Histaminylation alters collagen matrix mechanics and attenuates cardiac fibrosis post-myocardial infarction via mechanotransduction signaling axis

Figures

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  Fig. 1. The detection and functional analysis of histaminylation in type I collagen in cardiac tissue after AMI. a LC-MS/MS analysis of histaminylated type I collagen showed multiple histaminylation modifications on Gln(Q) residues. b Schematic model illustrating that histaminylation may inhibit TGM2-mediated collagen crosslinking by occupying Gln residues. c TEM images of cardiac collagen fibers from WT and Hdc−/− mice on day 7 post-AMI (scale bars: left, 2 μm; right, 500 nm), and quantification analysis showing no significant difference in the diameter of individual collagen fibrils between WT and Hdc−/− mice (randomly selected fibrils, n ≥ 30). d Both WT and Hdc−/− mice exhibited well-defined D-periods in collagen fibrils (scale bar: 200 nm). e The gap region of the D-periods was significantly shortened in collagen fibrils from Hdc−/− mice compared to WT mice, while the overlap region remained unchanged (randomly selected, n = 50; scale bar: 200 nm). f Representative and quantitative analysis of indentation curves showing significantly increased Young’s modulus in Hdc−/− infarcted myocardium scars (34.14 kPa) compared to WT controls (18.79 kPa) (n = 9). g AQMC analysis revealed a…

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  Fig. 2. In vitro reconstruction and analysis of the TGM2-crosslinked collagen matrix. a Schematic illustration of collagen matrix construction. b Representative image of the collagen matrix crosslinked by TGM2. c SEM images of the ultrastructure of collagen fibrils (scale bars, left, 10 μm, right, 2 μm). d Quantification of alignment dimension after crosslinking (randomly selected, n = 50). e Degradation curves of collagen matrices digested with 0.08% collagenase I (n = 3). f LC-MS/MS detection of HC revealed multiple histaminylation modification sites. g AQMC quantification analysis of collagen crosslinking. HC showed significantly decreased γ-Glu-ε-Lys levels (n = 3). h Representative indentation and stress-relaxation curves of NC (Young’s modulus = 17.19 Pa). i Representative indentation and stress-relaxation curves of UC (Young’s modulus = 84.67 Pa). j Representative indentation and stress-relaxation curves of HC (Young’s modulus = 50.19 Pa). k Quantification of Young’s modulus of NC, UC and HC (n ≥ 16). l Representative fitted stress-relaxation curves of UC and HC. m Statistical comparison of relaxation time constants (τ0.8) and residual force of UC and HC (n ≥ 16). For all…

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  Fig. 3. Fibroblasts cultured on UC and HC matrices under cyclic stretching combined with TGF-β stimulation exhibited marked differences in FMT and FA formation. a Representative and quantification of immunofluorescence staining of α-SMA and phospho-SMAD2 in NMCFs subjected to cyclic stretch on different matrices (n = 5) (Ctrl: uncoated soft-bottom plates, Young’s modulus = 1 kPa); scale bar, 50 µm. b Western blot analysis of the myofibroblast markers α-SMA and phospho-SMAD2 in NMCFs on different collagen matrices under stretch conditions (n = 4). c qRT-PCR data showing the expression of myofibroblast marker genes in NMCFs plated on UC and HC matrices (n = 6). d Representative and quantification of immunofluorescence staining of FA length (randomly selected, n ≥ 20) and intensity (n = 4) in NMCFs cultured on UC and HC matrices (scale bars, left 30 µm, right 10 µm). e SEM images showing the interaction between NMCFs and collagen matrices in the UC and HC groups (scale bars, left 2 µm, right 500 nm). f Quantification analysis of collagen alignment dimension of SEM images (n ≥ 20). g qRT-PCR data showing the expression of myofibroblast marker genes in human CF cell lines plated on UC and HC…

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  Fig. 4. RNA-seq and experimental validation suggest an association between FMT and PIEZO1 signaling. a KEGG enrichment analysis of differentially expressed genes (DEGs) between Hdc−/− and WT mouse hearts on day 7 post-AMI showed enrichment in focal adhesion and ECM-receptor interaction pathways. b Top 10 enriched GO pathways based on DEGs between Hdc−/− and WT mouse hearts. c Enriched pathways related to ECM and cell adhesion in further GO analysis. d Volcano plot highlighting DEGs from the GO cell-matrix gene set. e Expression profiles of integrins, FA markers, and Piezo1 in Hdc−/− and WT mouse hearts on day 7 post-AMI demonstrated the upregulation of multiple integrins in the Hdc−/− group. f Protein‒protein interaction prediction analysis of PIEZO1 and integrins by the GeneMANIA (https://genemania.org/) dataset. g qRT-PCR analysis of Itgb1 and Piezo1 mRNA levels in NMCFs cultured on UC and HC collagen matrices (n = 6). h Western blot analysis of ITGB1 and PIEZO1 protein levels in NMCFs on UC and HC matrices (n = 6). i Schematic representation of the uniaxial cyclic stretch setup under fluorescence microscopy. j Representative images and quantification of Fluo-4AM staining after 30 min…

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  Fig. 5. PIEZO1/ITGB1 modulates mechanotransduction and FMT in NMCFs cultured on different collagen matrices. a Immunofluorescence images and quantification of FA length (randomly selected, n ≥ 20) and intensity (n = 4) in NMCFs cultured on UC, HC, and HC supplemented with the PIEZO1 agonist Yoda (scale bar, left 50 µm, right 10 µm). b Western blot analysis of ITGB1, phospho-SMAD2, and α-SMA protein levels in NMCFs cultured on UC, HC, and HC+Yoda1 (n = 3). c Immunofluorescence analysis of FA length (randomly selected, n ≥ 20) and intensity (n = 4) in NMCFs cultured on UC and HC after siRNA-mediated knockdown of Piezo1 (scale bar, left 50 µm, right 10 µm). d Western blot analysis of ITGB1 and α-SMA protein expression in NMCFs cultured on UC and HC after Piezo1 knockdown by siRNA (n = 3). e Representative images and quantification analysis of Fluo-4AM staining after 30 min of cyclic uniaxial stretch in Itgb1 knockdown NMCFs cultured on UC and HC (n = 3; scale bar, 200 µm). f Western blot analysis of α-SMA protein expression in NMCFs cultured on UC and HC after Itgb1 knockdown by siRNA (n = 3). g Immunofluorescence analysis of FA length (randomly selected, n ≥ 20) and intensity (n = 4) in…

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  Fig. 6. Supplementation with histamine or inhibition of PIEZO1 significantly attenuates myocardial fibrosis in Hdc−/− mice post-AMI. a Construction of the HA-DA@histamine hydrogel. b Schematic of the Hdc−/− mouse AMI model treated with HA-DA@histamine and GsMTx4. c AQMC results of γ-Glu-ε-Lys in the cardiac tissue of WT, Hdc−/−, Hdc−/− treated with HA-DA@histamine and Hdc−/− treated with GsMTx4 mice 7 days post-AMI (n = 5). d Representative indentation curves show the difference between WT and Hdc−/− mice treated with HA-DA@histamine and GsMTx4. e Quantitative analysis of Young’s modulus in the scar region of WT mice, Hdc−/− mice, Hdc−/− mice treated with HA-DA@histamine and Hdc−/− treated with GsMTx4 mice 7 days post-AMI (n = 8). f Representative results and quantitative analysis of echocardiography on day 7 and day 21 post-AMI (n = 5). g ELISA results show the levels of IL-1β and IL-6 in the serum on day 1 and day 3 post-AMI (n = 6). h Representative results of Masson staining and quantitative analysis of cardiac fibrosis (n = 5; scale bar, top 1000 μm, bottom 250 μm). For all experiments, error bars represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001