Gata4 regulates fibroblast migration in a fibroblast-source-dependent manner under physiological matrix stiffness

Figures

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  Figure 1. Matrix stiffness alters the baseline cytoskeletal state of cardiac fibroblasts. (A, B) Schematic comparison of an 8 kPa PDMS substrate and conventional tissue-culture plastic (∼GPa). (C) Experimental timeline. Cardiac fibroblasts (CFs) were isolated from neonatal Gata4fl/fl mice and cultured on either substrate for 3–5 days before fixation and immunofluorescence staining. (D, E) Representative α-SMA images and quantification of fluorescence intensity per cell. (F, G) Representative Vimentin images and quantification. The outer images in panels D and F are enlarged views of the boxed regions in the corresponding whole-dish images. Images are stitched composites acquired at 10× magnification; fluorescence intensity per cell was quantified using ImageJ. Data are mean ± SEM; n = 5 biological preparations per group. Unpaired Student's t-tests; **p < 0.01 and ***p < 0.001. Scale bar, 1000 µm. Both sexes were included.

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  Figure 2. 8-kPa PDMS substrates preserve cardiac fibroblast migratory capacity. Cardiac fibroblasts isolated from neonatal Gata4fl/fl mice were cultured on 8 kPa PDMS or conventional tissue-culture plastic and imaged for 12–16 h. (A) Instantaneous speed, (B) path speed, (C) net displacement (line length), (D) line speed, (E) cumulative path length, (F) cell area, (G) segment length, and (H) directional persistence. Bars are mean ± SEM; dots are tracked cells from five biological preparations per condition. Unpaired Student's t-tests; **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant. Both sexes were included.

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  Figure 3. GATA4 abundance differs among fibroblast sources and is efficiently reduced by Ad-Cre. CFs, LFs, and TTFs isolated from neonatal Gata4fl/fl mice were cultured on 8 kPa PDMS. (A) Baseline Gata4 mRNA expression across fibroblast sources, normalized to CFs. (B) Gata4 mRNA expression after Ad-GFP (Control) or Ad-Cre-GFP (Gata4 KO) transduction. Gene expression was measured by qRT-PCR and normalized to Gapdh. Data are mean ± SEM; n = 5 biological preparations per group. Unpaired Student's t-tests; **p < 0.01, ***p < 0.001, ****p < 0.0001. Both sexes were included.

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  Figure 4. Gata4 deletion increases selected collagen mRNA levels across fibroblast sources. CFs, LFs, and TTFs were cultured on 8 kPa PDMS and transduced with Ad-GFP (Control) or Ad-Cre-GFP (Gata4 KO). qRT-PCR measurement of (A) Col1a1, (B) Col1a2, and (C) Col3a1 mRNA, normalized to Gapdh. Data are mean ± SEM; n = 5 biological preparations per group. Unpaired Student's t-tests; *p < 0.05, ***p < 0.001, ****p < 0.0001. Both sexes were included.

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  Figure 5. Gata4 deletion remodels fibroblast migration in a fibroblast-source-dependent manner. CFs, LFs, and TTFs isolated from neonatal Gata4fl/fl mice were cultured on 8 kPa PDMS and transduced with Ad-Cre-GFP (Gata4 KO) or Ad-GFP (Control). (A) Instantaneous speed, (B) path speed, (C) path distance, (D) line speed, (E) net displacement, (F) cell area, (G) segment length, and (H) directional persistence. Points are individual tracked cells; black bars show the interquartile range and white ticks the median. Metric-specific n values are displayed beneath each cell source as Control/KO. Statistical significance from two-way ANOVA with Sidak's multiple-comparisons test is indicated by *p < 0.05 and **p < 0.01; ns, not significant. Standardized effects and bootstrap confidence intervals are summarized in Figure 6. Both sexes were included.

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  Figure 6. Integrated effect-size analysis highlights fibroblast-source-dependent migration responses. (A) Heatmap of Hedges’ g calculated as Gata4 KO minus control for duration-comparable migration measurements and cell area. Negative values indicate lower measurements after Gata4 deletion; positive values indicate higher measurements. (B–D) Forest plots show Hedges’ g and 95% bootstrap confidence intervals for CFs, LFs, and TTFs. The vertical line at g = 0 indicates no difference. Cumulative path and line lengths were excluded from the integrated analysis because of their sensitivity to effective tracking duration. Effect sizes are descriptive summaries of cell-level distributions. (E) Track-matched relationship between mean cell area and instantaneous speed in Control and Gata4-knockout CFs. Each point represents one tracked cell. Spearman rank correlations were calculated separately for Control (n = 20) and Gata4-knockout CFs (n = 21).

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  Figure 7. Gata4 loss produces non-uniform remodeling of migration-related transcriptional programs. Publicly available adult cardiac fibroblast RNA-Seq data from GSE155358 were analyzed using control and single-Gata4-knockout samples (n = 3 per group). (A) NES values for 24 focused GO Biological Process gene sets grouped into motility/migration, actin remodeling, polarity/protrusion, and adhesion coupling. FDR values are labeled for terms with FDR < 0.05. (B) Distribution of NES values within each functional domain; black lines and numbers indicate domain medians. (C) Rank-based domain scores across individual control (C1–C3) and knockout (KO1–KO3) samples. Cell colors show within-domain standardized values; numbers show the underlying rank-based scores. Absence of significant enrichment is not interpreted as pathway suppression.

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  Figure 8. Gata4 loss differentially remodels actin cytoskeletal submodules. (A) Gata4-knockout-versus-control log2 fold changes for selected genes grouped into core actins, polymerization/branching, severing/turnover, contractility/stress fibers, adhesion coupling, and polarity/protrusion. (B) Gene-wise z-score heatmap across the three control (C1–C3) and three Gata4-knockout (KO1–KO3) samples. Individual-gene fold changes are displayed descriptively and are not assigned statistical significance from fold change alone.