Gut microbiota‐derived butyric acid regulates calcific aortic valve disease pathogenesis by modulating GAPDH lactylation and butyrylation

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  FIGURE 1. Aortic valve calcification is closely related to the gut microbiota. (A) Comparison of the degree of heart valve calcification in the difficult calcification and easy calcification groups of ApoE−/− mice after a high‐fat diet. (B) Flowchart of the dirty cage experiment and faecal microbiota transplantation. (C) The principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS‐DA) cluster analysis were used to group high‐fat diet‐fed mice (difficult calcification and easy calcification mice) on the basis of Von Kossa staining (IOD) of mouse aortic valves, valve thickness, flow velocity (mm/s) and transvalvular pressure difference (mmHg). (D–G) Neural network analysis was used to validate the results of PCA and OPLS‐DA cluster analyses. (H) Squirrel cages from the difficult calcification group (B group in Figure B) were utilized to feed other high‐fat‐fed ApoE−/− mice (Dirty group). ApoE−/− mice fed in clean cages were used as the control group (Normal group). (I) Faecal microbiota transplantation (FMT) was performed on other high‐fat‐fed ApoE−/− mice (C group in Figure B), the faeces collected from the difficult calcification group (A1 group…

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  FIGURE 2. Gut microbial sequencing identified Faecalibacterium prausnitzii (F. prausnitzii) as the key species in faeces that inhibits valve calcification. (A) Community structure analysis with statistical results of the number of species annotated to the phylum order family species level in each sample, in which the microbial species in the faeces of easy calcification (Easy) mice were significantly lower than those of difficult calcification (Diffct) mice. (B) Rank abundance (Abd) analysis showing differences in the abundance and homogeneity of the species contained in faeces samples of difficult calcification versus easy calcification mice. (C and D) Sample alpha diversity analysis. (E) Distance matrix after beta diversity analysis. (F) Taxonomic analysis via PCA. (G) Taxonomic analysis via NMDS. (H) Hierarchical clustering tree between samples. (I) Top 10 heatmaps of differential species abundance, Burkholderiales_bacterium (Burk bact) and Faecalibacterium prausnitzii (F. prausnitzii) detectable in the whole samples. (J) Multimetric correlation analysis between Burk bact strain abundance and valve calcification. (K) F. prausnitzii strain abundance and valve calcification multicriteria…

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  FIGURE 3. Metabolomic analysis of faeces and serum identified butyric acid as a key mediator of anticalcification in F. prausnitzii. (A–D) Cluster analysis of the metabolites PCA and OPLS‐DA in the faeces and serum of easy‐calcification and difficult‐calcification ApoE−/− mice (n = 24, 12 vs. 12). (E, F) Compositional analysis of the metabolites in the faeces (n = 24) and serum (n = 24). (G) 107 common differentially abundant metabolites were identified between the serum and faeces, of which 75 common differentially abundant metabolites were detected in all the samples. (H, I) Differences in the abundance of butyric acid in the faeces and serum of the mice. (J, K) Correlation analysis of the faecal and serum abundance of butyric acid with the abundance of F. prausnitzii. (L–N) Determination of butyric acid in the faeces, serum and heart of the easy calcification and difficult calcification ApoE−/− mice. (O) Determination of butyric acid in the serum of healthy and CAVD patients (n = 50). (P–R) Total ion flow and MS identification of the 13C‐labeled butyric acid detected in the faeces, serum and heart of the mice after oral gavage (BA: 13C‐labeled butyric acid oral administration group; HF:…

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  FIGURE 4. Butyric acid inhibited heart valve calcification through regulating glycolytic metabolism in hVICs. (A, B) Effects of butyric acid on calcium deposition in osteogenic medium (OM)‐induced calcific hVICs detected by alizarin red staining, scale bar: 100 μm. (C–F) Evaluation of calcification level in butyric acid‐treated human valve tissues ex vivo, including Von Kossa staining, and immunofluorescence staining of Runx2 and BMP2, scale bar: 500 μm, n = 5. (G–I) In‐cell western blot analysis of Runx2 and BMP2 in butyric acid‐treated hVICs under OM‐induced calcification, n = 3. (J–N) Evaluation of calcification level in butyric acid‐treated ApoE−/− mice after high‐fat‐feeding‐induced heart valve calcification, scale bar: 200 μm, n = 5. (O) PCA clustering analysis of butyric acid‐treated hVICs under OM‐induced calcification utilizing RNA‐seq, n = 3. (P) GSEA indicated that glycolysis metabolism was significantly different in hVICs under OM‐induced calcification between butyric acid‐treated and nonbutyric acid‐treated groups. (Q, R) Extracellular acidification rates (ECAR) and glycolysis levels in hVICs of the control, OM and OM + BA groups, n = 3. (S, T) PCA and OPLS‐DA clusting…

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  FIGURE 5. Butyric acid exerts antivalvular calcification effects by competitively inhibiting GAPDH lactylation. (A) In‐cell WB assay of global intracellular lactylation levels after treatment of osteogenic medium (OM)‐induced hVICs with butyric acid, n = 3. (B) The lactylation of GAPDH was most significantly inhibited by butyric acid, n = 3. (C) Sequencing analysis of lactylation levels of the glycolytic enzymes by butyric acid under OM induction, n = 3. (D, E) Histological analysis of hVICs subjected to OM induction revealed three significant lactylation sites in GAPDH. (F) Homology analysis of the three lactylated sites in different species. (G) Sequencing analysis of mutant plasmids of the three lactylated sites. (H) Alizarin red staining was used to analyze the role of three lactylated site mutations in regulating valve calcification, n = 5. Analysis of the role of three lactylated site mutations in regulating valve calcification via western blot analysis, n = 3 (I), in‐cell WB (J) and immunofluorescence staining (K) for Runx2 and BMP2 protein expression, scale bar: 200 μm, n = 5. (L) Immunoprecipitation analysis of butyric acid treatment affecting the butyrylation and lactylation…