HIF1α Attenuates Doxorubicin‐Induced Cardiotoxicity by Activating TEX264‐Associated ER‐phagy

Figure 1. HIF1α is dynamically regulated during DIC. A and B, AC16 cells were treated with increasing concentrations of doxorubicin (0, 0.2, 0.5, 1, 2 μM) for 48 h. (A) Representative Western blots and (B) quantitative analysis of HIF1α protein levels (n=4 per group). *P<0.05 vs 0 μM group. C and D, AC16 cells were treated with doxorubicin (0.5 μM) for the indicated time periods (0, 12, 24, 48, 72 h). (C) Representative Western blots and (D) quantitative analysis of HIF1α protein levels (n=6 per group). *P<0.05 vs 0 h group. E and F, Ventricular tissues were collected from mice at the indicated time points (1, 3, 5, 7, and 14 days) after doxorubicin injection. (E) Representative Western blots and (F) quantitative analysis of HIF1α protein levels (n=4 per group). *P<0.05 vs Day 0 group. Data are presented as mean±SEM. Statistical significance was determined by 1‐way ANOVA followed by Dunnett post hoc test. DIC indicates doxorubicin‐induced cardiotoxicity; and HIF1α, hypoxia‐inducible factor 1α.

Figure 2. HIF1α overexpression alleviates doxorubicin‐induced cardiotoxicity in vivo. A, Schematic diagram of the doxorubicin‐induced cardiotoxicity mouse model establishment and FG4592 intervention protocol. B, Representative images of M‐mode echocardiograms. C and D, Quantitative analysis of left ventricular ejection fraction (LVEF, C) and fractional shortening (FS, D) measured by echocardiography (n=6 per group). E, Representative images of hematoxylin and eosin staining of heart sections (scale bar: 1 mm; n=3–5 per group). F, heart weight/tibia length ratio (n=6 per group). G and H, Representative images of wheat germ agglutinin staining (G, scale bar: 20 μm) and quantitative analysis of cardiomyocyte cross‐sectional area (G) (n=6 per group). I and J, Representative images of TUNEL staining co‐stained with cardiac troponin T (I, scale bar: 20 μm) and quantitative analysis of TUNEL‐positive cardiomyocytes (J) (n=6 per group). K, Representative transmission electron micrographs of heart tissues, blue arrows indicate myofibril; black arrows indicate organized ER; red arrows indicate dilated ER; yellow arrows indicate ER‐phagy (scale bar: 500 nm). L through Q, Representative Western blots…

Figure 3. HIF1α overexpression alleviates doxorubicin‐induced cardiotoxicity in vitro. A, Schematic diagram of the in vitro experimental protocol. AC16 cells were treated with FG4592 (10 μM) for 4 h before exposure to doxorubicin (DOX, 1 μM) for 48 h. B and C, Representative images of TUNEL staining (B, scale bar: 20 μm) and quantitative analysis of TUNEL‐positive AC16 cells (C) (n=6 per group). D through I, Representative Western blots (D) and quantitative analysis of HIF1α (E), p‐JNK/JNK ratio (F), pro‐caspase 12 (G), CHOP (H), and LC3‐II (I) expression levels in AC16 cells. Protein expression was normalized to β‐actin (n=4 per group). J and K, Measurement of ER area. Representative images of ER labeled with mCherry‐ER‐3 plasmid (J, scale bar: 50 μm) and quantitative analysis of ER area per cell (K) (n=23–25 per group). L and M, ER‐phagy flux assay. Representative images of mCherry‐eGFP‐RAMP4 puncta in AC16 cells (L, scale bar: 50 μm) and quantification of ER‐phagy flux (M) expressed as the ratio of mCherry‐positive/eGFP‐negative RAMP4 puncta to mCherry‐positive/eGFP‐positive RAMP4 puncta per cell (n=23–26 per group). Data are presented as mean±SEM. Statistical significance was…

Figure 4. HIF1α deficiency exacerbates doxorubicin‐induced cardiotoxicity in vivo. A, Schematic diagram of the experimental protocol. Cardiomyocyte‐specific HIF1α knockout (HIF1α‐cKO) mice and their littermate controls (Hif1α fl/fl ) were subjected to doxorubicin treatment to establish the doxorubicin‐induced cardiotoxicity model. B, Representative images of M‐mode echocardiograms. C and D, Quantitative analysis of left ventricular ejection fraction (LVEF, C) and fractional shortening (FS, D) measured by echocardiography (n=6 per group). E, Representative images of H&E staining of heart sections (scale bar: 1 mm; n=3–5 per group). F, Heart weight/tibia length ratio (n=6 per group). G and H, Representative images of WGA staining (G, scale bar: 20 μm) and quantitative analysis of cardiomyocyte cross‐sectional area (n=6 per group). I and J, Representative images of TUNEL staining co‐stained with cardiac troponin T (cTnT) (I, scale bar: 20 μm) and quantitative analysis of TUNEL‐positive cardiomyocytes (J) (n=6 per group). K, Representative transmission electron micrographs of heart tissues, black arrows indicate organized ER; red arrows indicate dilated ER (scale bar: 500 nm; n=4 per group). L…

Figure 5. HIF1α knockdown promotes doxorubicin‐induced cardiotoxicity in vitro. A and B, Representative images of TUNEL staining (A, scale bar: 20 μm) and quantitative analysis of TUNEL‐positive AC16 cells (B) (n=6 per group). C through H, Representative Western blots (C) and quantitative analysis of HIF1α (D), p‐JNK/JNK ratio (E), pro‐caspase 12 (F), CHOP (G), and LC3‐II (H) expression levels in AC16 cells. Protein expression was normalized to β‐Actin (n=4 per group). I and J, Measurement of ER area. Representative images of ER labeled with mCherry‐ER‐3 plasmid (I, scale bar: 50 μm) and quantitative analysis of ER area per cell (J) (n=22–24 per group). K and L, ER‐phagy flux assay. Representative images of mCherry‐eGFP‐RAMP4 puncta in AC16 cells (K, scale bar: 50 μm) and quantification of ER‐phagy flux (L) expressed as the ratio of mCherry‐positive/eGFP‐negative RAMP4 puncta to mCherry‐positive/eGFP‐positive RAMP4 puncta per cell (n=25–27 per group). Data are presented as mean±SEM. Statistical significance was determined by 1‐way ANOVA followed by Tukey post hoc test. CHOP indicates C/EBP homologous protein; HIF1α, hypoxia‐inducible factor 1α; JNK, c‐Jun N‑terminal kinase; LC3‑II,…

Figure 6. HIF1α transcriptionally activates TEX264 expression. A, mRNA expression levels of ER‐phagy receptors in AC16 cells treated with doxorubicin alone or in combination with FG4592. The mRNA levels of candidate genes were measured by quantitative real‐time polymerase chain reaction (n=6 per group). B and C, Representative Western blots (B) and quantitative analysis (C) of TEX264 protein expression in heart tissues from the indicated groups (n=4 per group). D and E, Representative Western blots (D) and quantitative analysis (E) of TEX264 protein expression in AC16 cells from the indicated groups (n=4 per group). F, ChIP analysis of HIF1α binding to the TEX264 promoter in AC16 cells transfected with pcDNA3.1‐HIF1α (n=3 per group). G, Schematic diagrams of the putative HIF1α binding site and its mutation in the TEX264 promoter region. H, Dual‐luciferase reporter assay measuring the effect of HIF1α on wild‐type or mutant TEX264 promoter activity in AC16 cells (n=3 per group). Data are presented as mean±SEM. Statistical significance was determined by 1‐way ANOVA followed by Tukey post hoc test for (A, C, E) and unpaired 2‐tailed Student t‐test for (F, H). ATL3 indicates atlastin GTPase 3;…

Figure 7. TEX264 disruption abolishes the protective role of HIF1α in doxorubicin‐induced cardiotoxicity mice. A, Schematic diagram of the experimental protocol. Mice were injected with AAV‐NC or AAV‐Tex264 followed by doxorubicin and FG4592 treatment as indicated. B, Representative M‐mode echocardiograms. C and D, Quantitative analysis of left ventricular ejection fraction (C) and fractional shortening (D) (n=6 per group). E, Representative H&E staining of heart sections (scale bar: 1 mm; n=3–5 per group). F, Heart weight to tibia length (HW/TL) ratio (n=6 per group). G and H, Representative wheat germ agglutinin staining (F, scale bar: 20 μm) and quantification of cardiomyocyte cross‐sectional area (H) (n=6 per group). I and J, Representative TUNEL staining co‐stained with cardiac troponin T (I, scale bar: 20 μm) and quantification of TUNEL‐positive cardiomyocytes (J) (n=6 per group). K, Representative transmission electron micrographs of heart tissues, black arrows indicate organized ER; red arrows indicate dilated ER; yellow arrows indicate ER‐phagy (scale bar: 500 nm; n=4 per group). L through Q, Representative Western blots (L) and quantitative analysis of HIF1α (M), p‐JNK/JNK ratio…

Figure 8. TEX264 disruption abolishes the protective role of HIF1α in vitro. A and B, Representative images of TUNEL staining (A, scale bar: 20 μm) and quantitative analysis of TUNEL‐positive AC16 cells (B) (n=6 per group). C through H, Representative Western blots (C) and quantitative analysis of HIF1α (D), p‐JNK/JNK ratio (E), pro‐caspase 12 (F), CHOP (G), and LC3‐II (H) expression levels in AC16 cells. Protein expression was normalized to β‐actin (n=4 per group). I and J, Measurement of ER area. Representative images of ER labeled with mCherry‐ER‐3 plasmid (I, scale bar: 50 μm) and quantitative analysis of ER area per cell (J) (n=23–24 per group). K and L, ER‐phagy flux assay. Representative images of mCherry‐eGFP‐RAMP4 puncta in AC16 cells (K, scale bar: 50 μm) and quantification of ER‐phagy flux (L) expressed as the ratio of mCherry‐positive/eGFP‐negative RAMP4 puncta to mCherry‐positive/eGFP‐positive RAMP4 puncta per cell (n=23–24 per group). Data are presented as mean±SEM. Statistical significance was determined by 1‐way ANOVA followed by Tukey post hoc test. CHOP indicates C/EBP homologous protein; eGFP, enhanced green fluorescent protein; HIF1α, hypoxia‐inducible factor 1α; JNK,…