This is the peer reviewed version of the following article:    Panse KD, Felkin LE, Lopez‐Olaneta MM, Gomez‐Salinero J, Villalba M, Munoz L,  Nakamura K, Shimano M, Walsh K, Barton PJ, Rosenthal N, Lara‐Pezzi E. Follistatin‐Like 3  Mediates Paracrine Fibroblast Activation by Cardiomyocytes. J Cardiovasc Transl Res.  2012;5(6):814‐26    which has been published in final form at: https://doi.org/10.1007/s12265‐012‐9400‐9        Fstl3 mediates paracrine fibroblast activation Panse et al. Page 1 of 27 Title: Follistatin-like 3 mediates paracrine fibroblast activation by cardiomyocytes Authors: Kalyani D. Panse 1 , Leanne E. Felkin 1 , Marina M. López-Olañeta 2 , Jesús Gómez- Salinero 2 , María Villalba 2 , Lucía Muñoz 2 , Kazuto Nakamura 3 , Masayuki Shimano 3 , Kenneth Walsh 3 , Paul J.R. Barton 1,4 , Nadia Rosenthal 1,5 and Enrique Lara Pezzi 1,2,* . Affiliations: 1 Heart Science Centre, Imperial College London, Hill End Road, Middlesex UB9 6JH, United Kingdom; 2 Fundación Centro Nacional de Investigaciones Cardiovasculares, Melchor Fernandez Almagro, 3, 28029 Madrid, Spain; 3 Whitaker Cardiovascular Institute, Boston University Medical Campus, Boston, Massachusetts 02118, USA; 4 NIHR Cardiovascular Biomedical Research Unit, Royal Brompton and Harefield NHS Foundation Trust, London, United Kingdom; 5 Australian Regenerative Medicine Institute, Monash University, Melbourne, Australia. *Correspondance: Enrique Lara-Pezzi, Cardiovascular Development and Repair Department, Fundación Centro Nacional de Investigaciones Cardiovasculares, Melchor Fernandez Almagro, 3, 28029 Madrid, Spain. Tel.: +34-914531200, x3309. E-mail: elara@cnic.es Fstl3 mediates paracrine fibroblast activation Panse et al. Page 2 of 27 ABSTRACT Follistatins are extracellular inhibitors of TGF-β family ligands including activin A, myostatin and bone morphogenetic proteins. Follistatin-like 3 (FSTL3) is a potent inhibitor of activin signalling and antagonises the cardioprotective role of activin A in the heart. FSTL3 expression is elevated in patients with heart failure and is upregulated in cardiomyocytes by hypertrophic stimuli, but its role in cardiac remodelling is largely unknown. Here we show that production of FSTL3 by cardiomyocytes contributes to the paracrine activation of cardiac fibroblasts, inducing changes in cell adhesion, promoting proliferation and increasing collagen production. We found that FSTL3 is necessary for this response and for the induction of cardiac fibrosis. However full activation requires additional factors and we identify CTGF as a FSTL3 binding partner in this process. Together our data unveil a novel mechanism of paracrine communication between cardiomyocytes and fibroblasts that may provide potential as a therapeutic target in heart remodelling. Fstl3 mediates paracrine fibroblast activation Panse et al. Page 3 of 27 INTRODUCTION Initial compensation of the heart to increased systemic demands is provided in the form of cardiac hypertrophy, where individual cardiomyocytes grow in order to increase contractile function and reduce ventricular wall tension [1-3]. Physiological cardiac hypertrophy is commonly seen in response to exercise training or during pregnancy, where the increased workload is transitory and matched by preserved contractility [4]. In contrast, persistent hypertrophic growth due to arterial hypertension, aortic stenosis or regurgitation leads to pathological hypertrophy, fibrosis, heart remodelling and eventually contractile dysfunction and heart failure [2,5]. Cardiac structure is maintained by the extracellular matrix (ECM), which provides support for proper contractile function of the heart. The ECM is composed of fibrillar collagens (predominantly type I and type III), basement membrane components like fibronectin and laminin, proteoglycans and glucosaminoglycans [6]. Adaptation to myocardial stress induces a number of cellular and molecular alterations that lead to changes in the ECM composition and the collagen network as well as in the cardiac structure. During conditions of chronic hypertension and pressure overload, cardiac hypertrophy gradually decompensates and reactive interstitial fibrosis develops, increasing ventricular stiffness and progressively affecting contractility and diastolic chamber filling capacity [7-9]. It is becoming increasingly evident that heart remodelling as a result of hypertrophy and fibrosis is orchestrated by intercellular communication between cardiomyocytes and fibroblasts, both through direct contact and through paracrine mediators. However, these mechanisms are not well understood. TGF- is an example of such interaction. It is produced by different cell types in the heart and promotes both hypertrophy and fibrosis by activating distinct signalling pathways in fibroblasts and cardiomyocytes. Other members of the TGF-β family have also been widely implicated in the pathogenesis of heart failure. Activins are homodimeric cytokines that modulate diverse pathophysiological processes including inflammation and wound healing, cell proliferation, differentiation, metabolism, fibrosis and cardiac remodelling [10- 12]. In the heart, Activin A has a protective role following myocardial infarction and reduces cardiac hypertrophy after pressure overload [13,14]. Activin signalling is inhibited by the follistatin family of proteins. Both Follistatin (FST) and Follistatin-like 3 (Fstl3/FLRG/FSRP) Fstl3 mediates paracrine fibroblast activation Panse et al. Page 4 of 27 can bind activin with high affinity and neutralise its action by preventing its binding to the activin receptors [15]. On the other hand, both activin and TGF-β are known to transciptionally regulate the expression of FST and FSTL3 through Smad proteins in what appears to be a negative feedback loop [16,17]. We have recently shown that FSTL3 expression is induced in the myocardium of heart failure patients and correlates with poor cardiac function and disease severity [18]. Interestingly, elevated FSTL3 expression in serum has also been reported in response to heavy resistance training in humans [19]. We found a similar increase in FSTL3 expression in rodent hearts subjected to ischemic injury, pressure overload induced hypertrophy or hypertrophy resulting from infusion of the 2 adrenergic agonist clenbuterol [13,14,20]. FSTL3 in the hypertrophic heart is mainly produced by cardiomyocytes and interferes with the beneficial effects of activin. Activin-mediated protection against ischemic injury was abrogated by FSTL3, and knock-out of Fstl3 in the heart resulted in smaller cardiac infarct size following ischemia-reperfusion injury [13]. In addition, we observed that inhibition of Activin A by FSTL3 induces cardiac hypertrophy in a mouse model of pressure overload. Cardiac-specific Fstl3 knockout mice displayed reduced hypertrophy, improved function and lower levels of interstitial fibrosis than wild type mice [14]. However the role of FSTL3 in cardiac fibrosis was not explored in depth. Here we show that the role of FSTL3 in heart disease is more complex than previously anticipated. We investigated the transcriptome of cardiac-specific Fstl3 knockout myocardium and found that FSTL3 is necessary for full cardiac fibrosis to develop in response to pressure overload. We show that cardiomyocyte-derived FSTL3 promotes fibroblast proliferation and adhesion, and is necessary for paracrine activation of collagen production in fibroblasts. These findings may have important implications for the clinical management of cardiac fibrosis. Fstl3 mediates paracrine fibroblast activation Panse et al. Page 5 of 27 MATERIALS AND METHODS Patients and Human Samples Human myocardial samples were obtained from hearts isolated from transplanted end-stage heart failure patients and from control biopsies as previously described [21,22]. All the human samples used in this study conform to the Declaration of Helsinki. Mice and surgeries Fstl3 knockout (KO) mice and surgical procedures on these mice have been previously described [13,14]. Mice homozygous for a Fstl3 allele containing two loxP sites flanking exons 3-5 (Fstl3 flox/flox) were crossed with α-myosin heavy chain (α-MHC)-Cre transgenic mice on a C57BL/6 background. Recombination by Cre leads to excision of Fstl3 exons 3, 4 and 5 such that the progeny containing Fstl3 flox/flox as well as α-MHC-Cre lack Fstl3 expression in post-natal cardiomyocytes. Trans-aortic constriction (TAC) was performed in 6-8 week old Fstl3 KO mice and wild type (WT) littermate controls. Prior to induction of anesthesia, all mice were given buprenorphine (0.25 mg/kg s.c.). General anesthesia was induced by inhalation of isofluorane (3-4% (v/v) in oxygen) and maintained at 1-1.5%. Following chest hair removal and disinfection, an upper chest midline skin incision followed by median sternotomy was performed to open the chest cavity. A loose suture (7-0 silk) was placed around the aorta and a 27 gauge blunted needle, between the right innominate artery and left common carotid artery. The suture was tightened to fully constrict the aorta and the needle was removed, thereby leading to partial constriction of the transverse aorta. The chest cavity, the muscle and skin layer were closed, disinfected and the animals were allowed to recover in a warm chamber. Analgesia (0.1 mg/kg buprenorphine) was given up to twice daily for 2-3 days following surgery. The efficiency of the procedure and cardiac function measurements on these mice were previously reported [14]. All studies were approved by the Institutional Animal Care and Use Committee of Boston University. Fstl3 mediates paracrine fibroblast activation Panse et al. Page 6 of 27 Neonatal rat cardiomyocyte and fibroblast isolation Neonatal rat ventricular cardiomyocytes and fibroblasts were isolated as described by Toraason et.al. [23], with some modifications. Hearts from 1-4 day old rat pups were quickly excised and placed in ice-cold Ca 2+ and Mg 2+ free Hank‟s buffered salt solution (CMF – HBSS). Atria and surrounding connective tissue were removed, and the ventricles were minced into 1 mm 3 small pieces and digested with 50 μg/ml trypsin in 10 ml CMF-HBSS overnight at 4 o C. Trypsin was inactivated by adding 0.5 ml soybean trypsin inhibitor (Worthington Biochemical Corporation, USA) in CMF-HBSS and incubating at 37 o C in 5% CO2 (v/v) for 20 min. Samples were then digested with 750 units collagenase (Worthington Biochemical Corporation, USA) in Leibovitz L-15 serum-free medium (Sigma-Aldrich, UK) in a shaking water bath at 37 o C for 30-45 min. Cells were dislodged by triturating the suspension 10 times using a 10 ml serological pipette, washed once with equilibrated L-15 medium and filtered through a 0.70 μm cell strainer. Cells were then centrifuged and resuspended in 25 ml complete medium (Dulbecco‟s Modified Eagle Medium (DMEM) containing 4500 mg/L glucose, 200 mM L-glutamine, 10% [v/v] fetal calf serum (FCS), 100 units/ml penicillin and 0.1 mg/ml streptomycin; all from Sigma-Aldrich). The cell mixture was sequentially pre-plated thrice for 30, 30 and 20 minutes and the adherent fibroblasts were cultured further in complete medium while cardiomyocytes were transferred to a separate tube after the third pre-plating. Cells were plated in type I fibronectin-coated dishes (1 μg/cm2, Sigma-Aldrich). Cardiomyocytes obtained by this protocol routinely had a purity of 97-99%. Cardiac fibroblasts were allowed to multiply for 1 week, split 1:3, cultured for another week (P1) and passaged twice to remove contaminating endothelial cells. For experimental setup, both cardiomyocytes and fibroblasts were serum-starved overnight and incubated with 200 ng/ml FSTL3 (R&D Systems, USA) and/or 0.5 g/ml CTGF (eBioscience) as indicated in each experiment. Mechanical stretch was applied using Flexcell FX-4000 Tension Plus system (Flexcell International Corporation, USA). Cells were plated on fibronectin or collagen-coated BioFlex plates for 2 days in complete medium and serum- starved for 24 hours prior to experiments. The plates were loaded on 25 mm cylindrical loading posts and 10% or 15% equibiaxial stretch was applied at a frequency of 0.6Hz for 24 hours. Fstl3 mediates paracrine fibroblast activation Panse et al. Page 7 of 27 Cell Proliferation, Adhesion, Migration and Collagen Production To determine cell proliferation, cells were incubated in serum free medium for 24 h and then stimulated for 24 h with 200 ng/ml and/or 0.5 g/ml CTGF in serum free medium. During the last 18 h of the stimulation, cells were grown in the presence of bromodeoxyuridine (BrdU) according to the BrdU detection kit‟s instructions (Millipore, UK). The amount of BrdU incorporated into the newly synthesized DNA was determined using an anti-BrdU antibody followed by an HRP-linked secondary antibody as previously described [24]. Cell adhesion was analysed as we previously reported [25]. Briefly, cells were incubated in the presence of 100 ng/ml FSTL3 for 24 h, washed and detached using 10 mM EDTA. Cells were resuspended in serum free medium and allowed to adhere to uncoated tissue culture dishes for different periods of time. After fixation in 2% glutaraldehyde cells were stained with crystal violet and quantified in the spectrophotometer at 540 nm. Cell migration was assayed using a Transwell device with 8 m pore diameter [26]. A total of 105 fibroblasts were seeded onto the upper chamber of the device in serum-free medium. Cells were stimulated with 200 ng/ml FSTL3 for 1 h and then allowed to migrate towards the bottom chamber containing 2% FCS as a chemoattractant. After 6 h, cells in the bottom chamber were detached using trypsin and counted. Newly synthesized soluble collagen in the supernatant of fibroblast cell cultures was assayed using Sircol collagen assay (Biocolor Ltd., UK). Microarrays and qRT-PCR Total RNA was purified from myocardial tissue and cells using Trizol (Sigma-Aldrich) followed by RNAeasy (Qiagen, Netherlands) as previously described [27]. Whole transcriptome analysis was performed using the Gene 1.0 ST Array System (Affymetrix, Santa Clara, CA). Microarray data was analyzed using the GeneSpring GX software suite (Agilent Technologies, USA). After scanning, raw data in each chip was normalized to the 50 th percentile and to the median of the chip. Samples were then further normalized on a gene per gene basis to the values obtained for sham wild type hearts and gene lists were filtered for raw expression >50. Fold change lists comparing two conditions were generated using a cut- off value of 1.4-fold. For gene ontology (GO) analysis, the generated gene lists were compared to existing GO lists in GeneSpring as previously reported [20,24,28,29]. Only those GO lists showing a correlation p-value <0.01 with the studied gene list are shown. Fstl3 mediates paracrine fibroblast activation Panse et al. Page 8 of 27 Microarray results were validated by quantitative real-time PCR (qRT-PCR) using Taqman chemistry. Gene expression in all samples was normalized to values for 18S as previously described [30]. Yeast Two Hybrid A yeast two-hybrid assay was performed using the HybriZAP-2.1 Two-Hybrid pre-digested vector kit (Stratagene) to identify in vivo protein interaction partners for FSTL3. Truncated FSTL3 was used as a bait to test interaction with target proteins expressed artificially in the yeast from a neonatal rat cardiomyocyte cDNA library. FSTL3 was cloned in frame with the yeast Gal4 DNA-binding domain (BD) while the library proteins were expressed as hybrid proteins with the Gal4 transcriptional activation domain (AD). Neither hybrid protein was capable of initiating activation of the reporter gene by itself. When a specific interaction took place between the bait and target protein, the Gal4 BD and AD co-localised and activated expression of the reporter gene histidine (HIS3). Colonies which were positive for the reporter were selected and analysed for the sequence of the encoded target protein. The yeast host strain YRG-2 is defective in uracil, histidine, adenine, lysine, tryptophan and leucine. Immunoprecipitation and Western Blot Cardiac fibroblasts were lysed in 1 ml cold lysis buffer containing 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10 mM NaF, 1 mM Na3VO4, 1 µg/ml leupeptin, 1 mM PMSF and 1% Triton-X100. The lysate was kept on ice for 15 min. and pre- cleared using 50 μl protein A/G agarose beads (Santacruz Biotechnology, USA) by rotation at 4 o C for 1 h. Tubes were centrifuged at 2000 rpm at 4 o C for 5 min. and the supernatant was incubated with 4 μg goat polyclonal anti-CTGF immunoprecipitation antibody (Santacruz Biotechnology) or normal goat IgG (Santacruz Biotechnology) as control for 3 h at 4 o C with rotation. A total of 50 μl protein A/G agarose beads were added and tubes were incubated overnight at 4 o C. Samples were centrifuged at 2000 rpm at 4 o C for 5 min. and pellets were washed, resuspended in 100 μl 1X reducing loading buffer (Cell Signaling) and boiled at 95 o C for 5 min. The tubes were then kept on ice briefly and centrifuged at 2000 rpm at 4 o C for 5 minutes. The presence of FSTL3 and CTGF was detected using anti-FSTL3 antibody (R&D Systems) and anti-CTGF by western blot [28]. Fstl3 mediates paracrine fibroblast activation Panse et al. Page 9 of 27 Statistical Analysis Statistical analysis was carried out using Graphpad Prism. Data are expressed as mean ± standard error of the mean. Unpaired t-tests, one-way ANOVA and two-way ANOVA tests were used as indicated. p<0.05 was taken as significant. Fstl3 mediates paracrine fibroblast activation Panse et al. Page 10 of 27 RESULTS Cardiac-specific Fstl3 KO mice show reduced expression of fibrotic markers We have previously shown that genetically modified mice with cardiomyocyte-specific deletion of Fstl3 (KO) develop less hypertrophy and fibrosis in response to pressure overload [14]. In order to gain insight into the underlying mechanisms we explored the myocardial transcriptome of both KO and wild type (WT) mice 21 days after transaortic constriction (TAC) or a sham operation. In WT mice, microarray analysis showed increased expression (>1.4-fold) of heart failure markers, cytoskeleton encoding genes and genes related to cell adhesion and extracellular matrix (ECM) following pressure overload, paralleled by a decrease in genes associated with mitochondrial metabolism (Tables S1-S4). KO mice showed a similar trend, compared to knockout sham animals, although a weaker induction of ECM-related genes was detected and no clear pattern of down-regulated genes was observed (Tables S9-S12). Comparison between KO and WT mice under sham conditions revealed few differentially expressed genes, in agreement with our previous findings showing that FSTL3 is only induced in stressed cardiomyocytes [14,18]. However, following TAC KO hearts showed significantly lower overall ECM-related gene expression compared to WT (Tables S15, S16). It is interesting to note that around 50% of genes whose expression was reduced in knockout mice belonged to categories related to the extracellular matrix, rather than to growth or cytoskeleton. These results suggest that a major pathological role of FSTL3 in the heart is the regulation of fibrosis. To validate these results we carried out qRT-PCR analysis on sham and hypertrophic hearts. We first analysed the expression of heart failure markers in these mice and found that WT mice showed a strong increase in ANP, BNP, -skeletal actin and -myosin heavy chain 21 days after TAC, which were significantly decreased in KO mice (Fig. S1), in agreement with the reduced hypertrophy observed in these mice and with experiments in vitro using FSTL3 siRNA [14]. Importantly, whereas no clear changes were found in the expression of inflammatory mediators (Fig. S2), KO mice expressed significantly lower levels of ECM genes and fibrosis markers. As shown in Figure 1, collagen I α1 (Col1a1) and collagen III α1 (Col3a1) mRNA were strongly induced after TAC in WT mice. Similarly, the mRNA Fstl3 mediates paracrine fibroblast activation Panse et al. Page 11 of 27 expressions of lysyl oxidase (Lox), an enzyme cross-linking collagen fibres into mature collagen (C) [31], and fibronectin-1, which is crucial for collagen deposition (D) [32], were also elevated in the WT hearts. In contrast, KO mice showed reduced expression of these markers following TAC, compared to WT, indicating reduced collagen deposition and fibrosis and validating the microarray results. Analysis of pro-fibrotic growth factors showed that mRNA and protein expression of both TGF- β1 and CTGF was also elevated after TAC in WT mice compared to sham operated hearts, but remained unchanged in KO hearts after TAC (Fig. 1E-1H). Pressure-overload in the heart often leads to myocardial remodelling and progression to heart failure. Extracellular matrix components including matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play an active role in this process [33]. In order to check if FSTL3 was involved in the regulation of these molecules, the expression of typical markers of myocardial remodelling was examined using qRT-PCR. As shown in Figure 2, the expression of MMP2 and MMP9 increased in WT hearts in response to pressure overload. In contrast, KO hearts showed attenuated expression of both. Expression of ADAM12 – a disintegrin and metalloprotease – followed a similar pattern and showed reduced expression in KO hearts compared to WT hearts after TAC (Fig. 2C). Furthermore, expression of the tissue inhibitor of MMPs TIMP1 was elevated in WT mice but not in KO hearts following TAC (Fig. 2D). Together, these results suggest that FSTL3 is necessary for ECM deposition and remodelling in the hypertrophic heart. Fstl3 mediates paracrine activation of fibroblasts Since deletion of Fstl3 in cardiac myocytes significantly reduced fibrosis after pressure overload, we explored the possibility that FSTL3 might be mediating paracrine activation of fibroblasts. To address this question, we cultured cardiac fibroblasts in the presence of conditioned medium from cardiomyocyte cultures subjected to stretch. As shown in Figure 3, stretching induced FSTL3 production by cardiomyocytes and conditioned medium derived from these cells significantly induced collagen release by fibroblasts (Fig. 3B). To determine the role of FSTL3 in this paracrine effect, we performed similar experiments in the presence of a neutralizing anti-FSTL3 or an isotype control antibody. As shown in Figure 3C, blockade of FSTL3 prevented collagen release from fibroblasts stimulated with conditioned medium form stressed cardiomyocytes. Of note, stretch had no effect on collagen expression in Fstl3 mediates paracrine fibroblast activation Panse et al. Page 12 of 27 cardiomyocytes themselves, ruling out the possibility that collagen was being released by these cells (Fig. S3). These results define FSTL3 as a paracrine mediator of fibroblast activation. Fstl3 induces fibroblast proliferation and alters cell adhesion Our data demonstrate that cardiac myocytes can activate fibroblasts and that FSTL3 is required for this effect. To determine if FSLT3 alone is sufficient to elicit this response we investigated the effect of exogenous FSTL3 protein on fibroblasts in terms of proliferation, adhesion, migration and collagen gene expression. Fibroblasts treated with FSTL3 alone induced a mild but significant increase in cell proliferation as measured by BrdU incorporation (Fig. 4A). We next explored its effects on fibroblast adhesion, since FSTL3 has previously shown to increase adhesion of hematopoietic progenitors to fibronectin [34]. We treated fibroblasts with FSTL3 for 24 h and allowed them to adhere to a culture dish for different periods of time. As shown in Fig. 4B, FSTL3 induced an increase in cell adhesion already at early time points. This effect was abolished if cells were detached with trypsin prior to the adhesion assay (data not shown), indicating that the action of FSTL3 is dependent on membrane proteins, likely integrins. Cell migration was measured using a Transwell device. Fibroblasts in the upper chamber were stimulated with FSTL3 and allowed to migrate towards the bottom chamber containing FCS as a chemoatractant. In contrast to cell adhesion, we detected no changes in cell migration (Fig. 4C). Similarly, no differences were found in collagen I expression in resting fibroblasts incubated with FSTL3 (Fig. 4D). To test whether additional factors might be necessary for the action of FSTL3 on fibroblasts, mechanical stretch was applied to cultured cells. As shown in Figure 4E, Col1a1 expression was elevated when fibroblasts were stretched prior to FSTL3 stimulation, whereas stretch itself did not enhance collagen expression. Overall these data argue that FSTL3 acts to induce proliferation and adhesion but that other factors are required for full fibroblast activation. Fstl3 interacts with CTGF Fstl3 mediates paracrine fibroblast activation Panse et al. Page 13 of 27 FSTL3 is believed to act, at least in part, through interaction with components of the TGF-β family such as myostatin, Activin or BMPs. However, there has been no systematic study on FSTL3 interacting factors, or of potential intracellular binding proteins in the heart. To address this issue we carried out a yeast two-hybrid assay using FSTL3 linked to the DNA binding domain of Gal4 as a bait for a rat cardiomyocyte cDNA library linked to the transcription activation domain of Gal4. A total of 156 clones were selected based on their activation of a reporter gene and identified by sequencing. After eliminating probable false positives in the form of mitochondrial, ribosomal and cloning vector targets as well as out-of- frame sequences, 110 likely targets were identified. As shown in Table S17, a number of targets identified were encoded in multiple individual clones, thereby indicating high likelihood of interaction with FSTL3. Interestingly, fibronectin 1 was encoded in 5 separate clones. This served as a positive control as interaction of FSTL3 with type I domains of fibronectin has been previously reported, although not in the heart [34]. A majority of 34 clones encoded syntenin, a cytoplasmic syndecan binding protein. The second most abundant gene found was connective tissue growth factor (CTGF), a well-known fibrotic marker. A number of targets such as granulin, EFEMP2, fibronectin, laminins, fibulins or fibrillins are components of the extracellular matrix (ECM) and basement membranes. Thus, it seems likely that FSTL3 may interact with ECM-associated proteins to potentiate its downstream effects. From the collection of probable FSTL3 interaction partners, connective tissue growth factor (CTGF) seemed a most promising candidate due to its previously documented role in the regulation of cardiac hypertrophy and fibrosis [35-37]. To validate this interaction we immunoprecipitated CTGF from a cardiomyocyte lysate using an anti-CTGF antibody and observed co-immunoprecipitation of FSTL3 proteins by western blot (Fig. 5A). In order to test the functional consequences of this interaction we first investigated their effect on fibroblast proliferation either alone or in combination. As shown in Fig. 5B, both proteins enhanced cell proliferation on their own and showed a slightly stronger effect when combined. We next examined whether CTGF together with FSTL3 were sufficient to induce collagen expression by fibroblasts. We observed no significant differences in Col1a1 expression after stimulation with both proteins, either alone or in combination (Fig. 5C), suggesting that additional signals are necessary. We have previously identified elevated myocardial FSTL3 as a feature of human patients with end-stage heart failure [21]. We examined the same patients for levels of CTGF Fstl3 mediates paracrine fibroblast activation Panse et al. Page 14 of 27 and found a strong positive correlation was found between FSTL3 and CTGF (Fig. 6D). This correlation was also observed between FSTL3 and another of its interactors, Fibronectin 1, as we previously reported [21]. Fstl3 mediates paracrine fibroblast activation Panse et al. Page 15 of 27 DISCUSSION Cardiac fibrosis is often seen as part of the remodelling response of the hypertrophied myocardium and many interventional studies have noted simultaneous reduction or increase in both cardiac hypertrophy and fibrosis. In contrast, other studies have reported differential regulation of hypertrophy and fibrosis. TGF- triggers distinct signals in cardiomyocytes and fibroblasts to promote hypertrophy and fibrosis respectively [38]. Similarly, mice with constitutive active PI3K or dominant negative PI3K in the heart showed increased or decreased cardiac size respectively without any effect on fibrosis [39]. Cardiac specific Fstl3 KO mice displayed reduced interstitial collagen and cardiac remodelling which may be a direct effect of reduced hypertrophy following pressure overload [14]. However, microarray analysis in these mice suggested that FSTL3 may function mainly by modulation of extracellular matrix components rather than hypertrophic mediators. Moreover, cardiomyocytes being the main source of increased FSTL3 in the stressed heart suggested that FSTL3 may function through autocrine and/or paracrine actions on other cell types in the myocardium. This was further supported by in vitro observations demonstrating a necessary role for cardiomyocyte-derived FSTL3 in fibrosis development through paracrine actions. Recently, a number of studies have focused on the function of paracrine factors in mediation of cardiac hypertrophy and fibrosis. For example,. Cardiomyocyte-derived TGF-β has been recognized to act on fibroblasts in the presence of angiotensin II to enhance collagen production and increase cell adhesion [40,41]. Furthermore, it was recently reported that paracrine action of TGF-β from cardiomyocytes is necessary for the protective effects of TGF-β type II receptor inhibition in the form of attenuated hypertrophy as well as fibrosis in response to pressure overload [38]. Similarly, IL-6 family cytokines have also been shown to play a paracrine role in fibrosis and cardiomyocyte hypertrophy. Pro-hypertrophic angiotensin II signalling induced IL-6, cardiotrohpin-1 (CT-1) and LIF expression in cardiac fibroblasts, which activated gp-130 signalling in cardiomyocytes to induce hypertrophic responses [42]. CT-1 stimulation was also sufficient to induce cardiac fibroblast growth and collagen synthesis and exhibited cooperation with endothelin-1/ETA receptor signalling [43]. In this context, we show here that cardiomyocyte-derived FSTL3 induces paracrine fibroblast activation, promoting cell proliferation, adhesion and collagen production in conjunction with other factors. The reduction in interstitial fibrosis observed in cardiomyocyte-specific Fstl3 Fstl3 mediates paracrine fibroblast activation Panse et al. Page 16 of 27 KO mice [14] together with the decreased expression of pro-fibrotic factors and ECM remodelling genes observed in these mice (Fig. 1 and 2) further reinforce the role of FSTL3 as a paracrine pro-fibrotic factor. Additionally, FSTL3 may also act on endothelial and vascular smooth muscle cells in the myocardium, as has been previously reported for FSTL1 [44,45]. Increased FSTL3 expression was detected in the endothelium of failing hearts [18], and may have adverse effects in the response to injury, possibly by inducing perivascular fibrosis. The dependence of the paracrine action of FSTL3 on additional stretch-derived factors from the myocardium suggests a coordinated response of the heart undergoing remodelling. In this regard, endothelin-1 and angiotensin II have been described to be released by cardiac myocytes upon mechanical stretch [46,47]. Similar effects have been reported in neonatal rat cardiac fibroblasts subjected to mechanical loading and stimulation with IGF-1, in which increased expression of α1 collagen type I mRNA was observed [48]. Many humoral factors and cytokines including angiotensin II, endothelin-1, TNF-α and TGF- β have been known to be secreted by mechanically loaded fibroblasts and are crucial to collagen synthesis and ECM remodelling [49-52]. Although the identity of the factors cooperating with FSTL3 remains as yet unknown, it is possible that a cell surface receptor on fibroblasts may only be expressed or morphologically exposed upon mechanical stretch. This regulation has been shown for surface integrins on fibroblasts, whose expression is changed in response to mechanical stimuli and which have been known to act as receptor complexes for mechanosensitive signal transduction [53]. Using a yeast two-hybrid approach we identified connective tissue growth factor (CTGF) among other FSTL3 interactors. Most of these were ECM-associated proteins involved in homeostasis, remodelling and structural integrity. In addition, our microarray analysis showed that most proteins from the ECM were downregulated in response to pressure overload in Fstl3 KO hearts compared to WT and we recently showed that FSTL3 expression correlates with fibrosis markers and mediators, like fibronectin 1 and CTGF, in patients with end-stage heart failure [21]. Together these results suggest that FSTL3 may act through these factors to mediate its effect on cardiac fibrosis and maybe also hypertrophy. Although CTGF interaction with FSTL3 did not alter collagen synthesis in fibroblasts, it was found to promote mild fibroblast proliferation and may thus be an important pro-fibrotic mechanism in the heart. Due to the modular structure of CTGF and its interaction with several extracellular signalling proteins as well as cell surface molecules, it is possible that Fstl3 mediates paracrine fibroblast activation Panse et al. Page 17 of 27 FSTL3 may use CTGF to sequester itself to specific cell surface proteins to act in a context- specific manner. A cardioprotective role of CTGF was recently demonstrated in a mouse model of ischemia-reperfusion injury in which CTGF reduced scar size and protected the myocardium from ischemic injury [54]. Whether the FSTL3-CTGF interaction may interfere with the cardioprotective actions of CTGF remains to be explored, but in a similar context we have previously shown that FSTL3 abolishes the cardioprotective effects of Activin A [13,14]. Although the molecular mechanism through which FSTL3 promotes fibroblast activation remains partially unknown and indeed a receptor for FSTL3 has so far not been found, our data suggest that it may contribute to fibrosis by acting as a co-factor of other pro- fibrotic molecules. FSTL3 action on cardiomyocytes and fibroblasts and its role in their cross-talk represents an important target of therapeutic manipulation. In this regard, the action of FSTL3 from cardiomyocyte conditioned medium could be inhibited by the addition of a neutralising antibody. These have been recognized to be highly specific molecular tools to inhibit the action of important disease regulators. Antibody therapy against both TGF-β1 and TNF-α has been shown to effectively reduce cardiac fibrosis and progression to heart failure [55,56]. In this respect, FSTL3 inhibition by neutralising antibody may be used as an effective therapeutic strategy to minimize the harmful actions of FSTL3 on cardiomyocytes as well as fibroblasts, reducing the development of cardiac hypertrophy and fibrosis. Fstl3 mediates paracrine fibroblast activation Panse et al. Page 18 of 27 CLINICAL STATEMENT There is increasing evidence that cardiomyocytes and fibroblasts communicate through different soluble mediators. However, the role and the identity of these mediators in cardiac fibrosis and the progression of heart disease are not well understood. This project was born out of an interesting clinical observation: the strong induction of FSTL3 expression in myocardial samples from heart failure patients [328]. Here we show that FSTL3 is secreted by mechanically stimulated cardiomyocytes to activate cardiac fibroblasts. Cardiomyocyte- specific knockout of Fstl3 considerably reduces cardiac fibrosis, further underlining the importance of FSTL3 as a paracrine fibrotic mediator. Our data suggest that FSTL3 is a clinically-relevant target whose inhibition may have therapeutic potential for the treatment and/or prevention of cardiac fibrosis, remodeling and heart failure. Fstl3 mediates paracrine fibroblast activation Panse et al. Page 19 of 27 ACKNOWLEDGEMENTS This work was supported by a British Heart Foundation grant (PG/08/084/25827) to P.B., N.R. and E.L.P. In addition, PB was supported by Heart Research UK and by the National Institute for Health Research Cardiovascular Biomedical Research Unit at the Royal Brompton and Harefield NHS Foundation Trust and Imperial College. E.L.P. was supported by grants from the European Union (ERG-239158, ITN-289600), the Spanish Ministry of Science and Innovation (BFU2009-10016, CP08/00144) and the Regional Government of Madrid (S2010/BMD-2321 “Fibroteam”). Fstl3 mediates paracrine fibroblast activation Panse et al. Page 20 of 27 REFERENCES 1. Spann JF, Bove AA, Natarajan G, Kreulen T (1980) Ventricular performance, pump function and compensatory mechanisms in patients with aortic stenosis. Circulation 62:576- 582. 2. Grossman W, Jones D, Mclaurin LP (1975) Wall stress and patterns of hypertrophy in the human left ventricle. The Journal of Clinical Investigation 56:56-64. 3. 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Ahmed MS, Gravning J, Martinov VN, Von Lueder TG, Edvardsen T, Czibik G, Moe IT, Vinge LE et al (2011) Mechanisms of novel cardioprotective functions of CCN2/CTGF in myocardial ischemia-reperfusion injury. American Journal of Physiology - Heart and Circulatory Physiology 300:H1291-H1302. 55. Kadokami T, Frye C, Lemster B, Wagner CL, Feldman AM, Mctiernan CF (2001) Anti- Tumor Necrosis Factor-{alpha} Antibody Limits Heart Failure in a Transgenic Model. Circulation 104:1094-1097. 56. Kuwahara F, Kai H, Tokuda K, Kai M, Takeshita A, Egashira K, Imaizumi T (2002) Transforming Growth Factor-{beta} Function Blocking Prevents Myocardial Fibrosis and Diastolic Dysfunction in Pressure-Overloaded Rats. Circulation 106:130-135. Fstl3 mediates paracrine fibroblast activation Panse et al. Page 25 of 27 FIGURE LEGENDS Figure 1. Cardiac-specific Fstl3 KO mice show reduced expression of fibrosis markers. Total RNA was isolated from cardiac samples following TAC or sham procedures and qRT- PCR was used to measure the myocardial mRNA expression of Col1a1 (A), Col3a1 (B), Lox (C), fibronectin-1 (D), TGF-1 (E) and CTGF (F). Two-way ANOVA followed by Bonferroni post-tests were used to calculate statistical significance. **p<0.01, ***p<0.001 vs. WT Sham; ##p<0.01, ###p<0.001 vs. WT TAC. n = 3-6 per group. G, H, TGF- and CTGF protein expression was analysed in wild type and Fstl3 KO mice by western blot. Total Akt was determined to show equal sample loading. Figure 2. Reduced expression of markers of extracellular matrix remodelling in Fstl3 KO hearts. MMP2, MMP9, Adam12 and TIMP1 mRNA expression was analysed in ventricular samples from WT and Fstl3 KO hearts by qRT-PCR 3 weeks after TAC or sham operation. Two-way ANOVA followed by Bonferroni post-tests were used to calculate statistical significance. **p<0.01, ***p<0.001 vs. WT Sham; ##p<0.01, ###p<0.001 vs. WT TAC. n = 3-6 per group. Figure 3. Mechanically stretched cardiomyocytes express FSTL3 that induces collagen production by fibroblasts. A, Neonatal rat ventricular cardiomyocytes were subjected to 10% equibiaxial stretch for 24 h and FSTL3 mRNA expression was measured by qRT-PCR. B, Conditioned medium from stretched cardiomyocytes was added to neonatal rat cardiac fibroblasts and newly formed collagen released to the medium was measured using a colorimetric collagen assay. Data are expressed as mean ± SEM. Unpaired t-tests were used to calculate the p values both in (A) and (B). **p<0.01, ***p<0.001 compared to „no stretch‟ control samples. n = 6-7. C, Anti-Fstl3 neutralizing antibody or an isotype matched control IgG were added to conditioned medium from stretched cardiomyocytes and collagen release from fibroblasts was measured. Data are expressed as mean ± SEM. 2-way ANOVA followed by Bonferroni post-test was used to calculate the statistical significance. *p<0.05 Fstl3 mediates paracrine fibroblast activation Panse et al. Page 26 of 27 for 10% stretch vs. no stretch; # p < 0.05 for anti-FSTL3 vs. control IgG at 10% stretch. n = 4. Figure 4. FSTL3 enhances fibroblasts proliferation, adhesion and collagen expression. A, Neonatal cardiac fibroblasts were stimulated for 24 h with 200 ng/ml FSTL3 in serum-free medium and BrdU incorporation during the last 18 h was measured to determine cell proliferation. *p<0.05 FSTL3 vs control in unpaired t-test. n=4. B, Fibroblasts were incubated with 100 ng/ml FSTL3 for 24 h and allowed to adhere to an uncoated tissue-culture dish in stimulus-free medium for the indicated periods of time. Attached cells were fixed and stained with crystal violet and absorbance was measured at 540 nm. Data are expressed as mean ± SEM. 2-way ANOVA followed by Bonferroni post-test was used to calculate the statistical significance. *p<0.05, **<0.01 FSTL3 vs control. n = 3. C, Fibroblasts were plated on the upper chamber of transwell migration devices in serum-free medium, stimulated with 200 ng/ml FSTL3 and allowed to migrate to the lower chamber containing 2% FCS. After 6 h, cells in the bottom chamber were detached and counted. Data are expressed as mean ± SEM. D, Fibroblasts were treated with 200 ng/ml FSTL3 for 24 h and Col1a1 mRNA expression was measured by qRT-PCR. E, Fibroblasts were subjected to 15% equibiaxial stretch for 24 h and then stimulated with 200 ng/ml FSTL3 for another 24 h. Col1a1 mRNA expression was measured using qRT-PCR and expressed as mean fold induction ± SEM. 2- way ANOVA followed by Bonferonni post-test was used to calculate statistical significance. ***p<0.001 stretch vs. no stretch. ##p<0.01 for FSTL3 vs. control. n = 3. Figure 5. FSTL3 interacts with CTGF. A, CTGF was immunoprecipitated from fibroblast cell lysate using anti-CTGF antibody and the bound proteins detected by western blot. B, Fibroblasts were stimulated for 24 h with 200 ng/ml FSTL3, 500 ng/ml CTGF or both in serum-free medium and BrdU incorporation during the last 18 h was measured to determine cell proliferation. *p<0.05 stimulus vs control, unpaired t-test. n=4. C, Cells were stimulated for 24 h as in (B) and Col1a1 mRNA expression was analysed by qRT-PCR. D, FSTL3 and CTGF expression was determined by qRT-PCR in myocardial samples from heart failure patients (n=18) and Spearman correlation between both factors was calculated. Fstl3 mediates paracrine fibroblast activation Panse et al. Page 27 of 27 Figure 6. Schematic of FSTL3 action in the heart. FSTL3 is secreted from cardiomyocytes following mechanical loading and acts in an autocrine manner to induce cardiomyocyte hypertrophy. In coordination with unknown stretch-induced factors, FSTL3 acts in a paracrine manner on fibroblasts to promote collagen synthesis. In addition, FSTL3 interacts with CTGF to enhance fibroblast proliferation. Together these results suggest a role of FSTL3 in paracrine fibroblast activation. Figure 1 Click here to download high resolution image Figure 2 Click here to download high resolution image Figure 3 Click here to download high resolution image Figure 4 Click here to download high resolution image Figure 5 Click here to download high resolution image Figure 6 Click here to download high resolution image Fstl3 mediates paracrine fibroblast activation – Supplemental Material Panse et al. Page 1 of 2 SUPPLEMENTAL MATERIAL FIGURE LEGENDS Figure S1. Cardiac-specific Fstl3 KO mice show reduced expression of heart failure markers. Total RNA was isolated from cardiac samples following TAC or sham procedures and qRT-PCR was used to measure the myocardial mRNA expression of ANP/ANF (A), BNP (B), -skeletal actin/Acta1 (C) and -myosin heavy chain/Myh7 (D). Two-way ANOVA followed by Bonferroni post-tests were used to calculate statistical significance. **p<0.01, ***p<0.001 vs. WT Sham; #p<0.05, ##p<0.01, ###p<0.001 vs. WT TAC. n = 3-6 per group. Figure S2. Expression of inflammation markers in Fstl3 KO hearts. mRNA expression of cytokines IL-1 and IL6, chemokine CCL5/Rantes and the macrophage marker CD68 was analysed in ventricular samples from WT and Fstl3 KO hearts by qRT-PCR 3 weeks after TAC or sham operation. Two-way ANOVA followed by Bonferroni post-tests were used to calculate statistical significance. **p<0.01, vs. WT Sham. n = 3-6 per group. Figure S3. Mechanical stretched does not induce collagen expression in cardiomyocytes. A, Neonatal rat ventricular cardiomyocytes were subjected to 10% equibiaxial stretch for 24 h and Col1a1 and Col3a1 mRNA expression was measured by qRT-PCR. Data are expressed as mean ± SEM. Unpaired t-tests were used to calculate the p values. **p<0.01 compared to ‘no stretch’ control samples. n = 6-7. Supplemental figures and tables Click here to download Table: Panse suppl.pdf Fstl3 mediates paracrine fibroblast activation – Supplemental Material Panse et al. Page 2 of 2 SUPPLEMENTAL TABLES Tables S1-S16. Microarray analysis of cardiac hypertrophy. RNA extracted from WT and KO hearts 21 days after transaortic constriction (TAC) or Sham operation was analyzed by microarray as described in materials and methods. Odd number tables contain lists of genes whose expression changes after >1.4-fold, between the sample groups indicated in the title. Even number tables contain the Gene Ontology analysis of the preceding gene list. Table S17. Yeast two-hybrid analysis of FSTL3 interacting proteins. FSTL3 was used as a bait to identify possible interacting proteins expressed in yeast from a neonatal rat cardiomyocyte cDNA library. After eliminating probable false positives in the form of mitochondrial, ribosomal and cloning vector targets as well as out-of-frame sequences, 110 likely targets were identified. The number of clones that encoded each of them (Number of hits) is listed. Affy ID Fold Gene symbol Description 10398075 10.001 Serpina3n serine (or cysteine) peptidase inhibitor, clade A, member 3N 10598976 7.667 Timp1 tissue inhibitor of metalloproteinase 1 10492021 7.584 Postn periostin, osteoblast specific factor 10419934 6.169 Myh7 myosin, heavy polypeptide 7, cardiac muscle, beta 10358476 6.032 Prg4 proteoglycan 4 (megakaryocyte stimulating factor, articular superficial zone protein) 10586357 5.534 Cilp cartilage intermediate layer protein, nucleotide pyrophosphohydrolase 10541496 4.826 Mfap5 microfibrillar associated protein 5 10481627 4.813 Lcn2 lipocalin 2 10582592 4.735 Acta1 actin, alpha 1, skeletal muscle 10440091 4.726 Col8a1 collagen, type VIII, alpha 1 10510265 4.572 Nppa natriuretic peptide precursor type A 10401527 4.562 Ltbp2 latent transforming growth factor beta binding protein 2 10574023 4.359 Mt2 metallothionein 2 10376778 3.757 Mfap4 microfibrillar‐associated protein 4 10417212 3.732 Itgbl1 integrin, beta‐like 1 10355403 3.512 Fn1 fibronectin 1 10416181 3.506 Stc1 stanniocalcin 1 10462442 3.407 Il33 interleukin 33 10435641 3.232 Fstl1 follistatin‐like 1 10534667 3.207 Serpine1 serine (or cysteine) peptidase inhibitor, clade E, member 1 10484307 3.194 Frzb frizzled‐related protein 10517213 3.075 Cnksr1 connector enhancer of kinase suppressor of Ras 1 10513739 3.055 Tnc tenascin C 10572897 3.031 Hmox1 heme oxygenase (decycling) 1 10478048 2.986 Lbp lipopolysaccharide binding protein 10542355 2.885 Emp1 epithelial membrane protein 1 10513208 2.864 Svep1 sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1 10380419 2.854 Col1a1 collagen, type I, alpha 1 10554752 2.840 Nox4 NADPH oxidase 4 10424140 2.801 Col14a1 collagen, type XIV, alpha 1 10359851 2.763 Uck2 uridine‐cytidine kinase 2 10388430 2.638 Serpinf1 serine (or cysteine) peptidase inhibitor, clade F, member 1 10536220 2.534 Col1a2 collagen, type I, alpha 2 10586865 2.515 Aldh1a2 aldehyde dehydrogenase family 1, subfamily A2 10416215 2.427 Loxl2 lysyl oxidase‐like 2 10475517 2.417 AA467197 expressed sequence AA467197 10573979 2.336 Gnao1 guanine nucleotide binding protein, alpha O 10510260 2.319 Nppb natriuretic peptide precursor type B 10463070 2.245 Entpd1 ectonucleoside triphosphate diphosphohydrolase 1 10450242 2.219 C4b///C4a complement component 4B (Childo blood group)///complement component 4A (Rodgers blood group 10359849 2.215 Uck2 uridine‐cytidine kinase 2 10346015 2.210 Col3a1 collagen, type III, alpha 1 10582275 2.181 Slc7a5 solute carrier family 7 (cationic amino acid transporter, y+ system), member 5 10409464 2.176 Dbn1 drebrin 1 10401841 2.109 Dio2 deiodinase, iodothyronine, type II 10489878 2.097 Ptgis prostaglandin I2 (prostacyclin) synthase 10374083 2.079 Aebp1 AE binding protein 1 10434698 2.075 Fetub fetuin beta 10472426 2.061 Xirp2 xin actin‐binding repeat containing 2 10523175 2.044 Ereg epiregulin 10360764 2.032 Enah enabled homolog (Drosophila) 10464761 2.031 Syt12 synaptotagmin XII 10461721 2.018 Mpeg1 macrophage expressed gene 1 10407481 2.004 Pfkp phosphofructokinase, platelet 10429128 2.002 Sla src‐like adaptor 10562192 1.990 Fxyd5 FXYD domain‐containing ion transport regulator 5 10572398 1.974 Crlf1 cytokine receptor‐like factor 1 10467191 1.961 Ankrd1 ankyrin repeat domain 1 (cardiac muscle) 10600169 1.950 Bgn biglycan Supplemental Table 1 - WT TAC > WT Sham GO ID GO ACCESSION GO Term p‐value corrected p‐value Count in Selection % Count in Selection Count in Total % Count in Total 3394 GO:0005576 extracellular region 1.05E‐15 1.36E‐11 46 51.11111 1688 7.7477393 12843 GO:0031012 extracellular matrix 6.02E‐15 3.91E‐11 20 22.222221 292 1.3402488 3396 GO:0005578 proteinaceous extracellular matrix 2.45E‐13 1.06E‐09 18 20 274 1.2576307 18811 GO:0044420 extracellular matrix part 2.23E‐12 7.24E‐09 6 6.6666665 87 0.3993207 18812 GO:0044421 extracellular region part 9.11E‐11 2.36E‐07 20 22.222221 760 3.4883187 3351 GO:0005488 binding 8.26E‐08 1.78E‐04 58 64.44444 11058 50.75504 3365 GO:0005515|GO:0045308 protein binding 3.50E‐07 6.49E‐04 51 56.666668 5545 25.450956 4579 GO:0007015 actin filament organization 6.71E‐07 8.71E‐04 5 5.5555553 42 0.1927755 6431 GO:0009611|GO:0002245 response to wounding 5.57E‐07 8.71E‐04 1 1.1111112 359 1.6477716 25388 GO:0065008 regulation of biological quality 6.09E‐07 8.71E‐04 6 6.6666665 944 4.332859 3205 GO:0005201 extracellular matrix structural constituent 8.35E‐07 9.85E‐04 3 3.3333333 24 0.11015743 3420 GO:0005604 basement membrane 9.30E‐07 0.001005622 6 6.6666665 70 0.32129252 5391 GO:0008360|GO:0045788|GO:0045789 regulation of cell shape 1.94E‐06 0.001930834 6 6.6666665 50 0.22949465 11860 GO:0022603 regulation of anatomical structure morphogenesis 2.32E‐06 0.002150899 6 6.6666665 114 0.52324784 4699 GO:0007155 cell adhesion 2.98E‐06 0.002413551 14 15.555555 603 2.7677054 11867 GO:0022610 biological adhesion 2.98E‐06 0.002413551 14 15.555555 603 2.7677054 11976 GO:0030029 actin filament‐based process 3.74E‐06 0.002855842 8 8.888889 162 0.7435627 2128 GO:0003779 actin binding 6.50E‐06 0.004440153 11 12.222222 271 1.243861 11861 GO:0022604 regulation of cell morphogenesis 6.32E‐06 0.004440153 6 6.6666665 61 0.2799835 6425 GO:0009605 response to external stimulus 9.40E‐06 0.005808385 2 2.2222223 589 2.703447 8183 GO:0015629 actin cytoskeleton 1.10E‐05 0.006480557 5 5.5555553 185 0.8491302 11983 GO:0030036 actin cytoskeleton organization and biogenesis 1.60E‐05 0.008663518 7 7.7777777 148 0.6793042 Supplemental Table 2 - GO: WT TAC > WT Sham Affy ID Fold Gene symbol Description 10580635 7.043 Ces3 carboxylesterase 3 10598064 3.423 10441794 3.019 Mrgprh MAS‐related GPR, member H 10598062 3.008 10511363 2.946 Penk preproenkephalin 10598018 2.486 10386197 2.382 2210407C18Rik RIKEN cDNA 2210407C18 gene 10602385 2.301 Pfkfb1 6‐phosphofructo‐2‐kinase/fructose‐2,6‐biphosphatase 1 10579958 2.242 Il15 interleukin 15 10397763 2.238 9030617O03Rik RIKEN cDNA 9030617O03 gene 10398824 2.209 A530016L24Rik RIKEN cDNA A530016L24 gene 10534085 2.147 Phkg1 phosphorylase kinase gamma 1 10409999 2.119 Fbp2 fructose bisphosphatase 2 10462303 2.106 Kcnv2 potassium channel, subfamily V, member 2 10574488 2.055 Pdp2 pyruvate dehyrogenase phosphatase catalytic subunit 2 10433403 2.049 A2bp1 ataxin 2 binding protein 1 10468113 2.023 Kcnip2 Kv channel‐interacting protein 2 10413381 2.018 Dnahc12///Asb14 dynein, axonemal, heavy chain 12///ankyrin repeat and SOCS box‐containing 14 10436095 1.981 Retnla resistin like alpha 10536611 1.980 Kcnd2 potassium voltage‐gated channel, Shal‐related family, member 2 10422028 1.971 Tbc1d4 TBC1 domain family, member 4 10536697 1.940 Asb15 ankyrin repeat and SOCS box‐containing 15 10547153 1.936 Alox5 arachidonate 5‐lipoxygenase 10369806 1.897 1700040L02Rik RIKEN cDNA 1700040L02 gene 10537712 1.869 Gstk1 glutathione S‐transferase kappa 1 10419525 1.853 A930018M24Rik RIKEN cDNA A930018M24 gene 10480459 1.851 Hnmt histamine N‐methyltransferase 10428619 1.849 Enpp2 ectonucleotide pyrophosphatase/phosphodiesterase 2 10396079 1.842 Klhdc1 kelch domain containing 1 10357965 1.832 Lgr6 leucine‐rich repeat‐containing G protein‐coupled receptor 6 10454683 1.815 Pkd2l2 polycystic kidney disease 2‐like 2 10352133 1.813 Efcab2 EF‐hand calcium binding domain 2 10598077 1.806 10603746 1.789 Maob monoamine oxidase B 10348906 1.772 Gm6086 predicted gene 6086 10598023 1.771 10539143 1.770 Retsat retinol saturase (all trans retinol 13,14 reductase) 10415279 1.758 Fitm1 fat storage‐inducing transmembrane protein 1 10386020 1.758 Slc36a2 solute carrier family 36 (proton/amino acid symporter), member 2 10455146 1.744 3222401L13Rik RIKEN cDNA 3222401L13 gene 10463704 1.735 As3mt arsenic (+3 oxidation state) methyltransferase 10344725 1.728 Adhfe1 alcohol dehydrogenase, iron containing, 1 10422962 1.727 1110020G09Rik RIKEN cDNA 1110020G09 gene 10377245 1.717 Dhrs7c dehydrogenase/reductase (SDR family) member 7C 10606436 1.713 Hmgn5 high‐mobility group nucleosome binding domain 5 10545001 1.697 Ppm1k protein phosphatase 1K (PP2C domain containing) 10596718 1.696 Slc38a3 solute carrier family 38, member 3 10503484 1.679 Fam82b family with sequence similarity 82, member B 10474361 1.638 Mpped2 metallophosphoesterase domain containing 2 10433177 1.630 Gm9108 predicted gene 9108 10438017 1.627 Fgd4 FYVE, RhoGEF and PH domain containing 4 10601878 1.623 Tceal1 transcription elongation factor A (SII)‐like 1 10466903 1.621 4430402I18Rik RIKEN cDNA 4430402I18 gene 10409737 1.620 Agtpbp1 ATP/GTP binding protein 1 10542834 1.619 10348889 1.614 D2hgdh D‐2‐hydroxyglutarate dehydrogenase 10589368 1.613 Plxnb1 plexin B1 10517600 1.610 Pink1 PTEN induced putative kinase 1 10409118 1.610 Wnk2 WNK lysine deficient protein kinase 2 Supplemental Table 3 - WT TAC < WT Sham 10547410 1.609 LOC100048600///Erc1 ELKS/RAB6‐interacting/CAST family member 1 10456699 1.608 Acaa2 acetyl‐Coenzyme A acyltransferase 2 (mitochondrial 3‐oxoacyl‐Coenzyme A thiolase) 10569331 1.602 Gm14492 predicted gene 14492 10592061 1.598 Kcnj5 potassium inwardly‐rectifying channel, subfamily J, member 5 10382321 1.586 Kcnj2 potassium inwardly‐rectifying channel, subfamily J, member 2 10494388 1.585 Hist2h2be histone cluster 2, H2be 10574350 1.582 Mmp15 matrix metallopeptidase 15 10402117 1.582 Rps6ka5 ribosomal protein S6 kinase, polypeptide 5 10368720 1.578 Slc16a10 solute carrier family 16 (monocarboxylic acid transporters), member 10 10360053 1.576 Pcp4l1 Purkinje cell protein 4‐like 1 10587534 1.575 Bckdhb branched chain ketoacid dehydrogenase E1, beta polypeptide 10494114 1.557 Selenbp1///Selenbp2///LOCselenium binding protein 2///selenium binding protein 1 10423505 1.555 Cmbl carboxymethylenebutenolidase‐like (Pseudomonas) 10513692 1.550 Whrn whirlin 10529082 1.548 Gtf3c2///Mpv17 general transcription factor IIIC, polypeptide 2, beta///MpV17 mitochondrial inner membran 10497862 1.548 Trpc3 transient receptor potential cation channel, subfamily C, member 3 10478145 1.545 Ppp1r16b protein phosphatase 1, regulatory (inhibitor) subunit 16B 10575363 1.543 Zfp612 zinc finger protein 612 10420935 1.541 Ephx2 epoxide hydrolase 2, cytoplasmic 10533504 1.538 Ift81 intraflagellar transport 81 homolog (Chlamydomonas) 10411532 1.529 Mccc2 methylcrotonoyl‐Coenzyme A carboxylase 2 (beta) 10530819 1.526 Hopx HOP homeobox 10509204 1.525 Tcea3 transcription elongation factor A (SII), 3 10550638 1.517 Rtn2 reticulon 2 (Z‐band associated protein) 10406482 1.517 Ccnh cyclin H 10595205 1.517 2410127L17Rik RIKEN cDNA 2410127L17 gene 10401473 1.516 Aldh6a1 aldehyde dehydrogenase family 6, subfamily A1 10398751 1.511 Zfyve21 zinc finger, FYVE domain containing 21 10445214 1.510 Mut methylmalonyl‐Coenzyme A mutase 10424559 1.507 Khdrbs3 KH domain containing, RNA binding, signal transduction associated 3 10586368 1.503 Clpx caseinolytic peptidase X (E.coli) 10372988 1.499 Slc16a7 solute carrier family 16 (monocarboxylic acid transporters), member 7 10371332 1.496 Aldh1l2 aldehyde dehydrogenase 1 family, member L2 10513158 1.496 Ptpn3 protein tyrosine phosphatase, non‐receptor type 3 10551393 1.496 LOC100048123///Akt2 thymoma viral proto‐oncogene 2 10494085 1.495 Selenbp2 selenium binding protein 2 10405727 1.487 2410127L17Rik RIKEN cDNA 2410127L17 gene 10412481 1.486 2410127L17Rik RIKEN cDNA 2410127L17 gene 10529515 1.483 Sorcs2 sortilin‐related VPS10 domain containing receptor 2 10587871 1.482 Paqr9 progestin and adipoQ receptor family member IX 10482181 1.481 Strbp spermatid perinuclear RNA binding protein 10382573 1.479 2310067B10Rik RIKEN cDNA 2310067B10 gene 10510957 1.478 Pank4 pantothenate kinase 4 10461921 1.477 2410127L17Rik RIKEN cDNA 2410127L17 gene 10365408 1.476 Ric8b resistance to inhibitors of cholinesterase 8 homolog B (C. elegans) 10496605 1.468 Ccbl2 cysteine conjugate‐beta lyase 2 10501555 1.467 Amy1 amylase 1, salivary 10412909 1.466 Fdft1 farnesyl diphosphate farnesyl transferase 1 10474836 1.465 Ivd isovaleryl coenzyme A dehydrogenase 10593789 1.454 Etfa electron transferring flavoprotein, alpha polypeptide 10533345 1.453 Aldh2 aldehyde dehydrogenase 2, mitochondrial 10361926 1.449 Map3k5 mitogen‐activated protein kinase kinase kinase 5 10355084 1.448 Ndufs1 NADH dehydrogenase (ubiquinone) Fe‐S protein 1 10412882 1.447 Thrb thyroid hormone receptor beta 10408656 1.445 Peci peroxisomal delta3, delta2‐enoyl‐Coenzyme A isomerase 10495562 1.444 Lrrc39 leucine rich repeat containing 39 10420730 1.443 Fdft1 farnesyl diphosphate farnesyl transferase 1 10553935 1.443 Tm2d3///Tarsl2 threonyl‐tRNA synthetase‐like 2///TM2 domain containing 3 10346365 1.442 Sgol2 shugoshin‐like 2 (S. pombe) 10410743 1.441 Ankrd32 ankyrin repeat domain 32 10467162 1.441 Pank1 pantothenate kinase 1 10578810 1.441 Clcn3 chloride channel 3 10511382 1.438 Nsmaf neutral sphingomyelinase (N‐SMase) activation associated factor 10502982 1.436 Tnni3k TNNI3 interacting kinase 10546129 1.435 Kbtbd12 kelch repeat and BTB (POZ) domain containing 12 10524460 1.435 Acacb acetyl‐Coenzyme A carboxylase beta 10481474 1.433 Crat carnitine acetyltransferase 10494551 1.433 Acp6 acid phosphatase 6, lysophosphatidic 10455912 1.432 Isoc1 isochorismatase domain containing 1 10497309 1.430 Snx16 sorting nexin 16 10488322 1.430 Ralgapa2 Ral GTPase activating protein, alpha subunit 2 (catalytic) 10420804 1.429 4933401F05Rik RIKEN cDNA 4933401F05 gene 10492469 1.422 Mlf1 myeloid leukemia factor 1 10443391 1.422 Mapk14 mitogen‐activated protein kinase 14 10506154 1.422 Alg6 asparagine‐linked glycosylation 6 homolog (yeast, alpha‐1,3,‐glucosyltransferase) 10399882 1.421 Dus4l dihydrouridine synthase 4‐like (S. cerevisiae) 10346260 1.419 Osgepl1 O‐sialoglycoprotein endopeptidase‐like 1 10364030 1.418 Adora2a adenosine A2a receptor 10483521 1.414 Fastkd1 FAST kinase domains 1 10522160 1.413 N4bp2 NEDD4 binding protein 2 10582229 1.412 1110003O08Rik RIKEN cDNA 1110003O08 gene 10368162 1.409 Pex7 peroxisomal biogenesis factor 7 10511333 1.409 Plag1 pleiomorphic adenoma gene 1 10484207 1.407 2610301F02Rik RIKEN cDNA 2610301F02 gene 10355343 1.406 Abca12 ATP‐binding cassette, sub‐family A (ABC1), member 12 GO ID GO ACCESSION GO Term p‐value corrected p‐value Count in Selection % Count in Selection Count in Total % Count in Total 3518 GO:0005739 mitochondrion 5.96E‐10 5.24E‐06 30 39.473682 1393 6.393721 2134 GO:0003824 catalytic activity 1.92E‐09 8.43E‐06 34 44.736843 4937 22.660301 3517 GO:0005737 cytoplasm 8.18E‐07 0.002398932 60 78.947365 6718 30.834902 5226 GO:0008152 metabolic process 2.90E‐06 0.004247454 18 23.68421 6932 31.817139 3552 GO:0005777|GO:0019818 peroxisome 3.93E‐06 0.00432228 7 9.210526 102 0.4681691 17438 GO:0042579 microbody 3.93E‐06 0.00432228 7 9.210526 102 0.4681691 8779 GO:0016301 kinase activity 1.16E‐05 0.009255758 14 18.421053 794 3.644375 Supplemental Table 4 - GO: WT TAC < WT Sham Affy ID Fold Gene symbol Description 10481627 2.994 Lcn2 lipocalin 2 10574023 2.379 Mt2 metallothionein 2 10538802 2.323 A930038C07Rik RIKEN cDNA A930038C07 gene 10398075 2.232 Serpina3n serine (or cysteine) peptidase inhibitor, clade A, member 3N 10534667 2.011 Serpine1 serine (or cysteine) peptidase inhibitor, clade E, member 1 10416181 1.786 Stc1 stanniocalcin 1 10358476 1.639 Prg4 proteoglycan 4 (megakaryocyte stimulating factor, articular superficial zone protein) 10462442 1.636 Il33 interleukin 33 10555389 1.533 Ucp2 uncoupling protein 2 (mitochondrial, proton carrier) 10450242 1.525 C4b///C4a complement component 4B (Childo blood group)///complement component 4A (Rodgers blood group) 10566346 1.500 9230105E10Rik RIKEN cDNA 9230105E10 gene 10598976 1.496 Timp1 tissue inhibitor of metalloproteinase 1 10360985 1.494 Cenpf centromere protein F 10538082 1.487 Atp6v0e2 ATPase, H+ transporting, lysosomal V0 subunit E2 10601701 1.415 Tmem35 transmembrane protein 35 Supplemental Table 5 - KO Sham > WT Sham GO ID GO ACCESSION GO Term p‐value corrected p‐value Count in Selection % Count in Selection Count in Total % Count in Total No GO lists pass the cut Supplemental Table 6 - GO: KO Sham > WT Sham Affy ID Fold Gene symbol Description 10386197 1.676 2210407C18Rik RIKEN cDNA 2210407C18 gene 10510260 1.434 Nppb natriuretic peptide precursor type B Supplemental Table 7 - KO Sham < WT Sham GO ID GO ACCESSION GO Term p‐value corrected p‐value Count in Selection % Count in Selection Count in Total % Count in Total No GO lists pass the cut Supplemental Table 8 - GO: KO Sham < WT Sham Affy ID Fold Gene symbol Description 10510260 3.008 Nppb natriuretic peptide precursor type B 10419934 2.866 Myh7 myosin, heavy polypeptide 7, cardiac muscle, beta 10586357 2.733 Cilp cartilage intermediate layer protein, nucleotide pyrophosphohydrolase 10582592 2.718 Acta1 actin, alpha 1, skeletal muscle 10510265 2.567 Nppa natriuretic peptide precursor type A 10440091 2.506 Col8a1 collagen, type VIII, alpha 1 10492021 2.336 Postn periostin, osteoblast specific factor 10541496 2.309 Mfap5 microfibrillar associated protein 5 10376778 2.141 Mfap4 microfibrillar‐associated protein 4 10401841 2.027 Dio2 deiodinase, iodothyronine, type II 10448307 2.014 Tnfrsf12a tumor necrosis factor receptor superfamily, member 12a 10359851 1.989 Uck2 uridine‐cytidine kinase 2 10598976 1.943 Timp1 tissue inhibitor of metalloproteinase 1 10484307 1.934 Frzb frizzled‐related protein 10401527 1.880 Ltbp2 latent transforming growth factor beta binding protein 2 10513208 1.835 Svep1 sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1 10435641 1.833 Fstl1 follistatin‐like 1 10542355 1.820 Emp1 epithelial membrane protein 1 10359849 1.775 Uck2 uridine‐cytidine kinase 2 10554752 1.769 Nox4 NADPH oxidase 4 10417212 1.750 Itgbl1 integrin, beta‐like 1 10517213 1.749 Cnksr1 connector enhancer of kinase suppressor of Ras 1 10416215 1.740 Loxl2 lysyl oxidase‐like 2 10573979 1.666 Gnao1 guanine nucleotide binding protein, alpha O 10388430 1.637 Serpinf1 serine (or cysteine) peptidase inhibitor, clade F, member 1 10380419 1.624 Col1a1 collagen, type I, alpha 1 10472426 1.623 Xirp2 xin actin‐binding repeat containing 2 10407481 1.615 Pfkp phosphofructokinase, platelet 10490159 1.605 Pmepa1 prostate transmembrane protein, androgen induced 1 10360764 1.601 Enah enabled homolog (Drosophila) 10573747 1.597 Adcy7 adenylate cyclase 7 10346015 1.575 Col3a1 collagen, type III, alpha 1 10357043 1.562 Bcl2 B‐cell leukemia/lymphoma 2 10444591 1.556 Hspa1l heat shock protein 1‐like 10424140 1.553 Col14a1 collagen, type XIV, alpha 1 10548128 1.542 Tspan9 tetraspanin 9 10536220 1.532 Col1a2 collagen, type I, alpha 2 10500295 1.522 Plekho1 pleckstrin homology domain containing, family O member 1 10452485 1.512 Rab31 RAB31, member RAS oncogene family 10455461 1.475 Myot myotilin 10513739 1.454 Tnc tenascin C 10355403 1.437 Fn1 fibronectin 1 10445347 1.433 Clic5 chloride intracellular channel 5 10519140 1.430 Mmp23 matrix metallopeptidase 23 10467191 1.428 Ankrd1 ankyrin repeat domain 1 (cardiac muscle) 10381898 1.424 Mrc2 mannose receptor, C type 2 10386058 1.409 Sparc secreted acidic cysteine rich glycoprotein Supplemental Table 9 - KO TAC > KO Sham GO ID GO ACCESSION GO Term p‐value corrected p‐value Count in Selection % Count in Selection Count in Total % Count in Total 12843 GO:0031012 extracellular matrix 7.22E‐16 4.44E‐12 14 43.75 292 1.3402488 3396 GO:0005578 proteinaceous extracellular matrix 1.04E‐14 3.20E‐11 13 40.625 274 1.2576307 3394 GO:0005576 extracellular region 3.52E‐14 7.22E‐11 23 71.875 1688 7.7477393 18811 GO:0044420 extracellular matrix part 1.65E‐13 2.53E‐10 8 25 87 0.3993207 18812 GO:0044421 extracellular region part 2.25E‐11 2.76E‐08 14 43.75 760 3.4883187 4699 GO:0007155 cell adhesion 3.69E‐08 3.24E‐05 9 28.125 603 2.7677054 11867 GO:0022610 biological adhesion 3.69E‐08 3.24E‐05 9 28.125 603 2.7677054 3205 GO:0005201 extracellular matrix structural constituent 1.79E‐07 1.22E‐04 3 9.375 24 0.11015743 3400 GO:0005583 fibrillar collagen 1.75E‐07 1.22E‐04 2 6.25 6 0.027539358 3351 GO:0005488 binding 2.53E‐07 1.55E‐04 23 71.875 11058 50.75504 3420 GO:0005604 basement membrane 3.66E‐07 2.05E‐04 5 15.625 70 0.32129252 22049 GO:0048407 platelet‐derived growth factor binding 1.05E‐06 5.36E‐04 3 9.375 10 0.04589893 3365 GO:0005515|GO:0045308 protein binding 1.77E‐06 8.38E‐04 21 65.625 5545 25.450956 3401 GO:0005584 collagen type I 4.36E‐06 1.92E‐03 2 6.25 2 0.009179786 4795 GO:0007275 multicellular organismal development 6.53E‐06 0.002508909 4 12.5 2502 11.483912 3398 GO:0005581 collagen 7.03E‐06 2.54E‐03 3 9.375 18 0.08261807 11152 GO:0019838 growth factor binding 1.61E‐05 0.005227941 4 12.5 72 0.3304723 Supplemental Table 10 - GO: KO TAC > KO Sham Affy ID Fold Gene symbol Description 10580635 2.246 Ces3 carboxylesterase 3 10602385 2.211 Pfkfb1 6‐phosphofructo‐2‐kinase/fructose‐2,6‐biphosphatase 1 10598077 1.926 10511363 1.855 Penk preproenkephalin 10480459 1.845 Hnmt histamine N‐methyltransferase 10534085 1.804 Phkg1 phosphorylase kinase gamma 1 10441794 1.770 Mrgprh MAS‐related GPR, member H 10397763 1.749 9030617O03Rik RIKEN cDNA 9030617O03 gene 10606436 1.723 Hmgn5 high‐mobility group nucleosome binding domain 5 10386197 1.714 2210407C18Rik RIKEN cDNA 2210407C18 gene 10419525 1.708 A930018M24Rik RIKEN cDNA A930018M24 gene 10603746 1.704 Maob monoamine oxidase B 10574488 1.674 Pdp2 pyruvate dehyrogenase phosphatase catalytic subunit 2 10433177 1.651 Gm9108 predicted gene 9108 10462303 1.648 Kcnv2 potassium channel, subfamily V, member 2 10579958 1.633 Il15 interleukin 15 10369806 1.625 1700040L02Rik RIKEN cDNA 1700040L02 gene 10428619 1.613 Enpp2 ectonucleotide pyrophosphatase/phosphodiesterase 2 10601701 1.593 Tmem35 transmembrane protein 35 10360985 1.584 Cenpf centromere protein F 10436095 1.562 Retnla resistin like alpha 10481627 1.519 Lcn2 lipocalin 2 10512279 1.517 Cntfr ciliary neurotrophic factor receptor 10433403 1.507 A2bp1 ataxin 2 binding protein 1 10474361 1.505 Mpped2 metallophosphoesterase domain containing 2 10402117 1.497 Rps6ka5 ribosomal protein S6 kinase, polypeptide 5 10377245 1.495 Dhrs7c dehydrogenase/reductase (SDR family) member 7C 10536611 1.493 Kcnd2 potassium voltage‐gated channel, Shal‐related family, member 2 10466903 1.489 4430402I18Rik RIKEN cDNA 4430402I18 gene 10372988 1.488 Slc16a7 solute carrier family 16 (monocarboxylic acid transporters), member 7 10348906 1.486 Gm6086 predicted gene 6086 10409999 1.481 Fbp2 fructose bisphosphatase 2 10409118 1.478 Wnk2 WNK lysine deficient protein kinase 2 10544687 1.478 Cycs cytochrome c, somatic 10413381 1.473 Dnahc12///Asb14 dynein, axonemal, heavy chain 12///ankyrin repeat and SOCS box‐containing 14 10423505 1.468 Cmbl carboxymethylenebutenolidase‐like (Pseudomonas) 10422962 1.467 1110020G09Rik RIKEN cDNA 1110020G09 gene 10412909 1.459 Fdft1 farnesyl diphosphate farnesyl transferase 1 10532085 1.453 Tgfbr3 transforming growth factor, beta receptor III 10545001 1.451 Ppm1k protein phosphatase 1K (PP2C domain containing) 10398824 1.447 A530016L24Rik RIKEN cDNA A530016L24 gene 10575363 1.445 Zfp612 zinc finger protein 612 10592061 1.444 Kcnj5 potassium inwardly‐rectifying channel, subfamily J, member 5 10537712 1.443 Gstk1 glutathione S‐transferase kappa 1 10344725 1.435 Adhfe1 alcohol dehydrogenase, iron containing, 1 10495675 1.435 F3 coagulation factor III 10396079 1.434 Klhdc1 kelch domain containing 1 10501208 1.432 Gstm6 glutathione S‐transferase, mu 6 10601844 1.431 Bhlhb9 basic helix‐loop‐helix domain containing, class B9 10357965 1.430 Lgr6 leucine‐rich repeat‐containing G protein‐coupled receptor 6 10582229 1.430 1110003O08Rik RIKEN cDNA 1110003O08 gene 10494114 1.426 Selenbp1///Selenbp2///LOselenium binding protein 2///hypothetical protein LOC100044204///selenium binding  10494085 1.424 Selenbp2 selenium binding protein 2 10386020 1.418 Slc36a2 solute carrier family 36 (proton/amino acid symporter), member 2 Supplemental Table 11 - KO TAC < KO Sham GO ID GO ACCESSION GO Term p‐value corrected p‐value Count in Selection % Count in Selection Count in Total % Count in Total No GO lists pass the cut Supplemental Table 12 - GO: KO TAC < KO Sham Affy ID Fold Gene symbol Description 10580635 3.370 Ces3 carboxylesterase 3 10538802 2.322 A930038C07Rik RIKEN cDNA A930038C07 gene 10598064 2.276 10598018 2.188 10598062 1.880 10511363 1.874 Penk preproenkephalin 10441794 1.676 Mrgprh MAS‐related GPR, member H 10478145 1.550 Ppp1r16b protein phosphatase 1, regulatory (inhibitor) subunit 16B 10398824 1.541 A530016L24Rik RIKEN cDNA A530016L24 gene 10468113 1.538 Kcnip2 Kv channel‐interacting protein 2 10497862 1.520 Trpc3 transient receptor potential cation channel, subfamily C, member 3 10598023 1.517 10601878 1.501 Tceal1 transcription elongation factor A (SII)‐like 1 10542834 1.486 10534085 1.471 Phkg1 phosphorylase kinase gamma 1 10352133 1.440 Efcab2 EF‐hand calcium binding domain 2 10574488 1.429 Pdp2 pyruvate dehyrogenase phosphatase catalytic subunit 2 10397763 1.408 9030617O03Rik RIKEN cDNA 9030617O03 gene Supplemental Table 13 - KO TAC > WT TAC GO ID GO ACCESSION GO Term p‐value corrected p‐value Count in Selection % Count in Selection Count in Total % Count in Total No GO lists pass the cut Supplemental Table 14 - GO: KO TAC > WT TAC Affy ID Fold Gene symbol Description 10398075 3.851 Serpina3n serine (or cysteine) peptidase inhibitor, clade A, member 3N 10572897 3.263 Hmox1 heme oxygenase (decycling) 1 10358476 2.822 Prg4 proteoglycan 4 (megakaryocyte stimulating factor, articular superficial zone protein) 10492021 2.784 Postn periostin, osteoblast specific factor 10462442 2.741 Il33 interleukin 33 10416181 2.681 Stc1 stanniocalcin 1 10598976 2.637 Timp1 tissue inhibitor of metalloproteinase 1 10481627 2.441 Lcn2 lipocalin 2 10574023 2.394 Mt2 metallothionein 2 10355403 2.353 Fn1 fibronectin 1 10401527 2.168 Ltbp2 latent transforming growth factor beta binding protein 2 10475517 2.125 AA467197 expressed sequence AA467197 10513739 2.108 Tnc tenascin C 10419934 2.067 Myh7 myosin, heavy polypeptide 7, cardiac muscle, beta 10586357 2.006 Cilp cartilage intermediate layer protein, nucleotide pyrophosphohydrolase 10478048 1.918 Lbp lipopolysaccharide binding protein 10376778 1.864 Mfap4 microfibrillar‐associated protein 4 10523175 1.834 Ereg epiregulin 10463070 1.831 Entpd1 ectonucleoside triphosphate diphosphohydrolase 1 10380419 1.828 Col1a1 collagen, type I, alpha 1 10440091 1.827 Col8a1 collagen, type VIII, alpha 1 10541496 1.820 Mfap5 microfibrillar associated protein 5 10424140 1.816 Col14a1 collagen, type XIV, alpha 1 10534667 1.788 Serpine1 serine (or cysteine) peptidase inhibitor, clade E, member 1 10429128 1.768 Sla src‐like adaptor 10586865 1.744 Aldh1a2 aldehyde dehydrogenase family 1, subfamily A2 10434698 1.740 Fetub fetuin beta 10425066 1.731 Csf2rb colony stimulating factor 2 receptor, beta, low‐affinity (granulocyte‐macrophage) 10494271 1.721 Ctss cathepsin S 10435641 1.709 Fstl1 follistatin‐like 1 10536220 1.689 Col1a2 collagen, type I, alpha 2 10461721 1.684 Mpeg1 macrophage expressed gene 1 10354003 1.684 Mgat4a mannoside acetylglucosaminyltransferase 4, isoenzyme A 10422164 1.662 Ednrb endothelin receptor type B 10416215 1.659 Loxl2 lysyl oxidase‐like 2 10409464 1.651 Dbn1 drebrin 1 10417212 1.626 Itgbl1 integrin, beta‐like 1 10360028 1.620 Fcgr2b Fc receptor, IgG, low affinity IIb 10542355 1.604 Emp1 epithelial membrane protein 1 10365482 1.600 Timp3 tissue inhibitor of metalloproteinase 3 10484307 1.599 Frzb frizzled‐related protein 10462005 1.592 Tmem2 transmembrane protein 2 10526514 1.584 Cldn15 claudin 15 10586246 1.581 Dennd4a DENN/MADD domain containing 4A 10374083 1.580 Aebp1 AE binding protein 1 10346015 1.577 Col3a1 collagen, type III, alpha 1 10359851 1.575 Uck2 uridine‐cytidine kinase 2 10555389 1.565 Ucp2 uncoupling protein 2 (mitochondrial, proton carrier) 10596747 1.544 Sema3f sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3F 10517213 1.520 Cnksr1 connector enhancer of kinase suppressor of Ras 1 10494262 1.516 Ctsk cathepsin K 10398052 1.505 Serpina3h serine (or cysteine) peptidase inhibitor, clade A, member 3H 10388430 1.496 Serpinf1 serine (or cysteine) peptidase inhibitor, clade F, member 1 10401244 1.489 Actn1 actinin, alpha 1 10401781 1.478 Sptlc2 serine palmitoyltransferase, long chain base subunit 2 10513208 1.476 Svep1 sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1 10604961 1.474 Gabra3 gamma‐aminobutyric acid (GABA) A receptor, subunit alpha 3 Supplemental Table 15 - KO TAC < WT TAC 10527233 1.454 Cyth3 cytohesin 3 10464761 1.447 Syt12 synaptotagmin XII 10467420 1.433 Pdlim1 PDZ and LIM domain 1 (elfin) 10582275 1.432 Slc7a5 solute carrier family 7 (cationic amino acid transporter, y+ system), member 5 10607870 1.431 Tlr7 toll‐like receptor 7 10459421 1.415 Atp8b1 ATPase, class I, type 8B, member 1 10510265 1.410 Nppa natriuretic peptide precursor type A 10373902 1.409 Gatsl3 GATS protein‐like 3 10371321 1.406 Slc41a2 solute carrier family 41, member 2 10528268 1.405 Ptpn12 protein tyrosine phosphatase, non‐receptor type 12 GO ID GO ACCESSION GO Term p‐value corrected p‐value Count in Selection % Count in Selection Count in Total % Count in Total 3394 GO:0005576 extracellular region 1.10E‐21 7.20E‐18 34 100 1688 7.7477393 3396 GO:0005578 proteinaceous extracellular matrix 9.58E‐14 2.73E‐10 13 38.235294 274 1.2576307 18811 GO:0044420 extracellular matrix part 1.25E‐13 2.73E‐10 8 23.529411 87 0.3993207 12843 GO:0031012 extracellular matrix 2.29E‐13 3.76E‐10 13 38.235294 292 1.3402488 18812 GO:0044421 extracellular region part 8.18E‐13 1.07E‐09 13 38.235294 760 3.4883187 3420 GO:0005604 basement membrane 7.56E‐08 8.27E‐05 5 14.705882 70 0.32129252 3400 GO:0005583 fibrillar collagen 5.52E‐07 5.18E‐04 2 5.882353 6 0.027539358 3205 GO:0005201 extracellular matrix structural constituent 8.29E‐07 6.80E‐04 3 8.823529 24 0.11015743 22049 GO:0048407 platelet‐derived growth factor binding 3.28E‐06 0.002329905 3 8.823529 10 0.04589893 12309 GO:0030414 protease inhibitor activity 4.96E‐06 2.96E‐03 2 5.882353 142 0.6517648 3401 GO:0005584 collagen type I 9.32E‐06 5.10E‐03 2 5.882353 2 0.009179786 Supplemental Table 16 - GO: KO TAC < WT TAC Target Protein Number of hits Syntenin 34 Connective tissue growth factor (CTGF) 14 Granulin 12 EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) 11 Fibronectin 1 5 Dickkopf homolog 3 (DKK3) 4 Laminin gamma 1 4 Latent transforming growth factor beta binding protein 2 (LTBP2) 4 Proprotein convertase subtilisin/kexin type 6 3 NUFIP2 2 Laminin beta 2 1 Sorting nexin 3 (SNX3) 1 Y box binding protein 1 (YBX1) 1 CD63 antigen 1 Fibulin 5 1 Fibulin 2 1 Cyclophilin D 1 Ribophorin 1 1 Sprouty homolog 2 1 ZNF 330 1 Rabin 3 1 Lactate dehydrogenase A 1 NDRG 4 1 Ceramide synthase 2 1 Fibrillin 1 1 HLA-B associated transcript 1 1 Thrombospondin 1 1 Total 110 Table S17