Discoidin Domain Receptor 1 Promotes Myocardial Fibrosis by Suppressing Specificity Protein 1 Ubiquitination and Degradation in Male Spontaneously Hypertensive Rats

Figures

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  Figure 1. Upregulation of DDR1 and ROCK1 expression was observed in both SHR myocardial tissues and activated CFs. A, Volcano plot of all DEGs (|fold change|>0.2, P<0.05). Hierarchical clustering heatmap based on top 10 DEGs. Genes are clustered by row, and samples by column. Comparative gene expression analysis of DDR1 between the compensated cardiac hypertrophy group and the HF group on the basis of data from data set GSE19210 (n=6). B, Protein levels of α‐SMA, TGF‐β1, CTGF, collagen I, and collagen III in Wistar rats and SHRs were detected by western blotting and were quantitatively analyzed (n=6). C, Immunohistochemical staining for the detection of DDR1 (brown) and ROCK1 (brown) expression in Wistar rats and SHRs (scale bar: 20 μm) and quantitative analysis (n=6). D, Protein levels of DDR1 and ROCK1 in Wistar rats and SHRs were detected by western blotting and were quantitatively analyzed (n=6). E, NRCFs were stimulated with angiotensin II at varying concentrations (0, 1, 10, and 100 nM) for 24 hours, and with a fixed concentration (100 nM) for different durations (0, 6, 12, and 24 hours), followed by western blotting analysis of α‐SMA, TGF‐β1, CTGF, collagen I, and collagen III…

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  Figure 2. DDR1 promotes MF progression through positive regulation of ROCK1 and its downstream profibrotic signaling cascades. A, Western blotting was performed to detect protein levels of DDR1, α‐SMA, TGF‐β1, CTGF, collagen I, and collagen III (in the presence or absence of DDR1 siRNA), and ROCK1, α‐SMA, TGF‐β1, CTGF, collagen I, and collagen III (in the presence or absence of ROCK1 siRNA) in angiotensin II‐treated NRCFs, and (B) the results were quantitatively analyzed (n=6). C, Representative images of cellular immunofluorescence detection of ROCK1 (green) in angiotensin II‐treated NRCFs in the presence or absence of DDR1 siRNA (scale bar: 25 μm) and quantitative analysis (n=6). D, RT‐qPCR was performed to detect mRNA levels of ROCK1 (in the presence or absence of DDR1 siRNA) and DDR1 (in the presence or absence of ROCK1 siRNA) in angiotensin II‐treated NRCFs, and the results were analyzed quantitatively (n=3). E, Western blotting was performed to detect protein levels of DDR1 and ROCK1 in angiotensin II–treated NRCFs in the presence or absence of either DDR1 siRNA or ROCK1 siRNA, and the results were quantitatively analyzed (n=6). F, Western blotting was performed to detect protein…

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  Figure 3. SP1 links DDR1 and ROCK1 to mediate the positive regulation of DDR1 over ROCK1. A, Western blotting was performed to detect protein levels of DDR1, ROCK1, α‐SMA, TGF‐β1, CTGF, collagen I, and collagen III in angiotensin II–treated NRCFs in the presence or absence of either ROCK1 short hairpin RNA (+) or DDR1 short hairpin RNA (−), and the results were analyzed quantitatively (n=6). B, RT‐qPCR and western blotting were performed to detect mRNA and protein levels of SP1 in NRCFs treated with angiotensin II in the presence or absence of DDR1 siRNA, and the results were analyzed quantitatively (n=3). C, RT‐qPCR was performed to detect mRNA levels of DDR1 and ROCK1 in angiotensin II‐treated NRCFs in the presence or absence of SP1 siRNA, with quantitative analysis (n=3). D, Western blotting was performed to detect protein levels of DDR1, SP1, and ROCK1 in angiotensin II–treated NRCFs in the presence or absence of SP1 siRNA. E, ChIP‐PCR was performed to detect the DNA level of ROCK1 promoter in the presence or absence of SP1 siRNA in angiotensin II–treated NRCFs. RT‐qPCR was performed to detect mRNA levels of ROCK1 promoter in angiotensin II–treated NRCFs in the presence or absence of…

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  Figure 4. DDR1 inhibits SP1 ubiquitination‐mediated degradation by enhancing the interaction with SP1. A, Western blotting was performed to detect protein levels of SP1 in NRCFs treated with cycloheximide (10 μM) and angiotensin II for the indicated times in the presence or absence of DDR1 siRNA, and the results were analyzed quantitatively (n=3). B, After preincubation with MG132 (10μM) or chloroquine (10 μM) for 30 minutes, western blotting was performed to detect protein level of SP1 in the presence or absence of DDR1 siRNA in angiotensin II–treated NRCFs, and the results were analyzed quantitatively (n=3). C, Coimmunoprecipitation was performed to detect the ubiquitination level of SP1 in the presence or absence of DDR1 siRNA in angiotensin II–treated NRCFs (n=3). D, Representative images showing DDR1 (green) colocalizing with SP1 (red) in NRCFs by cellular immunofluorescence detection (scale bar: 25 μm) and quantitative analysis (n=3). E, Coimmunoprecipitation was performed on lysates from angiotensin II–treated or untreated NRCFs. Protein levels of DDR1 and SP1 were detected using specific antibodies and control IgG (n=3). F, Angiotensin II–treated or untreated HEK‐293T cells were…

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  Figure 5. Inhibiting DDR1 or ROCK1 expression improves cardiac structure and function in SHRs. A, Western blotting was performed to detect protein levels of cytoplasmic DDR1, nuclear DDR1, cytoplasmic SP1, and nuclear SP1 in angiotensin II–treated NRCFs with or without DDR1 siRNA, and quantitative analysis was conducted (n=3). B, Representative echocardiographic B‐mode and M‐mode images of Wistar rats and SHRs were acquired, and parameters including end‐diastole LVAW (d, mm), LVPW (d, mm), EF (%), FS (%),CO (mL/min), SV (μL), E/A, and IVRT (ms) were measured and quantitatively analyzed (n=7). C, The ratio of HW/BW in each group of Wistar rats and SHRs was measured (n=9). D, HE staining was performed overall (scale bar: 1 mm) and locally (scale bar: 20 μm) to observe the histostructural pathological changes of myocardium in each group of Wistar rats and SHRs. Masson's trichrome staining was used to evaluate the degree of MF in each group of rats (collagen fibers were blue; scale bar: 20 μm), and quantitative analysis was performed (n=9). Wheat germ agglutinin staining was performed to assess the degree of myocardial hypertrophy in each group of rats (scale bar: 20 μm), and quantitative…

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  Figure 6. DDR1 promotes myocardial fibrosis in SHRs via modulation of ROCK1 and its downstream profibrotic signaling cascades. A, Western blotting was performed to assay protein levels of DDR1, SP1, and ROCK1 in Wistar rats and SHR groups, and the results were analyzed quantitatively (n=8). B, Western blotting was performed to assay protein levels of α‐SMA, TGF‐β1, CTGF, collagen I, and collagen III in each group of Wistar rats and the SHRs, and the results were analyzed quantitatively (n=8). C, Immunohistochemical staining was performed to detect the expression of DDR1 (brown), SP1 (brown), and ROCK1 (brown) in each group of rats (scale bar: 20 μm), and quantitative analysis was conducted (n=9). D, Immunohistochemical staining was performed to detect the expression of α‐SMA (brown), TGF‐β1 (brown), CTGF (brown), collagen I (brown), and collagen III (brown) in each group of rats (scale bar: 20 μm), and quantitative analysis was conducted (n=9). GAPDH was used as a loading control. Data are expressed as mean±SEM. One‐way ANOVA and Tukey's post hoc test were used for analysis. AAV9 indicates adeno‐associated virus 9; CTGF, connective tissue growth factor; DDR1, discoidin domain receptor 1;…