Single-cell analysis reveals lysyl oxidase (Lox)+ fibroblast subset involved in cardiac fibrosis of diabetic mice

Abstract

Introduction: Myocardial fibrosis and cardiac dysfunction are the main characteristics of diabetic heart disease. However, the molecular mechanisms underlying diabetic myocardial fibrosis remain unclear.

Objectives: This study aimed to investigate the heterogeneity of cardiac fibroblasts in diabetic mice and its possible mechanism in the development of diabetic myocardial fibrosis.

Methods: We established a diabetic mouse model by injecting mice with streptozotocin. The overall cell profiles in diabetic hearts were analyzed using single-cell RNA transcriptomic techniques. Cardiac function was evaluated by echocardiography. Cardiac fibrosis was assessed by Masson's trichrome and Sirius red staining. Protein expression was analyzed using Western blotting and immunofluorescence staining.

Results: A total of 11,585 cells were captured in control (Ctrl) and diabetic (DM) hearts. Twelve cell types were identified in this study. The number of fibroblasts was significantly higher in the DM hearts than in the Ctrl group. The fibroblasts were further re-clustered into nine subsets. Interestingly, cluster 4 fibroblasts were significantly increased in diabetic hearts compared with other fibroblast clusters. Lysyl oxidase (Lox) was highly expressed in DM fibroblasts (especially in cluster 4). Beta-aminopropionitrile, a Lox inhibitor, inhibited collagen expression and alleviated cardiac dysfunction in the diabetic group. Lysyl oxidase inhibition also reduced high glucose-induced collagen protein upregulation in primary fibroblasts. Moreover, a TGF-β receptor inhibitor not only prevented an increase in Lox and Col I but also inhibited the phosphorylation of Smad2/3 in fibroblasts.

Conclusions: This study revealed the heterogeneity of cardiac fibroblasts in diabetic mice for the first time. Fibroblasts with high expression of Lox (cluster 4 fibroblasts) were identified to play a crucial role in fibrosis in diabetic heart disease. The findings of this study may provide a possible therapeutic target for interstitial fibrosis.

Keywords: Single-cell RNA sequencing; diabetic cardiomyopathy; fibroblasts; lysyl oxidase.

Graphical abstract

Fig. 1

Single-cell RNA sequencing showing cell profiles in DM mouse hearts. (A) Workflow for single-cell RNA transcriptome experiments. (B) t-SNE plot showing distinction of overall cell profiles between Ctrl (n = 4, shown in red) and DM (n = 4, shown in blue) groups. (C-D) t-SNE showing 20 cell clusters (C) and 12 cell types (D) in the integrated single-cell transcriptomes acquired from the Ctrl and DM groups. (E) Bar graph representing the frequency of cells acquired in each cluster. (F) Dot plot showing cell-specific markers. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Enrichment analysis of differently expressed genes in DM hearts. (A) Gene Set Enrichment Analysis of differently expressed genes between Ctrl and DM hearts from a single-cell RNA sequencing. (B) Top 10 GO enrichment pathways for up-regulated (red bars) and down-regulated (blue bars) genes. (C) Circos plot showing ligand-receptor interactions among various cell types. The thickness of ribbons is proportional to the number of interactions between two cells. (D) Fibroblasts were highlighted in orange color according to the expression of cell-specific marker (Col1a1, Col1a2, and Dcn). (E) Volcano plot showing up-regulated (red) and down-regulated (blue) differently expressed genes in DM hearts compared with Ctrl hearts. (F) Top 20 GO enrichment pathways of differently expressed fibroblasts genes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Re-clustering of fibroblasts and up-regulation of Lox in cluster 4 of fibroblasts in DM hearts. (A) Fibroblasts were re-clustered into nine subsets. (B) Specific markers used for fibroblast re-clustering. (C) Heatmaps showing distinct expression profiles of each fibroblast subset. (D) Bar graph representing the number of fibroblasts in each subset. (E-F) Top eight GO enrichment pathways for up-regulated (red bars) and down-regulated (blue bars) genes in cluster 2 (E) and cluster 4 (F). (G) t-SNE plot showing the difference in Lox mRNA expression between the Ctrl and DM groups. (H-I) Violin plot showing highly expressed Lox mRNA in fibroblasts (H), especially in cluster 4 (I). (J) Representative immunofluorescence image of Lox protein (red) in the Ctrl and DM hearts, stained with Col Ⅰ (green, fibroblast marker) and phalloidin (grey); scale bar = 10 µm. (K) Validation of Lox mRNA expression using qRT-PCR analysis (n = 6, **p < 0.001 vs Ctrl group). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Inhibition of Lox alleviated STZ-induced myocardial dysfunction. (A) Workflow of BAPN (an inhibitor of Lox) treatment in DM mice. (B-E) Effect of BAPN on body weight and glucose level in DM mice (**p < 0.01, DM vs Ctrl group). (F) Representative echocardiography M−mode images of mice hearts at 8 and 14 weeks after STZ injection. (G-H) Statistical analysis of echocardiogram. n = 7–10, **p < 0.01, *p < 0.05. (I-J) Measurement of serum levels of CK and LDH, n = 4, **p < 0.01, *p < 0.05. (K) Transmission electron microscopic analysis of hearts tissue at 14 weeks after STZ injunction. The Ctrl group image shows a well-organized structure and complete mitochondria. The DM group image shows damaged and ruptured mitochondria (red arrowheads). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Inhibition of Lox decreased collagen fibril production in DM hearts. (A) Representative immunofluorescence staining image displaying expression of Lox (red) stained with Col Ⅰ (green, fibroblast marker) and phalloidin (grey); scale bar = 10 µm. (B) Measurement of Lox activity of heart homogenates (n = 8, **p < 0.01). (C-D) Quantification of hydroxyproline (HYP) in heart tissues (n = 5) and serum (n = 8) (*p < 0.05, **p < 0.01). (E) Protein levels of Col Ⅰ and III by Western blotting. (F-G) Quantification of Col Ⅰ and III expression (n = 3, *p < 0.05, **p < 0.01). (H-K) Representative Masson and Sirius red staining images. In the Masson staining (H), the collagen area is blue, and the collagen area is red in the Sirius red staining (J). Quantification of results from Masson (I) and Sirius red staining (K) (n = 9, **p < 0.01). (L-P) Scatterplots showing correlation of Lox expression with HYP content, content of Col Ⅰ, content of Col III, and fibrosis area from the Masson and Sirius red staining. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Inhibition of Lox declined HG-induced collagen protein expression in primary fibroblasts.(A) Workflow of primary fibroblast culture. (B) Immunofluorescence staining showing the expression of Lox protein (Red), stained with Vimentin (green) and DAPI (blue); scale bar = 10 µm. (C) Western blot image showing the expression of Lox in fibroblasts. (D) Statistical analysis of Western blot results for Lox protein expression (n = 5, **p < 0.01). (E) Measurement of Lox activity in fibroblasts (n = 8, **p < 0.01). (F) Western blot image showing the expression of Col Ⅰ and III in the fibroblasts. (G-H) Quantification analysis of the expression of Col Ⅰ and III in fibroblasts (n = 5, *p < 0.05, **p < 0.01). (I-J) Scatterplots showing the correlation of Lox expression with Col Ⅰ or Col III protein content. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

HG induced Lox upregulation via TGF-β1 signaling pathway activation in fibroblasts. (A) Western blotting for Lox and Col Ⅰ protein. (B-C) Quantification analysis of Lox and Col Ⅰ protein (n = 4, *p < 0.05, **p < 0.01). (D) Western blotting for p-Smad3, Smad3, p-Smad2, and Smad2. (E-F) Statistical analysis of ratios of p-Smad3/smad3 and p-Smad2/smad2 (n = 4, **p < 0.01). (G) Western blotting for c-jun and p-c-jun. (H) Quantification analysis of p-c-jun/c-jun ratio (n = 4, *p < 0.05, **p < 0.01). (I-L) Representative immunofluorescence staining illustrated the expression of Lox, p-Smad3, p-Smad2, and p-c-jun (red) stained with phalloidin (green), vimentin (pink, I and J), Col Ⅰ (pink, K and L), and DAPI (blue); scale bar = 10 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)