Cyclic guanosine monophosphate-protein kinase G signaling attenuates aortic valve calcification through ULK1-mediated autophagy

Figures

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  Fig. 1. Dysregulation of the cGMP‒PKG signaling pathway contributes to the pathogenesis of CAVD. a KEGG pathway enrichment analysis of RNA-seq data from human calcified (CAVD) and non-calcified aortic valves (nonCAVD). b Volcano plot of RNA-seq data comparing CAVD versus nonCAVD valves; representative osteogenic-related genes and cGMP–PKG signaling-related genes are labeled. c, d Representative Western blot images and quantitative analysis demonstrating increased ALP and RUNX2 protein levels and decreased PKGI protein levels in calcified valves compared with noncalcified valves (n = 4 per group; data are expressed as the mean ± SEM; unpaired t-test). e, f Representative immunofluorescence images (e) and quantification (f) of PKGI (red) and α-SMA (green) in human aortic valve leaflets, with nuclei counterstained by DAPI (blue). Arrows indicate representative positive staining. Leaflet orientation is indicated (f, fibrosa; v, ventricularis). Fluorescence intensity was quantified using ImageJ and normalized to DAPI (n = 6 per group). Scale bar, 200 μm. Data are presented as mean ± SEM; unpaired two-tailed Student’s t-test. g Serum cGMP concentration measured in nonCAVD and CAVD subjects…

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  Fig. 2. PKGI haploinsufficiency exacerbates aortic valve calcification both in vivo and in vitro. a–c Echocardiographic assessment of transvalvular peak jet velocity (a, b) and LVEF (c) in sham-operated mice and mice subjected to AVWI with indicated genotypes (sham, n = 7; AVWI + WT, n = 8; AVWI+Prkg1+/−, n = 7). Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. d, e Representative Masson’s trichrome staining (d) and quantification of aortic valve leaflet thickness (e) from the indicated groups. Scale bar, 500 μm. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. f, g Representative Alizarin Red staining (g) and quantification of valve leaflet calcification (f) in the indicated groups. Scale bars, 500 μm (5×) and 100 μm (40×). Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. h, i Representative immunofluorescence images (h) and quantification (i) of RUNX2 (red) in aortic valve leaflets; nuclei were counterstained with DAPI (blue). Scale bar, 100 μm. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. j Serum cGMP concentrations measured by…

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  Fig. 3. Activation of the cGMP‒PKG signaling pathway attenuates osteogenic differentiation in hVICs and valve leaflet calcification ex vivo. a, b Representative images (a) and quantification (b) of Alizarin Red staining and ALP staining in hVICs cultured in OM and treated with an sGC stimulator (vericiguat, 500 nM), a PDE5 inhibitor (sildenafil, 100 nM), or a pGC activator (BNP, 10 μg/mL). or transfected with siRNA targeting PKGI. n = 6 independent experiments. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. c, d Representative immunoblot images (c) and quantification (d) of RUNX2 in hVICs treated as indicated. n = 3 independent experiments. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. e, f Ex vivo cultured aortic valve leaflets treated with OM with or without vericiguat, assessed by Von Kossa staining and Alizarin Red staining. scale bar: 2.5 mm for macro images; 250 µm for micro images; n = 3 independent experiments; mean ± SEM; unpaired t-test. g, h Representative images (g) and quantification (h) of Alizarin Red staining and ALP staining in hVICs cultured in OM and treated with vericiguat in the…

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  Fig. 4. Vericiguat treatment alleviates aortic valve calcification in murine models of CAVD. a, b Echocardiographic analysis of Ldlr−/− mice fed a normal chow diet (NCD), a high-cholesterol diet (HCD), or HCD and treated with low-dose vericiguat (3 mg/kg/day) or high-dose vericiguat (10 mg/kg/day) as indicated (n = 8–10 per group). Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. c–e Representative Von Kossa staining (c), quantification of valve calcification (d), and leaflet thickness (e) in Ldlr−/− mice fed HCD and treated as indicated. The white dashed outline indicates Von Kossa-positive calcified areas. Scale bars, 500 μm (5×) and 100 μm (40×). n = 8–10 per group. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. f, g Immunofluorescence staining of RUNX2 expression in Ldlr−/− HCD-fed mice compared with vehicle-treated control HCD-fed mice (scale bar: 100 µm; n = 6 per group; mean ± SEM; one-way ANOVA with Tukey’s multiple comparisons test). h, i Echocardiographic assessment of transvalvular peak jet velocity in mice subjected to AVWI and treated with vehicle or vericiguat as indicated (n = 8 per group).…

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  Fig. 5. Transcriptomic and functional analyses revealed that mitochondrial preservation and reduced oxidative stress are key mechanisms underlying vericiguat-mediated protection against osteogenic differentiation in hVICs. a PCA plot demonstrating distinct transcriptomic profiles between hVICs cultured in OM alone and those cultured in OM supplemented with vericiguat (OM + VER) (n = 3 replicates per group). b Volcano plot showing 681 DEGs between the OM and OM + VER groups, including 191 upregulated (red) and 490 downregulated (blue) genes (threshold: adjusted P value < 0.05, fold change > 1.0). c GO enrichment analysis of DEGs showing significant enrichment in pathways related to mitochondrial depolarization and ROS metabolic processes. The dot size represents the number of genes enriched, whereas color intensity indicates statistical significance (−log10 P value). d, e GSEA plots indicating significant enrichment in the ROS metabolic process (d) and oxidative phosphorylation (e) pathways in the vericiguat-treated group compared with those in the OM group. f, g Flow cytometric analysis (f) and quantification (g) of intracellular ROS levels in hVICs cultured under the indicated…

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  Fig. 6. cGMP-PKG signaling attenuates aortic valve calcification through enhancing autophagic flux. a Hierarchical clustering heatmap of differentially regulated proteins identified via TMT-based quantitative proteomics from hVICs cultured in control medium (NC), osteogenic medium (OM), OM plus vericiguat (VER), or OM plus vericiguat with a PKGI inhibitor (PKGi). b KEGG enrichment analysis of clusters 1, 3, and 5. c–e Representative immunoblot images (c) and quantification (d, e) of osteogenic markers (ALP and RUNX2) and autophagy-related proteins (p62 and LC3-II/LC3-I ratio) in hVICs treated with the PKGI activator 8-Br-cGMP in the presence or absence of bafilomycin, as indicated. n = 3 independent experiments. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. f Representative TEM images of human non-calcified and calcified aortic valve tissues. Scale bar, 1 μm. g, h Representative immunoblot images (g) and quantification (h) of LC3-II/I, p62, and Beclin in human non-calcified and calcified aortic valve tissues (n = 3 per group). Data are presented as mean ± SEM; unpaired two-tailed Student’s t-test. i Unsupervised clustering analysis identified 17…

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  Fig. 7. PKGI directly phosphorylates ULK1 at Ser556 to activate autophagy and inhibit the osteogenic differentiation of hVICs. a Overview of significantly up- and downregulated phosphosites across the control medium (NC), osteogenic medium (OM), OM plus vericiguat (VER), and OM plus vericiguat with PKGI inhibitor (PKGi) comparisons in hVICs via phosphoproteomic analysis (Student’s t-test, P < 0.05, fold change > 1.5). b Venn diagram showing the phosphosites oppositely regulated by vericiguat treatment and vericiguat plus PKGI inhibition. c, d Bioinformatics enrichment analyses (KEGG and ClueGO) highlighting the significant enrichment of differentially phosphorylated proteins in autophagy-related pathways. e LC‒MS/MS analysis identifying ULK1 as the top-ranked interacting protein of PKGI in FLAG-PKGI pulldown samples compared with IgG controls. f Protein–protein docking analysis indicating a strong and stable interaction between ULK1 and PKGI. g Scatter plot showed three phosphorylation sites on ULK1 (Ser477, Ser479, and Ser556) that exhibit opposite phosphorylation patterns between the Vericiguat-treated and PKGI-inhibited groups. h, i Co-IP assays confirming the direct interaction…

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  Fig. 8. The molecular mechanisms and experimental validation of cGMP-PKG signaling in CAVD. Clinical transcriptomic database analysis revealed impaired cGMP-PKG signaling during the progression of CAVD. The activation of cGMP-PKG signaling by vericiguat, a soluble guanylate cyclase (sGC) stimulator, has therapeutic potential against valve calcification. This protective effect was consistently demonstrated across multiple models, including hVICs subjected to osteogenic induction in vitro, LDL receptor knockout (Ldlr−/−) mice subjected to high-fat diet-induced metabolic stress, mice subjected to mechanical injury of the AVWI, and PKGI haploinsufficient (Prkg1+/−) mice combined with mechanical valve injury. Mechanistically, transcriptomic, proteomic, and phosphoproteomic analyses collectively revealed that the phosphorylation of ULK1 at serine556 by PKGI is a critical event that promotes autophagy. Enhanced autophagy inhibited the osteogenic differentiation of hVICs, thereby effectively preventing the progression of CAVD. Figure created with BioRender.com