Scleraxis And Fibrosis In The Pressureoverloaded Heart

In response to pro-fibrotic signals, scleraxis regulates cardiac fibroblast activation in vitro via transcriptional control of key fibrosis genes such as collagen and fibronectin; however, its role in vivo is unknown. The present study assessed the impact of scleraxis loss on fibroblast activation, cardiac fibrosis, and dysfunction in pressure overload-induced heart failure.

Scleraxis expression was upregulated in the hearts of non-ischemic dilated cardiomyopathy patients, and in mice subjected to pressure overload by transverse aortic constriction (TAC). Tamoxifen-inducible fibroblast-specific scleraxis knockout (Scx-fKO) completely attenuated cardiac fibrosis, and significantly improved cardiac systolic function and ventricular remodelling, following TAC compared to Scx+/+ TAC mice, concomitant with attenuation of fibroblast activation. Scleraxis deletion, after the establishment of cardiac fibrosis, attenuated the further functional decline observed in Scx+/+ mice, with a reduction in cardiac myofibroblasts. Notably, scleraxis knockout reduced pressure overload-induced mortality from 33% to zero, without affecting the degree of cardiac hypertrophy. Scleraxis directly regulated transcription of the myofibroblast marker periostin, and cardiac fibroblasts lacking scleraxis failed to upregulate periostin synthesis and secretion in response to pro-fibrotic transforming growth factor β.

Scleraxis governs fibroblast activation in pressure overload-induced heart failure, and scleraxis knockout attenuated fibrosis and improved cardiac function and survival. These findings identify scleraxis as a viable target for the development of novel anti-fibrotic treatments.

Structured Graphical Abstract

Scleraxis was required for pressure overload-induced (via transverse aortic constriction, TAC) recruitment of RNA polymerase II to pro-fibrotic genes, activation of cardiac fibroblasts to myofibroblasts, and cardiac fibrosis. Scleraxis gene deletion attenuated fibroblast activation and fibrosis to ameliorate cardiac functional loss regardless of whether deletion occurred prior to or following TAC.

See the editorial comment for this article ‘Targeting a transcriptional scler-axis to treat cardiac fibrosis’, by Alexander R. H. Hobby and Timothy A. McKinsey, /10.1093/eurheartj/ehac608.

Translational perspective

Persistent pressure overload induces extensive cardiac extracellular matrix remodelling following fibroblast activation to myofibroblasts, promoting fibrosis and subsequent heart failure. We demonstrate that scleraxis is highly expressed in the hearts of non-ischaemic dilated cardiomyopathy patients, and reveal the critical role of scleraxis in fibroblast activation in the pressure overloaded myocardium. Fibroblast-specific scleraxis knockout attenuates cardiac fibrosis, dysfunction, and remodelling, whether the deletion preceded or followed pressure overload. Scleraxis knockout also reduced death to zero from 33%. Scleraxis may be an important target for the development of novel anti-fibrosis treatments.

Cardiac fibrosis is both a pathological response to stress or injury and a natural consequence of aging characterized by excessive extracellular matrix (ECM) deposition following the activation of fibroblasts to myofibroblasts.1 Increased myocardial stiffness negatively impacts cardiac contractility, significantly increasing the risk of dysfunction and death.2 Fibrosis presence or progression in patients with dilated cardiomyopathy (DCM) predicts significantly increased all-cause mortality and major adverse cardiovascular events.3,4 Attenuation of cardiac fibrosis represents an important therapeutic target for improving outcomes in cardiovascular disease, and effective anti-fibrotic treatment is a specific knowledge gap.

Reducing cardiac fibrosis in cardiovascular disease patients remains an unrealized goal, with no currently available specific pharmacological therapies. Given the requirement for fibroblast activation in tissue fibrosis pathogenesis, preclinical studies have increasingly focused on blocking this process. The prominent role of transforming growth factor β1 (TGFβ1)/Smad signalling in fibroblast activation has made this pathway an interventional target, with some degree of success as its attenuation reduces fibrosis.5,6 However, the multiple roles of this pathway in tissues throughout the body increase the risk of off-target effects.7

We have shown that scleraxis, a transcription factor, plays a key role in cardiac fibroblast activation. Scleraxis is downstream of TGFβ/Smad-dependent and independent pro-fibrotic signalling, responds to mechanical stress to govern fibroblast activation, and regulates fibrillar collagen 1α2 (Col1α2), the fibronectin ED-A splice variant (EDA-Fn), α-smooth muscle actin and matrix metalloproteinase 2 (MMP2) gene expression.8–10 Scleraxis loss prevents cardiac fibroblast activation in vitro.8,9 While scleraxis expression is elevated in the rat cardiac infarct scar, and germline scleraxis deletion reduces cardiac fibroblast numbers, the role of scleraxis in cardiac fibrosis is unclear.8 Here, we examine the role of scleraxis in pressure overload-induced cardiac fibrosis and dysfunction.

Detailed methods are described in the Supplementary material online.

Scleraxis expression is elevated in human dilated cardiomyopathy and cardiac pressure overload in mice
Cardiac fibrosis is a common hallmark of DCM.3 Human healthy (NFC) and non-ischaemic DCM left ventricular heart samples (see Supplementary material online, Table S1) were analysed for scleraxis, type I and type III collagen expression, since scleraxis regulates collagen synthesis.8,11 These DCM patients presented with heart failure driven by systolic dysfunction including reduced ejection fraction and left ventricular (LV) dilation.12,13 Immunoblot revealed increased levels of scleraxis and both collagens in DCM samples compared to NCM (Figure 1A and B). Immunofluorescent labelling of cardiac tissues demonstrated that the percentage of scleraxis-positive nuclei doubled in DCM vs. non-failing control (Figure 1C).

Scleraxis expression is elevated in human dilated cardiomyopathy. (A, B) Non-ischaemic human dilated cardiomyopathy (DCM) and non-failing control (NFC) cardiac samples were assayed for expression of scleraxis and fibrillar collagen types 1 and 3 by western blot, n = 8 per group. (C) Immunofluorescence labelling of human cardiac tissue sections to identify scleraxis-positive nuclei (red vs. blue DAPI-labelled nuclei) in dilated cardiomyopathy vs. non-failing control samples (n = 4 per group, 3–4 fields per heart), with a dilated cardiomyopathy negative control lacking primary antibody; *P P P P The murine pressure overload model similarly presents with reduced ejection fraction and extensive remodelling including dilation, hypertrophy, and fibrosis. Transgenic mice harbouring a green fluorescent protein (GFP) reporter under the control of the scleraxis promoter14 were subjected to pressure overload by transverse aortic constriction (TAC) for 4 weeks. Compared to sham-operated controls, normalized heart weight was significantly elevated in TAC mice (see Supplementary material online, Figure S1A). TAC resulted in a nearly 8-fold increase in GFP expression and a significant increase in the myofibroblast marker periostin (Postn) (see Supplementary material online, Figure S1B). Cardiac tissue sections exhibited minimal GFP staining in sham hearts consistent with western blots but showed diffuse interstitial cell staining throughout the myocardium in TAC hearts similar to the pattern observed for fibrosis following TAC (see Supplementary material online, Figure S1C). Scleraxis is thus expressed at relatively low levels in the non-stressed myocardium and increases in both the human and mouse myocardium in conditions marked by cardiac fibrosis. Scleraxis gene deletion attenuates pressure overload-induced cardiac fibrosis Activation of fibroblasts to myofibroblasts is characteristic of cardiac fibrosis following pressure overload, leading to heart failure.15 We hypothesized that restricting fibroblast activation would limit cardiac fibrosis. We previously showed in vitro that scleraxis is both necessary and sufficient for stretch- or TGFβ1-mediated activation of fibroblasts to myofibroblasts.8,9 To determine the role of scleraxis in vivo, we employed tamoxifen-inducible cardiac fibroblast-specific scleraxis gene deletion in TAC-induced cardiac fibrosis. Adult animals underwent scleraxis gene deletion (Scx-fKO) or were left intact (Scx+/+) and then were subjected to sham or TAC surgery (Figure 2A). Following 8 weeks of pressure overload, Scx+/+ and Scx-fKO TAC animals exhibited similar levels of cardiac hypertrophy (Figure 2B; Supplementary material online, Figure S2A and S2B) with no evidence of hyperplasia (see Supplementary material online, Figure S2C); thus, scleraxis gene deletion does not impact hypertrophy in this model. Scleraxis gene deletion prevents pressure overload-induced cardiac fibrosis. (A) Study design schematic indicating timing of administration of tamoxifen (Tam) to induce scleraxis gene deletion or corn oil control, echocardiography (E), and pressure overload (transverse aortic constriction, TAC) or sham surgery. Tamoxifen administration to induce scleraxis deletion preceded surgery by one week, with the study ending at eight weeks post-surgery. (B) Trichrome and picrosirius red staining for cardiac fibrosis in control (Scx+/+) and fibroblast-specific scleraxis knockout sham or transverse aortic constriction hearts. Scale bar = 1 mm (25×) or 0.25 mm (100×). (C) Quantification of fibrosis area, n = 4 per group. (D) mRNA expression of Col1α1, Col1α2, Col3α1, and EDA-Fn, n = 10 per group. (E) Protein expression of collagen I, EDA-Fn and periostin (Postn) normalized to α-tubulin, n = 4–5 per group. (F) Chromatin immunoprecipitation using RNA Pol II-bound periostin or Col1α2 promoter, n = 3–4 per group. (G) Immunofluorescence imaging of periostin-positive cells (red vs. blue DAPI-labelled nuclei) in the myocardium of sham or transverse aortic constriction hearts. Scale bar = 50 μm. (H) Quantification of periostin-positive cells, n = 3 per group. (I) Representative images of echocardiographic assessment of left ventricular ejection fraction and fractional shortening with quantification, n = 10 per group; *P P P P +/+ Sham, ##P P P +/+ transverse aortic constriction. Masson’s trichrome and picrosirius red staining revealed extensive cardiac fibrosis in Scx+/+ TAC mice which was completely attenuated in Scx-fKO mice (Figure 2B and 2C; Supplementary material online, Figure S2D). Our previous in vitro studies demonstrated that scleraxis regulated the expression of cardiac fibrillar collagens Col1α1, Col1α2, Col3α1, and myofibroblast marker genes such as EDA-Fn, vimentin, and Postn.8 Col1α1, Col1α2, and Col3α1 were significantly elevated in Scx+/+ TAC mice, but not Scx-fKO, while EDA-Fn was significantly lower in Scx-fKO mice regardless of treatment (Figure 2D). Similarly, type I collagen, EDA-Fn, and Postn protein expression were elevated by TAC in Scx+/+ but not Scx-fKO mice (Figure 2E). Scleraxis is thus required for TAC-induced cardiac fibrosis. Scleraxis facilitates the assembly of transcription complexes at target gene promoters such as Col1α2.8 In response to TAC, RNA polymerase II was recruited to the Col1α2 and periostin promoters in Scx+/+ cardiac lysates (Figure 2F). In contrast, this recruitment did not occur in Scx-fKO hearts, and RNA polymerase II occupancy of the periostin promoter was effectively absent following TAC. Periostin is a key marker for cardiac myofibroblasts in fibrosis and not expressed in cardiac fibroblasts, thus we assessed fibroblast-to-myofibroblast activation by periostin immunolabeling.16 Periostin-positive cells significantly increased in number following TAC in Scx+/+ hearts, but not in Scx-fKO (Figure 2G and 2H). Thus, scleraxis deletion in cardiac fibroblasts attenuated fibrosis and myofibroblast production concomitant with failure to recruit RNA polymerase to fibrosis-associated genes Col1α2 and Postn following TAC, agreeing with our previous in vitro data showing a requirement for scleraxis in fibroblast activation.8 Systolic dysfunction is a feature of pressure overload.17,18 We confirmed that 8 weeks of pressure overload negatively impacted LV ejection fraction and fractional shortening in Scx+/+ hearts (Figure 2I). Both functional measures were significantly improved in Scx-fKO mice, and a similar trend was noted in endocardial systolic velocity (see Supplementary material online, Figure S2E). Cardiac dilation was induced by TAC: LV internal diameter at both end diastole and end systole was increased in Scx+/+ mice, but remained unchanged in Scx-fKO mice (see Supplementary material online, Figure S2F). Interestingly, an increase in LV posterior wall and septal thickness during diastole was observed only in Scx-fKO mice after TAC (Figure S2G), suggesting a remodelling process unique to these animals. Scleraxis deletion therefore improved cardiac systolic function and remodelling following TAC, ostensibly by preventing fibrosis. We measured early (E) and late (A) ventricular filling velocities, the E/A ratio, and mitral valve (MV) deceleration time to gain insight into potential diastolic functional changes. While A showed a non-significant trend to reduction following TAC in Scx+/+ mice, E was significantly reduced; neither measure was altered by TAC in Scx-fKO mice (see Supplementary material online, Figure S2H). E/A ratio was unaltered by TAC regardless of genotype (see Supplementary material online, Figure S2I); however, the reduction of E suggests that pseudonormalization occurred in Scx+/+ mice.19 A decrease in MV deceleration was significant only in Scx-fKO mice after TAC (see Supplementary material online, Figure S2J). Heart rate was not altered by TAC surgery or genotype (see Supplementary material online, Figure S2K). Thus, in this model, diastolic dysfunction after TAC was relatively modest, and the impact of scleraxis deletion on diastolic dysfunction was variable. Scleraxis deletion reverses cardiac fibrosis after established pressure overload The prevention of cardiac fibrosis by scleraxis deletion prior to TAC implicates scleraxis in the initiation of pathology; however, it is unclear if scleraxis impacts ongoing maintenance and expansion of cardiac fibrosis. We thus subjected mice to TAC or sham surgery to develop cardiac dysfunction over 4 weeks. Animals then received either tamoxifen to initiate cardiac fibroblast-specific scleraxis gene deletion or corn oil carrier alone to leave the gene intact and were followed for an additional 4 weeks (Figure 3A). After 8 weeks of pressure overload, cardiac hypertrophy developed similarly in Scx+/+ and Scx-fKO mice (see Supplementary material online, Figure S3A and S3B). Scx+/+ mice exhibited an increased lung-to-body weight ratio suggestive of congestion, but not Scx-fKO mice. Scleraxis deletion reverses established pressure overload-induced cardiac fibrosis. (A) Study design schematic indicating timing of administration of tamoxifen (Tam) to induce scleraxis gene deletion or corn oil control, echocardiography (E), and pressure overload (transverse aortic constriction, TAC) or sham surgery. Surgery preceded scleraxis deletion by four weeks, with four additional weeks to study end after knockout. (B) Trichrome and picrosirius red staining for cardiac fibrosis in control (Scx+/+) and fibroblast-specific scleraxis knockout sham or transverse aortic constriction hearts. Scale bar = 1 mm (25×) or 0.25 mm (100×). (C) Quantification of fibrosis area, n = 5 per group. (D) Protein expression of collagen 1α1, 1α2, EDA-Fn and periostin normalized to β-actin, n = 3–6 per group. (E) Immunofluorescence imaging of periostin-positive cells (red vs. blue DAPI-labelled nuclei) in the myocardium of sham or transverse aortic constriction hearts. Scale bar = 50 μm; (F) quantification of periostin-positive cells, n = 4 per group. (G) Immunofluorescence imaging of terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive cells (red vs. blue DAPI-labelled nuclei) in the myocardium of sham or transverse aortic constriction hearts, with quantification, n = 4–5 per group. Scale bar = 150 μm. (H) mRNA expression of caspase 3 and Bcl2 in P1 cardiac fibroblasts following treatment with adenovirus encoding shRNA targeting LacZ (AdshLacZ) or scleraxis (AdshScx), n = 3 per group. (I) Representative images of echocardiographic assessment of left ventricular ejection fraction and fractional shortening with quantification, n = 8–11 per group; *P P P P +/+ Sham or AdshLacZ, #P P P +/+ transverse aortic constriction, and P +/+ transverse aortic constriction at midpoint and endpoint, NS = not significant between fibroblast-specific scleraxis knockout transverse aortic constriction at midpoint and endpoint. Masson’s trichrome and picrosirius red sections of Scx+/+ TAC hearts displayed significant fibrosis after 8 weeks of pressure overload (Figure 3B). In contrast, Scx-fKO TAC hearts showed fibrosis comparable to sham controls (Figure 3B and 3C; Supplementary material online, Figure S3C). Type I collagen and EDA-Fn were upregulated by TAC in Scx+/+ but not Scx-fKO mice, and periostin was significantly reduced in Scx-fKO mice (Figure 3D). Periostin-positive cell number was highly increased after TAC in Scx+/+ but not Scx-fKO hearts (Figure 3E and 3F). Scleraxis is thus required for maintaining cardiac fibrosis following TAC. Myofibroblasts may be cleared from sites of damage or stress through apoptosis, but in the myocardium where tissue repair is blunted by cardiomyocyte terminal differentiation, myofibroblasts can persist indefinitely. We thus examined whether scleraxis deletion following pressure overload alters apoptosis in the heart by TUNEL labelling. Apoptosis was marginal in sham Scx-fKO mice and similarly low in all Scx+/+ samples (Figure 3G). In contrast, Scx-fKO TAC mice exhibited significantly increased apoptosis. Knockdown of scleraxis in activated cardiac fibroblasts by shRNA significantly increased expression of pro-apoptotic caspase 3 while decreasing expression of anti-apoptotic Bcl2 (Figure 3H). While identification of apoptotic cells in Scx-fKO TAC hearts was not performed, these results coupled with the virtually complete loss of periostin-positive myofibroblasts (Figure 3E and 3F) suggest the possibility that scleraxis may attenuate myofibroblast apoptosis. Cardiac function was assessed at the 4-week midpoint (prior to scleraxis deletion) and again at the 8-week endpoint post-TAC (Figure 3A). LV ejection fraction and fractional shortening were similar at the midpoint, but diverged at the endpoint as systolic function continued to decline in Scx+/+ mice while plateauing in Scx-fKO mice (Figure 3I). Notably, ejection fraction at the endpoint was significantly lower than midpoint in Scx+/+ but not Scx-fKO TAC mice. A similar trend was observed for endocardial systolic velocity (see Supplementary material online, Figure S3D). Chamber dimensions LVIDd, LVIDs, LVPWd, and LVSd were similar between Scx+/+ and Scx-fKO throughout the study, although a non-significant divergence in LVIDd and LVIDs was noted, with a significant increase in LVSd in Scx-fKO mice but not Scx+/+ (see Supplementary material online, Figure S3E–S3H). The diastolic functional parameters E and A showed a similar trend: values for Scx+/+ and Scx-fKO mice were similar at midpoint but diverged at endpoint, with Scx-fKO mice showing better numbers (see Supplementary material online, Figure S3I and S3J). E/A remained unchanged throughout the study (see Supplementary material online, Figure S3K), again suggesting pseudonormalization. MV deceleration time exhibited significant decreases with time post-TAC, but without a significant difference between Scx+/+ and Scx-fKO mice (see Supplementary material online, Figure S3L), and there was no difference in heart rate (see Supplementary material online, Figure S3M). Together, these data demonstrate that, in the setting of pre-existing cardiac remodelling due to TAC, scleraxis deletion ameliorates systolic dysfunction while reducing fibrosis. Heart failure and mortality are common in the TAC model at extended time points.17,20 To establish the beneficial effect of long-term scleraxis deletion in the setting of TAC, we repeated our study with follow-up to 12 weeks following pressure overload (Figure 4A). In surviving Scx+/+ and Scx-fKO mice, hypertrophy was similar at 12 weeks (see Supplementary material online, Figure S4A). In contrast, while LV fractional shortening was similar between Scx+/+ and Scx-fKO mice at 12 weeks post-TAC, LV ejection fraction was significantly improved by scleraxis deletion (Figure 4B). Notably, while Scx+/+ mortality commenced at 8 weeks post-TAC in our model, resulting in 67% survival by 12 weeks (8/12 surviving), no animals were lost in the Scx-fKO group (11/11 surviving; Figure 4C); thus, scleraxis deletion improved survival. No significant differences were noted in chamber dimensions (see Supplementary material online, Figure S4B and S4C) as a function of genotype, although a survivorship bias exists in the Scx+/+ group. In observing mice in both groups, we noted consistent differences in behaviour: following TAC, Scx+/+ mice showed progressive reduction in activity (reduced movement, grooming, and nesting), as well as hunching and rapid breathing, while Scx-fKO mice exhibited essentially normal activity (Sup. Video 1). The post-TAC reduction in cardiac fibrosis and improved function of Scx-fKO compared to Scx+/+ mice thus results in evidence of improved quality of life and survival. Scleraxis deletion attenuates mortality in existing long-term pressure overload. (A) Study design schematic indicating timing of administration of tamoxifen (Tam) to induce scleraxis gene deletion or corn oil control, echocardiography (E), and pressure overload (transverse aortic constriction, TAC) or sham surgery. Surgery preceded scleraxis deletion by four weeks, with eight additional weeks to study end after knockout. (B) Representative images of echocardiographic assessment of left ventricular ejection fraction and fractional shortening with quantification, n = 8–11 per group; *P +/+. (C) Mouse survival up to 12 weeks after the initiation of pressure overload (black, Scx+/+ 8 of 12 animals; red, fibroblast-specific scleraxis knockout 11 of 11 animals), P Scleraxis transcriptionally regulates periostin gene expression in cardiac myofibroblasts Scleraxis is upregulated during cardiac fibroblast activation, and scleraxis over-expression or loss either induces or blocks this process, respectively, while periostin is highly expressed only in myofibroblasts.8,11 We previously hypothesized that scleraxis coordinates this phenotypic switch by direct transcriptional regulation of fibrosis-associated genes and have demonstrated this relationship for collagen 1α2, α-smooth muscle actin, fibronectin, and MMP2.8,10,11 Given our observation that periostin expression mirrored scleraxis in our TAC studies, we examined whether scleraxis regulates periostin expression directly. Similar to scleraxis, periostin expression is significantly upregulated in cardiac myofibroblasts (twice-passaged, P2) compared to freshly isolated cardiac fibroblasts (zero-passaged, P0; Figure 5A). Activated cardiac fibroblasts exhibited increased expression of both scleraxis and periostin in response to TGFβ1, a potent cytokine that drives fibroblast activation (Figure 5B). TGFβ1 also induced periostin secretion as further evidence of conversion to myofibroblasts, since periostin is a key matricellular protein (Figure 5C). Scleraxis over-expression in activated cardiac fibroblasts induced periostin mRNA 20-fold and significantly increased periostin protein expression and secretion (Figure 5D–5F), similar to the effect of TGFβ1 and suggesting that periostin may be a direct gene target of scleraxis. Scleraxis transcriptionally regulates periostin gene expression in cardiac myofibroblasts. (A) Expression of periostin mRNA was assessed in primary adult rat cardiac fibroblasts (P0; within 6 h of initial plating) and myofibroblasts (P2; passaged twice), and normalized to Gapdh, n = 3 per group. (B) Expression of scleraxis and periostin mRNA was assessed in activated adult rat cardiac fibroblasts (P1; passaged once) treated with 10 ng/mL transforming growth factor β1 or vehicle for 24 h, n = 3 per group. (C) Periostin protein secretion to the cell culture medium was assayed following transforming growth factor β1 treatment as in (B), n = 4 per group. (D–F) Expression of scleraxis and periostin mRNA (D) and protein (E) was assessed in P1 cardiac fibroblasts treated with adenovirus encoding scleraxis (AdScx) or green fluorescent protein (AdGFP) for 48 h at a multiplicity of infection of 10, n = 3–4 per group. (F) Secreted periostin expression was assayed in conditioned medium collected from P1 rat cardiac fibroblasts treated with AdScx or AdGFP for 24 h, n = 4 per group. (G) Short (597 bp) or long (979 bp) regions of the human proximal periostin promoter, containing 2 or 4 putative E-boxes respectively, were subjected to luciferase assay in NIH3T3 fibroblasts co-transfected with a scleraxis expression vector (pcDNA-Scx) or empty vector control (pcDNA) with normalization to co-transfected Renilla luciferase, n = 4 per group. (H) Luciferase assays were repeated as in (G) following site-directed mutagenesis of E2 alone, or E1 and E2 together (black boxes), n = 4 per group. (I) Scleraxis-enriched HEK293 nuclear extracts were incubated with biotin-labeled DNA oligonucleotide probes containing the periostin promoter E-boxes E1 and E2, or oligonucleotides with mutated E-boxes (ΔE1/ΔE2). Lanes represent free probe (P), scleraxis nuclear extract (S), cold competition (CC), and complex incubation with anti-scleraxis (Scx) or non-specific (IgG) antibodies. The solid arrow depicts the shifted protein–oligonucleotide complex; the hollow arrow depicts the super-shifted protein–oligonucleotide–antibody complex. ns, non-specific complex. (J) Human cardiac myofibroblasts were treated with vehicle or transforming growth factor β1 (10 ng/mL) for 24 h followed by chromatin immunoprecipitation using anti-scleraxis antibody (Scx) or pre-immune serum (PS). qPCR was used to amplify the E1/E2-containing region of the periostin promoter to assess scleraxis enrichment at the promoter. Genomic DNA was used as a positive input control, n = 3 per group. (K) Primary mouse P1 cardiac fibroblasts were isolated from wild-type or scleraxis knockout hearts and treated with transforming growth factor β1 (10 ng/mL) or vehicle, followed by assessment of scleraxis or periostin mRNA expression, n = 4 per group. (L) Conditioned medium from wild-type or scleraxis knockout cardiac fibroblasts treated with transforming growth factor β1 (10 ng/mL) or vehicle was analysed for periostin protein secretion, with results normalized to WT + vehicle and Ponceau S-stained membranes, n = 4 per group. (M) Periostin secretion was assessed following adenovirus-mediated over-expression of scleraxis (AdScx, 48 h) in wild-type or knockout mouse cardiac fibroblasts, n = 4 per group; *P P P P H), ####P $$$$P L), ###P 1; in (M), ##P $$P As a basic helix-loop-helix transcription factor, scleraxis directly transactivates target gene promoters by binding to E-box consensus binding sites (CANNTG).21,22 Examination of the human POSTN promoter (−886 to +93 bp relative to the transcription start site) revealed four putative E-boxes (Figure 5G). We assessed scleraxis transactivation of this promoter via luciferase reporter assay. Both the full-length promoter and a shorter construct (−504 to +93 bp) excluding the two most distal E-boxes were significantly and equivalently transactivated by scleraxis (Figure 5G), suggesting that the two distal E-boxes are dispensable for scleraxis-mediated transactivation. The E-boxes E1 and E2 located in the short promoter are separated by only two nucleotides. To determine their role in scleraxis-mediated promoter transactivation, these sites were sequentially mutated to abrogate scleraxis binding. Site-specific mutation of E2 reduced scleraxis-mediated promoter transactivation by ∼50% (Figure 5H). Mutation of E2 and E1 together further reduced transactivation by >90% compared to the intact promoter—a significant reduction compared to mutation of E2 alone (Figure 5H) and demonstrating the critical importance of both E-boxes. To confirm the specific binding of scleraxis to E1 and E2, electrophoretic mobility shift assays were performed using oligonucleotides encompassing both sites. Incubation of scleraxis-enriched nuclear extracts with biotin-labelled oligonucleotides spanning E1 and E2 resulted in a shifted complex indicative of specific binding of scleraxis to this site (lane S left, Figure 5I). This complex was lost when competed by ‘cold’ unlabelled oligonucleotides (lane CC) and was super-shifted in the presence of a scleraxis-specific antibody but not a non-specific IgG antibody (lanes Scx and IgG), confirming the presence of scleraxis in the complex. Mutation of E1 and E2 to abrogate scleraxis binding (ΔE1/ΔE2) attenuated both shifted and super-shifted complex formation (Figure 5I right). These results corroborate the luciferase assay results, demonstrating that scleraxis transactivates the periostin promoter via specific binding at E1 and E2. TGFβ1 signalling upregulates scleraxis, which in turn increases scleraxis binding to its target promoters.11 We assessed scleraxis interaction with the human POSTN promoter in isolated human cardiac myofibroblasts treated with or without TGFβ1 by chromatin immunoprecipitation (ChIP) assay. Given the close proximity of E1 and E2, ChIP cannot discriminate specific binding to either site individually but can indicate scleraxis interaction with the region overall. TGFβ1 treatment induced a dramatic and significant enrichment of scleraxis at the POSTN promoter (Figure 5J), indicating a novel scleraxis-mediated mechanism for periostin upregulation by TGFβ1. Our laboratory previously demonstrated that scleraxis is an essential and integral regulator of TGFβ1-mediated fibrotic signalling mechanisms in cardiac fibroblasts and myofibroblasts. For example, scleraxis knockdown in primary cardiac myofibroblasts completely attenuates TGFβ1-induced cell contraction.8 To determine if a similar requirement for scleraxis exists in TGFβ1-mediated periostin upregulation, we employed a loss-of-function approach via experiments in activated cardiac fibroblasts isolated from wild-type (WT) or scleraxis germline knockout (KO) mice concomitant with vehicle or TGFβ1 treatment.8 While TGFβ1 induced a four-fold upregulation of periostin in WT cells, KO cells showed a complete failure to upregulate periostin in response to TGFβ1 (Figure 5K). Similarly, periostin secretion was very low in vehicle-treated WT cells and increased nearly 20-fold after TGFβ1 treatment (Figure 5L). In contrast, KO cells lacked the ability to increase periostin secretion in response to TGFβ1. Together, these results reveal a critical and absolute requirement for scleraxis in TGFβ1-mediated periostin expression; however, it is unclear if this is due to a permanent loss in the ability of KO cells to produce periostin. We therefore restored scleraxis expression in KO cells by adenoviral gene delivery. Scleraxis over-expression in WT activated cardiac fibroblasts induced a significant ∼11-fold increase in periostin protein secretion (Figure 5M). While scleraxis over-expression in KO cells was less potent than in WT cells, KO cells nonetheless exhibited a significant ∼5-fold upregulation of periostin compared to controls. Thus, KO cells retain the ability to produce periostin when scleraxis expression is restored. We observed a similar regulatory scheme governing MMP2 protein expression, suggesting a common mechanism of scleraxis activity across target genes.10 Discussion No specific drug for the therapeutic treatment of cardiac fibrosis is available, and fibrosis remains inadequately treated regardless of organ type. This lack of effective treatments stems from an incomplete understanding of underlying mechanisms and a paucity of exploitable drug targets. The identification of key players in persistent cardiac fibrosis, particularly in the heart, remains an unmet clinical need. The transcriptional control of cardiac fibrosis is not fully understood and represents a potential point of intervention. Persistent mechanical stress in the pressure overloaded myocardium triggers TGFβ1-Smad signalling, leading to fibroblast activation and excessive ECM synthesis.23 Pressure overload-associated interstitial ECM remodelling significantly impairs LV ejection fraction and is accompanied by altered chamber geometry with increased LV end systolic and diastolic volumes, leading to heart failure.23 Fibroblast activation to myofibroblasts is required for fibrosis to occur, and blockade of this step may arrest or possibly reverse cardiac fibrosis. Most studies to date have reported reduced fibrosis secondary to other improvements such as primary positive effects on myocyte function; thus, the specific impact of fibrosis reduction alone remains unclear. We previously reported that germline scleraxis deletion results in a 50% loss of cardiac fibroblasts which may be due to an impairment of developmental epithelial-to-mesenchymal transition.8,21 The residual fibroblasts were unable to convert to myofibroblasts unless scleraxis expression was restored, consistent with a failure of cardiac fibroblasts subjected to mechanical stretch to convert to myofibroblasts following scleraxis knockdown.9 These findings support a model in which scleraxis is required to govern the initiation and maintenance of the myofibroblast phenotype, but the in vivo impact of scleraxis loss in a cardiac pathology model could not be assessed using scleraxis global KO mice due to confounding phenotypic effects such as tendon malformations and reproductive impairment.24 Furthermore, it was unclear whether the role of scleraxis in fibroblast development and the synthesis of basal ECM would be similar in the pathological setting of greatly increased ECM production and cardiac fibroblast activation in cardiac fibrosis. Using conditional scleraxis deletion in cardiac fibroblasts in adult mice combined with the well-established pressure overload model, the present study provides two key insights into the role of scleraxis and fibrosis in cardiac pathology. First, scleraxis is required for cardiac fibrosis in pressure overload in our model, regardless of the timing of scleraxis deletion prior to or after the establishment of cardiac pathology. Second, the attenuation of cardiac fibrosis by scleraxis loss improves cardiac function and survival in the TAC model independent of any effects on hypertrophy. Our data support a model in which scleraxis is required for fibroblast activation in vivo, via direct transcriptional control of critical fibrosis-related genes such as periostin25 (Structured Graphical Abstract). The chronic stress of the pressure overloaded myocardium leads to systolic dysfunction.17 We confirmed this profile of dysfunction in our 8-week TAC model, concomitant with evidence of increased scleraxis gene activation. Scleraxis deletion prior to TAC reduced systolic dysfunction and chamber remodelling with attenuation of cardiac fibrosis, demonstrating a clear benefit of preventive reduction of scleraxis expression. As scleraxis deletion did not impact cardiac hypertrophy, we took this as indicating that a secondary effect on cardiomyocytes was unlikely. The salutary effect of scleraxis loss occurred even when the deletion was initiated at 4 weeks post-TAC, well after the establishment of adverse cardiac remodelling. Systolic functional improvements were observed 4 weeks after scleraxis deletion, i.e. 8 weeks post-TAC: cardiac function continued to decrease in Scx+/+ mice while further loss appeared to be attenuated in Scx-fKO mice. Thus, scleraxis may be required for chronic maintenance of the myofibroblast phenotype in the setting of pathological stress, as we noted attenuation of the number of periostin-positive myofibroblasts. It remains unclear if scleraxis loss leads to improvement and repair of cardiac function over extended time in this model, or merely arrests further decline. However, at 12 weeks post-TAC no mortality was seen in Scx-fKO mice compared to ∼33% loss of Scx+/+ mice, and animal behaviour appeared to be normal. These results support the possibility that therapeutic targeting of scleraxis in human disease may arrest cardiac functional decline. The development of cardiac fibrosis is an independent risk factor for morbidity and mortality in human disease. Multiple studies over the past decade have confirmed that cardiac fibrosis significantly increases all-cause mortality and risk of major adverse cardiac events in DCM.3,4 The factors driving cardiac fibrosis in humans are poorly understood compared to animal models. However, our finding of a significant elevation in scleraxis expression in non-ischaemic DCM patients compared to healthy controls, concomitant with increased collagen expression, suggests the possibility that similar mechanisms are at work. Furthermore, our data indicated that scleraxis transactivates the human periostin promoter, and TGFβ1 stimulates scleraxis binding to the promoter in isolated human cardiac fibroblasts. Our cases of non-ischaemic DCM reflect the degree of hypertrophy, LV dilation, and systolic dysfunction seen in the pressure overload murine model.12,13 We recently reported that serum scleraxis levels are elevated in patients with hypertrophic cardiomyopathy compared to healthy controls.26 Others have reported elevated serum and fibroblast scleraxis in samples from patients with idiopathic pulmonary fibrosis, systemic sclerosis, and hypersensitivity pneumonitis, with higher scleraxis correlating with higher disease burden.27 There is thus a growing body of evidence that indirectly ties scleraxis to human fibrotic diseases, and additional study in this area is needed. Our results are consistent with scleraxis driving a pro-fibrotic transcriptional programme in cardiac fibroblasts, effectively serving as a master regulator of cardiac fibrosis by controlling the expression of a host of fibrosis-related genes that essentially define the myofibroblast phenotype. In isolated cardiac myofibroblasts, scleraxis and the TGFβ1 effector Smad3 synergistically regulate Col1α2 gene expression, and scleraxis is necessary for the recruitment of Smad3 to the Col1α2 gene promoter, as a dominant-negative mutant of scleraxis dose-dependently attenuated assembly of an RNA polymerase II-containing transcriptional complex at the promoter.8,11 We have similarly noted a requirement for scleraxis in TGFβ-mediated regulation of genes encoding α-smooth muscle actin, fibronectin, and MMP2.8,10 After 8 weeks of TAC, we now report recruitment of RNA polymerase II to the Col1α2 promoters in Scx+/+ mice, consistent with the induction of this gene in cardiac fibrosis. In contrast, RNA polymerase II was not enriched at the Col1α2 promoter in Scx-fKO mice, indicating a failure to initiate transcription of this gene in the absence of scleraxis. A similar effect was observed at the periostin promoter wherein RNA polymerase II was not recruited to the promoter in Scx-fKO mice after 8 weeks of TAC, which corresponds with a lack of induction of periostin expression. Periostin expression is highly elevated after myocardial injury, pressure overload (in agreement with our study), and in the failing heart.16,28 Periostin is also used as a specific marker of myofibroblasts.16 Periostin shows evidence of a functional role in wound healing and fibrosis, as it promotes dermal fibroblast activation.29 Periostin-null mice exhibit reduced wound closure due to reduction of the myofibroblast population in granulation tissue, and periostin knockout attenuated kidney fibrosis in an ischaemic/reperfused kidney model.30,31 Periostin KO mice had a reduction in both cardiac fibrosis and hypertrophy following pressure overload and were more prone to rupture post-myocardial infarction.28 Periostin thus appears to play a causal role in fibrosis in multiple tissues. This study is the first to report direct transcriptional regulation of periostin expression by scleraxis, which has similarly been implicated in fibrosis or wound healing in multiple tissues including tendon, skin, and bone.32–34 Similar to other target genes reported by us and others, scleraxis induced periostin expression by binding to E-boxes located within the proximal human periostin gene promoter. TGFβ1-induced periostin expression and secretion were wholly dependent on scleraxis. Our study supports a transcriptional model placing scleraxis between TGFβ1 and periostin and provides a mechanism for numerous previous reports of TGFβ control over periostin production. Scleraxis thus may induce fibrosis both via direct transactivation of fibrotic genes (e.g. collagen, fibronectin, and α-smooth muscle actin), and upregulation of periostin to reinforce pro-fibrotic signalling. Fibrosis and wound healing show fundamental similarities in tissue repair processes, with growing evidence that fibrosis arises when conventional wound healing is disrupted or prolonged. Clearance of myofibroblasts at the site of injury by apoptosis appears to be a critical step in the resolution of wound healing without fibrosis. For example, myofibroblast persistence during corneal wound healing results in increased ECM secretion and corneal haze.35 Previous work has indicated that myofibroblasts can persist at cardiac injury sites for many years, and recent lineage-tracing studies have reported that myofibroblasts may become quiescent matrifibrocytes.15 Our results indicate that scleraxis is required for both fibroblast activation and myofibroblast maintenance since scleraxis deletion before or after the establishment of cardiac pressure overload was effective in reducing fibrosis and myofibroblast number while maintaining or improving cardiac function. Notably, we observed a significant increase in apoptosis in Scx-fKO but not Scx+/+ hearts following TAC, concurrent with the loss of periostin-positive myofibroblasts throughout the myocardium and preserved function, providing evidence that the lost cells are myofibroblasts. Loss of myocytes is unlikely, given the lack of apoptosis in scleraxis-intact hearts that exhibited continued functional loss. This finding suggests that an additional mechanism by which scleraxis may contribute to fibrosis is by promoting myofibroblast survival. The increased caspase 3 and decreased Bcl2 expression we observed following scleraxis knockdown in primary activated cardiac fibroblasts supports this possibility. Previous studies employing scleraxis over-expressing mesenchymal stem cells demonstrated that scleraxis promotes faster and stronger healing of damaged tendons compared to conventional stem cell treatment, and scleraxis is required for effective bone healing after fracture.32,33 Scleraxis may thus be a critical regulator of the balance between wound healing and fibrosis, with prolonged expression of scleraxis leading toward the latter. It remains to be determined whether scleraxis regulates other forms of cardiac fibrosis. We reported increased scleraxis expression in the cardiac infarct scar, but its specific role in this setting remains unclear.36 It is unknown whether over-expression of scleraxis in cardiac fibroblasts would be sufficient to induce fibrosis in the absence of other stimuli, but this presents an intriguing possibility to be examined. In pressure overload, however, our results clearly show that scleraxis plays a central role in fibrosis and that reducing scleraxis expression has beneficial effects on cardiac function and survival. In contrast, myofibroblast-specific knockout of Smad3 resulted in significantly impaired cardiac function post-TAC, suggesting that interference with TGFβ/Smad signalling results in deleterious off-target effects such as altered MMP expression and matrix remodelling.23 Thus, scleraxis may provide an optimal choice for the development of anti-fibrosis therapeutics in the myocardium. Supplementary material Supplementary material is available at European Heart Journal online. Acknowledgements We gratefully thank Drs. Eric Olson and Rhonda Bassel-Duby (University of Texas Southwestern Medical Center at Dallas) for the Tcf21tm3.1(cre/Esr1*) Eno mouse line. We are grateful to Dr. Depeng Jiang (University of Manitoba) for advice on statistical analysis. We thank Dr. Ronen Schweitzer (Oregon Health & Science University) for the Scxflox/flox mice. Funding This work was supported by the Canadian Institutes of Health Research (MOP and PJT to MPC, FRN to RAB and PJT37522 to ZK) and a scholarship from Research Manitoba to RSN. Data availability The data underlying this article will be shared upon reasonable request to the corresponding author. References 1 . Cardiac fibroblast diversity . ;:–. doi: 2 , , , , , et al. Impact of myocardial fibrosis on left ventricular remodelling, recovery, and outcome after transcatheter aortic valve implantation in different haemodynamic subtypes of severe aortic stenosis . ;:–. doi: 3 , , , , , , et al. Left ventricular midwall fibrosis as a predictor of mortality and morbidity after cardiac resynchronization therapy in patients with nonischemic cardiomyopathy . ;:–. doi: 4 , , , , , et al. Progression of myocardial fibrosis in nonischemic DCM and association with mortality and heart failure outcomes . ;:–. doi: 5 , , , , , , et al. Inhibition of TGF-beta signaling exacerbates early cardiac dysfunction but prevents late remodeling after infarction . ;:–. doi: 6 , , , , , et al. Postinfarction gene therapy against transforming growth factor-beta signal modulates infarct tissue dynamics and attenuates left ventricular remodeling and heart failure . ;:–. doi: 7 , , . Complexities of TGF-beta targeted cancer therapy . ;:–. doi: 8 , , , , , et al. The transcription factor scleraxis is a critical regulator of cardiac fibroblast phenotype . ;:. doi: 9 , , , , , , et al. Role of scleraxis in mechanical stretch-mediated regulation of cardiac myofibroblast phenotype . Am J Physiol Cell Physiol ;:–. doi: 10 , , , , , et al. Regulation of cardiac fibroblast MMP2 gene expression by scleraxis . ;:–. doi: 11 , . Synergistic roles of scleraxis and smads in the regulation of collagen 1alpha2 gene expression . ;:–. doi: 12 , , , , , et al. Cells of the adult human heart . ;:–. doi: 13 , , , , , , et al. The human explanted heart program: A translational bridge for cardiovascular medicine . Biochim Biophys Acta Mol Basis Dis ;:. doi: 14 , , , , . Generation of transgenic tendon reporters, ScxGFP and ScxAP, using regulatory elements of the scleraxis gene . ;:–. doi: 15 , , , , , et al. Specialized fibroblast differentiated states underlie scar formation in the infarcted mouse heart . ;:–. doi: 16 , , , , , , et al. Periostin expression is upregulated and associated with myocardial fibrosis in human failing hearts . ;:–. doi: 17 , , , , , et al. Resveratrol treatment of mice with pressure-overload-induced heart failure improves diastolic function and cardiac energy metabolism . ;:–. doi: 18 , , , , , , et al. Baicalin attenuates cardiac dysfunction and myocardial remodeling in a chronic pressure-overload mice model . ;:–. doi: 19 , , , , , et al. Physiologic and molecular characterization of a murine model of right ventricular volume overload . Am J Physiol Heart Circ Physiol ;:–. doi: 20 , , , , , , et al. The experimental model of transition from compensated cardiac hypertrophy to failure created by transverse aortic constriction in mice . ;:–. doi: 21 , , , , , . Scleraxis regulates Twist1 and Snai1 expression in the epithelial-to-mesenchymal transition . Am J Physiol Heart Circ Physiol ;:–. doi: 22 , , , , , et al. Scleraxis: a basic helix-loop-helix protein that prefigures skeletal formation during mouse embryogenesis . ;:–. doi: 23 , , , , , , et al. Protective effects of activated myofibroblasts in the pressure-overloaded myocardium are mediated through smad-dependent activation of a matrix-preserving program . ;:–. doi: 24 , , , , Jr, et al. Scleraxis is required for cell lineage differentiation and extracellular matrix remodeling during murine heart valve formation in vivo . ;:–. doi: 25 , , . Periostin in cardiovascular disease and development: a tale of two distinct roles . ;:. doi: 26 , , , , , et al. Scleraxis as a prognostic marker of myocardial fibrosis in hypertrophic cardiomyopathy (SPARC) study . ;:–. doi: 27 , , , , , , et al. The transcription factor SCX is a potential Serum biomarker of fibrotic diseases . ;:. doi: 28 , , , , , et al. Genetic manipulation of periostin expression reveals a role in cardiac hypertrophy and ventricular remodeling . ;:–. doi: 29 , , , . Periostin induces fibroblast proliferation and myofibroblast persistence in hypertrophic scarring . ;:–. doi: 30 , , , , , et al. Periostin modulates myofibroblast differentiation during full-thickness cutaneous wound repair . ;:–. doi: 31 , , , , , , et al. Periostin induces kidney fibrosis after acute kidney injury via the p38 MAPK pathway . Am J Physiol Renal Physiol ;:–. doi: 32 , , , , , et al. Transcription factor scleraxis vitally contributes to progenitor lineage direction in wound healing of adult tendon in mice . ;:–. doi: 33 , , , , , . Loss of scleraxis in mice leads to geometric and structural changes in cortical bone, as well as asymmetry in fracture healing . ;:–. doi: 34 , , , , , et al. Gene expression in human keloids is altered from dermal to chondrocytic and osteogenic lineage . ;:–. doi: 35 , , , . Nanomedicine approaches for corneal diseases . ;:–. doi: 36 , , , , , . The basic helix-loop-helix transcription factor scleraxis regulates fibroblast collagen synthesis . ;:–. doi: © The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Cardiology. All rights reserved. For permissions, please e-mail: © The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Cardiology. All rights reserved. For permissions, please e-mail: