The RyR2-R2474S Mutation Sensitizes Cardiomyocytes and Hearts to Catecholaminergic Stress-Induced Oxidation of the Mitochondrial Glutathione Pool

Figures

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  FIGURE 1. Baseline mitochondrial oxidation at higher beating rates in sTG-cardiomyocytes. (A) Representative time course of the redox biosensor I405/I488 nm ratio following repeated EFS-evoked 3 Hz beating trains (top) compared to the absence of EFS-pacing in WT control cardiomyocytes (bottom). Black bars indicate the start and end of each beating interval lasting 10, 30, and 60 s as summarized by the graphical protocol in Supplementary Figure 1. H2O2 (200 μM) was added at the end of each experiment to obtain the maximal level of mitochondrial oxidation. Of note, vertical “noise” may originate from aberrant cardiomyocyte contractions. (B) Dot plot showing the I405/I488 nm ratio of individual cells and mean values ± SEM. Measurements were obtained under baseline conditions and following EFS-pacing at 3 Hz for the indicated beating durations. Cell/heart numbers are indicated. Paired one-way ANOVA with Dunnett’s post-test. **#P = 0.006; **P = 0.002; ***P < 0.001. (C) Dot plot summarizing individual and mean ± SEM 405/488 nm normalized ratio values recorded at the indicated beating rates and train durations. Ratio values were normalized to baseline at the start of each experiment. Cell/heart…

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  FIGURE 2. Mitochondrial oxidation following prolonged 1 Hz beating in sTG-cardiomyocytes. (A) Representative time course of the redox biosensor I405/I488 nm ratio with pacing-evoked 1 Hz beating trains in a mito-Grx1-roGFP2 cardiomyocyte. Black bars indicate the start and end of each beating interval lasting 10, 30, and 60 s, respectively. H2O2 (200 μM) was added at the end of the experiment as indicated to confirm the maximal level of mitochondrial oxidation. The interval between 900 and 1,300 s was condensed to show the reversible signal changes after 30 s pacing. (B) Dot-bar plot showing the I405/I488 nm ratio of the individual cardiomyocytes and mean values ± SEM. Measurements were obtained under baseline conditions and following EFS-pacing at 1 Hz for the indicated beating durations. Cell/heart numbers are indicated. Paired one-way ANOVA with Dunnett’s post-test. **P = 0.007; ***P < 0.001.

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  FIGURE 3. Effect of contractile activity induced by electrical field stimulation (EFS) on the redox sensor signal in cardiomyocytes from cytosolic Grx1-roGFP2+/– mice. (A) Representative time course of the redox signal (ratio I405/I488) with contractile activity by EFS at 1 Hz (above) and 3 Hz (below). Marks indicate the start and stop of the stimulation interval which lasted 10, 30, and 60 s, respectively. H2O2 (200 μM) was added at the end of each experiment to obtain the maximal oxidized value. Up and down reflections of the signal represent low-frequency (<0.1 Hz) contractions of the myocyte. (B) Mean values ± SEM of ratio values obtained in cardiomyocytes under control conditions and after EFS at 1 Hz with a 10, 30, and 60 s duration. The signal values of each cardiomyocyte were obtained under control conditions and after the respective pacing. We used 11 cells from four mice as indicated. Statistical analysis was performed by paired one-way ANOVA with Dunnett’s post-test. n.s., non-significant. (C) Mean values ± SEM of ratio values obtained in cardiomyocytes under control conditions and after EFS at 3 Hz with a 10, 30, and 60 s duration. We used 16 cells from four mice as indicated.…

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  FIGURE 4. Preserved transverse (T-)tubule and mitochondrial network interfaces in sTG-cardiomyocytes. Confocal live-cell images showing a representative ventricular cardiomyocyte expressing mitochondrial Grx1-roGFP2 after staining with the membrane dye di-8-ANEPPS. Left (from top to bottom): (i) roGFP2 auto-fluorescent signals (green LUT) demonstrating the longitudinal chain pattern typical for inter-sarcomeric mitochondria. (ii) di-8-ANEPPS membrane signal (red LUT) confirming a preserved, regular TAT membrane network mainly composed of T-tubule components. (iii) Overlay of the Grx1-roGFP2 and TAT membrane network signals showing a preserved gap-arrangement and confirming network interfaces of T-tubules and mitochondrial membranes. (iv) Cell quality and membrane integrity was confirmed by transmission scanning microscopy with differential interference contrast detecting regular transverse striations and sharp brick-shaped cell edges. Scale bar 10 μm. Right (from top to bottom): zoom-in magnifications (i–iii) visualize the distinct mitochondrial and T-tubule compartments and interfaces. Scale bar 2 μm.

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  FIGURE 5. Increased mitochondrial oxidation in RyR2-WT vs. RyR2-R2474S dTG-cardiomyocytes following different beating rates. (A) Representative time course of the I405/I488 nm ratio following 3 Hz beating of RyR2-WT control (top) and RyR2-R2474S (bottom) dTG-cardiomyocytes. Black bars indicate the start and end of each beating interval lasting 10, 30, and 60 s. H2O2 (200 μM) was added at the end of each experiment to obtain the maximal oxidized value. (B,C) Dot plots showing individual and mean ± SEM normalized I405/I488 nm ratio values for the indicated durations either at the 1 Hz (B) or 3 Hz (C) beating rate. The fluorescent signal values of each cardiomyocyte were obtained under baseline conditions and after the indicated beating trains each lasting 10 s, 30 s or 60 s. Cell/heart numbers are indicated. Unpaired one-way ANOVA with Tukey’s post-test. ***P = 0.001; *P = 0.03; n.s., non-significant.

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  FIGURE 6. Increased mitochondrial oxidation in WT control and RyR2-R2474S dTG-cardiomyocytes following β-adrenergic stimulation and 3 Hz beating. (A) Representative time course of the I405/I488 nm ratio of RyR2-WT control (top) and RyR2-R2474S dTG (bottom)-cardiomyocytes upon/during isoproterenol (100 nM) stimulation. Black bar pairs indicate the start and stop of the pacing lasting 10, 30, or 60 s. H2O2 (200 μM) was added at the end of each experiment to obtain the maximal oxidized value. (B) Dot blot showing individual and mean values ± SEM of normalized ratio values obtained after EFS at 3 Hz with a 10, 30, and 60 s duration in the absence (open squares) or presence of isoproterenol (100 nM, closed squares) in cardiomyocytes from WT control mice. Numbers indicate the number of cells/number of mice used. Statistical analysis was performed by unpaired one-way ANOVA with Tukey’s post-test. **P < 0.009; n.s., non-significant. (C) Dot plot summarizing the individual and mean ± SEM values of the normalized 405/488 nm ratio obtained following 3 Hz beating for 10, 30, and 60 s in the absence (open squares) and presence of isoproterenol (100 nM, closed squares) in RyR2-R2474S dTG-cardiomyocytes.…

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  FIGURE 7. Comparison of the increased mitochondrial oxidation in RyR2-WT control vs. RyR2-R2474S dTG-cardiomyocytes following β-adrenergic stimulation. (A) Dot plot showing the individual and mean values ± SEM of the normalized I405/I488 nm ratio values after 3 Hz beating for 10, 30, and 60 s in the presence of isoproterenol (100 nM). Open circles and closed squares reflect the values of the single experiments from RyR2-WT control and Ryr2-R2474S dTG mice, respectively. Cell/heart numbers are indicated. Unpaired one-way ANOVA with Tukey’s post-test. ***P < 0.001; *P = 0.04; (B) Dot blot showing individual and mean values ± SEM of normalized ratio values obtained after EFS at 1 Hz with a 10, 30, and 60 s duration in the presence of isoproterenol (100 nM). Open circles and closed squares reflect the values of the single experiments from RyR2-WT control and Ryr2-R2474S dTG mice, respectively. Numbers indicate the number of cells/number of mice used. Statistical analysis was performed by unpaired one-way ANOVA with Tukey’s post-test. *P < 0.02; n.s., non-significant.

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  FIGURE 8. Glutathione redox potentials (EGSH) in cardiomyocytes from RyR2-WT control and RyR2-R2474S dTG mice. (A) Representative trace of the emitted fluorescent intensity signals at 405 and 488 nm excitation in a cardiomyocyte from a RyR2-R2474S dTG mouse (above) and a WT control mouse (below) in the presence of isoproterenol (100 nM). Bars indicate the start and stop of contractile activity induced by EFS at 3 Hz. H2O2 (200 μM) followed by DTT (1 mM) were applied at the end of the experiment to obtain the maximal and minimal signal values, respectively. (B) Calculated EGSH before and after contractile activity induced by EFS-pacing (3 Hz, 60 s, time point at 600 s) in the presence of isoproterenol (100 nM) in cardiomyocytes from RyR2-WT control and RyR2-R2474S dTG mice. Numbers indicate the number of cells used (five cells from two mice, each). Statistical analysis was performed by a paired (control vs. isoproterenol) or unpaired (RyR2-WT control vs. RyR2-R2474S dTG) Student’s t-test. **P = 0.001; *P = 0.02; *#P = 0.01; n.s., non-significant.

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  FIGURE 9. β-adrenergic stimulation induces mitochondrial oxidation in intact RyR2-R2474S hearts. (A,B) Color-coded I385/I470 nm ratio comparing a representative RyR2-R2474S dTG-heart (A) and a RyR2-WT heart (B). From left to right: (i) baseline control, (ii) following isoproterenol (33 nM) stimulation, (iii) treatment with H2O2 (200 μM) and iv) DTT (4 mM) (scale bar, 2.5 mm). (C) Representative time course of the normalized I385/I475 nm ratio comparing RyR2-WT and RyR2-R2474S dTG-hearts. Black lines indicate each perfusion with 33 nM isoproterenol, 200 μM H2O2, and 4 mM DTT. The baseline heart rate for RyR2-WT and RyR2-R2474S was 340 bpm and 275 bpm, respectively. In-between, we performed electrical pacing with 600 bpm and 900 bpm (red lines). Red arrows refer to time points for representative images shown in (A,B). (D) Paired dot-line plot showing EGSH calculated before and after application of isoproterenol each for RyR2-WT and RyR2-R2474S dTG-hearts. Each dot represents one heart/experiment. Paired Student’s t-test; *P = 0.013; n.s., non-significant.

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  FIGURE 10. Dantrolene diminishes mitochondrial oxidation in β-adrenergic stimulated RyR2-R2474S dTG cardiomyocytes. Dot plot showing the individual and mean ± SEM values of the normalized I405/I488 nm ratio recorded after 3 Hz beating for 10, 30, and 60 s in the presence of isoproterenol (100 nM) either with or without dantrolene pre-treatment (5 μM for 2 h). Triangles and squares represent individual experiments ± dantrolene as given by the legend. Cell/heart numbers are indicated. Unpaired one-way ANOVA with Tukey’s post-test. ***P < 0.0002; n.s., non-significant.