A Special Amino-Acid Formula Tailored to Boosting Cell Respiration Prevents Mitochondrial Dysfunction and Oxidative Stress Caused by Doxorubicin in Mouse Cardiomyocytes

Figures

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  Figure 1. HL-1 cell treatment.

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  Figure 2. In vivo mouse treatment.

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  Figure 3. Specific amino-acid mixtures prevent mitochondrial dysfunction in HL-1 cardiomyocytes acutely exposed to DOX. (A) Mitochondrial biogenesis marker expression: peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1-α), nuclear respiratory factor-1 (NRF1), transcription factor A (Tfam), cytochrome c (cyt c), and cytochrome c oxidase subunit IV (COX IV) mRNA levels were analysed by quantitative RT-PCR. Relative expression values for the untreated (CTRL) cells were taken as 1.0 (n = 5 experiments). (B) COX IV and cyt c protein levels were detected by immunoblot analysis. The relative values were determined by densitometric analysis relative to GAPDH levels; values for untreated (CTRL) cells were taken as 1.0 (n = 5 experiments). (C) Citrate synthase activity. The values were normalized to protein content (n = 3 experiments). * p < 0.05, ** p < 0.01, and ***p < 0.001 vs. untreated cells; † p < 0.01 vs. DOX-treated cells; § p < 0.01 vs. BCAAem-treated cells. All data are presented as the mean ± SD.

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  Figure 4. α5 Supplementation prevents DOX-induced oxidative stress in HL-1 cardiomyocytes. (A) Mitochondrial H2O2 release, (B) basal aconitase/total aconitase ratio, and (C) superoxide dismutase activity (SOD) in HL-1 cells (n = 3 experiments). (D) superoxide dismutase 1 (SOD1) and glutathione peroxidase 1 (GPX1) mRNA levels were analysed by quantitative RT-PCR. Relative expression values for the untreated (CTRL) cells were taken as 1.0 (n = 5 experiments). (E) Total DNA oxidative damage measured as 8-hydroxy-2’-deoxyguanosine (8-OHdG) production.

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  Figure 5. α5 formula prevents mitochondrial dysfunction in left ventricle of DOX-treated mice. (A,F) Mitochondrial biogenesis marker expression. (A) PGC1-α, NRF1, Tfam, cyt c, COX IV and (F) SOD1, superoxide dismutase 2 (SOD2), catalase (Cat), and GPX1 mRNA levels were analysed by quantitative RT-PCR. Relative expression values for the vehicle-treated (Veh) mice were taken as 1.0 (n = 5 experiments). (B) Mitochondrial DNA (mtDNA) amount was analyzed by quantitative RT-PCR. Relative units were expressed in comparison to those for the Veh-treated mice which were taken as 1.0 (n = 5 experiments). (C) COX IV and cyt c protein levels were determined by immunoblot analysis. The relative values were determined by densitometric analysis relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels; values for Veh-treated mice were taken as 1.0 (n = 5 experiments). (D) Citrate synthase activity. The values were normalized to protein content (n = 3 experiments). (E) Basal oxygen consumption rate. Mitochondria were isolated from left ventricle of mice treated or not with DOX and α5. Oxygen consumption rates were normalized to mitochondrial protein amount (n = 3 experiments). * p < 0.05, ** p <…

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  Figure 6. Different signaling pathways are implicated in the protective effects of α5 supplementation in DOX-treated mice. (A,E,F) Gene expression. (A) Endothelial nitric oxide synthase (eNOS), (E) Sestrin2, and (F) Krüppel-like factor 15 (KLF15) mRNA levels were analysed by quantitative RT-PCR. Relative expression values of the vehicle (Veh)-treated mice were taken as 1.0 (n = 5 experiments). (B,C,D, and G) Protein levels. (B) Phospho-eNOS, (C) phospho-S6, (D) phospho-mTOR, and (G) phospho-BCKDH protein levels were detected by immunoblot analysis. The relative values were densitometrically analyzed and reported as ratios to total eNOS, S6, mTOR, and BCKDH levels, respectively. Values for Veh-treated mice were taken as 1.0 (n = 5 experiments). * p < 0.05 and ** p < 0.01 vs. Veh-treated mice; † p < 0.05 vs. DOX-treated mice. All data are presented as the mean ± SD.

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  Figure 7. Different signaling pathways are implicated in the protective effects of α5 supplementation in DOX-treated HL-1 cardiomyocytes. (A, D, and F) Gene expression. (A) eNOS, (D) Sestrin2, (F) KLF15 mRNA levels were analysed by quantitative RT-PCR. Relative expression values for the untreated (CTRL) cells were taken as 1.0 (n = 5 experiments). (B, C, E–H) Protein levels. (B) Phospho-eNOS, (C) phospho-S6, (E) phospho-Akt, (F) KLF15, (G) PGC-1α and COXIV, and (H) phospho-S6 and phospho-BCKDH protein levels were detected by immunoblot analysis. The relative values were densitometrically analyzed and reported as ratios to total eNOS, S6, Akt, and BCKDH levels, respectively; KLF15, PGC-1α, and COXIV were normalized to GAPDH. Values for untreated HL-1 cells were taken as 1.0 (n = 5 experiments). (G) PGC1-α and COX IV protein levels were measured by immunoblot analysis in HL-1 cells transfected with either specific KLF15 siRNA or nontargeting siRNA, and treated with DOX or α5 alone or in combination. Values for untreated HL-1 cells were taken as 1.0 (n = 5 experiments). (H) S6 and BCKDH phosphorylation was measured by immunoblot analysis in HL-1 cells as in (H). * p < 0.05, ** p < 0.01, and…

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  Figure 8. A proposed model of α5 protective actions on mitochondrial damage induced by doxorubicin (DOX) in cardiomyocytes. Plus (+) and minus (−) symbol in red colour indicate stimulation or inhibition induced by DOX and, potentially, the α5 target sites. DOX reduces mitochondrial biogenesis and function, (1) binding to topoisomerase IIβ (TOP2B), which complexes to the promoters of Ppargc1a and Ppargc1b and blocks transcription of mitochondrial genes, including ROS defense genes, (2) reducing eNOS expression and mTORC1 activity—which are important regulators of mitochondrial physiology, (3) limiting KLF15 gene expression and presumably BCAA oxidation, with α-ketoacid and BCAA accumulation—which are toxic at high levels—beyond reduced TCA cycle intermediates and mitochondrial energy production. Mitochondrial accumulation of DOX is accompanied by increased ROS.