Fig 2: Generation of myofibroblast specific transforming growth factor ß receptor II gene (Tgfbr2) (r2)–ablated mice. A, Schematic representation of the different mouse lines used. Mice with cardiomyocyte-specific mutant a-B-crystallin expression (CryABR 120G) were crossed with Tgfbr2-loxP–containing gene targeted lines and those double transgenic (Tg) lines further crossed with mice containing the periostin locus (Postn) with a tamoxifen (TAM) regulated Mer-Cre-Mer cassette (Postnmcm) inserted into exon (E1). B, Experimental scheme where mice were fed a TAM diet starting from 6 to 8 weeks until they were euthanitized. C and D, Western blot and quantification for Tgfßr2 protein in purified fibroblasts (n=6 separate, total hearts). E and F, Expression and quantification of SMAD3 (phospho [p]-SMAD3/SMAD3) and (G and H) p38 signaling (p-p38/p38) in purified fibroblasts isolated from hearts of 3-month-old experimental cohorts fed with TAM chow (TAM+) or normal chow (-) (n=4–6 separate, total hearts). *P<0.05 vs uninduced Cre Cry ABR 120G /Postnmcm/r2 f/f animals. GAPDH indicates glyceraldehyde 3-phosphate; a-MHC, a-myosin heavy chain; SMAD3, small mothers against decapentaplegic homolog 3.

Fig 3: WWP2 regulates pro-fibrotic gene activity in primary cardiac fibroblasts. a Immunofluorescence images of LV-section staining from WT mice after AngII infusion, showing WWP2 expression (green, arrow) in non-myocytes (Left); WWP2 co-localization with FSP1-positive cells (red, arrow; Right). Scale bar: 40 µm. b t-SNE displaying LV single-cell RNA-seq data (508 cells detected) in WT mice after AngII infusion. Each dot corresponds to a single-cell, colored by Wwp2 expression level (Log2 normalized counts). Different cell subpopulations detected in mouse heart are indicated. c Primary cardiac (myo)fibroblasts were taken from LV in WT and WWP2Mut/Mut mice and modeled in vitro by incubation with TGFß1 (5 ng/ml, 72 h). d Relative mRNA expression (normalized to 18S) of ECM and pro-fibrotic marker genes (Acta2, Col1a1, Tcf21, Ctgf, Postn, and EDA-Fn) in primary cardiac (myo)fibroblasts (n = 5–6 for each group). e WB of ACTA2 and POSTN protein expression in WT and WWP2Mut/Mut cardiac (myo)fibroblasts. f Representative microscopy images (left) and quantification analysis (right) with immunostaining for ACTA2 and COL1A1 after TGFß1 stimulation in WT and WWP2Mut/Mut cardiac (myo)fibroblasts (five biological replicates, nine images each; box-and-whisker plots). g Representative WB of WWP2-FL, WWP2-N and WWP2-C isoforms expression in cardiac (myo)fibroblasts after 72 h TGFß1 stimulation. h WT cardiac (myo)fibroblasts were incubated with TGFß1 (72 h) and siRNA pools (SiRNA-Wwp2-N' and SiRNA-Wwp2-C') against 5'- or 3'-region of Wwp2 mRNA. i WT (myo)fibroblasts, SiRNAs-Wwp2-N'/C' attenuated TGFß1-induced pro-fibrotic responses, as shown by qPCR analysis of Acta2, Col1a1, and TGFß receptors (n = 5–6 for each group). j Representative WB showing the effect of siRNA-Wwp2-N' and siRNA-Wwp2-C' on each WWP2 isoform expression in WT (myo)fibroblasts and decrease of TGFß1-induced ACTA2 protein level by siRNA-Wwp2-N'. k Cultured human RV primary cardiac (myo)fibroblasts: siRNA-mediated knockdown of WWP2 reduced the expression of pro-fibrotic genes (COL1A1, COL1A2, and LUM). P1 tags both WWP2-N and WWP2-FL, P2 tags WWP2-FL only (n = 3 for each group). l Cardiac (myo)fibroblasts from WWP2Mut/Mut were transfected with either Wwp2-FL or Wwp2-N plasmid expression (separately) and incubated with TGFß1 (72 h). m, n After transfection of either Wwp2-FL or Wwp2-N, transcript levels of Acta2, Col1a1, and TGFß receptors were assessed by qPCR, in WWP2Mut/Mut (myo)fibroblasts (n = 5 for each group) and ACTA2 protein levels. These fibrogenic markers were enhanced by either Wwp2-FL or Wwp2-N transfection after 72 h TGFß1 treatment. P values calculated by Mann–Whitney U test; *P < 0.05, **P < 0.01, NS not significant; data reported as means ± SD

Fig 4: Effect of ECM maturation on aSMA expression in myofibroblasts.(A and B) Representative IHC images (A) and quantitation (B) of aSMA+ (red) Tcf21 lineage–traced (EGFP+) fibroblasts from the infarct region of hearts from Tcf21MCM/+;R26EGFP mice treated with BAPN or PBS as a control (Cont.). Nuclei are shown with DAPI (blue). Scale bars: 20 µm. Data are shown as mean ± SD (n = 3). ***P < 0.0001, 2-tailed t test. (C) Experimental scheme of tamoxifen treatment of PostnMCM/MCM;R26EGFP and PostnMCM/+;R26EGFP mice from day 2 to day 3 after MI by daily i.p. injections. Hearts were then harvested at 2 weeks after MI. (D) Representative IHC images from 3 separate hearts analyzed for Postn protein expression within the infarct region of hearts from PostnMCM/+;R26EGFP mice versus PostnMCM/MCM;R26EGFP mice 2 weeks after MI. Nuclei are shown with DAPI (blue). Scale bars: 20 µm. Postn lineage–traced cells (EGFP+) cells are also shown. (E) Representative IHC images from 3 separate hearts analyzed showing aSMA+ (red) and Postn lineage–traced (EGFP+) fibroblasts in the infarct region of hearts from PostnMCM/+;R26EGFP mice and PostnMCM/MCM;R26EGFP mice 2 weeks after MI. Nuclei are shown with DAPI (blue). Scale bars: 50 µm.

Fig 5: Periostin (Postn) is dramatically upregulated in adipose tissue during obesity. (a) mRNA expression levels of Postn in the liver and adipose tissue (epididymal fat [Epi-fat]) of C57BL/6J male mice after SD or HFD feeding for 16 weeks. n = 5 in each group. **p < 0.01 by unpaired t-test. (b) Protein levels of Postn in the liver and adipose tissue (subcutaneous fat [Sub-fat]) from diet-induced obese mice after 16 weeks of HFD feeding. n = 5, *p < 0.05, **p < 0.01 by one-way ANOVA. (c) Protein levels of Postn in adipose tissue from lean and obese mice after 16 weeks of SD or HFD feeding, respectively. n = 5, *p < 0.05 by unpaired t-test. (d) Time course of Postn and pro-inflammatory gene profiling in Epi-fat of WT SD-fed or HFD-fed mice for up to 36 weeks. n = 3 to 6, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus SD at each time point. Statistical comparisons were made by unpaired t-test.