The Metabolic Fingerprint of Doxorubicin-Induced Cardiotoxicity in Male CD-1 Mice Fades Away with Time While Autophagy Increases

Figure 1. Long-term vs. short-term effects after DOX on blood serum. The concentration of glucose, total protein, and albumin, along with the content of C reactive protein (CRP), protein carbonyl levels, and nitrotyrosine, as well as the activity of creatine kinase-MB (CK-MB) are depicted. A representative image of the slot-blot obtained is presented above the corresponding graph. For the complete blot obtained, see Supplementary Figure S1. Values are expressed as mean ± SD (n = 9 for CTRL1 and CTRL2 and n = 7 for DOX1 and DOX2 for glucose, total protein, albumin, and CK-MB; n = 5–6 for CRP, protein carbonyl levels, and nitrotyrosine). Statistical analyses were carried out using unpaired two-sided t-test (p < 0.05).

Figure 2. Long-term vs. short-term effects after DOX on the cardiac structure assessed by light microscopy. Representative light microscope micrographs obtained from three animals per group. Mice controls of each time-point (CTRL1 and CTRL2) were compared to DOX-treated animals (DOX1 and DOX2). DOX elicited, in both time-points, cardiac interstitial edema (white arrow), necrotic zones (blue arrow), vascular congestion (green arrow), inflammatory infiltration (cyan arrow), vacuolization (orange arrow), along with large and uncondensed nuclei (yellow arrow). Images were taken at 40× magnification (scale bar = 100 µm).

Figure 3. Long-term vs. short-term effects after DOX on heart metabolism and its regulation. The optical density of glucose transporter GLUT4 (GLUT4), phosphofructokinase (PFKM), electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO), ATP synthase subunit β (ATPB), sirtuin 3 (SIRT3), peroxisome proliferator-activated receptors α (PPARα) and γ (PPARγ), glycogen synthase kinase 3 β (GSK-3β), AMP-activated protein kinase (AMPK), and phosphorylated AMPK (pAMPK) are depicted along with the pAMPK-to-AMPK ratio. A representative image of the Western blot obtained is presented above each graph. For the complete blot obtained, see Supplementary Figure S1. Values are expressed as mean ± SD (n = 5–6). Statistical analyses were performed using unpaired two-sided t-test: * p < 0.05.

Figure 4. Long-term vs. short-term effects after DOX on heart redox/oxidative stress status. The optical density of protein carbonyl levels, nitrotyrosine, and manganese superoxide dismutase (MnSOD) are depicted. A representative image of the immuno-blot obtained is presented above each graph. For the complete blot obtained, see Supplementary Figure S1. Values are expressed as mean ± SD (n = 5–6). Statistical analyses were performed using unpaired two-sided t-test (p < 0.05).

Figure 5. Long-term vs. short-term effects after DOX on heart mitochondrial density and biogenesis. Citrate synthase (CS) activity was determined using a spectrophotometric assay. The optical density of peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α), mitochondrial transcription factor A (Tfam) and mitofusin1 (Mfn1) are depicted. A representative image of the Western blot obtained is presented above each graph. For the complete blot obtained, see Supplementary Figure S1. Values are expressed as mean ± SD (n = 5–6). Statistical analyses were performed using unpaired two-sided t-test: * p < 0.05, *** p < 0.001.

Figure 6. Long-term vs. short-term effects after DOX on heart autophagy and apoptosis. The optical density of Beclin1, autophagy protein 5 (ATG5), microtubule-associated protein 1 light chain 3 β (LC3B), Parkin, B-cell lymphoma-2 interacting protein 3 (BNIP3), B-cell lymphoma-2 associated X-protein (BAX), B-cell lymphoma-2 (BCL2), heat shock proteins 27 (HSP27) and 70 (HSP70) are depicted along with the BAX-to-BCL2 ratio. A representative image of the Western blot obtained is presented above each graph. For the complete blot obtained, see Supplementary Figure S1. Values are expressed as mean ± SD (n = 5–6). Statistical analyses were performed using unpaired two-sided t-test: * p < 0.05, ** p < 0.01.

Figure 7. Long-term vs. short-term effects after DOX on heart regeneration. The optical density of mast/stem cell growth factor receptor Kit (SCFR), CCAAT/enhancer-binding protein β (C/EBPβ) and Cbp/p300-interacting transactivator 4 (CITED4) are depicted. A representative image of the Western blot obtained is presented above each graph. For the complete blot obtained, see Supplementary Figure S1. Values are expressed as mean ± SD (n = 5–6). Statistical analyses were performed using unpaired two-sided t-test (p < 0.05).

Figure 8. Proposed schematic representation of the molecular pathways modulated by DOX at first and second time-points selected (one week vs. five months after DOX exposure). The modulation considering the first time-point, DOX1/CTRL1, is represented in white, while grey indicates the second time-point groups: DOX2/CTRL2. Proteins found increased or decreased are indicated by up or down arrows, respectively. The predisposition of proteins (0.05 < p-value < 0.1) to be increased or decreased is indicated by dashed arrows. At a short-term period, metabolism and apoptosis were impacted, whereas long-term after DOX exposure mainly decreased autophagy. For summarized changes over time of all biomarkers assessed, see Supplementary Table S1. Figure partly made with Servier medical art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license. 1,6BP: 1,6-bisphosphate, 6P: 6-phosphate, AMPK: AMP-activated protein kinase, ATG5: autophagy protein 5, ATPB: ATP synthase subunit β, BAX: B-cell lymphoma-2 associated X-protein, BCL2: B-cell lymphoma-2, BNIP3: BCL2 interacting protein 3, Casp: caspase, C/EBPβ: CCAAT/enhancer-binding protein β, CITED4: Cbp/p300-interacting…