Endoplasmic Reticulum Selective Autophagy Alleviates Anthracycline-Induced Cardiotoxicity

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  Figure 1. Monitoring Assay Systems for Endoplasmic Reticulum–Phagy in Cardiomyocytes (A) Western blotting was performed after indicated treatments on neonatal rat ventricular cardiomyocytes (NRVCs) expressing endoplasmic reticulum–phagy reporter proteins. The RFP-GFP band represents the total reporter protein, whereas the RFP band represents the digested fragment. (B) The same NRVCs prepared in Figure 1A were visualized on confocal microscopy. The intensity of digest RFP signals (arrows) were calculated. The scale bar represents 10 μm (n = 6, mean ± SEM, ∗∗P < 0.01, t-test). (C) Western blotting of homogenized heart or liver tissue of wild-type and Tg-tf-KDEL mice. (D) Western blotting of homogenized heart tissue of Tg-tf-KDEL mice. Mice were fed normally or starved (for 48 hours) before sacrifice (n = 3, mean ± SEM, ∗∗P < 0.01, t-test). (E) Frozen heart sections from the same mice in Figure 1D were visualized on confocal microscopy. RFP (red) puncta per 100 μm square are compared. The scale bar represents 10 μm (n = 6, mean ± SEM, ∗∗P < 0.01, t-test). EBSS = Earle’s balanced salt solution; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; RFP-GFP = red fluorescent protein - green…

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  Figure 2. Dox Strongly Induces Endoplasmic Reticulum–Phagy (A) Western blotting of tf-H9c2 cells treated with or without doxorubicin (Dox) (1 μmol/L for 24 h) (n = 4, mean ± SEM, ∗P < 0.05, t-test). (B) The same tf-H9c2 cells prepared in Figure 2A were visualized, and RFP intensity was calculated. The scale bar represents 10 μm (n = 6, mean ± SEM, ∗P < 0.05, t-test). (C) Live tf-H9c2 cells treated with Dox (1 μmol/L) were monitored on fluorescence microscopy. A set of time-lapse images is shown. The white arrow points to the same punctum, the color of which changes from yellow to red in 1 minute 3 seconds. The scale bar represents 5 μm. (D) Western blotting of homogenized heart tissue of Tg-tf-KDEL mice with indicated treatment (n = 3, mean ± SEM, ∗P < 0.05, t-test). (E) Frozen heart sections from saline- or Dox-treated Tg-tf-KDEL mice were visualized. RFP (red) puncta per 100 μm square were counted. The scale bar represents 10 μm (n = 7, mean ± SEM, ∗∗P < 0.01, t-test). Abbreviations as in Figure 1.

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  Figure 3. CCPG1 Regulated ER-Phagy Activity Induced by Dox Administration (A) Transcripts of H9c2 cells were collected after Dox administration (1 μmol/L for 24 hours) and the gene expressions of Ccpg1, Tex264, Fam134b, Sec62, and Atl3 are compared (n = 6, mean ± SEM, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, t-test). (B) The expressions of cell-cycle progression gene 1 (CCPG1) protein and transcripts from tf-H9c2 cells were compared after lentiviral transduction with indicated short hairpin RNA (shRNA) (n = 4, mean ± SEM, ∗∗∗P < 0.001, t-test). (C) Western blotting of tf-H9c2 cells, treated with Dox (1 μmol/L for 24 hours) after lentiviral transduction containing indicated shRNA (n = 3, mean ± SEM, ∗P < 0.05, t-test). (D) A schematic design for generating Tg-tf-KDEL-Ccpg1GT/GT mouse. (E) A set of representative confocal microscopic images of frozen heart sections from Dox-treated Tg-tf-KDEL and Tg-tf-KDEL-Ccpg1GT/GT. Only the merged images (GFP, RFP, and 4′,6-diamidino-2-phenylindole [DAPI]) are shown. RFP (red) puncta per 100 μm square are compared. The scale bar represents 10 μm (n = 4, mean ± SEM, ∗P < 0.05, t-test). Abbreviations as in Figures 1 and 2.

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  Figure 4. Ablation of CCPG1 Prompts ER Stress–Related Apoptosis With Dox Administration (A) The viability of H9c2 cells was assessed between 4 groups, which were treated as indicated (n = 6, mean ± SEM, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, analysis of variance with the Tukey post hoc test). (B) Terminal deoxy-nucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay was performed on H9c2 cells, which were treated with Dox (1 μmol/L for 24 hours) after the indicated lentiviral transduction. The rate of TUNEL-positive cells (arrows) is shown. The scale bar represents 100 μm (n = 3, mean ± SEM, ∗∗∗P < 0.001, t-test). (C) Lysates of H9c2 cells, prepared as in Figure 4B, were immunoblotted for the indicated antibodies. The protein expressions normalized to GAPDH or e-IF2α (for phosphorylated e-IF2α) are shown (n = 3 or 4, mean ± SEM, ∗P < 0.05, ∗∗P < 0.01, t-test). Abbreviations as in Figures 1 and 3.

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  Figure 5. CCPG1 Hypomorphic Mice Treated With Dox Demonstrated an Exacerbated Cardiomyopathic Phenotype (A) A representative image of echocardiography performed on wild-type and Ccpg1GT/GT mice at baseline and 8 weeks after the Dox injection. The scale bar represents 50 milliseconds. (B) Echocardiographic measurements and tissue weights, normalized to body weight, were compared (n = 8, mean ± SEM, ∗∗P < 0.01, t-test) (C) Representative MT-stained heart sections of Dox-treated wild-type and Ccpg1GT/GT mice are shown. The scale bar represents 500 μm and 50 μm (for upper and lower images, respectively). Fibrotic lesions, calculated as the blue area divided by the whole tissue area, are compared (n = 6, mean ± SEM, ∗∗P < 0.01, t-test). (D) Lysates of hearts from wild-type or Ccpg1GT/GT mice treated along with a high-dose Dox protocol were immunoblotted for the indicated antibodies (n = 4, mean ± SEM, ∗P < 0.05, t-test). BW = body weight; EF = ejection fraction; FS = fraction shortening; HW = heart weight; other abbreviations as in Figures 1 to 3.

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  Figure 6. TBK1 Physically Interacts With CCPG1 (A) Western blotting of tf-H9c2 cells transduced with CCPG1-harboring lentivirus or empty vector. (B) The tf-H9c2 cells, prepared in Figure 6A, were treated with Dox (1 μmol/L for 24 hours). The cell survival rate after Dox treatment was compared (n = 4, mean ± SEM, ∗P < 0.05, t-test) (C) Western blotting of tf-H9c2 cells prepared in Figure 6A. (D) Western blotting of HEK293 cells, which were transfected with plasmids coding GFP or GFP-CCPG1. (E) Lysates of HEK293 cells prepared in Figure 6D were immunoprecipitated with anti-GFP magnetic beads. Eluted samples from magnetic beads were immunoblotted for anti-CCPG1 antibody. (F) Pull-down assay. Recombinant TBK1, mixed with Myc or Myc-tagged CCPG1, was immunoprecipitated with Myc-tagged magnetic beads and subsequently immunoblotted for anti-TBK antibodies. An immunoblot result in triplicates is shown. For Figures 6A, 6C, 6D, and 6E, identical experiments were performed at least 3 times, and the representative images are shown. TBK1 = TANK-binding kinase 1; other abbreviations as in Figures 1 and 3.

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  Central Illustration. Endoplasmic Reticulum–Selective Autophagy Protects the Heart From Doxorubicin Toxicity The administration of doxorubicin (Dox) causes cardiotoxicity by inducing excessive endoplasmic reticulum (ER) stress, which results in a failure of the unfolded protein response in cardiomyocytes. Simultaneously, Dox administration increases the expression of cell-cycle progression gene 1 (CCPG1), one of the receptors of ER-phagy, provoking ER-phagy activation, which, in turn, mitigates cardiac dysfunction by suppressing excessive ER stress–induced cardiomyocyte apoptosis. TBK1 = TANK-binding kinase 1.