SLIT3 deficiency attenuates pressure overload-induced cardiac fibrosis and remodeling

In pulmonary hypertension and certain forms of congenital heart disease, ventricular pressure overload manifests at birth and is an obligate hemodynamic abnormality that stimulates myocardial fibrosis which leads to ventricular dysfunction and poor clinical outcomes. Thus, an attractive strategy is to attenuate the myocardial fibrosis to help preserve ventricular function. Here, by analyzing RNA-sequencing databases and comparing the transcript and protein levels of fibrillar collagen in wild-type and global knockout mice, we found that SLIT3 was predominantly present in fibrillar collagenproducing cells and that SLIT3 deficiency attenuated collagen production in the heart and other non-neuronal tissues. We then performed transverse aortic constriction or pulmonary artery banding in wild-type and knockout mice to induce left and right ventricular pressure overload, respectively. We discovered that SLIT3 deficiency abrogates fibrotic and hypertrophic changes and promotes long-term ventricular function and overall survival in both left and right ventricular pressure overload. Furthermore, we found that SLIT3 stimulated fibroblast activity and fibrillar collagen production, which coincided with the transcription and nuclear localization of the mechanotransducer YAP1. These results indicate that SLIT3 is important for regulating fibroblast activity and fibrillar collagen synthesis in an autocrine manner, making it a potential therapeutic target for fibrotic diseases, especially myocardial fibrosis and adverse remodeling induced by persistent afterload elevation. Research Cardiology Cell biology


Introduction 53
Neonatal pulmonary hypertension and certain forms of complex congenital heart disease subject the 54 cardiac ventricle to pressure overload early in life (1-3). While this hemodynamic stress is well tolerated 55 in the short term, chronic elevation in afterload can result in adverse ventricular remodeling characterized 56 by fibrosis and hypertrophy which then contributes to ventricular dysfunction (4,5). This is especially 57 important in systemic right ventricle congenital heart disease and pediatric primary pulmonary 58 hypertension where patient survival is determined by ventricular function (1, 6, 7). Therefore, in these 59 clinical conditions where chronic pressure overload is obligate and unavoidable, preventing the adverse 60 remodeling response would have an important clinical benefit (8). 61 Mammalian SLITs (SLIT1-3) are highly-conserved, secreted glycoproteins that were originally described 62 to mediate repulsive axonal guidance during central nervous system development by binding to 63 Roundabout (ROBO) receptors (9-12). Interestingly, SLIT3 is primarily expressed in non-neuronal tissues 64 (13)(14)(15)(16). The most striking manifestation of SLIT3 deficiency in mice is central congenital diaphragmatic 65 hernia (CDH) (13, 17) and osteopenia (14,18), while in humans, genetic variants and low SLIT3 serum 66 levels have been associated with height (19) and osteoporosis (18), respectively. 67 Fibrillar collagen is the major component of extracellular matrix (ECM) in a variety of load-bearing tissues 68 including the central diaphragm tendon, bone, and heart (20,21). In the developing heart, SLIT3 is 69 intensively expressed in mesenchymal cushions, which ultimately transforms into dense connective tissue, 70 including the membranous ventricular septum as well as the atrioventricular and semilunar valves (22). 71 Correspondingly, membranous ventricular septum defects, as well as atrioventricular and aortic valves 72 abnormalities, are exhibited in SLIT3 mutant mice (22,23). Congenital heart disease involving Tetralogy 73 of Fallot, septal and outflow tract defects is associated with SLIT3 variants in humans (24). 74 Considering the above-mentioned association between SLIT3 deficiency and these collagen-rich tissue 75 defects, we sought to further understand the function of SLIT3 in postnatal non-neuronal tissues, 76 especially in the heart, where excessive fibrillar collagen accumulation leads to myocardial fibrosis and 77 maladaptive remodeling. We found that SLIT3 is present at high levels in fibrillar collagen-producing cells, 78 and SLIT3 deficiency reduces Col1a1 and Col3a1 transcript levels as well as the total collagen content in 79 multiple non-neuronal tissues. More importantly, Slit3-targeted mice are markedly protected from pressure 80 overload-induced left and right ventricular fibrosis and remodeling as well as associated mortality. These 81 results identify the axon-guidance molecule SLIT3 as a fibroblast-secreted collagen regulating factor to 82 promote type I collagen expression and as a potential therapeutic target for attenuating fibrillar collagen 83 accumulation and adverse cardiac remodeling induced by pressure overload. 84

85
SLIT3 is present at high levels in fibrillar collagen-producing cells. Fibrillar collagen is a sub-group of the 86 collagen family, participates in the formation of striated fibrils, and provides robust mechanical strength 87 (25). Collagen types I and III are the most common fibrillar collagens and are the major components of 88 the myocardial collagen matrix (26). As the biosynthesis of fibrillar collagen is highly regulated at the 89 transcript level (27), and SLIT3 deficiency impacts collagen-rich tissues (13,14,17,18), we first 90 interrogated the cellular transcript levels of SLITs, ROBOs, COL1A1, and COL3A1 in the FANTOM5 91 (human) (28) and Tabula Muris (Mus musculus) (29) projects. We found that SLIT3 transcripts were 92 present at high levels in human fibroblasts, synoviocytes, tenocytes, adipocytes, mesenchymal cells, and 93 smooth muscle cells (SMCs) (Supplemental Table 1), all of which belong to the family of connective-tissue 94 cells distinguished by their ability to synthesize and deposit type I/III collagen (30). Linear regression 95 analysis between the transcript levels of SLIT3 and COL1A1 exhibited a positive correlation across 512 96 human cell lines (R² = 0.1764, P < 10 -15 ) ( Figure 1A and Supplemental Figure 1A). Also, in the single-cell 97 RNA sequencing (RNA-seq) study of mouse tissues, Slit3 transcripts were present at high levels in 98 mesenchymal stem cells (MSCs), stromal cells, and fibroblasts, and Slit3, Col1a1, and Col3a1 appeared 99 to be coexpressed in the same cell populations ( Figure 1B and Supplemental Figure 1B -C). 00 In order to confirm the above sequencing results by real-time PCR, we isolated and cultured primary aortic 01 adventitial, cardiac, and pulmonary fibroblasts, as well as aortic vascular smooth muscle cells from adult 02 wild-type (WT) CD-1 mice. Similar to the expression pattern of Col1a1, Slit3 transcript levels were low in 03 LV tissue and negligible in freshly isolated cardiomyocytes, fibroblasts from the heart and other tissues, 04 yet were significant in aortic vascular smooth muscle cells ( Figure 1C). Interestingly, the expression 05 pattern of Robo1, which is considered as the receptor for SLIT3-mediated effects on fibrillar collagen 06 formation in bone (14,18), is different from that of Slit3 by sequencing or real-time PCR. Compared to the 07 high intensity concentrated and overlapping expressions of Slit3 and Col1a1, the expression of Robo1 08 expands beyond these cell populations ( Figure 1A-C and Supplemental Figure 1A-C). In addition, we also 09 confirmed SLIT3 and ROBO1 protein expression in adult primary murine fibroblasts by 10 immunocytochemistry ( Figure 1D and Supplemental Figure 8). These results indicate that fibrillar 11 collagen-producing fibroblasts express SLIT3 and also possess its primary receptor ROBO1 (14,17,18,12 22,23,31), indicating a possible autocrine axis. 13 Phenotypic manifestations of Slit3 -/-CD1 mice. Due to the widespread distribution of fibrillar collagen-14 producing cells and the critical importance of fibrillar collagen in providing structural support for high-15 pressure structures such as the cardiac ventricles, we sought to investigate the influence of SLIT3 16 deficiency on the postnatal cardiovascular system. Since previous studies in inbred Slit3 -/-(knockout) mice 17 precluded analyses of the postnatal cardiovascular system because of the confounding phenotype of 18 diaphragmatic hernia (13,14,17,18), we utilized knockout mice on a CD-1 (outbred) background 19 (F:M=1:1), which have a lower penetrance of central CDH (approximately 5%, Supplemental Figure 2F) 20 and normal left ventricular (LV) systolic function in the first year of life (Supplemental Table 2). Compared 21 to age-matched WT mice, the body weight, tibia length, and hair follicle density of Slit3 -/mice were all 22 significantly reduced (Supplemental Figure 2A-E). Further, the amounts of fibrillar collagen fibers found in 23 the arterial adventitia and perivascular/interstitial regions of multiple non-neuronal organs (Figure 2A), 24 including that of the heart ( Figure 3A, E), were reduced in Slit3 -/mice while myocardial capillary density 25 was preserved ( Figure 3A). Interestingly, previous studies in inbred Slit3 -/mice described that the liver 26 was always found in the hernia sac and adhered to its wall without the formation of the falciform ligament 27 (13,17). However, we observed in a CD-1 knockout mouse that the stomach, rather than the liver, was 28 the only hernia sac content (Supplemental Figure 2F), which suggests that the formation of central CHD 29 is not necessarily related to liver or falciform ligament development. 30

SLIT3 deficiency reduces fibrillar collagen production in vivo and impacts LV biomechanical toughness. 31
Given the histological changes observed in Slit3 -/mice, we next evaluated Col1a1 and Col3a1 transcript 32 levels as well as total collagen protein levels in the aortic adventitia, lung, spleen, kidney, bone, skin, and 33 heart. Col1a1 and Col3a1 transcripts ( Figure 2B and Figure 3B), as well as hydroxyproline levels ( Figure  34 2D and Figure 3F) were all significantly reduced in Slit3 -/compared with WT mice. The transcript levels 35 of Slit3 and Col1a1 among individual WT mice exhibited a strong positive and linear correlation in all 36 analyzed tissues ( Figure 2C and Figure 3C). Interestingly, the indexed femur and heart weight (normalized 37 to tibia length), as well as cardiomyocyte size, were also decreased in Slit3 -/mice ( Figure 3I and 38 Supplemental Figure 6A). 39 Because cardiac fibroblasts are responsible for most of the fibrillar collagen production in the heart and 40 are widely distributed in the left and right ventricles that have different embryological origins and 41 physiological characteristics (32, 33). We next determined if there was a ventricular-specific expression 42 pattern for Slit3. Although the ventricles possessed widely varying levels of Slit3 transcripts between 43 animals, we found that Slit3 transcript levels were strongly correlated in the RV and LV in the same 44 individual (R 2 = 0.98, Figure 3D), indicating a tightly-regulated balance of Slit3 ventricular expression 45 which is also consistent with the common embryological origin of fibroblasts in both ventricles (34-36). As 46 SLIT3 deficiency reduces the myocardial fibrillar collagen content, which plays crucial mechanical roles 47 in the ventricle, we then investigated its effect on LV toughness i.e., the amount of energy absorbed prior 48 to irreversibly fracturing/tearing. Using gradual inflation of the cardioplegia-relaxed LV, the energy density 49 required to rupture the LVs from Slit3 -/mice was significantly reduced compared with WT mice (Figure  50 3G). Consistently, in 1-year-old mice, SLIT3 deficiency increased mitral E/A ratio, which is a sensitive 51 indicator of LV compliance ( Figure 3E-H) (37). To further confirm these findings, we evaluated WT and 52 knockout mice in a left anterior descending coronary artery ligation myocardial infarction (MI) model since 53 myocardial collagen is needed to maintain the integrity of the infarcted myocardium, thereby preventing 54 ventricular rupture (38, 39). We found that the 1-week survival rate was significantly reduced in Slit3 -/-55 compared with WT mice. Among twenty-five WT and twenty-four Slit3 -/mice after surgery, a total of seven 56 Slit3 -/mice died in the first week, six of which suffered LV rupture, characterized by a large amount of 57 blood in the thoracic cavity around the heart, and an area of transmural rupture in the ischemic area of 58 the LV ( Figure 3H). 59 SLIT3 deficiency attenuates LV fibrosis and adverse remodeling. Chronic pressure overload of the LV 60 and RV occurs in acquired and congenital cardiovascular disease and leads to adverse myocardial 61 remodeling characterized by fibrillar collagen accumulation and myocyte hypertrophy. To investigate the 62 role of SLIT3 in the LV and RV fibrotic response to pressure overload, we subjected WT and Slit3 -/body 63 weight matched mice to transverse aortic constriction (TAC) or pulmonary artery banding (PAB) with 64 similar initial peak pressure gradients ( Figure 4C and 5E, Supplemental Figure 3A, C, 4A and 5A). 65 Successful TAC in mice induced LV fibrillar collagen accumulation and cardiomyocyte hypertrophy, but 66 this pressure overload-induced cardiac remodeling was attenuated in Slit3 -/mice as compared to WT 67 mice. Perivascular fibrillar collagen area was decreased in Slit3 -/mice at 1, 3 and 8 weeks after TAC 68 ( Figure 4A-B), which corresponded to decreased transcript levels of fibrosis-related genes (Col1a1, 69 Col3a1, Ctgf, Fn1, and Acta2) at 1 and 3 weeks after TAC in these animals ( Figure 4D and Supplemental 70 Figure 4D). Unexpectedly, we also observed that myocardial hypertrophy was blunted in Slit3 -/mice after 71 TAC ( Figure 4A and Supplemental Figure 4C), consistent with a reduced heart weight/tibia length ratio, 72 LV cardiomyocyte cross-sectional area at 1, 3 and 8 weeks after TAC ( Figure 4A-B), LV end-diastolic 73 posterior wall thickness, mass, end-diastolic volume, and end-systolic volume at 3, 7 and 16 weeks 74 (Supplemental Figure 4E). In agreement with these observations, expression of hypertrophic-related 75 genes (Nppb, Nppa, and Myh7) was also reduced in Slit3 -/mice ( Figure 4D and Supplemental Figure 4D). 76 Further, the LV function was preserved at 7 and 16 weeks in Slit3 -/mice after TAC ( Figure 4F and 77 Supplemental Figure 4B). Most importantly, the long-term survival was significantly enhanced in Slit3 -/-78 mice where null mice remained viable over a 1-year time course compared with more than 60% mortality 79 in the control group ( Figure 4G). 80 SLIT3 deficiency attenuates RV fibrosis and adverse remodeling. Next, we subjected animals to PA 81 banding to determine the importance of SLIT3 in RV pressure overload. The effects of SLIT3 deficiency 82 were even more apparent in blunting the adverse remodeling response in the context of RV pressure 83 overload, a hemodynamic condition found in congenital heart disease that can lead to RV failure and has 84 no effective medical treatment besides heart transplantation (40). The RV free wall fibrosis area, 85 cardiomyocyte cross-sectional area, end-diastolic diameter and area, RV weight-to-LV plus septum 86 weight ratio (RV/LV+S), RV/LV end-systolic diameter ratio ( Figure 5A-D and Supplemental Figure 5B, D), 87 and transcript levels of Col1a1, Nppb, Col3a1, Nppa, and Myh7 ( Figure 5F and Supplemental Figure 5F) 88 were all reduced in Slit3 -/mice at 2 and 4 weeks, altogether indicating that knockout animals had a 89 decreased adverse fibrotic and hypertrophic response. In addition, after PAB, Slit3 -/mice had preserved 90 RV fractional shortening (FS) at 2 weeks, reduced liver congestion at 4 weeks, and markedly increased 91 survival ( Interestingly, the transcript levels of Slit3, as well as that of Col1a1, and Col3a1, were all increased 93 significantly at 1-2 weeks, and then returned to baseline levels in WT mice after TAC and PAB 94 (Supplemental Figure 3B, D) (41). Similarly, the peak aortic gradients in post-TAC WT mice increased 95 significantly from day 3 to day 21, as described previously and attributed to the development of 96 compensatory LV hypertrophy and the recovery of peak systolic wall stress index (42), and then returned 97 to initial levels after day 42 with the transition from compensated to decompensated heart failure as 98 indicated by the deterioration of LV function (Supplemental Figure 3A). By contrast, in Slit3 -/mice, peak-99 to-baseline ratios of fibrosis-related gene transcript levels were all decreased after TAC and PAB, and 00 changes in peak gradients and LV function were both relatively mild throughout the study period without 01 a time-dependent linear trend after TAC (Supplemental Figure 3C). Although, Col1a1 and Col3a1 02 transcript levels were significantly different between baseline WT and Slit3 -/mice, there was a genotypic 03 effect on the transcription of these fibrillar collagen genes in response to pressure overload surgery, and 04 Slit3 -/mice had a weaker fibrotic response to TAC and PAB than WT mice (two-way ANOVA, P<0.05, 05 Supplemental Figure 3E-F). 06 SLIT3 deficiency inhibits fibroblast activity. Growth factors such as transforming growth factor beta 1 07 (TGFβ1) and platelet-derived growth factor BB (PDGF-BB) are overexpressed and act as important 08 mediators of fibroblast activation in the pathogenesis of cardiac fibrotic and hypertrophic remodeling (8, 09 43-45). Therefore, TGFβ1 and PDGF-BB were used to activate primary adult fibroblasts in vitro. 10 Compared with passage-matched WT cells, Slit3 -/fibroblasts treated with or without either PDGF-BB or 11 TGFβ1 displayed significantly inhibited proliferation, migration and contraction activities ( Figure 6A-C). 12 Two-way ANOVA demonstrated significant interaction between SLIT3 and PDGF-BB in fibroblast 13 proliferation assays, although it accounted for only 4.5% of all variation. No other significant interactions 14 were observed in the contraction and migration assays. In the proliferation and contraction assays, PDGF-15 BB and TGFβ1 each contributed more than 82% of the total variation, whereas the genotypic effect of 16 Slit3 presence or absence in the fibroblasts only accounted for 5.9% or 12.3%, respectively ( Figure 6A-17 B). Overall, these functional results indicated that SLIT3's stimulation of fibroblast activity appears to be 18 relatively independent of rTGFβ1for the concentrations of the factors that we used in our experiments. To 19 further confirm the effect of SLIT3 on fibroblast biological activity in vivo outside of the heart, a splinted 20 skin excisional wound model was performed (46), and consistently, we found that dermal wound healing, 21 which is critically dependent upon fibroblast function and fibrillar collagen production (47), was also 22 delayed in Slit3 -/mice ( Figure 6D). 23

SLIT3 regulates YAP1 and fibrillar collagen production. Fibrillar collagen contributes to the biomechanical 24
properties of connective tissue (21), where resident fibroblasts are commonly quiescent (48) and  25 protected from persistent external loads by dynamically regulated ECM in vivo (49). Resident cardiac 26 fibroblasts are also quiescent cells with a low proliferation and ECM production under normal 27 hemodynamic conditions (50), and are the principal source of activated fibroblasts in injured mammalian 28 hearts (51, 52). We observed that acutely increasing ventricular wall stress with TAC or PAB resulted in 29 the activation of resident fibroblasts and increase of myocardial production of fibrous collagen and SLIT3 30 ( Figure 4E and Figure 5G) (53). The mechanical microenvironment is known to be an important 31 determinant of fibroblast activation in vitro and in vivo (54). To further investigate the effect of the 32 mechanical microenvironment on the role of SLIT3-induced fibroblast activation in vitro, aortic adventitial 33 fibroblasts (AAFs), cardiac fibroblasts (CFs), and lung fibroblasts (LFs) were cultured on stiff plastic tissue 34 surfaces or in soft collagen type I gel, as fibroblasts adaptate their internal stiffness to match that of their 35 substrate (55) and local stiffness acts as am important mechanical effector (56). Importantly, we found 36 that transcript levels of Acta2, Col1a1, Slit3 and Yap1 were all significantly increased by culturing 37 fibroblasts on a stiff surface ( Figure 7A-B), indicating that SLIT3 transcription may be regulated by the 38 surrounding mechanical microenvironment. 39 As the transcription factors YAP1/TAZ have been identified as an important mediator of 40 mechanotransduction signals in fibroblasts and widely regulates fibroblast biological activities, including 41 proliferation, migration and fibrillar collagen production (57-60), the potential association of SLIT3 and 42 YAP1/TAZ were then examined. We found that the transcript levels of Yap1, rather than Taz, and its 43 regulated gene Ctgf in the cardiac ventricles after TAC were reduced significantly in Slit3 -/compared with 44 WT mice ( Figure 7C and Supplemental Figure 6B) (61). In vitro, both the transcript levels of Yap1 and 45 Ctgf, as well as the nuclear translocation of YAP1, were reduced significantly in Slit3 -/as compared to 46 WT fibroblasts ( Figure 7D-F). Further, there was a strong linear association between the transcript levels 47 of Slit3 and Yap1 of fibroblasts cultured on both soft and stiff surfaces (R 2 =0.91, Figure 7G), and 48 recombinant SLIT3 (rSLIT3, aa 34-1116) treatment significantly increased the transcript levels of Yap1 as 49 well as its downstream gene targets Ctgf and Col1a1 in fibroblasts (62) ( Figure 7H), suggesting that SLIT3 50 has a positive regulatory effect on Yap1 transcription. In addition, although TGFβ1 treatment of fibroblasts 51 significantly reduced the transcript levels of Slit3, there was a genotypic effect on Ctgf expression 52 response to TGFβ1; i.e., Slit3 -/fibroblasts had a blunted response to TGFβ1 as compared to WT 53 fibroblasts (two-way ANOVA, P<0.001, Figure 7J). 54

Discussion 55
Since its original description as an axon guidance molecule (63), there has been mounting evidence that 56 SLIT3, a large, secreted glycoprotein (64), has other important extra-neuronal functions (14, 16, 17, 65-57 68). Here we provide evidence that SLIT3 plays an important role in the function of fibroblasts, which 58 express ROBO1 ( Figure 1D), also the principal Roundabout receptor for SLIT3-mediated osteogenesis 59 (14,18), and, consequently, the content of fibrillar collagen in the heart during conditions of homeostasis 60 as well as stress. Furthermore, our results demonstrate that SLIT3 also regulates fibroblasts and the 61 collagen content in extracardiac tissues, underscoring SLIT3's importance in collagen-rich tissues and, in 62 turn, their biomechanical properties. 63 Similar to previous reports, outbred SLIT3 deficiency mice have reduced bone mass ( Figure 3I). 64 Interestingly, for this phenotype, Xu et al (14) and Kim et al (18) reported contradictory mechanisms, and 65 mainly focused on the effective sources of SLIT3. However, using multiple lines of evidence, Li et al (69)  66 recently demonstrated that skeletal SLIT3 is mainly secreted by osteoblasts rather than osteoclasts. 67 Osteoblasts are differentiated from mesenchymal stromal cells and has also been regarded as a 68  (14), wound healing ( Figure 6D), and post myocardial infarction 74 ( Figure 3H); however, it may have beneficial effects in other contexts such as pressure-overload adverse 75 remodeling of the ventricle. After TAC or PAB, ventricular pressure overload increases peak wall stress 76 and induces reactive tissue remodeling that is thought to occur as a compensatory response to normalize 77 wall stress (42, 71). Within three weeks after TAC, the aortic peak gradient was positively correlated with 78 myocardial hypertrophy (Supplemental Figure 3G), and correspondingly, the early-stage remodeling was 79 compensatory in both WT and Slit3 -/mice ( Figure 4F) (42). Notably, different degrees of reactive 80 remodeling induced by similar initial hemodynamic load were observed in WT and Slit3 -/post-TAC mice 81 (71), as blunted fibrillar collagen synthesis together with a stable aortic gradients may have permitted the 82 recovery of LV wall stress in the absence of excessive hypertrophy in Slit3 -/mice. Furthermore, 83 suppressing reactive remodeling by SLIT3 deficiency yielded a long-term cardioprotective effect in the 84 setting of persistent afterload stress in LV, as the response in WT mice was compensatory, but also 85 excessive and ultimately maladaptive ( Figure 4F-G) (72, 73). 86 Unlike the more robust and pressure-resistant LV (74) Although several preclinical studies have demonstrated that inhibiting load-induced myocardial 98 hypertrophy might be beneficial in the short term despite persistence of the initiating stimulus (53,(83)(84)(85)(86)(87). 99 It is also evident that excessive inhibition of the required compensatory response to normalize increased 00 wall stress is associated with cardiomyocyte damage, depressed systolic function, and increased mortality 01 (42,71,88,89). This did not seem to be the case in SLIT3 deficient animals undergoing TAC as only 3 of 02 the total 13 (23%) animals demonstrated localized fibrosis possibly occurring after cardiomyocyte 03 apoptosis or necrosis (90) at the junctional area of the posterior ventricular septum and free wall and 1 04 animal (0.08%) suffered from transient cardiac dysfunction that recovered within 15 days while, in general, 05 the cardiac function of Slit3 -/mice was well preserved after TAC (Supplemental Figure 7A-F). These 06 findings indicate that the inhibitory effect of SLIT3 deficiency on the hypertrophy response induced by 07 pressure overload is moderate and may be secondary to defective fibroblast: cardiomyocyte crosstalk, 08 although a direct effect of SLIT3 on cardiomyocytes is also possible (86,(91)(92)(93). 09 On the premise of normalizing wall stress and preventing myocyte death, our results indicate that 10 minimizing, but not completely abrogating, the reactive remodeling, especially fibrosis induced by 11 pressure overload, is a promising strategy to pursue therapeutic long-term cardioprotective effects in both 12 the LV and RV. Our results indicate that global SLIT3 deficiency appears to yield such a beneficial effect, 13 achieving the "Goldilocks Zone" of tempering hypertrophy and fibrosis but not completely abolishing them. 14 While SLIT3 is mainly expressed in fibroblast and other connective tissue cell lineages, additional work 15 with lineage-specific and inducible knockout animals is needed to confirm that fibroblast mediated SLIT3 16 is responsible for the phenotype that we observed in our global knockout animals. Nonetheless, from a 17 therapeutics and translational perspective, targeting SLIT3 and its downstream signals via, for example 18 small molecule antagonists, will likely target most cells indiscriminately and thus may reflect the situation 19 in our global knockouts with the favorable phenotype in the setting of ventricular pressure overload. 20 It has been previously shown that pressure overload by TAC induces an acute increase in ventricular wall 21 stress that is later normalized within 10 days due to remodeling (42), which is nearly contemporaneous 22 with the peak expression of Slit3 ( Figure 4E and Figure 5G) and Col1a1 (Supplemental Figure 3B, D) that 23 we observed in our model (41). Interestingly, the signaling activity of TGFβ1, known to play an important 24 role in fibrosis (94, 95), has been demonstrated to start and progressively increase up to at least 9 weeks 25 post TAC (96). These results, taken together with our in vitro findings that the mechanical 26 microenvironment can stimulate Slit3 transcription, suggest that pressure overload may provide a 27 mechanical stimulus for Slit3 transcription. Further supporting a role for SLIT3 in mechanotransduction is 28 our finding that fibroblasts from SLIT3 deficient animals have decreased nuclear and total YAP1 content, 29 as well as the decreased transcription of TEAD target gene Ctgf ( Figure 7D-F). In addition, exposing 30 SLIT3-deficient fibroblasts to a ROBO1-binding rSLIT3 N-terminal fragment can acutely stimulate Yap1 31 and Ctgf transcription ( Figure 7H), indicating that SLIT3 deficiency attenuated the effect of mechanical 32 stimulation on fibroblasts by downregulating the transcript levels of Yap1, an important sensor and 33 mediator of mechanical cues (Graphical Abstract) (59). 34 The findings of this study are especially relevant to children with congenital heart disease or pulmonary 35 hypertension who are chronically exposed to either left or right ventricular pressure overload and whose 36 outcomes are dependent upon the maintenance of ventricular function. Importantly, children with pressure 37 overload of the RV have no effective medical treatment beyond heart transplantation (1, 6), and thus 38 targeting SLIT3 or fibroblast-specific YAP1 activation (58, 97, 98), rather than cardiomyocyte-specific 39 YAP1 activation (99), may a promising strategy to inhibit adverse remodeling and preserve ventricular 40 function by reducing fibrosis (100). Furthermore, myocardial infarction is rare in children, and hence 41 myocardial rupture after infarction as a potential side effect of SLIT3 inhibition would be unusual in children 42 with ventricular pressure overload. In sum, the results of this study identify SLIT3 as a potential therapeutic 43 target to prevent adverse remodeling and to preserve ventricular function in the setting of chronic 44 ventricular pressure overload. 45

Methods 46
replicates were downloaded from the FANTOM5 database (119 cell types). Analysis was performed as 48 described by Schafer et al (101). Briefly, the gene expression level was obtained by first adding all counts 49 that were assigned to a specific gene and then normalized by the library size to derive the transcripts per 50 million (TPM) for each gene. We determine the TPM for our genes of interest in 512 different primary cell 51 samples that included cell types from all lineages. 52 The mouse excisional wound splinting model was performed as previously described (46). After hair 65 removal, the dorsal skin of the chest was folded and punched with a 5 mm diameter biopsy punch to 66 create two full-thickness excisional wounds besides the midline. Donut-like splints with a 6 mm diameter 67 hole were glued and sutured to the skin around the wound. Digital photographs of individual wounds were 68 taken on days 0 and 15 after surgery. 69 Mouse left ventricle toughness. After anesthesia with inhalational isoflurane, a sternotomy was 70 performed, and the ascending aorta was clamped. The heart was removed, and the LV was perfused with 71 4°C histidine-tryptophan-ketoglutarate solution to obtain arrest in diastole. A balloon-tipped catheter was 72 inserted into the LV through the mitral valve, fixed by a purse-string suture, and gradually inflated until LV 73 rupture. The heart was then removed and weighed. Passive inflation was accomplished with a Gilson 74 MINIPULS 3 pump using a constant rate. During passive inflation of the LV, pressure was monitored with 75 a pressure transducer (ADInstruments MLT0670), and data was acquired with a PowerLab DAQ device 76 and recorded with LabChart software. Pressure-volume (PV) curves were then generated, and the area 77 under the PV curves after subtracting the PV curve obtained with the balloon only was calculated as the 78 energy of LV rupture, which was then normalized to the heart mass to obtain the energy density. 79 Echocardiography. Trans-thoracic echocardiography was performed by the University of Michigan 80 Cardiovascular Phenotyping Core, as previously described (103)  Tissue samples preparation and hydroxyproline assay. Mice were euthanized by cervical dislocation 91 under anesthesia with 2% isoflurane. Next, the chest, abdomen, and right atrium were opened, and the 92 LV was perfused with 20 ml 4°C PBS to flush the blood from the vessels. The heart, left lung, spleen, left 93 kidney, aorta, dorsal skin, and right femur were sequentially harvested and stored at 4°C in HBSS. Then 94 any attached adjacent tissues were removed from the organs with the aid of an operating microscope. 95 For the hydroxyproline assay, tissues were weighed (80-90 mg were utilized) and placed into pressure-96 tight 2.0 ml screw-top polypropylene tubes. For real-time qPCR, 5-10 mg tissues were placed into 1.5 ml 97 tubes. All samples were then immediately stored at -80°C. The hydroxyproline assay kit (Sigma, catalog 98 no. MAK008) was used to quantify the total tissue collagen content according to the manufacturer's 99 protocol. Briefly, 6 M HCl was added to the 2.0 ml screw-top polypropylene tubes as 20 μl per 1 mg tissue 00 and these samples were hydrolyzed for 24 hours under a tight lid at 110°C. Next, the tubes were mixed 01 and centrifuged at 10,000 x g for 3 minutes. 10 μl supernatant from the tubes of bone and skin was diluted 02 with 190 μl water in new 1.5 ml tubes. Then 20 μl (heart, kidney, and spleen), 10 μl (lung), and 20 μl 03 diluted (bone and skin) supernatants were transferred to 96-well plates, respectively. The 96-well plates 04 were placed in a 60°C oven to dry samples. Then 100 μl Chloramine T/Oxidation buffer mixture was 05 added to each reaction well and standard well. The plates were incubated at 25°C for 10 minutes. 100 μl 06 Diluted DMAB Reagent was added to each reaction well and standard well and then the plates were 07 incubated in a 60°C oven for 90 minutes. The absorbance of each well was measured at 560 nm with a 08 spectrophotometer. 09 Real-time qPCR (RT qPCR) gene transcript level analysis. Total RNA was extracted from frozen 10 tissues stored at -80°C or cells cultured in plates using RNeasy Mini Kit (Cat No./ID: 74106, QIAGEN). 11 About 0.5-1 μg of total RNA was used to synthesize the cDNA with the cDNA Synthesis kit (Lot 028755, 12 Quanta bio) and according to the manufacturer's protocol. Quantitative RT qPCR gene transcript level 13 analysis was performed on duplicate samples with SYBR green (Lot 028416, Quanta bio) technology 14 using a StepOnePlus™ (Applied Biosystem). The amplification protocol consisted of 40 cycles, including 15 denaturation at 95°C for 15 s, annealing, and extension at 60°C for 60 s. The gene expression levels were 16 normalized to that of the housekeeping gene GAPDH. The 2 -△△Ct method was used to calculate the fold 17 change. Sequences of the RT qPCR primer pairs are provided in the Supplemental Methods. 18 Western blot. Total protein was extracted from cultured cells in RIPA buffer supplemented with EDTA-19 free Halt protease inhibitor cocktail (Thermo Fisher Scientific). Cytoplasmic and nuclear proteins were 20 extracted using a NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) 21 according to the manufacturer's instruction. Equal amounts of protein were loaded onto a precast 22 NuPAGE 4% 12% Bis-Tris mini protein gel (Invitrogen) and transferred to a nitrocellulose membrane by 23 Trans-Blot Turbo transfer system (Bio-Rad). Transferred membranes were blocked for 1 hour in TBS 24 containing 5% non-fat dried milk followed by overnight incubation at 4°C in the corresponding primary 25 antibodies. The following antibodies purchased from Cell Signaling were used at 1:1000 dilution: β-tubulin, 26 Histone H3, YAP, phospho-YAP (Ser127). After three washes for 5 min in TBST, following incubation with 27 Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 680 and Goat anti-28 Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 800 (1:5,000; Thermo Fisher 29 Scientific) and then visualized by using an Odyssey CLx imaging system (LI-COR Biosciences). 30 Histology. Tissues from mouse models were fixed in 10% formalin, dehydrated and embedded in 31 paraffin, and sectioned at 5 µm. Masson's trichrome staining was used to detect collagen fibers, while 32 hematoxylin and eosin (H&E) staining was used to determine cardiomyocyte cross-sectional area, skin 33 hair follicle density and wound healing process. The images of sections were captured, scanned and then 34 analyzed with Aperio Image Scope (version 12.1) and Image-Pro Plus (version 7). 35 Immunofluorescence/Immunohistochemistry. Cells were fixed and permeabilized in 4% 36 paraformaldehyde (PFA) and 0.1% Triton in PBS, respectively, then blocked with 3% BSA in PBS for 30 37 minutes. Subsequently, cells were stained with primary antibodies targeting SLIT3 (SAB2104337: Sigma-38 Aldrich) or ROBO1 (ab7279: abcam) at 1:200 in blocking solution at 4°C overnight. Then, cells were 39 washed and incubated with the appropriate secondary antibody donkey anti-rabbit IgG (H+L) highly cross-40 adsorbed, Alexa Fluor Plus 488 (Invitrogen) in dark for 60 minutes at room temperature. Nuclei were 41 stained with 1 µg/mL DAPI. Images were obtained using a Nikon A1 confocal microscope. 42 Heart paraffin sections were deparaffinized with xylene and rehydrated with serial dilutions of ethanol. 43 After antigen retrieval, the sections were blocked with 5% donkey serum/3%BSA/0.1% Triton X-100/PBS 44 for 1 hour at room temperature. The following primary antibodies were used at 1:200 dilution overnight at 45 4°C: CD31 (AF3628: R&D Systems) and collagen type I (Abcam ab21286). After washing three times for 46 47 (Invitrogen) were incubated with a 1:500 dilution for 1 hour at room temperature. Sections were then 48 stained with DAPI. Images were acquired on an inverted Nikon A1 confocal microscope (Nikon, Japan). 49 Statistical analysis. All statistical analyses, including unpaired two-tailed student's t-test, one-way and 50 two-way ANOVA with following multiple comparisons test, linear regression and trend , as well as survival 51 curve were performed using GraphPad Prism software (version 8). Outliers were identified by GraphPad 52 outlier calculator (Alpha = 0.05) and removed before analysis. P values of less than 0.05 were regarded 53 as statistically significant.   with the predominant cell type composing each cluster (n = 44,949 individual cells from 20 mouse organs). 12 The clusters of cells expressing Slit3, Col1a1, and Robo1. (C) Transcript levels of Slit3, Col1a1, and 13 Robo1 in cardiac fibroblasts, Left ventricle, freshly isolated cardiomyocytes, aortic adventitial fibroblasts, 14 lung fibroblasts, aortic vascular smooth muscle cells (VSMC), aorta media, and aorta adventitia from WT 15 mice. Samples taken directly from or isolated from living tissue were marked blue and samples of purified 16 and cultured cells were marked red on panels. Each data point represents tissue/cells obtained from a 17 single animal. (D) Confocal immunofluorescence images of mouse aortic adventitial fibroblasts stained 18 with SLIT3 (green), ROBO1 (green) and, DAPI (blue) (n=2).. Scale bars, 50 μm. *P < 0.05, **P < 0.01, 19 ***P < 0.001 using the one-way ANOVA with Tamhane T2 multiple comparisons test (C). 20 female (F). *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT mice using the unpaired two-tailed Student's t-test 56 (B, E-G, I). 57 Slit3 −/− and WT aortic adventitial fibroblasts were seeded in 96-well plates (10% FBS media) and treated 97 with PBS or PDGF-BB (100 ng/ml). Absorbance was measured at 560 nm at 0 and 48 hours after 98 treatments (n=6-14 per group). (B) Floating collagen gel contraction assay. Representative images and 99 area quantification of collagen gel. Slit3 −/− and WT lung fibroblasts were seeded in PBS or TGFβ1 (5.0 00 ng/ml) added collagen gel (1mg/ml collagen type I, 0.5% FBS). The final to initial area ratio was 01 determined at 24 hours after floating (n=3 per group). Scale bars, 5 mm. (C) Scratch wound healing assay. 02 Representative images and migration distance quantification of scratch wound healing. Slit3 −/− and WT 03 lung fibroblasts were seeded in 6-well plates (1% FBS media) and treated with PBS or PDGF-BB (100 04 ng/ml). The migration distance was determined at 24 hours after scratching (n=4). Scale bars, 20 μm. (D) 05 Excisional wound healing assay. Representative gross and histological images (hematoxylin and eosin 06 staining) and quantification of wound area in 8-week-old Slit3 −/− and WT mice at the day of surgery (Day 07 0) and 15 days (Day 15) after surgery (n=19-21 per group). Scale bars, 1 mm and 700 μm from top to 08 bottom. Data are presented as mean±SD. In vitro experiments were performed at least 3 times 09 independently. *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT mice or cells using the unpaired two-tailed 10 Student's t-test (D) and two-way ANOVA with Two-stage step-up method of Benjamini, Krieger and 11 Yekutieli multiple comparisons test (A-C). AAFs and CFs in soft collagen gel (1mg/ml collagen type I, 1% FBS) for 24 hours (n=3 per group). (G) 23 Linear regression analysis between the transcript levels of Slit3 and Yap1 in WT AAFs cultured on stiff 24 surfaces (6-well plastic tissue culture plate, 10% FBS) or in soft collagen gel (1mg/ml collagen type I, 1% 25 FBS) for 24 hours (R 2 =0.9114, n = 18). (H) Transcript levels of Yap1, Ctgf, and Col1a1 in Slit3 −/− LFs 26 cultured in PBS or recombinant SLIT3 (rSLIT3, aa 34-1116, 1 µg/ml) added collagen gel (1mg/ml collagen 27 type I, 0.5% FBS) for 24 hours (n = 10 per group). (I) Transcript levels of Slit3 in WT AAFs and CFs 28 cultured in PBS or TGFβ1 (5.0 ng/ml) added collagen gel (1mg/ml collagen type I, 0.5% FBS) for 24 hours 29 (n = 3 per group). (J)Transcript levels of Yap1 and Ctgf n Slit3 −/− and WT CFs cultured in PBS or TGFβ1 30 (5.0 ng/ml) added collagen gel (1mg/ml collagen type I, 0.5% FBS) for 24 hours (n = 3 per group). Data 31 are presented as mean±SD, Number (n). In vitro experiments were independently performed at least 3 32 times unless indicated otherwise. *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT mice or cells using the 33 unpaired two-tailed Student's t-test (B, E-F, H-I) and two-way ANOVA with Tukey multiple comparisons 34 test (C, J). 35