Integrated multi-omics reveals acylcarnitine accumulation as an early driver of doxorubicin-induced cardiotoxicity targeted by chlorogenic acid

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  FIGURE 1. Transcriptomics reveals early metabolic disturbances in doxorubicin (Dox)-induced cardiotoxicity. (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs; (B) Heatmap of DEGs associated with the PPAR signaling pathway, Fatty acid oxidation, Citrate cycle, Oxidative phosphorylation, Glycolysis/Gluconeogenesis in Dox-induced cardiac injury, based on data from GSE233644; (C-H) Results of Gene Set Enrichment Analysis (GSEA) of Glutathione metabolism (C), fatty acid oxidation (D), Citrate cycle (E), Oxidative phosphorylation (F), Glycolysis/Gluconeogenesis (G) and PPAR signaling pathway (H). (I) Relative mRNA expression levels of PPARs (Ppara, Ppard, Pparg), Ppargc1a and Ppargc1b (upstream regulators of fatty acid metabolism) relative to controls; (J) Relative mRNA expression levels of genes associated with fatty acid oxidation. FPKM (fragments per kilobase of transcript per million fragments mapped reads) indicates the normalized RNA-seq expression values. Data are presented as mean ± SEM (n = 3). Statistical significance between two groups was determined using an unpaired Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

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  FIGURE 2. Lipidomic profiling uncovers perturbed sphingolipid metabolism and acylcarnitine accumulation in doxorubicin-induced cardiotoxicity. (A) Relative abundance of major lipid subclasses; (B) Changes in total lipid content; (C-F) Alterations in acylcarnitine (CAR, (C), free fatty acid (FFA, (D), monoacylglycerol (MG, (E) and diacylglycerol (DG, (F). (G) Lipid set enrichment analysis (MSEALipid) of differentially abundant lipids in the DOX group compared with Con group. Significant enrichment was observed for Sphingolipid (P < 0.001) and fatty acyls (P < 0.001); Bonferroni-corrected P values were calculated using the QEA module of MetaboAnalyst based on the globaltest algorithm and a generalized linear model to estimate the Q-stat for each lipid set. (H) Composition of Lipid subclasses among differentially abundant lipids in DOX group, relative to Con group. Each color represents a distinct lipid subclass, and the area of each colored segment indicates its relative proportion. (I) Heatmap of differential abundant lipids (P < 0.05, n = 3, Student’s test). Colors represent normalized lipid signal intensities, highlighting lipids increased or decreased in the DOX group, relative to the…

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  FIGURE 3. Mass spectrometry imaging analysis of metabolic patterns in doxorubicin (Dox)-induced cardiotoxicity. A,B. MSEAlipid of differentially abundant lipids in DOX group and Con group based on MSI data. (A) MSEAlipid of upregulated metabolites in CON group. (B) MSEAlipid of upregulated metabolites in DOX group. (C) Hematoxylin and Eosin (HE) staining and representative MSI of spatial distributions of lipid metabolites in mice heart of CON and DOX group. (D) Relative abundance of carnitine, acetylcarnitine and acylcarnitines in DOX group, relative to Con group. Data are presented as mean ± SEM (n = 3). Statistical significance between two groups was evaluated using an unpaired Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

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  FIGURE 4. Overlaping target genes between CGA and Cardiovascular disease (CD) and the drug-compound-target-disease network. (A) Venn diagram of intersection targets of CGA and CD. Blue represents the targets of CGA and yellow represents the targets of CD. There were 98 intersecting targets. (B) Network analysis of shared core targets between CGA and CD; (C) PPI network of hub protein related to CAG and CD; (D) The integrated drug-target-disease network analysis of CGA in relation to CD was visualized by Cytoscape. (E) Relative mRNA expression levels of selected core hub targets in H9c2 cells across different treatments. Data are presented as mean ± SEM (n = 3). Statistical significance among multiple groups was analyzed using one-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, P < 0.01, *P < 0.001 vs. Control; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. DOX.

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  FIGURE 5. CGA attenuated DOX-induced cardiac injury in mice by reducing apoptosis and oxidative stress. (A) Changes in mice body weight. CGA or ddH2O was administered via oral gavage starting on day 0. Ten days later, a single intraperitoneal injection of doxorubicin (15 mg/kg) or saline was given. Body weight declined significantly after doxorubicin injection, whereas CGA attenuated the weight loss. (B) Representative M-mode echocardiography images of mice in different groups. (C,D) Echocardiographic evaluation of Left Ventricular Ejection Fraction (C) and Fractional Shortening (D). (E–H) Serum levels of CK (E), CKMB (F), LDH (G), and LDH1 (H). (I) Representative fluorescence images of 4-HNE (upper panel) and TUNEL (lower panel) staining in cardiac tissues. (J,K) Quantification of the percentage of 4-HNE-positive cells (J) and TUNEL-positive cells (K). (L) Western blot analysis of Caspase 3, c-Caspase 3, and Bax protein expression in cardiac tissues following doxorubicin injection, with or without oral CGA treatment. (M–P) Quantification of Caspase 3 (M), cleaved Caspase 3 (N), the ratio of cleaved Caspase-3 to total Caspase-3 (O), and Bax (P) protein levels from replicate Western blots,…

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  FIGURE 6. CGA reduced acylcarnitine accumulation and alleviated oxidative stress in doxorubicin-induced cardiac injury. (A) The effect of different concentration of CGA on cell viability. Low-dose CGA showed no significant cytotoxicity, while high-dose CGA impaired cell viability. (B) Treatment with 100 μM CGA significantly enhanced the viability of H9C2 cells exposed to doxorubicin. (C) Representative images of DCFH-DA and MitoSOX staining. DCFH-DA was used to assess overall cellular ROS, while MitoSOX specifically detected mitochondrial ROS. (D) Quantification of fluorescence intensity and positive cell percentage for DCFH-DA and MitoSOX staining. (E) MSI analysis showed that CGA reduced doxorubicin-induced acylcarnitine accumulation. Representative H&E staining and MSI images display the spatial distribution of acylcarnitines, carnitine and acetylcarnitine in mice heart. (F) Comparison of carnitine, acetylcarnitine, and acylcarnitine levels between DOX and CGA + DOX groups, expressed relative to the control group. (G) Relative mRNA expression levels of genes associated with PPARs. (H) Western bolt analysis of = PPARs protein expression in cardiac tissue following doxorubicin injection,…

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  FIGURE 7. Pharmacological blockade of the PPARα-FAO axis and exogenous acylcarnitine accumulation abolish the cardioprotective and metabolic effects of CGA. (A) Representative images of mitochondrial ROS production detected by MitoSOX (red) and nuclei stained by Hoechst 33,342 (blue) in H9c2 cells. Cells were treated with DOX (5 μM), CGA (100 μM), the CPT1 inhibitor Etomoxir (Eto, 40 μM), and Palmitoylcarnitine (Pal-c, 25 μM) as indicated. (B) Representative fluorescence images of JC-1 staining for assessing mitochondrial membrane potential (ΔΨm). Red fluorescence represents JC-1 aggregates (normal ΔΨm), while green fluorescence indicates JC-1 monomers (depolarized ΔΨm). (C) Cell viability across different treatment groups determined by CCK-8 assay. (D) Quantification of normalized MitoSOX fluorescence intensity. (E-F) Quantification of normalized JC-1 monomers (E) and JC-1 aggregates (F). (G) Absolute acylcarnitine content (mg/mL) in H9c2 cells under different treatment conditions including the specific PPARα antagonist GW6471. Acylcarnitine levels were calculated as the difference between total carnitine and free carnitine concentrations. (H) Direct measurement of the Fatty Acid…