Butyrate ameliorates doxorubicin-induced heart failure by inhibiting cardiomyocyte ferroptosis through the gut-heart axis

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  Figure 1. The gut microbiota plays a crucial role in modulating the DIHF (A) The schematic diagram shows the experimental design and timeline of the rat DIHF model. (B) Representative echocardiograms and quantitative analysis of LVEF (%), LVFS (%) in the DIHF-LS (n = 8), DIHF-HS (n = 9), Control (n = 6) groups. (C) ɑ-diversity (Chao index) of gut microbiota derived from the DIHF-LS, DIHF-HS, and control groups by 16S rRNA V3-V4 NGS. (D) Microbiota dysbiosis index (MDI) of gut microbiota derived from the DIHF-LS, DIHF-HS, and control groups. (E and F) Representative images (E), quantitative analysis (F) of EUB338 bacterial probe count in cardiac tissue of the DIHF-LS, DIHF-HS, and control groups via FISH (scale bars, 20 μm). (G) The Pearson’s correlation analysis of EUB338 probe count and LVEF decrease rate in the DIHF-LS and DIHF-HS groups. Statistical significance was determined using the simple linear regression of Correlation. (H) The schematic illustration for the experimental design and timeline of FMT from the DIHF-LS and DIHF-HS groups. (I) Representative echocardiograms and quantitative analysis of LVEF (%), LVFS (%) of FMT-DIHF-LS, FMT-DIHF-HS group (n = 8 per group) by…

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  Figure 2. The abundance of butyrate-producing bacteria and butyrate levels were significantly reduced, and lipid metabolism was dysregulated in the DIHF-HS rats (A) Principal component analysis (PCA) of fecal non-targeted metabolomics (LC-MS) profiling in the DIHF-HS and the DIHF-LS group. (B) A volcano plot shows differential abundance of fecal metabolites in the DIHF-HS and the DIHF-LS group (significant p < 0.05 and OPLS-DA VIP >1). (C) Serum butyrate concentration (μg/mL) was determined by colorimetric assay in the DIHF-LS, DIHF-HS, and control group. (D) Firmicutes/Bacteroidota ratio of the DIHF-LS, DIHF-HS, and control group. (E) KEGG enrichment analysis of the DIHF-HS group, compared with the DIHF-LS group. DIHF-HS group n = 9, DIHF-LS group n = 8, Control group n = 6. Error bars represent the mean ± SEM. For normally distributed data with homogeneous variances, statistical analyses were performed using one/two-way ANOVA for multiple comparisons. For non-normal/heteroscedastic data, the Wilcoxon rank-sum test was applied for two groups. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001.

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  Figure 3. NaB reduced cardiomyocyte toxicity in vitro and ameliorated DIHF in vivo (A) Cell viability of H9C2 cardiomyocytes co-cultured with NaB by CCK-8 detection in vitro (n = 8 per group). (B) Cell viability of H9C2 cardiomyocytes co-cultured with DOX by CCK-8 detection in vitro (n = 8 per group). (C) Cell viability of H9C2 cardiomyocytes co-cultured with DOX for 24 h, followed by NaB for 24 h, 48 h by CCK-8 detection in vitro (n = 8 per group). (D) Cell viability of AC16 human cardiomyocytes co-cultured with DOX for 24 h, followed with NaB for 24 h, 48 h by CCK-8 detection in vitro. (E) Representative images and quantitative analysis of reactive oxygen species (ROS) in H9C2 cardiomyocytes in the DOX+NaB, DOX, and control group by DCFH-DA fluorescent probe test (n = 8 per group) (scale bars, 50 μm). (F) Representative images and quantitative analysis of cell death of H9C2 cardiomyocytes in the DOX+NaB, DOX group by Propidium iodide (PI) staining (n = 8 per group) (scale bars, 100 μm). (G) The schematic illustration for the experimental design and timeline of the DOX+NaB and DOX+NaCl groups in the DIHF model. (H) Representative echocardiograms and quantitative analysis of LVEF (%), LVFS…

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  Figure 4. NaB reduced DOX-induced gut microbiota dysbiosis and lowered serum total cholesterol (TC) levels (A) ɑ-diversity (Shannon index) of gut microbiota in the DOX+NaB and DOX+NaCl groups. (B) PCA of gut microbiota in the DOX+NaB and DOX+NaCl groups on the genus level. (C) Venn diagram shows differences in gut microbiota composition on the genus level between the DOX+NaB, DOX+NaCl group. (D) Linear discriminant analysis (LDA) displays the differential gut microbiota in the DOX+NaB and DOX+NaCl groups (LDA score>2). (E) MDI of gut microbiota in the DOX+NaB, DOX+NaCl group. (F) PCA of serum metabolites in the DOX+NaB and DOX+NaCl groups by LC-MS profiling. (G) Volcano plot shows differential serum metabolites in the DOX+NaB, DOX+NaCl group. (H) Serum appearance and serum total cholesterol concentration in the DOX+NaB, DOX+NaCl group. DOX+NaB, DOX+NaCl group n = 8 per group. Error bars represent the mean ± SEM. For normally distributed data with homogeneous variances, statistical analyses were performed using Student’s t tests for two groups and two-way ANOVA for multiple comparisons. For non-normal/heteroscedastic data, the Wilcoxon rank-sum test was applied for two groups. ∗∗p < 0.01;…

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  Figure 5. NaB reduced bacterial translocation and LPS levels by enhancing the barrier function of the colonic and cardiac tissues (A) Histopathological damage of colonic tissue in the DOX+NaB, DOX +NaCl group by Hematoxylin and Eosin (H&E) staining (scale bars, 50 μm). (B and C) Representative images, quantitative analysis of Claudin-1 (B) and ZO-1 (C) of colonic tissue in the DOX+NaB, DOX+NaCl group by immunofluorescence (scale bars, 50 μm). (D) Representative images of goblet cell changes of colonic tissue in the DOX+NaB, DOX+NaCl group by AB-PAS staining (scale bars, 50 μm). (E) The mRNA expression levels of Muc2 and Tff3 in the colonic tissue via qRT-PCR assay in the DOX+NaB and DOX+NaCl groups. (F) Serum and cardiac tissue LPS concentrations in the DOX+NaB and DOX+NaCl groups via ELISA. (G and H) Representative images (G), quantitative analysis (H) of EUB338 bacterial probe count in cardiac tissue of the DOX+NaB and DOX+NaCl groups by FISH (scale bars, 20 μm). (I) The Pearson’s correlation analysis of EUB338 probe count and LVEF decrease rate in the DOX+NaB and DOX+NaCl groups. Statistical significance was determined using the simple linear regression of Correlation. (J)…

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  Figure 6. NaB significantly alters the cardiac bacteria composition, leading to bacterial function enhancement and Fe2+ level decrease in favor of inhibiting ferroptosis (A) Principal co-ordinates analysis (PCoA) of bacterial composition in cardiac tissue of the DOX+NaB and DOX+NaCl groups by 5R 16s rRNA sequencing. (B) LDA shows differential bacteria in cardiac tissue of the DOX+NaB and DOX+NaCl groups (LDA >3, p < 0.05). (C) Venn diagram shows differential bacterial composition in the cardiac tissue of the DOX+NaB and DOX+NaCl groups on the genus level. (D) Venn diagram shows shared bacteria between gut microbiota and cardiac tissue microbiota on the genus level. (E) The relative abundance of translocated gut microbiota within cardiac tissue in the DOX+NaB and DOX+NaCl groups. (F) PICRUSt2 PFAM analysis for the prediction of bacterial functions in cardiac tissue in the DOX+NaB and DOX+NaCl groups. (G) Representative images, quantitative analysis of Fe2+ levels in cardiac tissue in the DOX+NaB, DOX+NaCl group. DOX+NaB and DOX+NaCl groups (n = 8 per group) (scale bars, 10 μm). Error bars represent the mean ± SEM. For normally distributed data with homogeneous variances, statistical…

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  Figure 7. NaB reduced DOX-induced cardiomyocyte ferroptosis via the GPX4/GSH pathway (A) Representative images and quantitative analysis of GPX4 expression levels in H9C2 cardiomyocytes treated with DOX alone, or DOX combined with NaB (100, 250, 500 μg/mL), detected by Western blot assay (n = 3 independent experiments). (B) Representative images and quantitative analysis of GPX4 expression levels in H9C2 cardiomyocytes treated with Erastin alone or Erastin combined with NaB for 24 h and 48 h, detected by Western blot assay (n = 3 independent experiments). (C) Representative images and quantitative analysis of ROS in Erastin+NaB, Erastin, and control group via DCFH-DA fluorescent probe test (n = 8 per group). Statistical significance was determined using the One-way ANOVA test (scale bars, 100 μm). (D) Representative images and quantitative analysis of GPX4 expression levels of cardiac tissue in the DOX+NaB, DOX+NaCl group by Western blot assay. (E) GSH concentration in cardiac tissue of the DOX+NaB and DOX+NaCl groups. (F) MDA concentration in cardiac tissue of the DOX+NaB and DOX+NaCl groups. (G) Change of pro-inflammatory cytokines IL-1β, IL-6 in cardiac tissue of the DOX+NaB and…