Increased RyR2 activity is exacerbated by calcium leak-induced mitochondrial ROS

Fig.1. Mitochondrial ROS scavenging reduces caffeine-mediated SR Ca2+ leak in rat VMs. a Representative images of a cultured rat VM 48 h after adenoviral infection with G-CEPIAer intra-SR Ca2+ biosensor (left image, green). Using anti-RyR antibody, VMs were probed for RyR2 expression (center image, red). A merged image is shown on the right, indicating correct probe localization. b Representative images of infected VM treated with thapsigargin (20 µmol/L) and high-dose caffeine (20 mmol/L) to achieve minimal fluorescence, demonstrating G-CEPIAer sensitivity. c Representative time-dependent profiles (F/F0) of G-CEPIAer fluorescence from infected VMs. Myocytes were either treated with ISO (50 nmol/L), ISO and mitoTEMPO (MT, 20 μmol/L, 10 min pretreatment), ISO and caffeine (CAFF, 200 μmol/L), or ISO, CAFF and mitoTEMPO. Myocytes were exposed to SERCA inhibitor thapsigargin (TG, 10 μmol/L) after 1 min of recording, and the time constant of decay (τ, s) was calculated as a measure of SR Ca2+ leak. Signal was normalized to minimum fluorescence obtained by application of high-dose caffeine (10 mmol/L). The graph in d depicts mean data ± SEM for rate of decay (min−1). N = 5–9 animals, n =…

Fig. 2. Increased SR Ca2+ leak increases mitochondrial ROS emission in rat VMs. a Representative image of a cultured rat VM 48 h after adenoviral infection with OMM-HyPer mito-ROS biosensor (left image, green). Myocytes were stained with mitochondrial dye MitoTracker Red (center image, red). A merged image is shown on the right, indicating correct probe localization. b Representative images of infected VM treated with DTDP (200 µmol/L) to achieve maximal fluorescence, demonstrating OMM-HyPer sensitivity. c Representative traces of OMM-HyPer fluorescence from infected VMs. Myocytes were paced at 2 Hz for 5 min (black bars), and treated with ISO (50 nmol/L, left trace) or ISO and caffeine (CAFF, 200 µmol/L, right trace) before further pacing. The signal was normalized to minimum fluorescence obtained by application of DTT (5 mmol/L) and maximum fluorescence by application of DTDP (200 µmol/L). The graph in d depicts mean data ± SEM for maximum normalized fluorescence (%) after pacing and application of ISO, or application of ISO and CAFF. N = 3–4 animals, n = 7–16 VMs. *p < 0.05 vs. ISO group, two-sample Student’s t test

Fig. 3. Enhanced RyR2 activity in rat VMs leads to SR oxidative stress and RyR2 oxidation. a Representative images of a cultured rat VM 48 h after adenoviral infection with ERroGFP_iE oxidative stress biosensor (left image, green). Using anti-RyR antibody, VMs were probed for expression of RyR2 (center image, red). A merged image is shown on the right, indicating correct probe localization. b Representative images of infected VM treated with DTDP (200 µmol/L) to achieve maximal fluorescence, demonstrating ERroGFP_iE sensitivity. c Representative traces of ERroGFP_iE fluorescence from infected VMs. Myocytes were paced at 2 Hz for 5 min (black bars) and treated with ISO (50 nmol/L, left trace) or ISO and caffeine (CAFF, 200 µmol/L, right trace). Signal was normalized to minimum fluorescence obtained by application of DTT (5 mmol/L) and maximum fluorescence by application of DTDP (200 µmol/L). The graph in d depicts mean data ± SEM for maximum normalized fluorescence (%) after pacing and application of ISO, or application of ISO and CAFF. N = 3–4 animals, n = 15–16 VMs. *p < 0.05 vs. ISO group, two-sample Student’s t test. e Immunoprecipitated RyR2 from freshly isolated rat VMs was…

Fig. 4. Enhancement of RyR2 activity in rat VMs diminishes the ability of mitochondria to accumulate and retain Ca2+. a Representative image of a cultured rat VM 48 h after adenoviral infection with mtRCamp1h mitochondrial Ca2+ biosensor (left image, red), and mitochondrial-targeted GFP (mtGFP, center image, green). A merged image is shown on the right, indicating correct probe localization. b Representative images of infected VM treated with EGTA (2 mmol/L) to achieve minimal fluorescence, and Ca2+ (20 µmol/L) to achieve maximal fluorescence, demonstrating mtRCamp1h sensitivity. c Representative trace of mtRCamp1h fluorescence from infected VM. Myocytes were paced at 2 Hz for 5 min (black bars), before treatment with ISO (50 nmol/L), or ISO and caffeine (CAFF, 200 µmol/L) and further pacing. Myocytes were saponin permeabilized (SAP) before treatment with EGTA (2 mmol/L) to obtain minimum fluorescence, followed by Ca2+ (20 µmol/L) to obtain maximal fluorescence. d Representative traces of [Ca2+]m (µmol/L) during pacing in VMs treated with ISO (black line) or ISO and CAFF (red line). Graphs depict mean data ± SEM of ISO and ISO + CAFF groups for: e baseline [Ca2+]m (μmol/L); f peak…

Fig. 5. Enhancement of RyR2 activity reduces cytosolic Ca2+ transient amplitude and mitochondrial membrane potential. a Representative Fluo-3 fluorescence (F/F0) images (upper images) and profiles (lower images) of ISO treated (50 nmol/L) rat VM undergoing standard pacing protocol (arrows) of 2 Hz for 5 min, before and after treatment with low-dose caffeine (CAFF, 200 µmol/L) or mitoTEMPO (20 µmol/L) and caffeine. Line scan images were recorded during the last minute of pacing. The graph in b depicts mean data ± SEM for Ca2+ transient amplitude (ΔF/F0). N = 2–4 animals, n = 12–15 VMs. *p < 0.05, one-way ANOVA with Bonferroni post hoc test. c Representative images of a rat VM before and after treatment with ISO. Myocyte ΔΨm was monitored by TMRM fluorescence. d Representative recording of changes in ΔΨm in response to pacing at 2 Hz (black bar) for 5 min, followed by application of ISO and further pacing. The signal was normalized to minimum fluorescence obtained by application of FCCP (50 μmol/L) and represented as percentage of baseline. e Representative images of a TMRM-stained rat VM before and after treatment with ISO and CAFF. f Representative recording of changes in ΔΨm in response…

Fig. 6. Inhibition of MCU Ca2+ uptake with MCU-DN reduces mito-ROS emission in rat VMs. a Representative western blots from VMs probed for MCU expression. The control lane (Ctrl) represents VMs infected with virus carrying an empty vector, and the MCU-DN lane represents VMs infected with virus carrying the dominant-negative MCU subunit. The graph in b depicts quantification of normalized MCU signal (%). N = 4 animals. *p < 0.05 vs. Ctrl group, two-sample Student’s t test. c Representative traces of mtRCamp1h fluorescence from control virus (black line) or MCU-DN virus (pink line) infected VMs. Myocytes were paced at 2 Hz for 5 min (black bar) after treatment with ISO (50 nmol/L) or ISO and low-dose caffeine (CAFF, 200 µmol/L). No mitochondrial Ca2+ uptake is observed with expression of MCU-DN. d Representative traces of OMM-HyPer fluorescence from infected VMs. Myocytes were paced at 2 Hz for 5 min (black bars), treated with ISO, and paced again at 2 Hz. The black line indicates control VM signal, while the pink line indicates MCU-DN infected VM. The signal was normalized to minimum fluorescence obtained by application of DTT (5 mmol/L) and maximum fluorescence by application of DTDP…

Fig. 7. Proarrhythmic SCWs in CPVT mice VMs with genetically increased RyR2 activity can be attenuated by mito-ROS scavenging. a Representative confocal line scan images of Ca2+ transients and b Fluo-3 fluorescence (F/F0) profiles of ISO treated (100 nmol/L) WT and CPVT mouse VMs undergoing 0.5 Hz pace-pause protocol (arrows) to induce SCWs. Myocytes from CPVT mice were pretreated with mitoTEMPO (20 µmol/L, 20 min, CPVT + MT group). Graphs in cand d depict mean data ± SEM for WT, CPVT and CPVT + MT groups of Ca2+ transient amplitude (ΔF/F0) and SCW latency (seconds), respectively. N = 3 WT, 5 CPVT animals, n = 12–16 VMs. *p < 0.05 vs. WT group, **p < 0.05 vs. CPVT group, one-way ANOVA with Bonferroni post hoc test. e Graph depicting the percentage (%) of cells exhibiting waves. N = 3 WT, 5 CPVT animals, n = 20–25 VMs. *p < 0.05 vs. WT group, **p < 0.05 vs. CPVT group, Fisher’s exact test with Freeman-Halton extension. f Representative Fluo-3 fluorescence profiles (F/F0) of caffeine-induced Ca2+ transients (10 mmol/L) used to determine SR Ca2+ content. The graph in g depicts mean data ± SEM of caffeine-sensitive SR Ca2+ content (ΔF/F0). N = 3 WT, 3 CPVT animals, n = 4–8 VMs. *p < 0.05 vs.…

Fig. 8. Myocytes of CPVT mice have increased mito-ROS emission rates and RyR2 oxidation in comparison to controls. a Representative recording of ROS production measured with MitoSOX in ISO-treated (100 nmol/L) WT and CPVT VMs. Signal was normalized to maximum fluorescence obtained on application of DTDP (200 µmol/L). The ROS production rate (%) was calculated by normalization to the maximum rate of ROS emission observed on application of DTDP, and rates for these representative recordings are indicated. The graph in b depicts mean data ± SEM for the normalized ROS production rate of WT and CPVT VMs. N = 3 WT, 5 CPVT animals, n = 7–13 VMs. *p < 0.05 vs. WT group, paired Student’s t test. c Representative images of immunoprecipitated RyR2 from freshly isolated ISO-treated WT and CPVT VMs, immunoblotted for RyR2 signal and oxidation using DNP antibody. The graph in d depicts quantification of normalized DNP signal (%). N = 6 animals per group. *p < 0.05 vs. WT group, two sample Student’s t test. e Representative western blots from WT and CPVT VMs probed for MCUb and MCU expression. The graph in f depicts quantification of normalized MCUb/MCU signal (%). N = 5 animals per group. *p < 0.05…