CCR2 deficiency protects against doxorubicin-induced cardiac dysfunction through enhanced IL12B-dependent autophagy

Figures

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  Figure 1. CCR2 upregulation in DIC and its protective role in acute cardiac injury. (A) CCR2 mRNA relative levels in mouse, rat, and pig hearts following DOX treatment based on publicly available datasets (GSE97643, n = 5; GSE218397, n = 6; GSE291450, n = 3). (B) Western blot results of CCR2 protein in mouse hearts following vehicle or DOX treatment (n = 9). (C) Quantitative PCR analysis of CCR2 mRNA levels in mouse hearts after vehicle or DOX administration (n = 10). (D) Flow cytometry results of CCR2+ cell populations in DOX-exposed hearts compared with control hearts (n = 5). (E) Representative M-mode echocardiograms of WT and CCR2⁻/⁻ mouse 7 days post vehicle or DOX injection. Scale bar: 0.1 s, 2 mm. (F, G) Quantitative analysis of EF and FS in WT and CCR2⁻/⁻ mouse following vehicle or DOX treatment (n = 10). (H–J) The levels of cardiac injury markers in serum, including cTnI, CK-MB, and LDH-L (n = 10).

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  Figure 2. Hematopoietic CCR2 is necessary and sufficient for DIC. (A) Schematic illustration of the BMT strategy: lethally irradiated WT recipients were reconstituted with either WT or CCR2-/- donor bone marrow. After an 8-week period for bone marrow reconstitution, mouse was subjected to the acute DIC model. Cardiac function was then evaluated, and tissues were collected. (B) Representative PCR genotyping of bone marrow cells from chimeric mouse confirming successful reconstitution. (C) Representative M-mode echocardiographic tracings. Scale bar: 2 mm, 0.1 s. (D–E) Quantitative analysis of EF and FS in irradiated WT mouse reconstituted with WT or CCR2-/- bone marrow treated with vehicle or DOX (n = 8). (F–H) The levels of cardiac injury markers in serum, including cTnI, CK-MB, and LDH-L (n = 8). (I) Strategy of reciprocal BMT experiment: CCR2-/- recipients were reconstituted with either WT or CCR2-/- donor bone marrow. (J) Representative PCR genotyping confirming successful reconstitution. (K) Representative M-mode echocardiographic tracings. Scale bar: 2 mm, 0.1 s. (L-M) Quantitative analysis of EF and FS in irradiated CCR2-/- mouse reconstituted with WT or CCR2-/- bone marrow treated…

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  Figure 3. CCR2 deficiency enhances cardiomyocyte autophagy in DIC. (A) Western blot results of autophagy markers ATG12, LC3B-II, P62, and GABARAPL1 in cardiac heart from WT and CCR2-/- mouse following vehicle or DOX treatment (n = 7). (B) Quantitative PCR analysis of autophagy genes Atg12, Atg5, Atg7, Atg3, and Gabarapl1 in cardiac heart from the same groups (n = 6). (C) Autophagic flux assessment using bafilomycin A1 pretreatment confirms increased autophagic flux in CCR2-/- hearts (n = 7). (D) Immunofluorescence staining and quantitative analysis of LC3B puncta in cardiac sections. Scale bar: 50 μm. (n = 6). (E) In vivo autophagic flux monitoring using an AAV9-delivered mRFP-GFP-LC3 reporter. Representative confocal images and quantification of autophagosomes (yellow puncta) and autolysosomes (red puncta). Scale bar: 10 μm. (n = 7). (F) Ultrastructural analysis by transmission electron microscopy. The arrows indicate characteristic autolysosomes containing electron dense material. Scale bar: 1 μm, 0.5 μm. (n = 6).

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  Figure 4. Autophagy dependency of CCR2-mediated cardioprotection in DIC. (A) Experimental design for autophagy inhibition studies. CCR2-/- mouse received daily injections of autophagy inhibitor 3-MA (15 mg/kg, i.p) and a single dose of DOX (15 mg/kg, i.p), and cardiac assessment performed after 7 days. (B) Western blot results demonstrating that 3-MA treatment effectively suppressed autophagy in CCR2-/- hearts (n = 7). (C–E) Representative echocardiographic images (Scale bar: 2 mm, 0.1 s) and quantitative results of EF and FS in CCR2-/- mouse treated with vehicle, DOX, 3-MA and DOX + 3-MA (n = 7). (F–H) The levels of cardiac injury markers in serum, including cTnI, CK-MB, and LDH-L (n = 7). (I) Experimental timeline for autophagy activation studies. (J) Western blot verification of successful autophagy induction by rapamycin in WT hearts (n = 7). (K–M) Representative echocardiographic images (Scale bar: 2 mm, 0.1 s) and quantitative results of EF and FS (n = 7) in WT mouse treated with vehicle, DOX, rapamycin and DOX + rapamycin (n = 7). (N‒P) The levels of cardiac injury markers in serum, including cTnI, CK-MB, and LDH-L (n = 7).

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  Figure 5. IL12B is upregulated in CCR2-deficient hearts and activates autophagy through PI3K/Akt/mTOR pathway inhibition. (A) Western blot results of IL12A and IL12B protein in heart from WT and CCR2-/- mouse (n = 6). (B) ELISA quantification of IL12A and IL12B levels in heart lysates from WT and CCR2-/- mouse (n = 6). (C) Western blot results of IL12B protein in BMDMs differentiated from WT and CCR2-/- mouse (n = 3 independent experiments). (D) Immunofluorescence co-staining of IL12B (green) and the macrophage marker CD68 (red) in heart sections from WT and CCR2-/- mouse. Scale bar: 5 μm. (E) Western blot results of autophagy markers (LC3B-II and P62) in H9C2 cells treated with recombinant IL12B protein (n = 3 independent experiments). (F) Quantitative PCR analysis of autophagy-related genes Atg12, Atg5, Atg7, Atg3, and Gabarapl1 in H9C2 cells following vehicle or IL12B treatment (2 ng/mL) (n = 3 independent experiments). (G) Western blot results of autophagy-related signaling pathway in H9C2 treated with vehicle or IL12B (2 ng/mL) (n = 3 independent experiments). (H) Western blot results of pPI3K, PI3K and autophagy markers (LC3B-II, ATG12, GABARAPL1, and P62) in H9C2 treated with…

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  Figure 6. IL12B promotes cardiomyocyte autophagy in vitro. (A) Cell viability assay of H9C2 treated with DOX (1 μM) in the presence or absence of recombinant IL12B proteins (2 ng/mL) (n = 3 independent experiments). (B) Western blot analysis of autophagy markers (LC3B-II, ATG12, GABARAPL1, and P62) in the same groups (n = 3 independent experiments). (C) Immunofluorescence staining and quantitative analysis of LC3B puncta in the same groups (n = 3 independent experiments). Scale bar: 20 μm. (D) Tandem fluorescence microscopy of H9C2 cells expressing the mRFP-GFP-LC3 reporter. Yellow puncta represent autophagosomes, and red puncta represent autolysosomes. Scale bar: 10 μm (n = 3 independent experiments). (E) Transmission electron microscopy of H9C2. Arrows indicate autolysosomes. Scale bar: 2 μm, 0.5 μm (n = 3 independent experiments).

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  Figure 7. Recombinant IL12B administration protects against DIC through autophagy activation in vivo. (A) Experimental design for in vivo IL12B administration. (B) Representative M-mode echocardiographic tracings of mouse from the vehicle, DOX alone, and DOX + IL12B groups. Scale bar: 0.1 s, 2 mm. (C, D) Quantitative analysis of EF and FS in the indicated groups (n = 6). (E–G) The levels of cardiac injury markers in serum, including LDH-L, CK-MB, and cTnI (n = 6). (H) Western blot analysis of autophagy markers (LC3B-II, ATG12, GABARAPL1, and P62) in heart tissues from the same groups (n = 6). (I) Quantitative PCR analysis of autophagy-related gene (Atg12, Atg5, Atg7, Atg3, and Gabarapl1) in heart tissues from the same groups (n = 6). (J) Immunofluorescence staining and quantitative analysis of LC3B puncta in cardiomyocytes from the same groups. Scale bar: 50 μm (n = 6). (K) Transmission electron microscopy analysis of autolysosome density from the same groups. Arrows indicate autolysosomes. Scale bars: 1 μm and 0.5 μm (n = 6).

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  Figure 8. IL12B neutralization abolishes CCR2 deficiency-mediated cardioprotection in vivo. (A) Experimental design of IL12B neutralization in CCR2-/- mouse. (B) Representative M-mode echocardiographic tracings of CCR2-/- mouse treated with vehicle, DOX alone, or DOX + anti-IL12B Neu. Scale bar: 0.1 s, 2 mm. (C, D) Quantitative results of EF and FS in the indicated groups (n = 6). (E–G) The levels of cardiac injury markers in serum, including LDH-L, CK-MB, and cTnI (n = 6). (H) Western blot results of autophagy markers (LC3B-II, ATG12, GABARAPL1, and P62) in heart tissues from CCR2-/- mouse treated with vehicle, DOX alone, or DOX + anti-IL12B Neu (n = 6). (I) Quantitative PCR analysis of autophagy-related gene (Atg12, Atg5, Atg7, Atg3, and Gabarapl1) in heart tissues from the same groups (n = 6). (J) Immunofluorescence staining and quantitative analysis of LC3B puncta in cardiomyocytes from the same groups. Scale bar: 50 μm (n = 6). (K) Transmission electron microscopy analysis of autolysosome density in cardiomyocytes from the same groups. Arrows indicate autolysosomes. Scale bars: 1 μm and 0.5 μm (n = 6).

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  Figure 9. Pharmacological CCR2 inhibition protects against DIC without compromising the antitumor efficacy of doxorubicin in 4T1 breast cancer model. (A) Cell viability assay of 4T1 breast cancer cells treated with DOX or in combination with CCR2 antagonist4 (n = 3 independent experiments). (B, C) Tumor growth curves and representative tumor images at the study endpoint in a syngeneic 4T1 breast cancer model. Scale bar: 1 cm (n = 6). (D) Representative M-mode echocardiographic tracings of tumor bearing mouse treated with vehicle, DOX alone, CCR2 antagonist4 alone or DOX + CCR2 antagonist4. Scale bar: 0.1 s, 2 mm. (E, F) Quantitative results of EF and FS in the same groups (n = 6). (G–I) The levels of cardiac injury markers in serum, including LDH-L, CK-MB, and cTnI (n = 6). (J) Western blot results of autophagy markers (LC3B-II, ATG12, GABARAPL1, and P62) in heart tissues from the same groups (n = 6). (K) Quantitative PCR analysis of autophagy-related gene (Atg12, Atg5, Atg7, Atg3, and Gabarapl1) in heart tissues from the same groups (n = 6). (L) Working model. DOX administration triggers cardiomyocyte dysfunction, ultimately leading to heart failure. In contrast, loss of function of CCR2…