Melatonin Mitigates Doxorubicin‐Induced Cardiotoxicity by Targeting the USP18/PKM2 Axis Without Compromising Antitumor Efficacy

Figures

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  FIGURE 1. Melatonin protects against chronic doxorubicin‐induced cardiomyopathy. (A) Chemical structure of melatonin. (B) Schematic diagram of the experimental protocol for the chronic DIC model. (C) Kaplan–Meier survival curves of mice treated with DOX or DOX+Mel. (D) HW/TL ratio. (E) Representative echocardiographic images. (F) Quantification of EF% and FS%. (G) Representative WGA staining and quantification of cardiomyocyte cross‐sectional area. (H) qPCR analysis of Bnp mRNA expression. (I) Representative Masson and quantification of fibrotic area. (J) qPCR analysis of Collagen III mRNA expression. (K) Representative DHE staining and quantification of oxidative stress. (L) qPCR analysis of Nox2 mRNA expression. (M) Serum levels of SOD and MDA. (N) Serum LDH levels.

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  FIGURE 2. Melatonin inhibits DOX‐induced myocardial injury via MT1 and MT2 receptors. (A) Representative WGA staining and quantification of cardiomyocyte area. (B) qPCR analysis of Bnp mRNA. (C) Masson's trichrome staining and fibrosis quantification. (D) qPCR analysis of Collagen III mRNA. (E) DHE staining and quantification of oxidative stress. (F) qPCR analysis of Nox2 mRNA. (G) Serum SOD and MDA levels. (H) Serum LDH levels.

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  FIGURE 3. USP18 is identified as a key downstream target mediating melatonin's cardioprotection. (A) Heatmap showing differentially expressed USP family genes in AC16 cells. (B) Volcano plot of USP genes (DOX vs. Saline). (C) Volcano plot of USP genes (DOX+Mel vs. DOX). (D, E) Western blot analysis of USP18 protein in AC16 and NRCMs after DOX treatment. (F) Immunofluorescence staining of USP18 (green) in NRCMs; nuclei stained with DAPI (blue). (G) qPCR analysis of USP18 mRNA in cardiomyocytes treated with DOX or Mel. (H) WGA staining and quantification of cardiomyocyte area in mice treated with DOX, DOX+Mel, or DOX+Mel+AAV9‐USP18. (I) qPCR of Bnp mRNA. (J) IF of Collagen III staining and quantification. (K) qPCR of α‐SMA mRNA. (L) DHE staining and quantification of oxidative stress. (M) qPCR of Nox2 mRNA. (N, O) Serum SOD and MDA levels. (P) Serum LDH levels.

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  FIGURE 4. Cardiomyocyte‐specific deletion of USP18 directly alleviates doxorubicin‐induced cardiotoxicity. (A) Survival curves of WT and cardiomyocyte‐specific Usp18 knockout mice treated with DOX. (B, C) qPCR analysis of Il‐1β and Nox2 mRNA. (D) Echocardiographic EF%. (E) H&E staining and quantification of vacuolization. (F) Masson's trichrome staining (upper) and Collagen III immunofluorescence (lower) with quantification. (G) WGA staining and quantification of cardiomyocyte area. (H) DHE staining and quantification of oxidative stress.

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  FIGURE 5. Disruption of the melatonin‐USP18 axis protects the heart without compromising antitumor efficacy in tumor‐bearing mice. Syngeneic melanoma‐bearing mice were assigned to four groups: Tumor only, Tumor+DOX, Tumor+DOX+Mel, and Tumor+DOX+Usp18 cKO. (A) Echocardiographic EF%. (B) Masson's trichrome staining and fibrosis quantification. (C) H&E staining for vacuolization score. (D) WGA staining and cardiomyocyte cross‐sectional area. (E) Tumor growth curves during the experimental period. (F–H) Tumor volume and weight at endpoint. (I) Immunohistochemical staining for PCNA and quantification.

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  FIGURE 6. STAT1 mediates DOX‐induced USP18 transcriptional upregulation. (A–C) DNA pull‑down assay with the biotinylated Usp18 promoter probe. Nuclear extracts from cardiomyocytes were incubated with control probe (non‑biotinylated) or Usp18 promoter probe. Protein‑DNA complexes were pulled down with streptavidin beads, resolved by SDS‑PAGE, and visualized by silver staining. The specific band in the Usp18 promoter probe lane (arrow) was excised and subjected to mass spectrometry (MS). MS identified STAT1 as the top candidate transcription factor binding to the Usp18 promoter. (D, E) Western blot analysis of p‐STAT1 and total STAT1 in cardiomyocytes after DOX treatment. (F) Western blot analysis of p‐STAT1 and total STAT1 in cardiomyocytes after DOX, Mel, and Luzindole treatment. (G) qPCR of STAT1 mRNA following STAT1 knockdown using three independent siRNAs (575, 945, and 2406). (H) qPCR of USP18 mRNA after STAT1 Knockdown. (I) qPCR of STAT1 mRNA following STAT1 overexpression. (J) qPCR of USP18 mRNA after STAT1 overexpression. (K) Western blot analysis of usp18 in cardiomyocytes after DOX, AGO, and si‐STAT1 treatment.

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  FIGURE 7. USP18 Interacts with PKM2 and Inhibits Its Ubiquitination. (A) Co‐IP was performed to detect the interaction between endogenous USP18 and PKM2 in AC16 cells. (B) Schematic diagram of molecular docking between USP18 and PKM2. (C) IF co‐localization assay was performed to detect the co‐localization of USP18 and PKM2 in NRCMs. (D) CHX chase assay was performed to detect the protein stability of PKM2 in cardiomyocytes with or without USP18 overexpression. (E, F) 293T cells were co‐transfected with Flag‐USP18 and Myc‐PKM2. Reciprocal co‐IP assays using anti‐Flag or anti‐Myc antibodies. (G, H) 293T cells were co‐transfected with Myc‐PKM2, HA‐Ubiquitin, and either Flag‐USP18 or shUSP18 to detect the effect of USP18 overexpression or knockdown on PKM2 ubiquitination. (I) AC16 cells were co‐transfected with Myc‐PKM2, HA‐Ubiquitin, and treated with Mel or Flag‐USP18 to detect the effect of USP18 overexpression on Mel‐induced PKM2 ubiquitination by WB. (J) Schematic diagram of USP18 truncation mutants. (K‐N) Co‐IP was performed in 293T and AC16 cells co‐transfected with Myc‐PKM2 and Flag‐tagged USP18 truncation mutants to map the PKM2‐binding domain on USP18.

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  FIGURE 8. USP18 Alters PKM2 Ubiquitination, Facilitating Nuclear Translocation, and Inhibiting Lysosomal Degradation. (A) Ubiquitination assay was performed in 293T cells co‐transfected with Myc‐PKM2, Flag‐USP18, and HA‐tagged ubiquitin mutants to detect the ubiquitin linkage types regulated by USP18. (B) Ubiquitination assay was performed in 293T cells transfected with K27R and K33R ubiquitin mutants to confirm the specific ubiquitin linkage sites targeted by USP18. (C) Cardiomyocytes were stimulated with DOX, Mel, and USP18‐overexpressing plasmids; cytoplasmic and nuclear fractions were isolated to examine PKM2 subcellular distribution. (D, E) Cardiomyocytes were transfected with K27 or K27R plasmids together with USP18 overexpression, and PKM2 levels in cytoplasmic and nuclear fractions were detected by Western blot (D) and immunofluorescence (E). (F, G) Immunofluorescence staining of LAMP1 and PKM2 was performed in cardiomyocytes with USP18 overexpression (F) or knockdown (G) to evaluate lysosomal marker expression and PKM2 nuclear translocation. (H) Cardiomyocytes were treated with MG132 or Baf‐A1, and PKM2 protein levels were detected by Western blot to determine the degradation…

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  FIGURE 9. PKM2 inhibition rescues the detrimental effects of USP18 overexpression, confirming PKM2 as a downstream effector of the melatonin‐USP18 axis. (A) WGA staining and quantification of cardiomyocyte area. (B) qPCR of BNP mRNA. (C) Masson's trichrome staining and fibrosis quantification. (D) qPCR of Collagen III mRNA. (E) DHE staining and oxidative stress quantification. (F) qPCR of Nox2. (G) Immunofluorescence for γH2AX and quantification. (H) Serum LDH levels. (I, J) Serum SOD and MDA levels. (K) Western blot analysis of NLRP3, GPX4, and FTH.