WISP1 drives a mechanically active immune modulatory and proliferative cardiac myofibroblast state

Figures

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  Figure 1.. WISP1 is induced by TGFβ1 and is sufficient to drive α-SMA expression in cardiac fibroblasts. (A)Wisp1 mRNA expression in primary adult mouse cardiac fibroblasts following TGFβ1 (10 ng/mL) treatment for 24–72 h. (B) Representative immunofluorescence images of α-SMA (green), vimentin (red), and DAPI (blue) after 72 h treatment with vehicle (Ctrl), WISP1 (500 ng/mL), TGFβ1 (10 ng/mL), or WISP1 + TGFβ1. (C, D) Representative Western blot (C) and densitometric quantification (D) of α-SMA normalized to GAPDH. Data are mean ± SEM from ≥3 independent isolations. Statistical analysis by one-way ANOVA with Tukey’s post hoc test; *p < 0.05. Data are mean ± SEM from ≥3 independent isolations. Statistical analysis by one-way ANOVA with Tukey’s post hoc test; *p < 0.05.

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  Figure 2.. WISP1 enhances collagen expression and secretion in primary cardiac fibroblasts. (A–B) Total collagen production measured by picrosirius red elution (A540) following 72 h treatment (n = 14 isolations; 7 male, 7 female). (C) Col1a1 mRNA expression by qPCR. (D–E) Representative Western blot (D) and densitometric quantification (E) of collagen I normalized to GAPDH. Data are mean ± SEM. Statistical analysis by one-way ANOVA; *p < 0.05 versus Ctrl.

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  Figure 3.. WISP1 enhances cardiac fibroblast contractility and accelerates wound closure (A–B) Primary adult mouse cardiac fibroblasts were embedded in floating collagen gels and treated for 24 h with vehicle (Ctrl), WISP1 (500 ng/mL), TGFβ1 (10 ng/mL), or WISP1 + TGFβ1. (A) Representative gel images at 0 h and 24 h. (B) Quantification of gel area (% contraction; n = 4 isolations, 2 males & 2 females). (C–D) Confluent fibroblast monolayers were scratched and imaged every 2 hours for 24 hours using the same treatment conditions. (C) Representative bright-field images at time point 0 and 14. (D) Quantification of wound closure (% of gap closed relative to time 0; n = 8 isolations, 4 male & 4 female). Data are mean ± SEM. One-way ANOVA with Tukey’s multiple comparisons; *p ≤ 0.05.

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  Figure 4.. WISP1-dependent myofibroblast activation is p38 MAPK–dependent and uncoupled from periostin expression. (A) Periostin (Postn) expression assessed by qPCR in primary cardiac fibroblasts treated for 72 h. (B-C) Representative Western blot (B) and densitometry normalized to GAPDH and Ctrl (C) of Periostin across 6 biological replicates. (D-E) Representative Western blots for α-SMA (D) and densitometric quantification (E) following treatment with p38 MAPK inhibitor SB203580 (10 μM). (F-G) Representative Western blots for periosotin (F) and densitometric quantification (G) following treatment with p38 MAPK inhibitor SB203580 (10 μM). (H) Total soluble collagen secretion measured by Picrosirius Red. Data are mean ± SEM. Statistical significance was determined by one-way ANOVA or two-way ANOVA as appropriate.

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  Figure 5.. WISP1 establishes a proliferative and immune-inflammatory transcriptomic program distinct from TGFβ1-driven myofibroblasts. (A) Heatmap of Hallmark gene set normalized enrichment scores (NES) across treatments. (B–C) Dot plot representation of proliferation (B) and inflammation (C) pathway enrichment. (D) Aggregated mean NES across functional categories. (E) Myofibroblast and ECM module scores relative to control. Data presented as mean ± SEM or box-and-whisker plots.

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  Figure 6.. Compartment-resolved proteomics reveals WISP1-driven intracellular proliferation and ECM-associated immune remodeling. (A) Heatmap of pathway NES across global (soluble), ECM, and ECM-enriched proteomes. (B–C) Dot plot analysis of proliferation and inflammation pathway enrichment in global (B) and ECM (C) fractions. Dot size represents significance as adjusted p-value. (D–F) Myofibroblast and ECM module scores across global (D), ECM (E), and ECM-enriched (F) proteomes. Box plots show median with interquartile range.