Hyperuricemia suppresses lumican, exacerbating adverse remodeling after myocardial infarction by promoting fibroblast phenotype transition

Figures

• 

  Fig. 1. Hyperuricemia aggravates adverse cardiac remodeling after MI. (A) Schedule for establishing hyperuricemia and MI in a mouse model. (B) Serum levels of uric acid (UA) were measured in potassium oxonate-treated mice subjected to MI or sham surgery on day 14 (n = 6). (C) Kaplan–Meier survival curves for each group of mice. (D) Representative M-mode images of heart tissue from mice obtained on day 14. (E, F) Quantification of the ejection fraction and fractional shortening measured via echocardiography on day 14 (n = 8). (G) Representative H&E images of cardiac tissues obtained from hyperuricemic mice on day 14 after MI or sham surgery. (H, I) Quantitative analysis of infarct size and wall thickness (n = 6). (J) Representative images of Masson’s trichrome-stained cardiac tissues obtained from hyperuricemic mice after MI or sham surgery on day 14. (K) Areas positive for Masson’s trichrome staining were quantified to determine the collagen density in the hearts of the mice in the indicated groups (n = 6). (L, M) Representative immunofluorescence images and quantitative analysis of TUNEL and nuclear 4’-6-diamidino-2-phenylindole (DAPI) in c-TnT-positive cardiomyocytes obtained from…

• 

  Fig. 2. Decreased lumican levels are associated with enhanced cardiac remodeling in hyperuricemia-related MI. (A) Volcano plots showing differentially expressed genes in patients with gout, patients with heart failure, and MI mice compared with controls through microarray datasets or single-cell RNA sequencing. (B, C) Heatmaps depicting DEGs between patients with gout and healthy individuals (B) and between patients with heart failure and healthy individuals (C). (D) Venn analysis of DEGs from patients with gout, heart failure, and MI mice compared with controls. (E) Lumican protein levels in the infarct area of hyperuricemic mice after MI surgery determined by western blotting on day 14. (F) Quantitative analysis of lumican expression (n = 6). (G) Lumican mRNA expression in the infarct area of hyperuricemic mice after MI surgery was assessed by qPCR on day 14 (n = 6). (H) Plasma levels of lumican were evaluated by ELISA (n = 10). (I) Immunofluorescence staining for lumican in the infarct area of hyperuricemic mice on day 14 after MI surgery (n = 6). The data are expressed as the means ± standard errors. Statistical analysis was performed via Student’s t test. *p < 0.05. DEG,…

• 

  Fig. 3. Lumican accelerates adverse cardiac remodeling by inhibiting TGFβ/Smad signaling. (A, B) KEGG enrichment analysis of DEGs between patients with heart failure and healthy individuals (A) and between patients with gout and healthy individuals (B), (C) Serum levels of active TGF-β1 detected by ELISA in hyperuricemic mice after MI surgery (n = 6). (D) Western blot analysis of p-Smad2, total Smad2, p-Smad3, total Smad3, and Smad4 in the infarcted area of hyperuricemic mice after MI surgery. (E, F) Quantitative analysis of p-SMAD2 and p-SMAD3 expression (n = 6). (G) α-SMA mRNA expression in the infarcted area of hyperuricemic mice after MI surgery was assessed via qPCR. (H) Western blot analysis of α-SMA, collagen 1, and fibronectin in the infarct area of hyperuricemic mice after MI surgery. (I, J) Immunofluorescence staining and quantification of α-SMA, collagen 1, and fibronectin levels in the infarct areas of hyperuricemic mice after MI surgery. The data are expressed as the means ± standard errors. Statistical analysis was performed via Student’s t test. *p < 0.05. DEG, differentially expressed gene; KEGG, Kyoto Encyclopedia of Genes and Genomes; MI, myocardial infarction

• 

  Fig. 4. Lumican is derived from cardiac myofibroblasts. (A) GO enrichment analysis of DEGs between MI and control mice. (B) UMAP of 11 main cell types in the infarct area after MI surgery at different time intervals. (C) Percentages of different cell types identified in the infarct area tissues. (D) UMAP of lumican expression differences in different cell types identified in the infarct area tissues. (E) Representative images of lumican (green), α-SMA (red), CD68 (purple), Ly6G (blue), CD31 (yellow), and c-TnT (dark blue) immunostaining in hyperuricemic murine hearts after MI surgery on day 14. (F) α-SMA, collagen 1, and fibronectin expression in uric acid-treated cells evaluated by western blotting in rat primary cardiac fibroblasts treated with uric acid (100 mg/L) for 24 h. (G, H) Representative immunostaining image and quantitative analyses of lumican (green) and α-SMA (red) in uric acid-treated rat primary cardiac fibroblasts. The data are expressed as the means ± standard errors. Statistical analysis was performed via Student’s t test. *p < 0.05. DEG, differentially expressed genes; GO, Gene Ontology; MI, myocardial infarction; UMAP, uniform manifold approximation and projection…

• 

  Fig. 5. Lumican from cardiac fibroblasts regulates fibrotic phenotype conversion. (A) UMAP plot of fibroblasts in the infarct area tissues after MI surgery on day 14, color-coded for 13 subclusters. (B) Violin plots showing the normalized expression of lumican in each subcluster. (C, D) GO and KEGG enrichment analyses of DEGs between fibroblasts with high (Lumicanhigh) and low (Lumicanlow) lumican expression. (E) Western blotting and quantitative analyses of α-SMA, collagen 1, and fibronectin expression in lumican-knockdown rat cardiac fibroblasts treated with 10 ng/mL TGFβ1 for 24 h. (H, I) Representative images of lumican (green), α-SMA (red, F), and collagen 1 (red, F) immunostaining in lumican-knockdown rat cardiac fibroblasts after TGFβ1 treatment. (J) α-SMA, collagen 1, and fibronectin expression in rat cardiac fibroblasts treated with 10 ng/mL TGFβ1 and 100 nM recombinant lumican for 24 h. (K) Representative image of α-SMA immunostaining in rat cardiac fibroblasts after treatment with TGF-β1 and recombinant lumican. The data are expressed as the means ± standard errors. Statistical analysis was performed via Student’s t test. *p < 0.05. DEG, differentially expressed gene; GO, Gene…

• 

  Fig. 6. Allopurinol ameliorates hyperuricemia-mediated adverse cardiac remodeling in MI mice. (A) Schedule for establishing hyperuricemia and MI in a mouse model with allopurinol treatment (125 mg/L). (B) Serum uric acid levels in hyperuricemic mice after MI surgery, with and without allopurinol treatment, on day 14 (n = 6). (C) Kaplan–Meier survival curves for each experimental group. (D) Representative M-mode images of mouse hearts obtained from each group on day 14. (E, F) Quantification of the ejection fraction and fractional shortening measured via echocardiography on day 14 (n = 8). (G, H) Representative H&E images and quantitative analysis of cardiac tissues obtained from hyperuricemic mice on day 14 after MI or sham surgery (n = 7). (I, J) Levels of c-TnT and CK-MB in mouse plasma as assessed by ELISA (n = 6). (K) Representative images of fluorescein isothiocyanate-conjugated WGA-stained cardiac tissues from hyperuricemic mice after MI with or without allopurinol treatment on day 14. (L) Representative immunofluorescence images of TUNEL (red) and nuclear DAPI (blue) in c-TnT-positive cardiomyocytes (green) obtained from hyperuricemic mice after MI with or without allopurinol…

• 

  Fig. 7. Schematic illustration of the regulatory mechanisms of the lumican/TGF-β pathway in hyperuricemia-mediated adverse cardiac remodeling