Aldehyde Dehydrogenase 2 Protects Against Post-Cardiac Arrest Myocardial Dysfunction Through a Novel Mechanism of Suppressing Mitochondrial Reactive Oxygen Species Production

Figures

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  Figure 1. The flowchart of the animal study.

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  Figure 2. The effect of ALDH2 activation by Alda-1 on post-cardiac arrest myocardial dysfunction and survival rate. (A) Left ventricular cardiac output (CO) and ejection fraction (EF) evaluated by Millar pressure-volume catheter within 4 h after ROSC in protocol 1 of the ALDH2 activation study (n = 6 animals per group). (B) Left ventricular EF and fractional shortening (FS) evaluated by echocardiography within 4 h after ROSC in protocol 2 of the ALDH2 activation study (n = 9 animals per group). (C) Left ventricular EF and FS evaluated by echocardiography at 72 h after ROSC in the two protocols of the ALDH2 activation study (n = 3–9 animals per group). (D) Kaplan-Meier survival curves within 72 h after ROSC in the two protocols of the ALDH2 activation study. Myocardial function between groups was compared by Student’s t-test and time-based measurements within each group were compared by repeated-measures ANOVA. The survival rate between groups was compared by the log-rank test. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 versus CA-CPR group.

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  Figure 3. The effect of ALDH2 activation by Alda-1 on post-cardiac arrest cardiomyocyte death, mitochondrial structural damage, and cardiac ROS levels. (A) Representative photographs of cardiomyocyte apoptosis by TUNEL staining at 4 h after ROSC and quantification at 4 h and 72 h after ROSC (n = 3–4 animals per group). Scale bar = 100 μm. (B) The plasma levels of CK-MB at 4 h and 72 h after ROSC (n = 3–4 animals per group). (C) Representative photographs and quantification of mitochondrial morphology by TEM examination at 1 h after ROSC (n = 3 animals per group). Scale bar = 1 μm. (D) Representative photographs of dihydroethidium (DHE) staining at 4 h after ROSC and quantification at 4 h and 72 h after ROSC (n = 3–4 animals per group). Scale bar = 100 μm. (E) Representative immunoblots and quantification of 4-HNE-protein adducts at 4 h and 72 h after ROSC (n = 3–4 animals per group). Group comparisons were performed by ANOVA with Tukey’s post hoc test. Data are presented as mean ± SEM. **P < 0.01, ***P < 0.001 versus Con group; #P < 0.05, ###P < 0.001 versus CA-CPR group.

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  Figure 4. The effect of cardiac overexpression of ALDH2 on post-cardiac arrest myocardial dysfunction and mitochondrial structural damage. (A) Representative immunoblots and quantification of ALDH2 expression in the liver, skeletal muscle, and heart tissue (n = 5 animals per group). **P < 0.01 versus AAV9-Veh group. (B) Left ventricular EF within 4 h after ROSC in AAV9-Veh+CA-CPR and AAV9-ALDH2+CA-CPR group (n = 7–8 animals per group). *P < 0.05 versus AAV9-Veh+CA-CPR group. (C) Representative photographs and quantification of mitochondrial morphology by TEM examination at 1 h after ROSC (n = 3 animals per group). Scale bar = 1 μm. ***P < 0.001 versus Naïve group; ###P < 0.001 versus AAV9-Veh+CA-CPR group. (D) Representative photographs of dihydroethidium (DHE) staining at 4 h after ROSC (n = 4 animals per group). Scale bar = 100 μm. *P < 0.05, ***P < 0.001 versus Naïve group; ###P < 0.001 versus AAV9-Veh+CA-CPR group. (E) Representative immunoblots and quantification of 4-HNE-protein adducts at 4 h after ROSC (n = 4 animals per group). Myocardial function between groups was compared by Student’s t-test and time-based measurements within each group were compared by repeated-measures ANOVA.…

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  Figure 5. The effect of Alda-1 on mitochondrial ROS levels and mitochondrial energy generation capacity during hypoxia/reoxygenation (H/R). (A–C) Data are from H9c2 cells and are presented as mean ± SEM. (A) Representative photographs and quantification of mitochondrial ROS levels (n = 3 samples per group). Scale bar = 250 μm. ***P < 0.001 versus Con group; ###P < 0.001 versus H/R group. (B) The levels of cellular ATP (n = 3 samples per group). **P < 0.01, ***P < 0.001 versus Con group; ##P < 0.01 versus H/R group. (C) Mitochondrial respiratory function evaluated by OCR and quantification of maximal OCR (n = 3 samples per group). The maximal OCR was calculated by subtracting non-mitochondrial respiration rate (the final rate after Rot/Ant) from the maximal FCCP rate. *P < 0.05 versus H/R group. (D, E) Data are from primary cardiomyocytes isolated from adult male WT mice and ALDH2 KO mice, and are presented as mean ± SEM. (D) Representative photographs and quantification of mitochondrial ROS levels (n = 3 samples per group). Scale bar = 250 μm. ***P < 0.001 versus WT group; ###P < 0.001 versus WT+H/R group. (E) Mitochondrial respiratory function evaluated by OCR and quantification of…

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  Figure 6. The effect of 4-HNE on mitochondrial ROS levels, succinate accumulation, mitochondrial membrane potential and SDH carbonylation. (A) Representative immunoblots and quantification of 4-HNE-protein adducts under hypoxia/reoxygenation (H/R) (n = 3 samples per group). (B) Representative photographs and quantification of mitochondrial ROS levels under the treatment of 4-HNE (40 μM) and 4-HNE (40 μM) +dimethyl malonate (DMM) (n = 3 samples per group). (C, D) The mitochondrial ROS levels after the addition of succinate or dimethyl malonate (DMM) during hypoxia/reoxygenation (H/R) (n = 4 samples per group). (E) The levels of succinate under the treatment of 4-HNE (n = 3 samples per group). (F) The quantification of mitochondrial membrane potential under the treatment of 4-HNE (40 μM) (n = 3 samples per group). (G) Representative immunoblots and quantification of SDH carbonylation under the treatment of 4-HNE (40 μM) (n = 3 samples per group). Group comparisons were performed by ANOVA with Tukey’s post hoc test or Student’s t-test. Data are presented as mean ± SEM. *P < 0.05, ***P < 0.001 versus Con group; #P < 0.05 versus Con+4-HNE group or H/R group; ###P < 0.001 versus H/R group.

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  Figure 7. The proposed molecular mechanisms of the protective effect of ALDH2 on post-cardiac arrest myocardial dysfunction. The aldehydes, e.g. 4-HNE, which are accumulated during ischemia/reperfusion result in mitochondrial ROS (mROS) burst. The latter triggers a series of orchestrated events including reduced mitochondrial respiratory capacity, reduced ATP generation, increased mitochondrial structure damage, and cardiomyocyte death, thereby contributing to myocardial dysfunction. Evidence from this study show that targeting ALDH2 would provide an important approach for treating post-cardiac arrest myocardial dysfunction through inhibiting mitochondrial ROS production.