figure 1. Pharmacological modulation of MCU Ca uptake exerted divergent effects on Ca wave development in CPVT2 and FFD. (A) Representative traces and fluorescent images of Fluo-3 fluorescence (F/F0) for WT, CPVT2, and FFD both with and without kaempferol. Five still images are shown for each trace: the pacing-induced Ca transient and baseline fluorescence (first two images), and frequency of Ca waves (last three images). The time point for each image is labelled in the representative trace using a red triangle. (B) Mean ± SEM of average of frequency of Ca waves after pacing at 2 Hz for 5 s, with or without MCU activator kaempferol, n = 62–145 cells, N = 4–6 mice, *P < 0.05, unpaired Student’s t-test between the different groups of cells. (C) Representative traces and fluorescent images of F/F0 for CPVT2 and FFD both with and without Ru360. (D) Mean ± SEM of average of frequency of Ca waves after pacing at 2 Hz for 5 s, with or without MCU inhibitor Ru360, n = 71–98 cells, N = 4 mice, *P < 0.05, unpaired Student’s t-test between the different groups of cells.

Figure 2. MCU activator kaempferol modulated mitochondria membrane potential and ROS generation differently in CPVT2 and FFD. Mean ± SEM of average of mitochondria ROS production rate and representative images of MitoSOX Red loaded cells treated, both with and without kaempferol, are shown in (A), (B), and (C) for WT, CPVT2, and FFD, respectively, n = 33–59 cells, N = 4–5 mice *P < 0.05, unpaired Student’s t-test between the different groups of cells. (D) Normalized representative trace showing decrease in ΔΨm upon perfusion of kaempferol into cells for 11 min. (E) Mean ± SEM of average level of TMRE fluorescence (F/F0) after kaempferol perfusion, as normalized to the starting level, and (F) Mean ± SEM of average time to reach 50% decrease in TMRE fluorescence, n = 55–101 cells, N = 4–5 mice, *P < 0.05, one-way ANOVA between the different groups of cells.

Figure 3. CPVT2 and FFD displayed intrinsic differences in mitochondrial Ca handling. (A) Representative trace showing Rhod-2 fluorescence (F/F0) in permeabilized myocytes upon addition of 2 µM free Ca to the bath. (B) Mean ± SEM of average of maximal Rhod-2 signal at 12 min after addition of Ca to permeabilized myocytes and (C) mean ± SEM of average time to 50% max signal, n = 41–66 cells, N = 4 mice, *P < 0.05, one-way ANOVA between the different groups of cells. Mean ± SEM of average and representative traces of mitochondrial Ca content, indicated by change in Fluo-3 F/F0, in WT, CPVT2, and FFD, were shown in (D) and (E) for baseline conditions and in the presence of 100 nM ISO, respectively. Cells were paced at 0.5 Hz for 10 s before solution was switched to a Li Tyrode solution containing 10 µM thapsigargan for 60 s. Solution was switched to solution with 1 µM FCCP and oligomycin for 45 s to induce mCa release into the cytosol, then 10 mM caffeine was applied. n = 23–66 cells, N = 3–4 mice, *P < 0.05, one-way ANOVA between the different groups of cells.

Figure 4. Ablation of MCU was lethal in CPVT2. (A and B) mitochondria swelling assay, N = 4 mice. (A) Normalized trace showing decrease in relative absorbance at 540 nm due to 200 µM Ca challenge in isolated mitochondria. (B) Mean ± SEM of average decrease in relative absorbance (540 nm) after addition of 200 µM Ca, *P < 0.05, unpaired Student’s t-test between the different groups of mice. (C) Western blot of MCU expression in cardiac mitochondria. The COX4 subunit of mitochondrial complex IV was used as a loading control. (D) Survival curve of MCUCKO vs. CPVT2-MCUCKO 25 days post-induction, *P < 0.05, log-rank test (N = 14, N = 28 mice, respectively). (E) Mean ± SEM of heart weight normalized to body weight and representative picture of hearts in CPVT2 and CPVT2-MCUCKO (N = 4–5 mice), *P < 0.05, one-way ANOVA. (F) Representative surface ECG traces of CPVT2 (N = 8) and CPVT2-MCUCKO (N = 5) mice after catecholamine challenge (2 mg/kg EPI and 120 mg/kg caffeine, IP). (G) Mean ± SEM of arrhythmia score, *P < 0.05, unpaired Student’s t-test between the different groups of mice.

Figure 5. CsA inhibiton of mPTP affected frequency of Ca wave generation and rate of ROS generation differently in CPVT2 and FFD. (A) Representative traces and fluorescent images of Fluo-3 fluorescence (F/F0) for WT, CPVT2, and FFD both with and without CsA. Five still images are shown for each trace: the pacing-induced Ca transient and baseline fluorescence (first two images), and frequency of Ca waves (last three images). The time point for each image is labelled in the representative trace using a red triangle. (B) Mean ± SEM of average of frequency of Ca waves after pacing at 2 Hz for 5 s, with or without CsA, n = 84–154 cells, N = 4–5 mice, *P < 0.05, unpaired Student’s t-test between the different groups of cells. Mean ± SEM of average of mitochondria ROS production rate and representative images of MitoSOX Red loaded cells treated, both with and without CsA, are shown in (C), (D), and (E) for WT, CPVT2, and FFD, respectively, n = 29–65 cells, N = 4–5 mice, *P < 0.05, unpaired Student’s t-test between the different groups of cells.

Figure 6. CPVT2-CypD−/− mice show altered Ca handling, increased mtROS generation, and exacerbated ventricular arrhythmias. (A) Mean ± SEM of average of mitochondria ROS production rate and representative images of MitoSOX Red loaded cells after ISO perfusion in CPVT2 and CPVT2-CypD−/−, n = 39–48 cells, N = 4–5 mice, *P < 0.05, unpaired Student’s t-test between the different groups of cells. (B) Representative traces and fluorescent images of Fluo-3 fluorescence (F/F0) for CPVT2 and CPVT2-CypD−/− with 100 nM ISO perfusion. Five still images are shown for each trace: the pacing-induced Ca transient and baseline fluorescence (first two images), and frequency of Ca waves (last three images). The time point for each image is labelled in the representative trace using a red triangle. (C) Representative trace of SR Ca content (F/F0) after application of 20 mM caffeine. (D) Mean ± SEM of average Fluo-3 amplitude (ΔF/F0), frequency of Ca waves, and SR Ca content (ΔF/F0) in CPVT2 and CPVT2-CypD−/− cells after ISO perfusion (n = 26–38, n = 61–64, n = 12–14 cells, respectively), N = 4–5 mice, *P < 0.05, unpaired Student’s t-test between the different groups of cells. (E) Representative surface ECG…

Figure 7. CPVT2 cells showed higher level of transient mPTP openings as compared with WT and FFD. (A) Representative image of a MitoWink, whereby mitochondria undergo transient depolarization in membrane potential. (B) Mean ± SEM of average number of MitoWinks per cell per 20 min (n = 41–58 cells, N = 3 mice), (C) Mean ± SEM of average duration of MitoWinks in seconds in WT, CPVT2, and FFD, n = 13–18 cells, N = 3 mice, *P < 0.05, one-way ANOVA between the different groups of cells. (D) The percentage of cells exhibiting MiniWinks in WT, CPVT2, and FFD. Numbers in the grey bars represent cells without MiniWinks, and numbers in black bars represent those that exhibited MiniWinks. *P < 0.05, one-way ANOVA between the different groups of cells. (E) Representative trace of TMRE fluorescence (F/F0) showing transient depolarization for both MiniWinks (blue arrows) and MitoWinks (black arrow).