Follistatin‐like 1 promotes cardiac fibroblast activation and protects the heart from rupture

Abstract Follistatin‐like 1 (Fstl1) is a secreted protein that is acutely induced in heart following myocardial infarction (MI). In this study, we investigated cell type‐specific regulation of Fstl1 and its function in a murine model of MI. Fstl1 was robustly expressed in fibroblasts and myofibroblasts in the infarcted area compared to cardiac myocytes. The conditional ablation of Fstl1 in S100a4‐expressing fibroblast lineage cells (Fstl1‐cfKO mice) led to a reduction in injury‐induced Fstl1 expression and increased mortality due to cardiac rupture during the acute phase. Cardiac rupture was associated with a diminished number of myofibroblasts and decreased expression of extracellular matrix proteins. The infarcts of Fstl1‐cfKO mice displayed weaker birefringence, indicative of thin and loosely packed collagen. Mechanistically, the migratory and proliferative capabilities of cardiac fibroblasts were attenuated by endogenous Fstl1 ablation. The activation of cardiac fibroblasts by Fstl1 was mediated by ERK1/2 but not Smad2/3 signaling. This study reveals that Fstl1 is essential for the acute repair of the infarcted myocardium and that stimulation of early fibroblast activation is a novel function of Fstl1.

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A qPCR analysis of TNF-a and IL-1b mRNA expression in heart tissues. The heart was harvested at 7 days after the surgery. Error bars represent mean AE SEM (n = 16 and 15 for WT and cfKO sham group, n = 15 and 14 for WT and cfKO MI group, respectively). Statistical analysis was performed by two-way ANOVA. Post hoc test was performed by Tukey's test. B Detection of macrophage by F4/80 staining (AbD Serotec, Clone A3-1) in the ischemic myocardium of WT and cfKO mice at day 7 after MI. DAB substrate was used for detection. Counter staining was performed using hematoxylin stain. F4/80-positive cell density in the infarcted area was measured as pixel at high magnification. Scale bar indicates 100 lm. Error bars represent mean AE SEM (n = 12 and 14 for WT and cfKO, respectively). Statistical analysis was performed by unpaired t-test (two-tailed).
Source data are available online for this figure. A The expression of p-Smad2, p-Smad3, Smad2, and Smad3 in Fstl1-cfKO and WT hearts was assessed by immunoblotting. Hearts were harvested at 7 days after the surgery. Error bars represent mean AE SEM (n = 3 for each sham group and n = 4 for each MI-IA group). Statistical analysis was performed by two-way ANOVA. Post hoc test was performed by Tukey's test. B The effect of recombinant Fstl1 protein on TGF-b1-stimulated Smad2/3 signaling pathway in neonatal cardiac fibroblasts (NRCFbs). Recombinant Fstl1 protein (50 ng/ml) or control vehicle was added to serum-deprived (24 h) NRCFbs at 30 min prior to TGF-b1 protein (2 ng/ml) stimulation. Samples were harvested at 15 min after TGF-b1 stimulation. Phosphorylation of Smad 2/3 and Smad 2/3 expression in cell lysates was assessed by immunoblotting. Tubulin was used for internal control. Error bars represent mean AE SEM (n = 3 for each group). Statistical analysis was performed by ordinary one-way ANOVA and Tukey's test for Smad 2, Kruskal-Wallis test and Dunnett's T3 test for Smad 3. Three independent experiments were performed.
Source data are available online for this figure.   A Western blot analysis of mouse Fstl1 protein in cell lysates and secreted from cardiomyocytes and cardiac fibroblasts. Neonatal rat cardiomyocytes and cardiac fibroblasts were infected with adenovirus encoding mouse Fstl1 (50 MOI) for 24 h. Culture media was changed from FBS 10% contained DMEM/F-12 to 0% FBS for CM and 0.5% FBS for FB. Cells were cultured with or without tunicamycin (1 lg/ml) for 16 h. Conditioned media was concentrated by Amicon Ultra filter 10k device (14,000× g, 10 min). Mouse Fstl1 protein was detected by Western blotting. B Molecular size of multiple Fstl1 recombinant proteins was assessed by Western blotting. Detailed information for each recombinant protein is listed in Table EV5. Equal amount of proteins (5 ng/lane) were loaded to 4-12% TGX gel and transferred to PVDF membrane. Fstl1 proteins were detected using human and mouse Fstl1 polyclonal antibodies (both from R&D Systems). C Bioactivity of Fstl1 recombinant proteins from different cell sources was assessed using a cardiac fibroblast migration assay. This scratch assay was performed as described in the main manuscript. Error bars represent mean AE SEM (n = 10 for each group). Statistical analysis was performed by ordinary one-way ANOVA and Fisher's LSD test for post hoc analysis.
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