ADAMTS4 mediates LPS‐induced cardiac injury and myocardial fibrosis through proteolytic cleavage of TSP1

Figures

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  FIGURE 1. Adamts4 expression is increased in fibroblasts of septic mice. (A) Results of Reactome pathway enrichment analysis based on RNA‐seq data of mouse hearts after LPS stimulation (n = 6). (B) Volcano plot of differentially expressed genes in LPS‐induced septic cardiomyopathy. Matrisome‐related genes are highlighted, with representative ECM‐remodelling genes, including Adamts4, Vcan, Timp1, Mmp14, and Mmp9, specifically labelled. (C) Representative Western blot images and statistical analysis of Adamts4 protein expression in mouse hearts 24 h after LPS treatment (n = 6). (D) The level of Adamts4 mRNA relative to NS group in mouse hearts 24 h after LPS treatment (n = 6). (E) Representative Western blot images and statistical analyses of the expression of ADAMTS4 protein in mouse fibroblasts following 24 h of LPS treatment (n = 6). (F) The relative mRNA level of Adamts4 in mouse fibroblasts 24 h after LPS treatment (n = 6). (G) Representative immunofluorescence images showing α‐SMA, ADAMTS4 and DAPI staining in heart sections from NS‐ and LPS‐treated mice. Arrowheads indicate ADAMTS4‐positive signals overlapping with or spatially associated with α‐SMA‐positive regions. Quantification…

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  FIGURE 2. Tcf21‐lineage Adamts4‐deficient mice exhibit attenuated fibrosis and cardiac dysfunction in response to lipopolysaccharide (LPS) administration. (A) Tcf21‐lineage Adamts4‐deficient mice (Adamts4 cKO) were generated by crossing mice with conditional knockout alleles of Adamts4 (Adamts4fl/fl) with Tcf21‐iCreERT2 mice. (B) Representative Western blot images and statistical analysis of Adamts4 protein expression in CFs isolated from Adamts4 cKO mice and littermate controls (n = 6). (C) Survival rate of mice challenged with LPS (15 mg/kg) (n = 12). (D and E) Changes in ejection fraction (EF) and fractional shortening (FS) in different groups of mice after LPS stimulation (n = 12). (F) Changes in left ventricular end‐systolic diameter (LVESD) after LPS stimulation in different groups of mice (n = 8). (G and H) Changes in levels of LDH and CK‐MB in mouse serum after LPS stimulation in different groups of mice (n = 6). (I) Representative echocardiograms of different groups of mice. (J) H&E staining. The black line indicates the thickness of myofibrils (n = 6), and myofibrils become thinner after LPS stimulation. (K) PSR staining. The area of cardiac tissue fibrosis in Adamts4 cKO mice…

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  FIGURE 3. Tcf21‐lineage Adamts4‐deficient mice display attenuated myocardial inflammation in response to lipopolysaccharide (LPS) administration. (A) The expression levels of cytokines such as IL‐6, TNF‐α, IL‐1β, IL‐10, MCP1 and IL‐8 in samples from Adamts4fl/fl mice (control group) and Adamts4 cKO mice were detected, and the right‐side scale shows the relative expression fold (n = 5). (B–E) The protein expression of inflammation‐related indicators IKKβ, phosphorylated IKKβ (p‐IKKβ), IκBα, phosphorylated IκBα (p‐IκBα), p65, and phosphorylated p65 (p‐p65) was detected (n = 6). (F–I) The protein expression levels of apoptosis‐related indicators Bax, Bcl‐2, Caspase‐3 and Cleaved‐Caspase‐3 were detected (n = 6). (J) Representative images of DHE distribution detected by immunofluorescence, and the DHE fluorescence intensity was counted (n = 6). (K) The TUNEL method was used to detect the apoptosis of cells in myocardial tissue sections of mice in different groups, and the cell apoptosis rate was calculated (n = 6). Data are presented as mean ± SEM. Normality was assessed using the Shapiro–Wilk test. All tests were two‐sided. For two‐group comparisons (Figure 3B), an unpaired two‐tailed…

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  FIGURE 4. Postn‐lineage Adamts4‐deficient mice exhibit attenuated LPS‐induced cardiac dysfunction and myocardial fibrosis. (A and B) By crossing Adamts4 knockout mice (Adamts4fl/fl) with Postn‐CreER mice, we successfully developed Postn‐lineage Adamts4‐deficient mice. The figure displays representative Western blot images and statistical analysis results of Adamts4 protein expression in cardiac fibroblasts (CFs) isolated from Adamts4 mfKO mice and littermate Adamts4fl/fl controls (n = 6). (C) Survival curves of mice challenged with LPS (15 mg/kg) (n = 12). (D and E) Dynamic changes in ejection fraction (EF) and fractional shortening (FS) in different groups of mice after LPS stimulation (n = 12). (F and G) Changes in serum levels of lactate dehydrogenase (LDH) and creatine kinase‐MB (CK‐MB) in different groups of mice after LPS stimulation (n = 6). (H) Representative immunofluorescence images showing DHE distribution and quantification of DHE fluorescence intensity (n = 6). (I) TUNEL staining and quantification of apoptotic cells in myocardial sections from different groups of mice (n = 6). (J) PSR staining showing reduced cardiac fibrosis area in Adamts4 mfKO mice after LPS stimulation (n…

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  FIGURE 5. Tcf21‐lineage fibroblast Adamts4 overexpression exacerbates lipopolysaccharide (LPS)‐induced myocardial fibrosis and cardiac dysfunction. (A and B) Conditional Tcf21‐lineage fibroblast Adamts4 overexpression mice (Adamts4 cTg) were generated by crossing Adamts4 Tgfl/fl conditional transgenic with Tcf21‐iCreERT2 mice. Representative Western blot images and statistical analysis of Adamts4 protein expression in cardiac fibroblasts (CFs) isolated from Adamts4 cTg mice and littermate controls (n = 6). (C) Survival rate of mice challenged with LPS (15 mg/kg) (n = 12). (D and E) Changes in ejection fraction (EF) and fractional shortening (FS) in different groups of mice after LPS stimulation (n = 12). (F) Changes in left ventricular end‐systolic diameter (LVESD) in different groups of mice after LPS stimulation (n = 8). (G and H) Changes in levels of LDH and CK‐MB in mouse serum in different groups of mice after LPS stimulation (n = 6). (I) H&E staining: Black lines indicate the thickness of myofibrils (n = 6). Myofibrils in Adamts4 cTg mice were thinner after LPS stimulation. (J) PSR staining: After LPS stimulation, the fibrosis area of cardiac tissue in Adamts4 cTg mice increased (n =…

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  FIGURE 6. ADAMTS4 promotes cardiac fibroblast activation through direct interaction with and cleavage of TSP1. (A) Mouse cardiac fibroblasts (CFs) were transfected with shRNA targeting Adamts4 (shAdamts4) or control shRNA and stimulated with LPS. Protein expression of α‐SMA was assessed by Western blotting and quantified relative to GAPDH. (B) CFs with Adamts4 knockdown were stimulated with LPS, and α‐SMA mRNA levels were measured by qPCR. (C) CFs were infected with lentivirus encoding full‐length Adamts4 (oeAdamts4) or control vector and stimulated with LPS. Protein expression of α‐SMA was analysed by Western blotting and quantified relative to GAPDH. (D) CFs overexpressing Adamts4 were stimulated with LPS, and α‐SMA mRNA levels were measured by qPCR. (E) Co‐immunoprecipitation (Co‐IP) combined with LC‐MS analysis was performed using heart tissue lysates from LPS‐treated mice. Representative high‐confidence candidate proteins are listed, with thrombospondin‐1 (TSP1) identified as a potential interacting partner of ADAMTS4. (F) Reciprocal Co‐IP assays were performed in heart tissue lysates using antibodies against ADAMTS4 or TSP1, followed by immunoblotting to validate the interaction…

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  FIGURE 7. ADAMTS4‐mediated cleavage of TSP1 at residues 236–246 drives fibroblast activation via TGF‐β/Smad and NF‐κB signalling. (A) Schematic representation of full‐length mouse TSP1 highlighting predicted ADAMTS4 cleavage sites within the N‐terminal region. Two candidate cleavage regions (Site1 and Site2) identified by protease prediction analysis are indicated. (B) Alanine substitution strategy for candidate cleavage motifs within TSP1. Three regions selected based on prediction and structural accessibility (236–246 aa, 298–308 aa, and 322–332 aa) were mutated to alanine. (C) Sequence alignment of candidate cleavage regions across Homo sapiens, Mus musculus and Rattus norvegicus, demonstrating conservation of key residues. (D) Cleavage of TSP1 was assessed by Western blotting using antibodies against HA (full‐length and fragment), Flag (ADAMTS4), and GAPDH. (E) Full‐length intracellular proteins were normalized to GAPDH, whereas secreted TSP1‐derived fragments in conditioned media were normalized to total membrane protein staining. The resulting ratios were quantified and statistically analysed. (F) NIH/3T3 fibroblasts were transfected with wild‐type ADAMTS4 or the catalytically…