COX5A Alleviates Doxorubicin-Induced Cardiotoxicity by Suppressing Oxidative Stress, Mitochondrial Dysfunction and Cardiomyocyte Apoptosis

Figures

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  Figure 1. COX5A expression in the failing hearts. (A–D) Western blot analysis of COX5A, COX5B, and SOD2 expression in rejected healthy donor hearts and end-stage heart failure patients with DCM. Data are expressed as mean ± SEM. n = 4, * p < 0.05 vs. the normal control (NC) group.

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  Figure 2. COX5A expression is decreased by DOX. (A,B) Western blot analysis of COX5A and SOD2 expression in mice heart tissues following DOX treatment for 4 weeks and 6 weeks, n = 6. (C) Western blot analysis of COX5A and SOD2 expression in H9c2 cells after treatment with DOX at various concentrations for 24 h, n = 6. Data are expressed as mean ± SEM. * p < 0.05 vs. the control (CON) group.

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  Figure 3. COX5A protects against DOX-induced cardiomyopathy in mice. (A) Representative M-mode echocardiograms for each group 6 weeks after DOX injection. (B) Echocardiographic quantification of LVEF and FS. (C) Body weight alterations with DOX treatment for different durations. (D) Statistical analysis of HW/TL. (E) Representative WGA-Alexa Fluor 488 conjugate stained heart sections and analysis of cardiomyocyte cross-sectional areas (CSA). Scale bar = 100 μm. (F) Representative histopathological findings of heart sections stained with H&E. Black arrows indicate extensive cytoplasmic vacuolization. Scale bar = 50 μm (G) Representative histopathological manifestations of heart sections stained with Sirius Red. Scale bar = 100 μm. (H) Analysis of serum level of cTnT by Elisa. (I,J) Biochemical determination of CK-MB and LDH serum levels. Data are expressed as mean ± SEM. * p < 0.05.

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  Figure 4. COX5A attenuates DOX-induced oxidative stress and apoptosis in mice. (A) Representative DHE staining images. Oxidized DHE intercalates into DNA and the nuclei appear in red. Scale bar = 100 μm. (B) Quantitative analysis of DHE-positive nuclei. (C) Representative IHC staining images of 4-HNE. (D–I) Western blot analysis of the protein level of COX5A, SOD2, Bax/Bcl-2, cleaved caspase-3/caspase-3, and p-Akt(S473)/t-Akt. Data are expressed as mean ± SEM. * p < 0.05.

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  Figure 5. COX5A improves myocardial energy metabolism in mice. (A) Representative images of myocardial glucose uptake, determined by PET/MRI using 18F-FDG. (B) Quantitative analysis of SUV value. (C) The ultrastructure of mitochondria in the heart of mice. (D) Quantitative analysis of cytochrome c oxidase (COX) activity of mitochondria in the heart of mice. (E) Quantitative analysis of ATP production of myocardium of mice. Data are expressed as mean ± SEM. * p < 0.05.

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  Figure 6. COX5A alleviates DOX-induced oxidative stress and cardiomyocyte apoptosis in vitro. (A) Expression of COX5A in H9c2 cells after transfection of lentivirus-COX5A and lentivirus-NC by Western blot. (B) DCFH-DA staining to detect ROS level. Nuclei were counterstained using Hoechst 33,342. Scale bars = 100 μm. (C) The morphology of mitochondria in H9c2 cells were stained with MitoTracker Green probe. (D) Annexin V-FITC staining followed by flow-cytometric analysis. (E) Quantitative analysis of Annexin V positive ratio. (F–I) Western blot analysis of the protein level of SOD2, p-Akt(T308)/t-Akt, and p-Akt(S473)/t-Akt. Data are expressed as mean ± SEM. * p < 0.05.

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  Figure 7. PI3K/Akt signaling is responsible for COX5A-mediated protective role in DOX-induced cardiotoxicity. (A) DCFH-DA staining to detect ROS level. Scale bars = 100 μm. (B) Cell viability detected by CCK-8 assay. n = 8. (C) Annexin V-FITC/PI staining followed by flow-cytometric analysis. (D) Quantitative analysis of of Annexin V positive ratio. n = 4. (E) The morphology of mitochondria in H9c2 cells stained with MitoTracker Green probe. (F) OCR measurements by means of the SeahorseTM XFe96 extracellular flux analyzer at baseline and after addition of oligomycin, FCCP, and rotenone + antimycin A. (G–J) Western blot analysis of the protein level of p-Akt(S473)/t-Akt, cleaved caspase-3/t-caspase-3, and SOD2. Data are expressed as mean ± SEM. * p < 0.05.