ICG-001 Provides Cardioprotection Against Doxorubicin-Induced Cardiotoxicity and Enhances Cancer Cytotoxicity

Figures

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  Figure 1. Validation of CD36hi hiPSC-CM Model (A) Schematic of differentiation and CD36hi cardiomyocyte (CM) isolation. Human induced pluripotent stem cell (iPSC)-CMs were stained with mitotracker and BODIPY dyes, and anti-CD36 antibody. (B) Cells were gated as CD36hi and CD36lo. Isotype control in red, CD36 in blue. (C) Normalized fluorescence intensities of mitotracker and BODIPY staining, and forward scatter (FSC) and size scatter (SSC) measurements, n = 5, 4, 5, 5. (D-G) CD36hi CMs were treated with solvent control (CTRL), doxorubicin (DOX) (1 μmol/L, 26 hours), ± dexrazoxane (DRZ) (200 μmol/L, 1-hour pretreatment, followed by cotreatment with DOX). (D) Mitotracker dye staining. Percentage (%) of cells with elongated (asterisk), punctate (arrowhead), and perinuclear (arrow) mitochondria, n = 12. Significance is shown relative to DOX. (E) γH2AX staining, n = 3. (F) Redox potential measured using the PrestoBlue assay, n = 7. (G) % CMs with condensed nuclei, n = 4. Data are presented as mean ± SEM. Statistical significance was calculated using the 1- or 2-way analysis of variance with Tukey’s post hoc test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Scale bar = 20 μm.

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  Figure 2. ICG-001 Protects Against DOX-Induced Cardiotoxicity in CD36hi CMs CD36hi CMs were treated with solvent CTRL, DOX (1 μmol/L, 26 hours) ± ICG-001 (ICG, 1-hour pretreatment). (A) Mitotracker dye staining. Percentage (%) of cells with elongated (asterisk), punctate (arrowhead), and perinuclear (arrow) mitochondria. Significance is shown relative to DOX. n = 10. (B) Redox potential measured using the PrestoBlue assay, n = 11. (C and D) Graph and images of γH2AX staining n = 8. (E) α-actinin staining, n = 4 and (F) MLC2V staining, n = 4. Arrow indicates striations and asterisk indicates disorganized structure. (G) Normalized caspase 3/7 activity, n = 12. (H) Percentage (%) of cells with condensed nuclei, n = 6. Data are presented as mean ± SEM. Statistical significance was calculated using the 1- or 2-way analysis of variance with Tukey’s post hoc test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Scale bar = 20 μm. Abbreviations as in Figure 1.

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  Figure 3. ICG-001 Protects Against Daunorubicin-Induced Cardiotoxicity CD36hi CMs were treated with solvent control (CTRL), daunorubicin (DNX: 2 μmol/L, 26 hours) ± ICG-001 (ICG: 2 μmol/L) (A) Mitotracker dye staining, n = 5. (B) Redox potential measured using the PrestoBlue assay, normalized to control and presented as fold change (FC), n = 10. (C) Percentage (%) of cells with condensed nuclei, n = 3. Data are presented as mean ± SEM. Statistical significance was calculated using the 1-way analysis of variance with Tukey’s post hoc test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Scale bar = 10 μm.

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  Figure 4. ICG-001 Is Comparable to Dexrazoxane in Cardioprotection CD36hi CMs were treated with solvent CTRL, DOX (1 μmol/L, 26 hours), ± ICG-001 (ICG: 2 μmol/L), or DRZ (200 μmol/L). (A) Percentage (%) of CD36hi CMs with elongated, punctate and perinuclear mitochondria were quantified, n = 8. Significance is shown relative to DOX. (B) ATP assay normalized to control, n = 6. (C) Redox potential was measured using the PrestoBlue assay, normalized to control and presented as FC, n = 7. (D) The intensity of γH2AX staining, n = 3. (E) % cells with condensed nuclei, n = 6. Data are presented as mean ± SEM, and n refers to biological replicates. Statistical significance was calculated using the 1- or 2-way analysis of variance with Tukey’s post hoc test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Abbreviations as in Figures 1 and 3.

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  Figure 5. ICG-001 Protects CD36hi CMs by Inhibiting DRP1 and Preserving the Mitochondria CD36hi CMs were treated with DOX (1 μmol/L, 26 hours) ± ICG-001 (ICG: 2 μmol/L). Gene ontology analysis of differentially expressed genes between DOX vs ICG+DOX. (A) Cellular components and (B) biological processes and pathways. Enrichment is indicated by −log(p), where p refers to P value. Large −log10(p) implies significant enrichment. (C) Ingenuity Pathway Analysis showing upstream regulators (blue), which positively (triangles) or negatively (diamonds) regulate downstream targets (oval shapes) involved in metabolism (green), Ca2+ handling (pink), contraction (purple), and electrophysiology (yellow). (D) qPCR analysis of Wnt target genes. Expression was normalized to glyceraldehyde 3-phosphate dehydrogenase and to control, n = 3. (E) Quantification and representative images of DRP1 staining, normalized to control and presented as FC, n = 4. CD36hi CMs were treated with DOX ± mdivi-1 (MD). (F) Fluorescence images of mitotracker dye staining (green). The percentage (%) of cells with elongated (asterisk), punctate (arrowhead) and perinuclear (arrow) mitochondria were quantified, n=13. Significance is…

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  Figure 6. ICG-001 Can Protect Against DOX-Induced Cardiotoxicity In Vivo (A) Schematic of treatment. C57BL/6 mice were intraperitoneally injected with DOX ± pretreatment (1 hour) of ICG or DRZ, n = 4-7. (B) Change in body weight (BW) relative to Day 0. Significance was calculated based on %BW on day 28, n = 4-7. (C) Heart weight (HW) normalized to tibial length (TL). (D) Examination of cardiac pathology by H and E staining. Degeneration of the myocardial tissue (black arrow), sarcoplasmic vacuolation (yellow arrow), inflammatory cell infiltrations (red arrow). (E) Fibrosis was measured by Masson’s Trichrome staining and quantified using image analysis platform. Echocardiography examinations were performed on day 28. (F) Representative tracings. (G) Ejection fraction (EF). (H) Fractional shortening (FS). Left ventricular internal diameter (LVID) at the end of (I) systole (s) and (J) diastole (d). (K) qPCR analysis of Wnt target genes, normalized to glyceraldehyde 3-phosphate dehydrogenase and to control mice, n = 5. (L and M) Gene set enrichment analysis (GSEA) of RNA-sequencing data. (L) Top 5 biological processes and cellular components altered in DOX vs control and ICG-001+DOX vs DOX…

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  Figure 7. ICG-001 Suppresses Human Cancer Cells by Inhibiting the Wnt Signaling Pathway MV4-11 (A-E, H-J) and SKOV3 (F and G) cells were treated with solvent CTRL, DOX (MV4-11: 0.2 μmol/L and SKOV3: 1 μmol/L, 24 hours) ± ICG-001 (ICG: 2 μmol/L unless otherwise stated) ± DRZ (200 μmol/L) ± mdivi-1 (MD). (A-C, F, G, and J) Normalized viability measured using the PrestoBlue assay, presented as FC, n = 4-7. (D) %Trypan blue+ cells, n = 8. (E) Representative phase-contrast of MV4-11 cells after treatment. (H) qPCR analysis of Wnt target genes, normalized to glyceraldehyde 3-phosphate dehydrogenase and control, n = 6. (I) Western blot of DRP1, normalized to β-actin and control, n = 3. (J) The effect of mdivi-1 (MD) measured using the PrestoBlue assay, normalized to control, n = 4. Data are presented as mean ± SEM. Statistical significance was calculated using the 1-way analysis of variance with Tukey’s or Dunnett’s post hoc test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Significance is shown relative to CTRL (A and F) or DOX (B and G) or both (C and D, H-J). Abbreviations as in Figures 1 and 3.