FAM114A1 influences cardiac pathological remodeling by regulating angiotensin II signaling

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  Figure 1. Increased FAM114A1 expression in activated myofibroblasts in human and mouse failing hearts. (A) FAM114A1 mRNA expression is increased in failing human hearts (n = 18) compared with nonfailure hearts (n = 8). 18S rRNA was used as a normalizer. (B) FAM114A1 protein expression is increased in failing human hearts (n = 20) compared with nonfailure hearts (n = 8). GAPDH protein was used as a normalizer. (C and D) FAM114A1 mRNA and protein expression is induced in the hearts from mice with Ang II infusion (4 weeks) compared with hearts from vehicle-treated mice (n = 6 for both groups). 18S rRNA and Gapdh mRNA were used as normalizers for mRNA and protein quantification, respectively. (E and F) IF analysis and quantification of FAM114A1 protein expression in human failing hearts and Ang II–treated mouse heart tissue sections. n = 100–115 cells were counted from multiple sections from murine hearts (n = 6). Scale bar: 10 μm (E); 5 μm (F). (G) Fam114a1 mRNA expression is induced in the hearts from mice with MI surgery at infarct zone (IZ), border zone (BZ), and remote zone (RZ) compared with the hearts with Sham surgery (20 days) (n = 6). Data are presented as mean ± SEM. Comparisons of…

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  Figure 2. Deletion of FAM114A1 mitigates Ang II–induced cardiac remodeling and heart failure in mice. (A) Fam114a1–/– mice show reduced HW/TL (heart weight/tibia length) ratio after 4 weeks of Ang II infusion compared with WT mice. n = 6 for each group (male mice). (B) WGA staining of heart tissue sections of WT and Fam114a1–/– mice under Ang II or vehicle treatment. Cross-sectional area of CMs was measured and quantified. n = 6 hearts per group with 250–270 CMs measured per heart. Scale bar: 20 μm. (C) Picrosirius red staining of heart tissue sections of WT and Fam114a1–/– mice under Ang II or vehicle treatment. Scale bar: 1 mm. n = 6 for each group. (D) qPCR measurement of cardiac fibrosis and hypertrophy marker gene expression in WT and Fam114a1–/– hearts after 4 weeks of Ang II versus vehicle infusion. Rpl30 mRNA was used as a normalizer. n = 3 for each group. (E) Representative echocardiographic images suggest improved cardiac function in Fam114a1–/– mice compared with WT mice after Ang II or vehicle infusion. Quantification of ejection fraction (EF) and fractional shortening (FS) was shown. n = 6 for each group. Data are presented as mean ± SEM. Statistical significance was confirmed…

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  Figure 3. Deletion of FAM114A1 reduces cardiac remodeling and improves cardiac function after MI surgery. (A) Fam114a1–/– mice show reduced HW/TL ratio 20 days after MI surgery compared with WT mice. n = 6 for each group (male and female mice). (B) WGA staining of heart tissue sections of WT and Fam114a1–/– mice 20 days after MI surgery. n = 6 (each group) with 100–150 CMs in the border zone measured per heart. Scale bar: 20 μm. (C) Quantification of Picrosirius red staining of heart tissue sections of WT and Fam114a1–/– mice 20 days after MI surgery. n = 5 for each group. (D) Quantification of PDGFRα protein expression (% of the area) and percentage of PDGFRα+BrdU+ cells in BrdU+ cells in heart tissue sections of WT and Fam114a1–/– mice 20 days after MI or Sham surgery. n = 4 for each group, and ~100–150 positive cells were counted. (E) TUNEL assay for heart tissue sections (border zone) of WT and Fam114a1–/– mice 20 days after surgery. Scale bar: 20 μm. n = 5 for each group. (F) Representative echocardiographic images of WT and Fam114a1–/– mice at baseline and 2 weeks after surgery. n = 8 for each group. Quantification of EF and FS was shown. Data are presented as mean ± SEM. Statistical…

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  Figure 4. FAM114A1 deletion antagonizes Ang II–induced inflammation and oxidative stress. (A) qPCR measurement of mRNA expression levels of Il6, Tnfa, Cxcl1, and Arg1 in WT and Fam114a1–/– mice. n = 4 for each group. Gapdh mRNA was used as normalizer. (B) Representative IF images show CD45 (green) and α-SMA (red) in heart tissue sections of WT and Fam114a1–/– mice after 4 weeks of Ang II infusion. Scale bar: 20 μm. n = 5 for each group. (C) DHE staining of cultured CF cells isolated from WT and Fam114a1–/– mice. Scale bar: 100 μm. n = 4 for each group. (D) qPCR measurement of mRNA expression level of NADPH oxidase genes. Gapdh mRNA was used as a normalizer. n = 4 for each group. Data are presented as mean ± SEM. Statistical significance was confirmed by 2-way ANOVA with Tukey’s multiple-comparison tests for A–D. *P < 0.05; **P < 0.01; ***P <0.001;.****P <0.0001.

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  Figure 5. FAM114A1 deletion abolishes Ang II–induced cardiac fibroblast proliferation, myofibroblast activation, and migration. (A) Fam114a1 mRNA expression in PMCFs and CMs. Gapdh mRNA was used as normalizer. n = 4 for each group. (B) Endogenous FAM114A1 protein expression in PMCFs and CMs detected by Western blot. GAPDH protein was used as a loading control. n = 3 for each group. (C) Ang II treatment (1 μM) induces FAM114A1 mRNA and protein expression in PMCFs. n = 3 for qPCR and n = 100–120 cells per treatment for IF. 18S rRNA was used as normalizer for mRNA measurement. Scale bar: 20 μm. (D) qPCR measurement of mitotic cyclin gene expression in CFs from WT and Fam114a1–/– mice after Ang II treatment. n = 3 for each group. 18S rRNA was used as a normalizer. (E and F) Representative images of IF staining and quantification of normalized intensity of MF activation markers in CFs of WT and Fam114a1–/– mice after vehicle or Ang II treatment. n = 150–200 cells were analyzed for each group. Scale bar: 20 μm. (G) Representative images of migrating CFs from WT and Fam114a1–/– mice after vehicle or Ang II treatment. Quantification of scratch closure after 24 hours was shown. n = 6 biological…

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  Figure 6. FAM114A1 depletion stabilizes AGTRAP, enhances AT1R degradation, and influences Ang II signaling in cardiac fibroblasts. (A) Immunoprecipitation and immunoblot confirm the interaction of FAM114A1 with AGTRAP in the mouse heart tissue lysates. This experiment was repeated 3 times, and representative images are shown. (B) IF detection of overexpressed EGFP-tagged FAM114A1 and Myc-tagged AGTRAP in mouse NIH/3T3 fibroblast cells. Protein expression changes were observed after 3 hours and 24 hours after Ang II or vehicle treatment. Scale bar: 50 μm. This experiment was repeated 3 times, and representative images are shown. (C) Western blot measurement and quantification of FAM114A1, AGTRAP, and AT1R in vehicle or Ang II–treated primary CF cells isolated from WT and Fam114a1–/– mice. Separate gels were run for detecting FAM114A1 and AGTRAP compared with AT1R and GAPDH. (D) Western blot measurement and quantification of p-ERK1/2, ERK1/2, p-AKT, AKT, p-p38, and p38 in Ang II– or vehicle-treated CFs (24 hours) from hearts of WT and Fam114a1–/– mice (n = 3). (E) Representative images of IF staining and quantification of normalized intensity of MF activation markers in CFs of WT and…

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  Figure 7. ADAMTS15, a differentially regulated gene in Fam114a1-null cardiac fibroblasts, is required for CF-to-MF activation. (A) Heatmap of gene expression in CFs isolated from WT and Fam114a1–/– mice at baseline analyzed by RNA-Seq. P60 male mice, n = 3 per group, Padj < 0.05. (B) Volcano plot of differentially expressed genes in CFs isolated from WT and Fam114a1–/– mice at baseline analyzed by RNA-Seq. (C) Gene Ontology analysis of enriched pathways of downregulated genes in RNA-Seq. The top pathways are listed with enriched gene sets (cellular component). (D) qPCR validation of multiple downregulated genes in Fam114a1–/– mice derived CFs. 18S rRNA was used as a normalizer (n = 3). (E) Gene Ontology analysis of enriched pathways of upregulated genes in RNA-Seq (biological process). (F) IF quantification of COL1A1 and α-SMA protein expression after knockdown of Adamts15 in TGF-β–treated PMCFs. n = 100–120 cells from 3 biological replicates were analyzed. (G) Knockdown of Adamts15 reduced PMCF cell migration indicated by scratch assays (n = 6). Data are presented as mean ± SEM. Statistical significance was confirmed by unpaired Student t test for D and 1-way ANOVA with Tukey’s…