Cardiac Molecular Remodeling by Anticancer Drugs: Doxorubicin Affects More Metabolism While Mitoxantrone Impacts More Autophagy in Adult CD-1 Male Mice

Figures

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  Figure 1. Effect of DOX and MTX on heart structure assessed by light microscopy. Representative light micrographs obtained through hematoxylin and eosin staining from CTRL, DOX, and MTX mice are depicted (representative images of three animals analyzed per group). The CTRL group showed normal morphology and structure. DOX group presented cellular oedema (pink arrow), necrotic zones (green arrow), large and uncondensed nucleus (yellow arrow), vacuolization (red arrow), inflammatory infiltration (black arrow), and vascular congestion (blue arrow). MTX group showed cellular oedema (pink arrow), necrotic zones (green arrow), vacuolization (red arrow), inflammatory infiltration (black arrow), and vascular congestion (blue arrow). Images were taken at 40× magnification (scale bar = 100 µm). For additional quantitative data, please consult references [35] and [36] for DOX and MTX, respectively.

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  Figure 2. Effect of DOX and MTX on heart metabolism markers when assessed by Western blot in cardiac homogenates. The protein content displayed was normalized to total protein using Ponceau S staining. Representative images of the Western blot obtained are presented, and the Ponceau S staining for each protein is presented in Supplementary Figure S2. Values are expressed as mean ± SD (n = 5–6) of relative content, and statistical analyses were performed with one-way ANOVA followed by Tukey’s multiple comparisons test: * p < 0.05, ** p < 0.01, *** p < 0.001. GLUT4: glucose transporter GLUT4, PFKM: phosphofructokinase, GAPDH: glyceraldehyde-3-phosphate dehydrogenase, ETFDH: electron transfer flavoprotein-ubiquinone oxidoreductase, ATPB: ATP synthase subunit β.

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  Figure 3. Effect of DOX and MTX on the cardiac acylcarnitine profile assessed by MS/MS. Values are expressed as mean ± SD (n = 5–6) of concentration, and statistical analyses were performed with two-way ANOVA followed by Tukey’s multiple comparisons test: * p < 0.05, **** p < 0.0001. C0: carnitine, C2: acetylcarnitine, C4: butyrylcarnitine, C6: hexanoylcarnitine, C8: octanoylcarnitine, C10: decanoylcarnitine, C10:1: decenoylcarnitine, C10:2: decadienoylcarnitine C12: dodecanoylcarnitine, C12:1: dodecenoylcarnitine, C14: myristoylcarnitine, C14:1: tetradecenoylcarnitine, C14:2: tetradecadienoylcarnitine, C16: palmitoylcarnitine, C16:1: palmitoleoylcarnitine, C18: stearoylcarnitine, C18:1: oleoylcarnitine, C18:2: linoleylcarnitine.

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  Figure 4. Effect of DOX and MTX on the activity of cardiac enzymes involved in FA oxidation assessed through the ratios among acylcarnitine species: carnitine (C0), acetylcarnitine (C2), octanoylcarnitine (C8), palmitoylcarnitine (C16), stearoylcarnitine (C18) and oleoylcarnitine (C18:1). Values are expressed as mean ± SD (n = 5–6), and statistical analyses were performed with one-way ANOVA followed by Tukey’s multiple comparisons test: * p < 0.05, ** p < 0.01.

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  Figure 5. Effect of DOX and MTX on cardiac amino acids profile, when assessed by MS/MS. Values are expressed as mean ± SD (n = 5–6) of concentration, and statistical analyses were performed with one-way ANOVA followed by Tukey’s multiple comparisons test: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

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  Figure 6. Effect of DOX and MTX on heart metabolism regulation when markers were assessed by Western blot in cardiac homogenates. The protein content displayed was normalized to total protein using Ponceau S staining. Representative images of the Western blot obtained are presented, and the Ponceau S staining for each protein is presented in Supplementary Figure S2. Values are expressed as mean ± SD (n = 5–6) of relative content, and statistical analyses were performed with one-way ANOVA followed by Tukey’s multiple comparisons test: * p < 0.05, ** p < 0.01. AMPK: AMP-activated protein kinase, pAMPK: AMPK phosphorylated, PPARα: peroxisome proliferator-activated receptor α, PPARγ: peroxisome proliferator-activated receptor γ, GSK-3β: glycogen synthase kinase 3 β, SIRT3: sirtuin 3.

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  Figure 7. Effect of DOX and MTX on heart mitochondrial density and biogenesis when markers were assessed by a spectrophotometric assay (CS activity) and Western blot in cardiac homogenates. The protein content displayed was normalized to total protein using Ponceau S staining. Representative images of the Western blot obtained are presented, and the Ponceau S staining for each protein is presented in Supplementary Figure S2. Values are expressed as mean ± SD (n = 5–6) of activity or relative content, and statistical analyses were performed with one-way ANOVA followed by Tukey’s multiple comparisons test: * p < 0.05. CS: citrate synthase, Tfam: mitochondrial transcription factor A, PGC-1α: peroxisome proliferator-activated receptor γ coactivator 1 α, Mfn1: mitofusin1.

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  Figure 8. Effect of DOX and MTX on heart autophagy and apoptosis markers when assessed by Western blot in cardiac homogenates. The protein content displayed was normalized to total protein using Ponceau S staining. Representative images of the Western blot obtained are presented, and the Ponceau S staining for each protein is presented in Supplementary Figure S2. Values are expressed as mean ± SD (n = 5–6) of relative content, and statistical analyses were performed with one-way ANOVA followed by Tukey’s multiple comparisons test: * p < 0.05, ** p < 0.01. ATG5: autophagy protein 5, LC3B: microtubule-associated protein light chain 3, BNIP3: BCL2 interacting protein 3, BAX: BCL2 associated X-protein, BCL2: B-cell lymphoma-2, HSP27: heat shock protein 27 kDa, HSP70: heat shock protein 70 kDa.

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  Figure 9. Schematic representation of the molecular pathways modulated by DOX and MTX. Proteins searched in this study are indicated in green, while the ones in grey belong to the pathway but were not investigated. Proteins found upregulated (up arrows) or downregulated (down arrows) by DOX or MTX compared to CTRL are indicated with red or blue arrows, respectively. Figure was made with Servier medical art. ACAD: acyl-CoA dehydrogenases, AMP: adenosine monophosphate, AMPK: AMP-activated protein kinase, ATG: autophagy protein, ATP: adenosine triphosphate, ATPB: ATP synthase subunit β, BAX: B-cell lymphoma-2 associated X-protein, BCAA: branched-chain amino acids, BCL2: B-cell lymphoma-2, BNIP3: BCL2 interacting protein 3, C2: acetylcarnitine, C-I: nicotinamide adenine dinucleotide-ubiquinone oxidoreductase, C-II: succinate-ubiquinone oxidoreductase, C-III: ubiquinone-cytochrome c oxidoreductase, C-IV: cytochrome c oxidase, C-V: F1Fo ATP synthase, Casp: caspase, CoQ: ubiquinone, CPT: carnitine palmitoyl-transferase, CS: citrate synthase, Cyt c: cytochrome c, ETF: electron transfer flavoprotein, ETFDH: ETF-ubiquinone oxidoreductase, FA: fatty acids, FAD: flavin adenine dinucleotide, FADH2:…