Deletion of Cd248 in Postn+ myofibroblast fails to attenuate pressure-overload induced cardiac remodeling and fibrosis in mice

Figures

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  Fig. 1. Single-cell RNAseq reveals co-expression of Cd248 and Postn in cardiac fibroblasts and mural cells following pressure overload. (A) Uniform Manifold Approximation and Projection (UMAP) of cardiac interstitial cells from sham and TAC-operated mice, annotated by cell type and experimental group. (B–D) Quantification of the percentage of cells positive for Cd248, Postn, or both within each major cell lineage. Data are presented as percentages for each group (salmon = Sham, teal = TAC). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 2. Knockdown of Cd248 blunts TGF-β1-induced fibroblast activation, migration, and proliferation in vitro. (A) Representative immunofluorescence of cultured cardiac fibroblasts transfected with control siRNA (si-Scrambled) or siRNA targeting Cd248 (si-Cd248) and stimulated with vehicle or TGF-β1 (5 ng/mL) for 24 h. Myofibroblast activation is indicated by the formation of α-SMA stress fibers (green); nuclei are counterstained with Hoechst (blue). Scale bar, 50 μm. (B) Quantification of myofibroblast activation, showing the percentage of cells with organized α-SMA stress fibers and the relative mRNA expression of Acta2 (α-SMA). Two-way Analysis of Variance (ANOVA) was performed, followed by Tukey's multiple comparisons post-hoc test. *P < 0.05 vs. the corresponding vehicle control; #P < 0.05 vs. si-Scrambled with TGF-β1 stimulation. (D) Scratch-wound assay demonstrating impaired fibroblast migration following Cd248 knockdown. Quantification shows the percentage of wound closure at 24 h relative to the initial scratch. *P < 0.05 vs.si-Scrambled, determined by an unpaired Student's t-test. (E-G) Analysis of fibroblast proliferation via EdU incorporation. Representative images (E) and…

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  Fig. 3. Postn+ myofibroblast-specific deletion of Cd248 does not alter cardiac fibrosis after pressure overload. (A) Representative low-magnification images of HE-stained long-axis heart sections 8 weeks after transverse aortic constriction (TAC). Scale bar, 1.25 mm. (B) High-magnification images of Picro-Sirius Red-stained heart section of control (Postn-MCM) and Postn+myofibroblast-specific Cd248 knockout (Cd248-cKO) mice are shown. Collagen is stained red, and myocardium appears yellow. Detailing interstitial fibrosis (top) and perivascular fibrosis (bottom) for each genotype were shown. Scale bar, 50 μm. (C) Quantification of the fibrotic area. Data are presented as mean ± SEM; n = 5, each dot represents an individual animal. Statistical analysis was performed as described in the Methods. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 4. Postn+ myofibroblast-specific deletion of Cd248 does not alter cardiomyocyte hypertrophy or microvascular density in response to pressure overload. (A) Representative confocal images of left ventricular cross-sections from control and Cd248-cKO mice 8 weeks after TAC surgery. Capillaries are labeled with Isolectin-B4 (IB4, green), cardiomyocyte membranes with Wheat Germ Agglutinin (WGA, red), and nuclei with DAPI (blue). Scale bar, 50 μm. (B–D) Quantification of morphometric parameters. No significant differences were observed between control (blue bars) and Cd248-cKO (red bars) mice in (B) capillary density, (C) cardiomyocyte cross-sectional area, or (D) the ventricle weight to body weight ratio. (E) Representative images of arterioles identified by α-smooth muscle actin (α-SMA) immunohistochemistry (arrows). Scale bar, 50 μm. Quantification of α-SMA+ arterioles, showing no significant differences in total arteriolar density (left) or in the distribution of arteriolar sizes (right) between genotypes. Data are presented as mean ± SEM; n = 4–10, each dot represents an individual animal. Statistical analysis was performed as described in the Methods. (For interpretation of the…

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  Fig. 5. Postn+ myofibroblast-specific deletion of Cd248 does not alter cardiac function or dimensions after pressure overload. (A) Representative echocardiographic recordings from control and Cd248-cKO mice at baseline and 8 weeks after TAC. Images shown are M-mode, pulsed-wave Doppler of transmitral inflow, and tissue-Doppler imaging (TDI) of the mitral annulus. (B–I) Quantification of key echocardiographic parameters at baseline and 8 weeks post-TAC for control (blue bars) and Cd248-cKO (red bars) mice. Parameters shown include left ventricular internal dimensions at diastole and systole (LVID;d, LVID;s), systolic function (ejection fraction [EF], fractional shortening [FS]), diastolic indices (E/A, E'/A', and E/E') and posterior wall thickness (LVPW;d, LVPW;s). No significant differences were observed between genotypes. Data are presented as mean ± SEM; n = 5–7, each dot represents an individual animal. Two-way Analysis of Variance (ANOVA) was performed, followed by Tukey's multiple comparisons post-hoc test. *P < 0.05 vs. baseline. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 6. Single-cell transcriptomic analysis reveals distinct biological programs in fibroblast subpopulations from pressure-overloaded hearts. (A) UMAP visualization of cardiac fibroblasts isolated from mice 14 days after TAC, showing unsupervised clustering into distinct subpopulations (0−10). (B) Cd248−Postn− (grey), Cd248−Postn+ (green), Cd248+Postn− (red)and their co-expression (yellow) within the fibroblast UMAP. (C–D) Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs) from the TAC fibroblast population. Dot plots show the top enriched biological programs comparing (C) Postn−Cd248+ versus Postn+Cd248+ fibroblasts and (D) Postn+Cd248+ versus Postn+Cd248− fibroblasts. Dot size corresponds to the number of DEGs mapped to each term, and colour indicates the false discovery rate (FDR). (BP, Biological Process; CC, Cellular Component; MF, Molecular Function). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 7. Transcriptomic profiling reveals Cd248 regulates TGF-β1-induced actomyosin organization and angiogenic programs in vitro. (A) Venn diagram and schematic strategy for identifying Cd248-dependent transcriptional changes. Differentially expressed genes (DEGs) induced by TGF-β1 (si-Scrambled TGF-β1 vs. si-Scrambled) were intersected with DEGs modified by Cd248 knockdown (si-Cd248 TGF-β1 vs. si-Scrambled TGF-β1). This analysis identified 158 “Reverse-response genes” (genes where Cd248 knockdown antagonized the TGF-β1 effect) and 239 “Amplified-response genes” (genes where knockdown enhanced the TGF-β1 effect). (B) Gene Ontology (GO) enrichment analysis of the Reverse-response genes. The dot plots display the top enriched terms for Biological Process (BP), Cellular Component (CC), and Molecular Function (MF), highlighting pathways involved in actomyosin structure organization and extracellular matrix. (C) Heatmap of representative Reverse-response genes enriched in the “Actomyosin structure organization” pathway (e.g., Myl9, Cnn1, Wnt11). The colour scale indicates that the TGF-β1-induced upregulation of these genes (red in si-Scrambled TGF-β1) is attenuated by Cd248 knockdown…