Concomitant Administration of Dantrolene is Sufficient to Protect Against Doxorubicin-Induced Cardiomyopathy

Figures

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  Figure 1. DOX-Induced Ca2+ Spark Increase Is Suppressed by RYR2 Stabilization (A) Representative line scan images of Fluo-4 AM fluorescence in intact wild-type (WT) and ryanodine receptor 2 (RYR2) V3599K-KI cardiomyocytes. (B) Data summary. Control (CTL): no doxorubicin (DOX); DOX: 1 μmol/L; dantrolene (DAN): 1 μmol/L; N-acetyl cysteine (NAC): 1 mmol/L. Mean ± SEM (n = 50-66 cells from 7 hearts). ∗∗∗P < 0.001 by 1-way analysis of variance with Tukey's post hoc test. KI = knock-in.

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  Figure 2. RYR2 Stabilization Suppresses DOX-Induced Oxidative Stress (A) Representative images of DCFH-DA fluorescence in intact WT and RYR2 V3599K-KI cardiomyocytes. (B) Data summary. Scale bar: 100 μm. CTL: no DOX; DOX: 1 μmol/L; DAN: 1 μmol/L; NAC: 1 mmol/L. Mean ± SEM (n = 113-155 cells from 6 hearts). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 by 1-way analysis of variance with Tukey's post hoc test. Abbreviations as in Figure 1.

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  Figure 3. RYR2 Stabilization Maintains RYR2-Bound CaM Despite DOX-Induced Dissociation (A) Representative images of endogenous calmodulin (CaM) and RYR2 in intact WT and RYR2 V3599K-KI cardiomyocytes fixed 5 minutes after adding DOX (1 μmol/L), DOX (1 μmol/L) + DAN (1 μmol/L), or DOX (1 μmol/L) + NAC (1 mmol/L). RYR2 is shown in red; CaM is shown in green. (B) Data summary. Scale bar: 10 μm. Mean ± SEM (n = 45-62 cells from 7 hearts); ∗∗∗P < 0.001 by 1-way analysis of variance with Tukey's post hoc test; not significant (n.s.) by unpaired Student’s t-test. Abbreviations as in Figure 1.

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  Figure 4. Effects of DOX on Ca2+ Transient and Sarcomere Shortening (A) Representative recordings of Fluo-4 AM fluorescence signals at 2-Hz pacing in intact WT and RYR2 V3599K-KI cardiomyocytes 30 minutes after observation. (B) Data summary (mean ± SEM; n = 34-42 cells from 6 hearts). (C) Representative recordings of sarcomere length at 2-Hz pacing in intact WT and RYR2 V3599K-KI cardiomyocytes 30 minutes after observation. (D) Data summary. CTL: no DOX; DOX: 1 μmol/L; DAN: 1 μmol/L; NAC: 1 mmol/L. Mean ± SEM (n = 35-47 cells from 6 hearts). ∗∗∗P < 0.001 by 1-way analysis of variance with Tukey's post hoc test; n.s. by unpaired Student’s t-test. F/F0 = fluorescent intensity/baseline fluorescent intensity; other abbreviations as in Figures 1 and 3.

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  Figure 5. Intracellular DOX Fluorescence Over Time Intracellular DOX fluorescence assessed in isolated cardiomyocytes from different mice at each time point after DOX administration. Scale bar: 50 μm. Mean ± SEM (n = 30-35 cells from 5 hearts); ###P < 0.001 vs autofluorescence by 1-way analysis of variance with Dunnett’s post hoc test. Abbreviations as in Figure 1.

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  Figure 6. DAN Prevents DOX-Induced Cardiac Dysfunction in Chronic Model Mice (A) Body weight (BW) and heart weight (HW) normalized to BW (n = 9). (B) Echocardiographic images and (C) summary of data (n = 9-11). (D) Collagen fibers in the left ventricle (LV) evaluated by Picro-Sirius Red staining and (E) data summary. Collagen is shown in red; muscle fibers in yellow (n = 9). Scale bar: 100 μm. (F) TUNEL staining images at low power (LP) (scale bar: 100 μm) and high power (HP) (scale bar: 10 μm) fields, and (G) data summary (n = 9). Arrowheads indicate TUNEL-positive nuclei. Data acquired as shown in Supplemental Figure 7A. ∗∗P < 0.01 and ∗∗∗P < 0.001 by 1-way analysis of variance with Tukey's post hoc test. Values for individual mice are plotted as mean ± SEM. bpm = beats/min; HR = heart rate; other abbreviations as in Figure 1.

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  Figure 7. Evaluation of Isolated Cardiomyocytes in Chronic Model Mice (A) Representative images of DCFH-DA fluorescence in isolated cardiomyocytes and (B) summary of data. Scale bar: 100 μm. Mean ± SEM (n = 65-78 cells from 8 hearts). (C) Representative line scan images of Fluo-4 AM fluorescence in isolated cardiomyocytes and (D) data summary. Mean ± SEM (n = 42-63 cells from 8 hearts). (E) Representative recordings of sarcomere length at 2-Hz pacing in isolated cardiomyocytes and (F) data summary. Mean ± SEM (n = 43-59 cells from 8 hearts). Data acquired as shown in Supplemental Figure 7A. ∗∗∗P < 0.001 by 2-way analysis of variance with Tukey's post hoc test; n.s. by unpaired Student’s t-test. Abbreviations as in Figures 1, 3, and 4.

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  Figure 8. Evaluation of ER Stress Markers Changes in endoplasmic reticulum (ER) stress markers under control conditions or with DOX (1 μmol/L, 4 hours), DAN (1 μmol/L, 24 hours), DAN (short) (1 μmol/L, 4 hours), or NAC (1 mmol/L, 24 hours). (A) Representative immunostaining images of GRP78 and (B) data summary. Scale bar: 50 μm. Mean ± SEM (n = 52-58 cells from 6 hearts). (C) Western blots of GRP78, P53, caspase-12, and GAPDH in lysates from cardiomyocytes cultured for 24 hours and (D) data summary. Mean ± SEM (n = 5 hearts). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 by 1-way analysis of variance with Tukey's post hoc test. Abbreviations as in Figure 1.

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  Figure 9. Evaluation of Ferroptosis Markers (A) Representative fluorescence images of mitochondrial lipid peroxidation in cultured cardiomyocytes under control conditions or with DOX (1 μmol/L, 4 hours), DAN (1 μmol/L, 24 hours), DAN (short) (1 μmol/L, 4 hours), or NAC (1 mmol/L, 24 hours). Mitochondrial lipid peroxidation was evaluated using MitoPeDPP (green, middle panels), and mitochondria were double-stained with Mito Tracker Red (red, upper panels). (B) Data summary. Scale bar: 50 μm. Mean ± SEM (n = 33-48 cells from 6 hearts). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 by 1-way analysis of variance with Tukey's post hoc test. Abbreviations as in Figure 1.

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  Central Illustration. RYR2 Stabilization Protects the Heart From Doxorubicin Cardiotoxicity Apoptosis from endoplasmic reticulum (ER) stress and ferroptosis from reactive oxygen species (ROS), both induced by doxorubicin (DOX), are associated with Ca2+ leakage and calmodulin (CaM) dissociation. Stabilizing ryanodine receptor 2 (RYR2) suppresses these effects. Dantrolene (DAN), a RYR2 stabilizer, provides sufficient cardioprotective effects even when administered only during DOX treatment. SR = sarcoplasmic reticulum.