Distinct Effects of Mitochondrial Na ⁺ /Ca ²⁺ Exchanger Inhibition and Ca ²⁺ Uniporter Activation on Ca ²⁺ Sparks and Arrhythmogenesis in Diabetic Rats

Figures

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  Figure 1. [Ca2+]m measurements in intact myocytes. A, WT and HIP myocytes expressing the FRET‐based mitochondrial Ca2+ sensor mitoCam were field‐stimulated at 0.1, 0.3, and 1 Hz in the presence of 100 nmol/L isoproterenol. [Ca2+]m was measured as the FRET ratio FYFP/FCFP. B, Mean peak [Ca2+]m at 1 Hz stimulation. Measurements were performed in 22 WT myocytes (n=4 rats, 4–7 myocytes/rat) and 29 HIP myocytes (n=5 rats, 3–7 myocytes/rat). Bars show mean±SEM. Statistical significance was determined using a mixed model nested t test. ****P<0.0001. [Ca2+]m indicates mitochondrial Ca2+concentration; FRET, fluorescence resonance energy transfer; FCFP, fluorescence emitted by CEP (FRET donor in mitoCam); FYFP, fluorescence emitted by yellow fluorescent protein (FRET acceptor in mitoCam); HIP, rats with type 2 diabetes; mitoCAM, mitochondrial Ca2+ probe 4mtD3cpv Cameleon; and WT, wild type.

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  Figure 2. Mitochondrial Ca2+ uptake and extrusion in saponin‐permeabilized myocytes. A, Example of [Ca2+]m measurements using Rhod‐2 in a HIP myocyte. Following permeabilization and equilibration in a 10 mmol/L Na+, 0 Ca2+ internal solution, cytosolic Ca2+ was increased to 1 μmol/L. After 10 minutes, myocytes were exposed to Ca2+‐free/25 mmol/L Na+ solution. This Ca2+ loading/unloading cycle was repeated with 1 μmol/L CGP‐37157 present in the 0 Ca2+ internal solution. B, [Ca2+]m decline in the 0 Ca2+ internal solution, normalized to the initial [Ca2+]m, in myocytes from HIP and WT rats, with and without CGP‐37157. C, Rate of mitoNCX‐mediated Ca2+ extrusion as a function of [Ca2+]m. Data were averaged over 15 HIP myocytes (n=5 rats, 2–5 cells/rat) and 10 WT cells (n=4 rats, 2–4 myocytes/ rat). D, Mean [Ca2+]m during the mitochondrial Ca2+ loading phase. Experiments were performed in 67 HIP rat myocytes (n=9 rats, 5–11 cells/rat) and 59 WT cells (n=5 rats, 9–15 myocytes/rat). E, Average [Ca2+]m values at the end of the 10‐min Ca2+ uptake period for experiments shown in panel (D). F, Rate of the initial increase in [Ca2+]m upon raising cytosolic Ca2+ from 0 to 1 μmol/L with mitoNCX inhibited…

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  Figure 3. [Na+]m measurements in saponin‐permeabilized myocytes. A, Representative example in a WT myocyte. B, Mean [Na+]m after myocytes were equilibrated in 0 Ca2+ internal solution, which largely inactivates mitoNCX (green circle in Figure 3A). C and D, Mean [Na+]m when mitoNCX is activated by mitochondrial Ca2+ loading with 1 μmol/L Ca2+ in the cytosolic solution (C; yellow circle in Figure 3A) followed by a return to 0 Ca2+ in the cytosolic solution (D; blue circle in Figure 3A). Measurements were performed in 14 myocytes from 6 WT rats (1–6 cells/rat) and 12 myocytes from 4 HIP rats (2–4 cells/rat). Bars show mean±SEM. Statistical significance was determined using a mixed model nested t test. HIP indicates rats with type 2 diabetes; F340/F380, ratio between fluorescence excited at 340 nm and 380 nm; mitoNCX, mitochondrial Na+/Ca2+ exchanger; [Na+]m, mitochondrial; SBFI, fluorescent Na+ indicator; and WT, wild type.

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  Figure 4. Effect of mitoNCX inhibition on net ROS production and Ca2+ spark frequency in myocytes from control and T2D rats. A, Representative example of ROS measurement in a diabetic HIP rat myocyte in the absence (Ctl) and in the presence of 1 μmol/L CGP‐37157. The cell was first at rest, then stimulated at 1 Hz for 2 minutes, followed by addition of 1 μmol/L isoproterenol (ISO). The slope of the fluorescence signal in the absence and in the presence of ISO (purple lines) was taken as the measure of net mitochondrial ROS production in the 2 conditions. B and C, Net mitochondrial ROS production in the absence (B) and in the presence of ISO (C) in myocytes from WT and HIP rats under control conditions and in the presence of either 1 μmol/L CGP‐37157 or 25 μmol/L MitoTempo. WT: n=4 rats, 9–12 cells/group, 1–5 cells per rat; HIP: n=4 rats, 13–15 cells/group, 2–5 cells per rat. D, Representative linescans showing spontaneous Ca2+ sparks in HIP rat myocytes under control conditions (Ctl, top) and in the presence of 1 μmol/L CGP‐37157 (bottom). Myocytes were initially paced at 1 Hz and Ca2+ sparks were measured upon cessation of the electrical stimulation. The preceding 2 Ca2+ transients are…

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  Figure 5. Effect of MCU activation on net ROS production and Ca2+ sparks. A and B, Net mitochondrial ROS production in myocytes from WT and HIP rats under control conditions (Ctl) and with MCU activated by 10 μmol/L SB‐202190. ROS production was measured in cells stimulated at 1 Hz in the absence (A) and in the presence (B) of 1 μmol/L isoproterenol. Measurements were performed in 27 myocytes from 7 WT rats (3–5 cells per rat) and 24 myocytes from 7 HIP rats (2–5 cells per rat). C, Representative linescan recordings of spontaneous Ca2+ sparks in myocytes from WT and HIP rats under control conditions (Ctl) and in the presence of 10 μmol/L SB‐202190. Myocytes were preconditioned by electrical stimulation at 1 Hz, and then stimulation was stopped to record the spontaneous sparks. The preceding 2 Ca2+ transients are also shown. D, Ca2+ spark frequency in myocytes from WT and HIP rats under control conditions and in the presence of SB‐202190. WT: n=3 rats, 96 to 103 cells, 26 to 38 cells per rat; HIP: n=3 rats, 59 to 86 cells, 11 to 36 cells per rat. Myocytes were preincubated with SB‐202190 for 20 minutes. Bars show mean±SEM. Data in panels A, B, and D were analyzed with 2‐way ANOVA followed…

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  Figure 6. Effect of mitoNCX inhibition and MCU activation on Ca2+ waves and arrhythmogenesis. A, Representative example of Ca2+ waves measurements (HIP rat myocyte); top: linescan, bottom: fluorescence intensity. Red arrows indicate electrically stimulated Ca2+ transients. B and C, Ca2+ wave frequency in control conditions and following 20 minutes preincubation with CGP‐37157 (CGP) and SB‐202190 (SB). Experiments were performed in >8 myocytes per rat per group and individual symbols show wave frequency/rat. D, Examples of electrical rhythms recorded in a HIP rat under control conditions. E and F, Duration of ventricular arrhythmias in HIP rats during a 40‐minute period following a stress test with caffeine and dobutamine. Experiments were first performed under control conditions (Ctl) and were repeated a week later following two 1‐hour apart injections with either CGP‐37157 (0.1 mg/kg body weight) or SB‐202190 (1 mg/kg body weight). Bars show mean±SEM. Statistical significance was determined using Friedman nonparametric test followed by Dunn's multiple comparisons post‐test for data in panels (B and C) and paired t test for panels (E and F). *P<0.05. Ctl indicates control conditions; HIP,…

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  Figure 7. Mitochondrial function and gene expression. A, Mitochondrial membrane potential measurements in intact myocytes using TMRM. Myocytes were first equilibrated in normal Tyrode's solution and then exposed to 5 μg/mL oligomycin for 5 minutes. B, Relative TMRM fluorescence at the end of the 5 minutes of perfusion with oligomycin. Measurements were performed in 34 myocytes from 4 WT rats (3–13 cells/rat) and 50 myocytes from 3 diabetic HIP rats (9–6 cells/rat). C, ATP level in lysates of myocytes isolated from WT and HIP rats (WT: n=9 rats; HIP: n=8 rats). D, Volcano plot showing expression of genes that encode mitochondrial proteins in hearts from HIP vs WT rats (6 rats/group). Genes were considered differentially expressed if the fold change was >2 and adjusted P<0.05. Data are shown as mean±SEM. Statistical significance was determined using repeated‐measures ANOVA for data in panel (A), a mixed model nested t test for panel (B), and unpaired t test for panels (C and D) (with False Discovery Rate step up method used to correct for multiple comparisons in D). *P<0.05, ****P<0.0001. HIP indicates rats with type 2 diabetes; TMRM, tetramethylrhodamine; and WT, wild type.