Inactivation of Sox9 in fibroblasts reduces cardiac fibrosis and inflammation

Inactivation of Sox9 in fibroblasts reduces cardiac fibrosis and inflammation Gesine M. Scharf, … , Johann Bauersachs, Joerg Heineke JCI Insight. 2019;4(15):e126721. https://doi.org/10.1172/jci.insight.126721. Fibrotic scarring drives the progression of heart failure after myocardial infarction (MI). Therefore, the development of specific treatment regimens to counteract fibrosis is of high clinical relevance. The transcription factor sex-determining region Y box 9 (SOX9) functions as an important regulator during embryogenesis, but recent data point toward an additional causal role in organ fibrosis. We show here that SOX9 is upregulated in the scar after MI in mice. Fibroblast-specific deletion of Sox9 ameliorated MI-induced left ventricular dysfunction, dilatation, and myocardial scarring in vivo. Unexpectedly, deletion of Sox9 also potently eliminated persisting leukocyte infiltration of the scar in the chronic phase after MI. RNA-Seq from the infarct scar revealed that Sox9 deletion in fibroblasts resulted in strongly downregulated expression of genes related to extracellular matrix, proteolysis, and inflammation. Importantly, Sox9 deletion in isolated cardiac fibroblasts in vitro similarly affected gene expression as in the cardiac scar and reduced fibroblast proliferation, migration, and contraction capacity. Together, our data demonstrate that fibroblast SOX9 functions as a master regulator of cardiac fibrosis and inflammation and might constitute a novel therapeutic target during MI. Research Article Cardiology Find the latest version: http://jci.me/126721/pdf 1insight.jci.org https://doi.org/10.1172/jci.insight.126721 RESEARCH ARTICLE Authorship note: GMS and KK contributed equally to this work. Conflict of interest: The authors have declared that no conflict of interest exists. Copyright: © 2019, American Society for Clinical Investigation. Submitted: December 10, 2018 Accepted: July 9, 2019 Published: July 16, 2019. Reference information: JCI Insight. 2019;4(15):e126721. https://doi.org/10.1172/jci. insight.126721. Inactivation of Sox9 in fibroblasts reduces cardiac fibrosis and inflammation Gesine M. Scharf,1,2 Katja Kilian,1 Julio Cordero,3 Yong Wang,1,4 Andrea Grund,1,2 Melanie Hofmann,1 Natali Froese,1 Xue Wang,1 Andreas Kispert,5 Ralf Kist,6 Simon J. Conway,7 Robert Geffers,8 Kai C. Wollert,1,4 Gergana Dobreva,3,9 Johann Bauersachs,1 and Joerg Heineke1,2,9 1Department of Cardiology and Angiology, Hannover Medical School, Hannover, Germany. 2Department of Cardiovascular Research and 3Department of Anatomy and Developmental Biology Center for Biomedicine and Medical Technology Mannheim, European Center for Angioscience, Medical Faculty Mannheim of Heidelberg University, Mannheim, Germany. 4Division of Molecular and Translational Cardiology, Department of Cardiology and Angiology, and 5Institute of Molecular Biology, Hannover Medical School, Hannover, Germany. 6Institute of Genetic Medicine, Faculty of Medical Sciences, and Centre for Oral Health Research, School of Dental Sciences, Newcastle University, Newcastle upon Tyne, United Kingdom. 7HB Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana, USA. 8Helmholtz Center for Infection Research, Braunschweig, Germany. 9German Center for Cardiovascular Research partner site Mannheim/Heidelberg, Heidelberg, Germany. Introduction Chronic heart failure (CHF) emerges most commonly after myocardial infarction (MI) in the Western world (1). Despite some recent advances in treatment strategies, the mortality of patients with CHF remains unacceptably high. Cardiac fibrosis is a major driver of disease progression in CHF (2). Although the deposition of extracellular matrix (ECM) is needed to replace dead cardiomyocytes after MI and to prevent ventricular rupture, excessive fibrosis causes large infarct scars resulting in cardiac dilatation and reduced cardiac function (2–4). Within the first days after MI, scar formation coincides with the reduction of leukocyte infiltration and inflammation, which is initially needed to clear necrotic cardiomyocytes (5). Adequate termination of the inflammatory response, however, is required in the myocardium to prevent left ventricular dilatation, which (besides large MI size) is the main contributor to heart failure development and poor prognosis (6). Although initial infarct size after MI can be therapeutically reduced by immediate percutaneous coronary interventions to restore patency of the infarcted coronary vessel, ECM deposition and related scar expansion as well as persisting myocardial inflammation cannot be targeted by current therapies (6, 7). In this regard, it would be especially desirable to modulate myocardial scar tissue in a way that reduces matrix deposition and inflammatory signals. However, neither the predominantly responsible cell type(s) nor lineage-restricted nodal molecular regulators that could be addressed by future therapies are well defined. Fibrotic scarring drives the progression of heart failure after myocardial infarction (MI). Therefore, the development of specific treatment regimens to counteract fibrosis is of high clinical relevance. The transcription factor sex-determining region Y box 9 (SOX9) functions as an important regulator during embryogenesis, but recent data point toward an additional causal role in organ fibrosis. We show here that SOX9 is upregulated in the scar after MI in mice. Fibroblast-specific deletion of Sox9 ameliorated MI-induced left ventricular dysfunction, dilatation, and myocardial scarring in vivo. Unexpectedly, deletion of Sox9 also potently eliminated persisting leukocyte infiltration of the scar in the chronic phase after MI. RNA-Seq from the infarct scar revealed that Sox9 deletion in fibroblasts resulted in strongly downregulated expression of genes related to extracellular matrix, proteolysis, and inflammation. Importantly, Sox9 deletion in isolated cardiac fibroblasts in vitro similarly affected gene expression as in the cardiac scar and reduced fibroblast proliferation, migration, and contraction capacity. Together, our data demonstrate that fibroblast SOX9 functions as a master regulator of cardiac fibrosis and inflammation and might constitute a novel therapeutic target during MI. 2 insight.jci.org https://doi.org/10.1172/jci.insight.126721 RESEARCH ARTICLE Resident cardiac fibroblasts are considered the main homeostatic producer of ECM in the heart, although other cells, for example cardiomyocytes, are also able to make and secrete collagens (8). In response to pathological overload or ischemic injury, quiescent fibroblasts become activated, proliferate, migrate, and differentiate toward a pathological myofibroblast phenotype, which deposits excess ECM and exerts contractile properties (8). Sex-determining region Y box 9 (SOX9) is a transcription factor that plays an essential role during mammalian development, where it crucially regulates chondrogenesis and sex differentiation (9–12). Heterozygous mutations of the SOX9 gene lead to the phenotype of campomelic dysplasia, a human skeletal malformation syndrome with XY sex reversal that is usually lethal within the neonatal period (13, 14). As an important regulator of ECM genes, SOX9 plays a significant role in the pathogenesis of various fibrotic diseases, such as liver fibrosis, glomerulosclerosis, and heart valve calcification (15–17). Recently, SOX9 was suggested for the first time as a potential regulator of cardiac fibrosis in mice after ischemia/reperfusion injury (18). The functional relevance of SOX9 in pathological cardiac fibrosis, the cardiac cell type it acts in to promote ECM deposition, as well as its effect on the entire fibroblast transcriptome, however, remain currently unknown. Here, we describe that fibroblast-specific downregulation of Sox9 reduces ECM deposition in a mouse model of MI in vivo and that it prevents the activation toward a proliferative and migrating fibroblast phenotype in cell culture in vitro. In addition, we demonstrate that reduced Sox9 expression in fibroblasts ameliorates persisting inflammation within the infarct scar, prevents cardiac dilatation, and improves cardiac function after MI. Results Sox9 is expressed in cardiomyocytes and cardiac fibroblasts and is upregulated after MI in mice. Immunofluorescence staining for SOX9, platelet-derived growth factor receptor–α (PDGFR-α, a marker for cardiac fibroblasts) (19), and cardiac troponin T (as a cardiomyocyte marker) showed expression of SOX9 in nuclei of cardiac fibroblasts and cardiomyocytes in adult mouse ventricles (Figure 1A). Quantification of mRNA levels in isolated cardiomyocytes and isolation of cardiac fibroblasts by quantitative PCR (qPCR) from uninjured mouse hearts confirmed Sox9 expression in both cell types, although mRNA levels of Sox9 were markedly higher in fibroblasts compared with cardiomyocytes (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.126721DS1). Analysis of SOX9 by immunoblotting showed a significant upregulation of SOX9 in the infarct zone early after MI (day 3), which partially decreased over time in the scar region 7 and 28 days after MI but remained significantly elevated compared with uninjured sham hearts or the remote zone after MI. In contrast, ventricular samples from the remote area of the same MI-injured mice showed no significant difference in SOX9 protein levels compared to sham-operated mice (Figure 1, B–F). To investigate the functional relevance of Sox9 in activated fibroblasts in vivo, B6;129Sv-Sox9tm1Gsr (Sox9fl/fl) mice were bred with mice expressing Cre recombinase linked to the periostin (Postn) promoter to obtain mice with specific Sox9 knockout in activated fibroblasts (PostnCre/+ Sox9fl/fl; for short, Sox9fl-Per– Cre; Figure 2A). Postn is expressed in various structures under normal conditions during cardiac development (e.g., in the developing cardiac cushions), but its expression in quiescent fibroblasts or cardiomyocytes of the uninjured adult heart is negligible. However, the Postn-Cre allele is robustly upregulated in adult activated fibroblasts following cardiac injury (20–24). Accordingly, immunofluorescence staining of SOX9 in the infarct area of Sox9fl-Per–Cre mice 7 days after MI revealed a marked reduction of SOX9+ fibroblasts (stained with PDGFR-α) compared with littermate controls (Sox9fl/fl; for short, Sox9fl/fl; see Figure 2, B and C). Moreover, immunoblot analysis of Sox9fl-Per–Cre mice after MI revealed a marked reduction of SOX9 protein levels compared with control mice only in the cardiac scar but not in the remote region of the myocardium (Figure 2, D–F). Because the scar is composed mainly of fibroblasts with only a few residual myocytes, these results suggested decreased expression of SOX9 in fibroblasts within the scar region after MI, but not in the remote area, which predominantly (at least with regard to cell volume) consists of cardiomyocytes. Hence, Sox9fl-Per–Cre mice exerted reduced fibroblast SOX9 levels within the scar but not in the cardiomyocyte compartment in the remote myocardium. Fibroblast-specific Sox9 deletion improves cardiac function after MI. To analyze the impact of fibroblast SOX9 after MI, Sox9fl-Per–Cre and Sox9fl/fl (control mice) underwent left anterior descending coronary artery (LAD) ligation or sham surgery. After 42 days, we found no difference in heart weight/body 3 insight.jci.org https://doi.org/10.1172/jci.insight.126721 RESEARCH ARTICLE weight (HW/BW) ratio between Sox9fl-Per–Cre and control mice, while it significantly increased in both genotypes after MI versus sham-operated mice (Figure 3A). The lung weight/body weight (LW/BW) ratio was not different between Sox9fl-Per–Cre and control mice, and there was no difference between the MI and sham cohorts, indicating that the animals were in a compensated state of heart failure without pulmonary edema (Figure 3B). Transthoracic echocardiography revealed a significantly better systolic cardiac function (measured as left ventricular ejection fraction) of the Sox9fl-Per–Cre compared with control mice 7 days after MI, which was maintained until 42 days after surgery (Figure 3, C and D). Moreover, the left ventricular chambers of Sox9fl-Per–Cre mice were significantly less dilated (i.e., exerted a reduced LVEDA) 7 and 42 days after MI compared with control mice (Figure 3, E and F). No difference occurred between both genotypes at any time point after sham surgery, and no anatomical abnormalities were observed in Sox9fl-Per–Cre mice. Deletion of Sox9 in fibroblasts in vivo reduces myocardial scarring and fibroblast proliferation after MI. To identify the underlying cause for the preservation of cardiac function after MI upon Sox9 deletion in fibroblasts, we quantified the extent of myocardial scar formation in hearts 7 days after MI. Masson’s trichrome staining revealed significantly smaller myocardial scar areas in Sox9fl-Per–Cre compared with control mice (Figure 4, A and B). Immunofluorescence staining for the proliferation marker Ki67 with PDGFR-α as a fibroblast marker suggested reduced fibroblast proliferation in the scars of Sox9fl-Per–Cre compared with control mice 7 days after MI (Figure 4, C and D). To investigate whether Figure 1. Cardiac SOX9 protein level after MI. Adult wild-type mice underwent MI by permanent ligation of the left anterior descending artery. (A) Representative immunofluorescence images show SOX9 (green) in cardiac fibroblasts (stained with PDGFR-α, red) and cardiomyocytes (stained with troponin T, red) in the remote area; nuclei are stained with DAPI (blue). Arrows indicate SOX9+ cardiac fibroblasts; arrowheads indicate SOX9+ cardiomyocytes. Scale bar: 50 μm. (B–F) Western blot analysis (B) and densitometric quantification (C–F) of SOX9 protein level in heart tissue of the remote or infarct area and sham controls isolated from wild-type mice 3, 7, or 28 days after sham operation or MI. Ponceau S (range 40–55 kDa) was used as a loading control. For comparison of SOX9 protein level in the infarct area at different time points (F), data were normalized to 3-day sham as a reference. Data are shown as mean ± SEM. n = 3–4 mice/group; 1-way ANOVA with Tukey’s multiple-comparisons test; *P < 0.05, ***P < 0.001, ****P < 0.0001. 4 insight.jci.org https://doi.org/10.1172/jci.insight.126721 RESEARCH ARTICLE Sox9 deletion prevents the formation of myofibroblasts, we performed immunofluorescence staining for smooth muscle actin (α-SMA) in infarct hearts of Sox9fl-Per–Cre compared with control mice. We found a clear increase of α-SMA deposition in the infarct area of Sox9fl-Per–Cre mice as well as littermate controls compared with the remote zone, with a trend toward higher α-SMA deposition in the infarct area of control mice, although a high variability of staining intensity existed within the scars and between mice (Figure 4E). Quantification of Acta2 mRNA level in the remote and infarct area of Sox9fl-Per–Cre and control mice 7 days after MI confirmed the increase of Acta2 in the infarct area compared with control. Although there was a trend toward lower Acta2 mRNA level in the infarct tissue of Sox9fl-Per–Cre compared with control mice, we did not find a significant reduction of Acta2 mRNA levels upon Sox9 deletion in our in vivo model (Figure 4F). Deletion of Sox9 in vitro reduces proliferation, migration, and gel contraction of cardiac fibroblasts but has no direct effect on α-SMA formation. To investigate the direct effects of Sox9 deletion on the phenotype of cardiac fibroblasts, we extracted fibroblasts from hearts of adult Sox9fl/fl mice and treated them with adenoviruses expressing Cre recombinase (AdCre) or control virus (Adβ-gal). qPCR and Western blotting revealed highly efficient downregulation of Sox9 mRNA (by about 90%) and protein expression (by about 60%) in AdCre-treated fibroblasts in vitro (Supplemental Figure 1B and Figure 5, A and B). Cell proliferation was assessed with a BrdU incorporation immunoassay, which revealed significantly less proliferation in Sox9-deficient fibroblasts compared with control cells (Figure 5C), thereby confirming the in vivo results shown in Figure 4, C and D. To measure the fibroblast contractile capacity, which is a hallmark of cell differentiation into myofibroblasts, the cells were infected with AdCre or Adβ-gal and Figure 2. Fibroblast-specific Sox9 deletion in vivo. (A) Schematic illustration showing the interbreeding of B6;129Sv-Sox9tm1Gsr (Sox9fl/fl) with Tg(Postn-Cre)1Sjc (Per-Cre) mice to obtain mice with specific Sox9 deletion in activated cardiac fibroblasts (Sox9fl-Per–Cre). (B and C) Representative IHC images (B) and quantification (C) of SOX9+ fibroblasts from the scar area of Sox9fl/fl and Sox9fl-Per–Cre heart tissue 7 days after MI by permanent ligation of the left anterior descending artery showing SOX9 (green) in cardiac fibroblasts (stained for PDGFR-α, red); nuclei are stained with DAPI (blue). Scale bars: 50 μm. A total of n = 6 hearts per group were stained, and at least 4 images per heart were analyzed for C. (D–F) Western blot analysis (D) and densitometric quantification (E and F) illustrating a highly efficient deletion of SOX9 protein from Sox9fl-Per–Cre mouse cardiac scar tissue 42 days after MI. SOX9 protein levels in remote samples after MI show no significant differences. GAPDH was used as a loading control. Data are shown as mean ± SEM. n = 3 mice/group. Two-tailed Student’s t test was used for comparison of 2 groups; *P < 0.05, **P < 0.01. 5 insight.jci.org https://doi.org/10.1172/jci.insight.126721 RESEARCH ARTICLE poured into a collagen gel matrix. After 48 hours, contraction was quantified as the difference between the gel area after 48 hours and 0 hours, which showed that gel matrices with Sox9-deficient fibroblasts were significantly less contracted (indicating less myofibroblast abundance) than the control samples (Figure 5D). Moreover, cell migration was analyzed under baseline conditions or after stimulation with angiotensin II, TGF-β1, or FBS. The migration level, quantified as closure of a defined scratch area within 24 hours, was reduced in Sox9-deficient cardiac fibroblasts under baseline conditions as well as after stimulation with angiotensin II, TGF-β1, or FBS (Figure 5, E–H). Overall, we found that Sox9 deficiency causes less active cardiac fibroblasts compared with control cells in vitro. Next, we analyzed the formation of stress fibers as a key marker of myofibroblast differentiation in isolated cardiac fibroblasts with Sox9 deletion compared with control cells, under baseline conditions or after stimulation with TGF-β1 for 24 hours to induce myofibroblast differentiation. Unexpectedly, fluorescence staining for filamentous actin with phalloidin and consecutive quantification of stress fiber–positive fibroblasts failed to reveal differences between Sox9-deficient cardiac fibroblasts and control cells (Supplemental Figure 1, C and D). Moreover, mRNA levels of key myofibroblast differentiation markers, particularly Acta2 and others (Fmn1, Cnn1, Tagln), were similarly expressed between Sox9-deficient cardiac fibroblasts and control cells (Supplemental Figure 1, E–H). Fibroblast SOX9 controls ECM formation and inflammation in the chronic stage after MI in vivo. Next, we investigated the effect of fibroblast-specific Sox9 deletion in the late phase after MI when the formation of the chronic scar is completed and myofibroblasts are further differentiated into quiescent matrifibrocytes (25). Figure 3. Improved cardiac function of mice with fibroblast-specific Sox9 deletion after MI. Morphological analysis and functional evaluation of Sox9fl/fl and Sox9fl-Per–Cre mice 7 days and 42 days after MI by permanent ligation of the left anterior descending artery. (A and B) Quantification of the HW/BW ratio (A) shows an increase in Sox9fl/fl and Sox9fl-Per–Cre mice 42 days after MI; the LW/BW ratio (B) shows no significant differences. (C–F) Echocardiography showed an improved ejection fraction in Sox9fl-Per–Cre mice compared with Sox9fl/fl mice 7 (C) and 42 (D) days after MI. Moreover, Sox9fl-Per–Cre mice exerted less left ventricular dilatation, i.e., increased left ventricular end-diastolic area (LVEDA), in relation to Sox9fl/fl mice 7 (E) and 42 (F) days after MI. Sham-operated mice had no impairment of cardiac function. Data show mean ± SEM. n = 4 (Sox9fl/fl and Sox9fl-Per–Cre; sham), n = 7 (Sox9fl/fl; MI), and n = 11 (Sox9fl-Per– Cre; MI). Two-way ANOVA with Holm-Šídák’s multiple-comparisons test; *P < 0.05, **P < 0.01, ***P < 0.001. 6 insight.jci.org https://doi.org/10.1172/jci.insight.126721 RESEARCH ARTICLE Masson’s trichrome staining of the infarcted hearts 42 days after MI revealed that the scar area of Sox9flPer–Cre mice was still significantly smaller compared with controls at this late stage (Figure 6, A and B). To identify SOX9-dependent gene regulation in the chronic myocardial scar, we performed a genome-wide transcriptomic analysis of cardiac scar tissue from Sox9fl-Per–Cre and Sox9fl/fl mice 42 days after MI by RNA-Seq. Genes with significant differential regulation between both conditions (at least 50% upor downregulated) are demonstrated in the heatmap (Figure 6C and Supplemental Figure 2A). In the chronic infarct scar of Sox9fl-Per–Cre mice, 2611 genes were downregulated and belonged to the GO (biological process) classes cartilage development, cell chemotaxis, extracellular matrix organization, immune response, and proteolysis (Figure 6C). Highly downregulated genes in the myocardial scar of Sox9fl-Per–Cre mice included mainly collagen genes (Col2a1, Col9a2, Col11a1), inflammatory mediators (Cxcl13, Il6, Tnfsf15), and proteases (Adam32, Prss23, Capn6; other example genes are listed in Figure 6C and in Supplemental Figure 2, B–D). In the scar of Sox9fl-Per–Cre mice, 1218 genes were upregulated, which were related mainly to oxidative cellular metabolism (GO classes ATP metabolic process, tricarboxylic acid cycle, and fatty acid metabolic process), for example, Atp5b, Idh3a, and Acadl, but we also found genes of the category muscle contraction, for example, Myh6, Tnni3, and Ttn (more example genes are listed in Figure 6C) within this group. We subsequently Figure 4. Fibroblast-specific Sox9 deletion reduces the scar area and attenuates the activation of cardiac fibroblasts after MI. (A and B) Masson’s trichrome staining of representative midventricular sections (A) and quantification of the scar area (B) of Sox9fl-Per–Cre (n = 9) compared with Sox9fl/fl (n = 7) mouse hearts 7 days after MI. Scale bars: 1 mm. (C and D) Representative IHC images showing Ki67 (green) and PDGFR-α (red) as markers for fibroblast proliferation in the infarct region of Sox9fl/fl and Sox9fl-Per–Cre mouse hearts 7 days after MI (C) and quantification (D). Nuclei are shown with DAPI (blue). Arrows indicate exemplary Ki67+ fibroblasts. Scale bars: 100 μm. A total of 5 hearts per group were stained, and 4 images per heart were analyzed for D. (E) Representative IHC images showing SOX9 (green) and α-SMA (red) in the remote and infarct area of Sox9fl/fl and Sox9fl-Per–Cre mouse hearts 7 days after MI. Nuclei are shown with DAPI (blue). Representative images of 5 hearts per group are shown. Scale bars: 100 μm. (F) mRNA expression of Acta2 measured with reverse transcription PCR in samples of the remote and infarct area of Sox9fl/fl and Sox9fl-Per–Cre mouse hearts 7 days after MI. Data were normalized to Acta2 in the remote area of Sox9fl/fl mice 7 days after MI. n = 5 (Sox9fl/fl) and 6 (Sox9fl-Per–Cre). Data are shown as mean ± SEM. Student’s t test was used for comparison of 2 groups (B and D); 2-way ANOVA with Holm-Šídák’s multiple-comparisons test was used for F. P value for Acta2 in the infarct area of Sox9fl/fl and Sox9fl-Per–Cre was 0.11 (indicated as n.s. in F). **P < 0.01, ****P < 0.0001. 7 insight.jci.org https://doi.org/10.1172/jci.insight.126721 RESEARCH ARTICLE focused on the genes that were downregulated because of Sox9 deletion in Sox9fl-Per–Cre mice because for these genes direct regulation by SOX9 was more likely. To investigate whether these downregulated genes might be direct targets of SOX9, we intersected our RNA-Seq data with SOX9 ChIP-Seq results from developing mammalian chondrocytes (26). In that study, SOX9 binding regions were characterized throughout the genome, and SOX9 was found to bind close to the promoter in an indirect manner (“class I engagement,” found mostly in genes with general cellular functions) or to bind directly to DNA in enhancer regions of cartilage-related genes, which were in large part identical to the ECM genes in our study. Intersection with the downregulated genes from our data set revealed that 50.5% of the downregulated genes exerted direct SOX9 binding, whereby 43% showed class II engagement or both class I and class II engagement. GO term analysis in these 43% of downregulated genes reflected very similar GO classes (leukocyte migration, cell chemotaxis, extracellular matrix organization; Supplemental Figure 3A) as observed among all downregulated genes in the myocardial scar of Sox9fl-Per–Cre mice. KEGG pathway analysis revealed slightly more class I engagement alone but Figure 5. In vitro Sox9 deletion in cardiac fibroblasts reduces migration, proliferation, and contractility. Isolated cardiac fibroblasts from uninjured Sox9fl/fl mouse hearts were infected with adenovirus expressing Cre recombinase (AdCre) to generate in vitro Sox9 loss of function or control adenovirus (Adβ-gal). (A and B) Efficiency of SOX9 deletion is demonstrated on the protein level by Western blot (A) and densitometric quantification (B). (C) Proliferation of cardiac fibroblasts with Sox9 deletion compared with control cells was measured by BrdU incorporation (n = 24 samples/group). (D) Contraction capacity of cardiac fibroblasts quantified as area change of a collagen matrix containing fibroblasts with Sox9 deletion or control cells after 48 hours compared with baseline at 0 (n = 6 samples/group). (E–H) Migration of Sox9fl/fl fibroblasts treated with either Adβ-gal (β-gal) or AdCre (Cre) under baseline conditions (unstimulated, E) or in the presence of profibrotic stimuli angiotensin II (Ang II, F), TGF-β1 (G), or 10% FBS (H), quantified as scratch closure after 24 hours. Representative pictures are shown directly after scratch and 24 hours later; n = 12 samples/group. Scale bars: 400 μm. Data are shown as mean ± SEM. Two-tailed Student’s t test was used for comparison of 2 groups; **P < 0.01; ***P < 0.001. 8 insight.jci.org https://doi.org/10.1172/jci.insight.126721 RESEARCH ARTICLE identified otherwise similar functional classes (cytokine-cytokine receptor interaction, ECM-receptor interaction, protein digestion and absorption; Supplemental Figure 3B). Supplemental Figure 4 shows exemplary results for class I and class II binding of SOX9 to selected ECM genes (Col2a1, Col9a2, and Fn1; Supplemental Figure 4, A–C), inflammatory genes (Cxcl13 and Il6; Supplemental Figure 4; D and E), and a proteolysis gene (Mmp2; Supplemental Figure 4F) in conjunction with our RNA-Seq results. These data imply a direct interaction of SOX9 with the promoter or enhancer regions of ECM, proteolysis, or inflammatory genes identified in this study. Because RNA-Seq showed decreased expression of inflammatory mediators in the scar region of Sox9fl-Per–Cre mice, we investigated leukocyte abundance 6 weeks after MI, which was strongly diminished upon reduced fibroblast SOX9 levels both in the scar as well as in the remote area, although in the scar leukocyte abundance was generally higher in both control and Sox9fl-Per–Cre mice (Figure 7, A and B). As one example of the ECM-related gene expression, we confirmed reduced COL2A1 protein abundance in the scar of Sox9fl-Per–Cre mice by Western blot and immunofluorescence staining (Figure 7, C–E). Figure 6. Fibroblast-specific Sox9 deletion reduces the scar area and inhibits expression of proinflammatory and ECM genes after long-term MI. (A and B) Masson’s trichrome staining of representative midventricular sections (A) and quantification of the scar area (B) (n = 6–11) of Sox9fl-Per–Cre compared with Sox9fl/fl mouse hearts 42 days after MI. Scale bars: 1 mm. (C) Gene expression in scar area tissue of Sox9fl-Per–Cre compared with Sox9fl/fl mouse hearts 42 days after MI was analyzed with RNA-Seq. Heatmap of differentially regulated genes in the MI scar of Sox9fl/fl and Sox9fl-Per–Cre mice (n = 3) 42 days after MI is shown on the left of C. Gene Ontology (GO, biological process) classification of highly regulated processes and selected example genes are demonstrated on the right. 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