Scoparone alleviates Ang II‐induced pathological myocardial hypertrophy in mice by inhibiting oxidative stress

Abstract Long‐term poorly controlled myocardial hypertrophy often leads to heart failure and sudden death. Activation of ras‐related C3 botulinum toxin substrate 1 (RAC1) by angiotensin II (Ang II) plays a pivotal role in myocardial hypertrophy. Previous studies have demonstrated that scoparone (SCO) has beneficial effects on hypertension and extracellular matrix remodelling. However, the function of SCO on Ang II‐mediated myocardial hypertrophy remains unknown. In our study, a mouse model of myocardial hypertrophy was established by Ang II infusion (2 mg/kg/day) for 4 weeks, and SCO (60 mg/kg bodyweight) was administered by gavage daily. In vitro experiments were also performed. Our results showed that SCO could alleviate Ang II infusion‐induced cardiac hypertrophy and fibrosis in mice. In vitro, SCO treatment blocks Ang II‐induced cardiomyocyte hypertrophy, cardiac fibroblast collagen synthesis and differentiation to myofibroblasts. Meanwhile, we found that SCO treatment blocked Ang II‐induced oxidative stress in cardiomyocytes and cardiac fibroblasts by inhibiting RAC1‐GTP and total RAC1 in vivo and in vitro. Furthermore, reactive oxygen species (ROS) burst by overexpression of RAC1 completely abolished SCO‐mediated protection in cardiomyocytes and cardiac fibroblasts in vitro. In conclusion, SCO, an antioxidant, may attenuate Ang II‐induced myocardial hypertrophy by suppressing of RAC1 mediated oxidative stress.


| INTRODUC TI ON
Pathological myocardial hypertrophy is the common feature of various cardiovascular diseases, such as hypertension, valve disease, myocardial infarction and congenital heart disease. It is characterized by cardiac hypertrophy and interstitial fibrosis. Long-term poorly controlled myocardial hypertrophy often leads to heart failure and sudden death in patients with the above-mentioned diseases. 1,2 The mechanism of myocardial hypertrophy is very complicated; however, angiotensin II (Ang II) has been demonstrated to play a pivotal role in the pathogenesis. 3,4 Ras-related C3 botulinum toxin substrate 1 (RAC1) is a member of the Rho family of GTPases, that cycles between active (GTP-bound) and inactive (GDP-bound) states. 5 Activation of RAC1 is required for Ang II-induced cardiac hypertrophy, fibrosis and intracellular oxidation in adult hearts. [6][7][8][9] In the cardiovascular system, RAC1 is a major regulator of NADPH oxidase (NOX) activity. In cardiac fibroblasts and cardiomyocytes, Ang II can activate RAC1 (increasing expression of RAC1-GTP) and NADPH oxidase (NOX). 7,10,11 NOX is a family of seven enzymes that participate in generating O 2− by catalysing the electron transfer from NADPH to O 2 . Among the NOX family, NOX2 and NOX4 are mainly expressed in cardiomyocytes and cardiac fibroblasts 12 and are involved in cardiac hypertrophy and fibrosis. 13,14 Scoparone (6,7-dimethoxycoumarin, SCO) is one of the primary active ingredients isolated from the shoot of Artemisia capillaries. In the cardiovascular system, SCO was reported to be a potent hypotensive drug, with a probable mechanism of vascular dilatory action based on calcium mobilization regulation. 15 SCO could inhibit Ang II-induced extracellular matrix remodelling in cardiac fibroblasts in vitro at least partly by inhibiting TGF-β1/Smad signalling. 16 SCO also showed a protective effect against ischaemia-reperfusion-induced myocardial injury by counteracting oxidative stress. 17 However, the effect of SCO on Ang II-induced myocardial hypertrophy is still unknown.
In this study, we discovered that SCO down-regulated Ang II-induced activation of RAC1 and oxidative stress both in cardiomyocytes and cardiac fibroblasts. We also demonstrate that SCO-mediated inhibition of RAC1 alleviates Ang II-induced cardiac hypertrophy and fibrosis in vitro and in vivo. To our knowledge, this is the first report to describe SCO as an inhibitor of RAC1 and agent of anti-myocardial hypertrophy.

| Materials
Scoparone (E-0358) was purchased from Tauto (Shanghai, China).  Ang II-induced oxidative stress in cardiomyocytes and cardiac fibroblasts by inhibiting RAC1-GTP and total RAC1 in vivo and in vitro. Furthermore, reactive oxygen species (ROS) burst by overexpression of RAC1 completely abolished SCO-mediated protection in cardiomyocytes and cardiac fibroblasts in vitro. In conclusion, SCO, an antioxidant, may attenuate Ang II-induced myocardial hypertrophy by suppressing of RAC1 mediated oxidative stress.

K E Y W O R D S
angiotensin II, fibrosis, hypertrophy, oxidative stress, scoparone housed in an air-conditioned room (temperature 20 ± 1℃, humidity 60 ± 10%, a light cycle of 12 hours). All mice had free access to food and water. All mouse experiments in this study were performed in accordance with the National Institutes of Health in CMC solution daily as previously reported 19 ; and (d) SCO group mice (n = 7) were treated with the same amount of SCO and subcutaneous delivery of vehicle saline as described above. Blood pressure and bodyweight were measured every week.

| Histology and immunohistochemistry
Euthanized mice were perfused with saline to eliminate blood.
Hearts were harvested, dried on gauze, weighed, dissected and fixed in 10% formalin or frozen in liquid nitrogen. After fixing for 24 hours, heart tissues were paraffin-embedded and cut into 5 μm  were quantified with IPP 6.0 and expressed as a relative value.

| Measurement of malondialdehyde (MDA)
MDA is a marker of lipid peroxidation and oxidative stress. The level of MDA in the heart tissue was detected using a thiobarbituric acid (TBA)-based assay kit. MDA can react with TBA to form a colorimetric product, proportional to the MDA present. The intensity of the colour was measured spectrophotometrically at 532 nm. 21

| Western blot
Total protein was extracted and quantified from cells and tissues using the method described previously. 3 Equal amounts of protein were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred onto 0.2 μm polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% skimmed milk solution and incubated with the primary and secondary antibodies. Specific protein expression levels were normalized to β-actin protein levels in total cell lysates.

| Reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR (Q-PCR)
Total RNAs from heart tissues and neonatal rat cardiomyocytes (NRCMs) were extracted using TRIzol reagent, and reverse transcription reactions were performed with 0.5 µg RNA using a First Strand cDNA Synthesis Kit. Expression levels of target genes were normalized by concurrent measurement of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels. Primers used for Q-PCR are summarized in Table S1.

| Isolation and culture of neonatal rat cardiomyocytes and fibroblasts
NRCMs and neonatal rat fibroblasts (NRCFs) were isolated and cultured as previously described. 3 The isolated cardiomyocytes were seeded onto gelatin-coated plastic culture dishes at a density of 5 × 10 4 cells/cm 2 in low glucose (1 g/L) Dulbecco's modified Eagle's medium (DMEM) supplemented with 8% horse serum, 5% new-born calf serum, penicillin (100 U/mL) and streptomycin (100 mg/mL). The isolated fibroblasts were maintained in low glucose (1 g/L) DMEM supplemented with 10% foetal bovine serum, penicillin (100 U/mL) and streptomycin (100 mg/mL). Cardiac fibroblasts at passage one were used in cell growth curve assay, and passage two or three cells were used for the other experiments.

| 3 H-leucine incorporation
NRCMs seeded in 24-well plates were treated with different stim-

| Measure of reactive oxygen species (ROS)
Cells were cultured in 6-well plates and treated for the indi-

| LDH release assay
Death of NRCMs and NRCFs in vitro was spectrophotometrically measured by LDH release assay with Cytotoxicity Detection Kit, which was measured in optical density (OD). Briefly, cells were cultured in serum-free DMEM with stimulations for 48 hours, and

| Statistical analysis
Data are presented as the mean ± SD. Statistical analyses were performed by one-way ANOVA followed by the Student-Newman-Keuls test with SPSS10.0 (SPSS Inc, Chicago, IL, USA). Probability (P) values < 0.05 were considered statistically significant.

| SCO attenuates Ang II-induced pathological cardiac hypertrophy in vivo
Ang II infusion is a classic method to establish a myocardial hypertrophy model in mice. Long time Ang II infusion results in cardiac hypertrophy. As shown in Figure 1A,B, SCO treatment significantly reduced the heart weight /bodyweight ratio (HW/ BW), heart weight/tibia length ratio (HW/TL) and cardiomyocyte cross section area (CSA) in mice subjected to Ang II infusion. Meanwhile, compared to the Ang II group, SCO treatment decreased LVPWd and IVSd and increased LVIDd and LVIDs ( Figure 1C). In addition, Ang II infusion up-regulated the levels of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and beta-myosin heavy chain (β-MHC) in the hearts of mice, and SCO treatment decreased these changes ( Figure 1D). In addition, SCO significantly lowered Ang II-elevated blood pressure (SBP, F I G U R E 1 Scoparone inhibits Ang II infusion-induced pathological cardiac hypertrophy in vivo. A, Heart weight /bodyweight ratio (HW/ BW), heart weight/tibia length ratio (HW/TL) in mice from the four groups (n = 7 or 8 per group). B, Representative photographs of wheat germ agglutinin (WGA) stained sections and data of cardiomyocytes cross-sectional area (CSA, n = 4).C, Echocardiographic parameters, including FS (fractional shortening), LVPWd (left ventricular posterior wall thickness end diastole), LVIDd (left ventricular internal diameter end diastole), LVIDs (left ventricular internal diameter end systole) and IVSd (interventricular septum thickness end diastole) were evaluated and calculated (n = 7 or 8). D, Real-time PCR analysis of ANP (atrial natriuretic peptide), BNP (brain natriuretic peptide) and β-myosin heavy chain (β-MHC) mRNA level (n = 4).*P < 0.05 versus control group; # P < 0.05 versus Ang II group | 3141 LYU et aL. DBP and MAP) at different time-points, but it was not able to normalize the blood pressure ( Figure S1).

| SCO attenuates Ang II-induced cardiomyocytes hypertrophy in vitro
To further explore the anti-hypertrophic effect of SCO, neonatal rat cardiomyocytes (NRCMs) were isolated and cultured, and Ang II was used to induce cardiomyocyte hypertrophy in vitro. In the LDH assay, 10-200 μmol/L SCO showed no toxicity to NRCMs ( Figure S2A).
The 3 H-leucine incorporation rate reflects the efficiency of protein synthesis in cells and is a quantifiable index of cardiomyocyte hypertrophy in vitro. 22,23 Figure S2B,C shows that SCO attenuated Ang II-induced cardiomyocyte 3 H-leucine incorporation and cell surface enlargement in a dose-dependent manner. These results indicate that SCO can block Ang II-induced cardiac hypertrophic response in vitro.

| SCO attenuates Ang II-induced cardiac fibrosis in vivo
In Ang II-induced cardiac remodelling, cardiac hypertrophy and fibrosis are often accompanied. As shown in Figure 2A 4). E, Rac1-GTP immunofluorescence staining in cardiac fibroblasts (CFs, vimentin positive) in vivo (n = 4).*P < 0.05 vs control group; # P < 0.05 vs Ang II group fibroblast, which has a stronger pro-fibrogenic ability. In Figure 2B, immunofluorescent staining of α-SMA and vimentin showed that Ang II infusion significantly increased the population of α-SMA and vimentin-positive cells in mouse hearts, and SCO administration blocked the switch of fibroblasts to myofibroblasts in vivo.
Accordingly, SCO treatment also dramatically reduced the protein levels of α-SMA, CTGF, collagen I and collagen III in the hearts of Ang II-infused mice ( Figure 2C).

| SCO attenuates Ang II-induced cardiac fibroblasts differentiation and collagen synthesis in vitro
As Figure S3A showed, 10-200 μmol/L SCO had no cytotoxicity to NRCFs. Next, we assessed the anti-fibrotic effect of SCO on NRCFs.
In Figure S3B,C, decreased expression of α-SMA indicating that SCO suppressed Ang II-induced NRCFs differentiation from fibroblasts to myofibroblasts in vitro. In agreement with these results, SCO significantly inhibited Ang II-induced expression of CTGF, collagen I and collagen III in NRCFs ( Figure S3C).

| SCO alleviates Ang II-induced oxidative stress of cardiomyocytes and cardiac fibroblasts in vivo
To investigate the anti-oxidative effect of SCO in Ang II-induced myocardial oxidative stress, immunohistochemical staining of 4-HNE and measurement of MDA were performed. 4-HNE and MDA are markers of lipid peroxidation and oxidative stress. 20,24 The results ( Figure 3A,B) showed that Ang II leads to an increase in 4-HNE and MDA levels in the hearts, whereas the effect was significantly suppressed by SCO treatment in the Ang II + SCO group. Meanwhile, as shown in Figure 3C, in response to the sustained Ang II stimulation, the levels of RAC1-GTP, RAC1-GTP/ F I G U R E 4 Scoparone inhibits Ang II-induced cardiomyocytes oxidative stress by suppressing Rac1. NRCMs were treated with Ang II (1 μmol/L), scoparone (SCO, 50 μmol/L), control or RAC1 overexpression adenovirus (10 moi) for 48 h. A, Western blot of RAC1-GTP, t-RAC1, NOX2 and NOX4 (n = 3); B, Immunofluorescent staining of RAC1-GTP (n = 3); C.ROS assay (n = 3). *P < 0.05 vs control group; # P < 0.05 vs Ang II group; † P < 0.05 vs Ang II + SCO group total-RAC1 and downstream proteins of RAC1, including NOX2 and NOX4, were up-regulated. SCO treatment suppressed the elevated levels of RAC1-GTP, RAC1-GTP/total-RAC1, NOX2 and NOX4. The level of total RAC1 was not changed by Ang II infusion, but SCO administration inhibited the level of total RAC1 in mouse hearts.
To distinguish the expression of Rac1-GTP in cardiomyocytes and cardiac fibroblasts in hearts, RAC1-GTP was co-immunostained separately with α-actinin and vimentin. Figure 3D,E shows that SCO treatment alleviated Ang II-induced RAC1-GTP both in cardiomyocytes and cardiac fibroblasts in vivo.

| Protein level of NOX2 and NOX4 could be regulated by RAC1 in cardiomyocytes and cardiac fibroblasts
Increasing RAC1-GTP/total RAC1 ratio manifests activation of RAC1. As shown in Figure S4A,B, Ang II increased the levels of RAC1-GTP/total RAC1, RAC1-GTP, NOX2 and NOX4 in NRCMs and NRCFs in a concentration-dependent manner. This is consistent with previous reports. The role of RAC1 in protein level regulation of NOX2 and NOX4 is still not clear. To explore the role of RAC1 in regulating the expression of NOX2 and NOX4, three doses of RAC1 overexpression adenovirus (1-10 moi) were applied.
The results showed that overexpression of RAC1 increased the protein levels of NOX2 and NOX4 in NRCMs and NRCFs ( Figure   S5A,B).

| SCO inhibits Ang II-induced cardiomyocytes hypertrophy by suppressing RAC1 mediated oxidative stress
According to the results in

| SCO inhibits Ang II-induced cardiac fibroblasts differentiation and collagen synthesis by suppressing Rac1 mediated oxidative stress
Scoparone decreased Ang II-induced oxidative stress and expression of NOX2 and NOX4 by inhibiting Rac1 protein levels in cardiac fibroblasts ( Figure 6). As shown in Figure 7, SCO (50 μmol/L) blocked Ang II-induced NRCF collagen synthesis and differentiation, and RAC1 overexpression prevented SCO from the above anti-fibrotic effects.
Overall, these findings indicate that SCO inhibits Ang II-induced cardiac fibroblast differentiation and collagen synthesis by suppressing Rac1-mediated oxidative stress.
This finding would help us deepen our understanding of the relationship between RAC1 and NOXs. In Figure 3, SCO showed strong anti-oxidative ability in the hearts of Ang II-infused mice. SCO could also inhibit the expression of RAC1-GTP and total-RAC1, NOX2 and NOX4 in Ang II-infused mice hearts. Figures 3, 4 and 6 show that, both in vivo and in vitro, SCO could suppress RAC-1 GTP and total-RAC1 expression in cardiomyocytes and cardiac fibroblasts. RAC1 is necessary for the development of myocardial hypertrophy. In Ang II-stimulated mice, cardiomyocyte-specific knockout of RAC1 resulted in decreased NADPH oxidase activity, cardiac hypertrophy and myocardial oxidative stress. 6 Overexpression of a dominant-negative mutant form of RAC1 (N17RAC1) suppresses phenylephrine-induced cardiomyocyte oxidative stress and hypertrophy in vitro. 40 Figure 5 shows that SCO could inhibit Ang II-induced cardiac hypertrophy in vitro, and overexpression of RAC1 eliminated the above effect. Therefore, SCO may block Ang II-induced cardiac hypertrophy by inhibiting RAC1-mediated oxidative stress. It is well known that RAC-GTP is also involved in cardiomyocytes hypertrophy by regulating mitogen-activated protein kinases (MAPKs), apoptosis signal-regulating kinase (ASK) 1 and a transcriptional factor, nuclear factor-B (NF-B). 41,42 Therefore, the anti-hypertrophic effect of SCO based on the inhibition of RAC1 is not only by ROS regulation, but also via anti-inflammation.
In T1DM mice, RAC1 deficiency reduced myocardial fibrosis and hypertrophy, resulting in improved myocardial function. 43 In Ang IIstimulated NRCFs, knockdown of RAC1 decreased the expression of CTGF. 8 Figure 7 shows that SCO could inhibit Ang II-induced cardiac fibroblast differentiation and collagen synthesis in vitro, and overexpression of RAC1 could eliminate the above effect. Hence, SCO blocks Ang II-induced cardiac fibrosis by inhibiting RAC1 mediated oxidative stress.
In conclusion, we have demonstrated a novel function of SCO in the protective effect of Ang II-induced myocardial hypertrophy both in vitro and in vivo, and the effect may be mediated by inhibition of RAC1-dependent oxidative stress ( Figure S6). Our study provides insight into the future treatment of myocardial hypertrophy through the application of SCO.

ACK N OWLED G EM ENTS
We thank Dr Zhang Wei (The Affiliated Hospital of Shandong University of Traditional Chinese Medicine) for his assistance F I G U R E 7 Scoparone inhibits Ang IIinduced cardiac fibroblasts differentiation and collagen synthesis by suppressing Rac1. NRCFs were treated with Ang II (1 μmol/L), scoparone (SCO, 50 μmol/L), control or RAC1 overexpression adenovirus (10 moi) for 48 h. A, Immunofluorescent staining of α-SMA (n = 3). B, Western blot of α-SMA, CTGF, collagen I and collagen III (n = 3). *P < 0.05 vs control group; # P < 0.05 vs Ang II group; † P < 0.05 vs Ang II + SCO group