Initial Phase of Anthracycline Cardiotoxicity Involves Cardiac Fibroblasts Activation and Metabolic Switch

Figures

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  Figure 1. Cardiac function immediately after the completion of DOX treatment. (A) Assessment of diastolic function using Doppler measurements of transmitral inflow: peak velocities of the E wave and A wave, the E/A ratio, deceleration time of E wave (E wave Dec t), and isovolumetric relaxation time (IVRT). (B) The measurement of systolic function using ejection fractioning (EF) and fractional shortening (FS). * p < 0.05 vs. CTRL.

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  Figure 2. Myocardial fibrosis immediately after the completion of DOX treatment. (A) Myocardial expression of matrix metalloproteinase (MMP)-9 and MMP-2, Galectin-3, connective tissue growth factor (CTGF), and transforming growth factor-β (TGF-β) via Western blot. (B) Representative Western blot bands and Ponceau staining as loading condition. * p < 0.05 vs. CTRL. (C) Myocardial section of DOX-treated heart stained with α-smooth muscle actin (α-SMA, red), vimentin (green), and α-sarcomeric actin (α-SA, white); DAPI-labeled nuclei in blue; scale bar 20 μm. The uncropped blots are shown in Figures S1 and S2.

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  Figure 3. Phenotype of CFs from animals receiving a single DOX injection. (A) Protein expression analysis of Collagen I, phospho-SMAD 2-3, α-smooth muscle actin (α-SMA), and transforming growth factor-β (TGF-β) in N-CFs compared with DOX-CFs based on Western blot. (B) Representative Western blot bands. (C) Primary DOX-CFs stained with α-SMA (red) and vimentin (green) (left panel) or TGF-β (green) and Collagen I (red) (right panel); DAPI-labeled nuclei in blue; scale bars 20 μm (left panel), 40 µm (right panel). (D) Protein expression analysis of NADPH oxidase-2 (NOX-2) in N-CFs compared with DOX-CFs based on Western blot. (E) Representative Western blot bands. (F) Wound-healing assay. For each graph, data points indicate the number of biological replicates. * p < 0.05 vs. N-CFs. The uncropped blots are shown in Figures S3–S5.

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  Figure 4. Effect of DOX on primary CFs. (A) Growth curves of untreated naïve (N-CFs) and CFs exposed to DOX in vitro. (B–E) Protein expression analysis of phospho-SMAD 2-3, transforming growth factor-β (TGF-β), α-smooth muscle actin (α-SMA), and NADPH oxidase-2 (NOX-2) in CFs exposed to DOX in vitro (N-CFs+DOX) based on Western blot; representative Western blot bands. TGF-β as a positive control of myofibroblast induction (N-CFs+TGF-β). * p < 0.05 vs. N-CFs. For each graph, data points indicate the number of biological replicates. The uncropped blots are shown in Figures S5–S7.

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  Figure 5. Metabolic changes in CFs. (A) Basal oxygen consumption rate (OCR) and basal extracellular acidification rate (ECAR) in untreated and DOX-treated CFs. (B) OCR and ECAR profile by Mito stress test in N-CFs and N-CFs+DOX. (C) Energy phenotype profile of N-CFs and N-CFs+DOX: baseline phenotype indicated by open markers and stressed phenotype indicated by filled markers. (D) Basal OCR and ECAR in N-CFs vs. DOX-CFs. (E) Energetic phenotype shown as basal OCR/ECAR ratio. (F) Expression of pyruvate kinase (PKM2) and lactate dehydrogenase A (LDHA) in N-CFs and DOX-CFs. * p < 0.05 vs. N-CFs. Data points indicate the number of biological replicates (A,D,F). (G) Fluorescence intensity of intracellular glucose analog 2-NBDG in N-CFs and N-CFs+DOX.