Angiotensin II promotes cardiac differentiation of embryonic stem cells via angiotensin type 1 receptor

Differentiation 86 (2013) 23–29

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Angiotensin II promotes cardiac differentiation of embryonic stem cells via angiotensin type 1 receptor$ Liyuan Wu a, Zhuqing Jia b,c, Lihui Yan a,1, Weiping Wang b,c, Jiaji Wang b, Yongzhen Zhang a,n, Chunyan Zhou b,c,nn a

Department of Cardiology, Peking University Third Hospital, Beijing 100191, China Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Peking University, Beijing 100191, China c Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, Peking University, Beijing 100191, China b

art ic l e i nf o

a b s t r a c t

Article history: Received 22 January 2013 Received in revised form 30 May 2013 Accepted 28 June 2013 Available online 7 August 2013

As embryonic stem cells (ESCs) represent an attractive candidate cell source for obtaining cardiomyocytes to be used in cell replacement therapy, it is thus of considerable importance to understand the mechanism by which cardiac differentiation is regulated. In previous studies, we have shown that angiotensin type 1 receptor (AT1R) expressed in cardiomyocytes derived from mouse embryonic stem cells. However, little is known about the role of AT1R in cardiac differentiation, which plays a key role in cardiac physiology and pharmacology. In the present study, we demonstrated that AT1R agonist significantly enhanced cardiac differentiation as determined by increased percentage of beating embryoid bodies and a higher expression level of cardiac markers. On the contrary, AT1R agonist stimulated differentiation was reversed in the presence of AT1R antagonist. In addition, by administering selective inhibitors we found that the effect of AT1R was driven via extracellular-signal regulated kinase, c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase pathways. These findings suggest that AT1R signaling plays a key role in cardiac differentiation of ESCs. & 2013 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Keywords: Angiotensin type 1 receptor Cardiac differentiation Embryonic stem cell Extracellular-signal regulated kinase c-Jun NH2-terminal kinase P38 mitogen-activated protein kinase

1. Introduction Embryonic stem cells (ESCs), because of their tremendous capacity for expansion and cardiac differentiation potential, represent an attractive candidate cell source for obtaining cardiomyocytes for cardiac repair. Furthermore, the understanding of mechanisms that involved in the differentiation of ESCs into cardiomyocytes may lead to a method for the large-scale

Abbreviations: AT1R, angiotensin type 1 receptor; Ang II, angiotensin II; ESCs, embryonic stem cells; ERK, extracellular-signal regulated kinase; JNK, c-Jun NH2terminal kinase; MAPK, mitogen-activated protein kinase. ☆ Competing interests: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors declare no conflicts of interest. n Correspondence to: Department of Cardiology, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, China. Tel.: +86 10 82265549; fax: +86 10 82074373. nn Correspondence to: Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Haidian District, Beijing 100191, China. Tel./fax: +86 10 82802417. E-mail addresses: [email protected] (Y. Zhang), [email protected] (C. Zhou). 1 Current address: Key Laboratory of Hormones and Development (Ministry of Health), Institute of Endocrinology, Metabolic Disease Hospital, Tianjin Medical University, Tianjin 300070, China.

production of cardiomyocytes from ESCs. Recently studies have reported that many signaling pathways and transcriptional factors are essential for cardiac differentiation, such as TGF-β (Yook et al., 2011), BMP4 (Taha et al., 2007), FGF (Marques et al., 2008), WNT (Paige et al., 2010), NOTCH (Chau et al., 2006) signaling pathways and ISL1 (Cai et al., 2003). We previously showed that angiotensin type 1 receptor (AT1R) was expressed in cardiomyocytes derived from mouse embryonic stem cells (Cui et al., 2010), but the effect of AT1R signaling on cardiac differentiation is still unclear. AT1R inhibitor Losartan is widely used in the clinical treatment of myocardial infarction. Therefore, it is necessary to know its effects on cardiac differentiation of ESCs and the underlying molecular mechanisms. Angiotensin II (Ang II) is the main effector peptide in the reninangiotensin system. It has systemic and local effects, favoring cell growth and differentiation through four types of receptors of which types 1 is the most important subtype. Stimulation of AT1R leads to the activation of intracellular pathways that finally lead to vasoconstriction, inflammation and proliferation. It was reported that AT1R might play a role in the development of cardiac dysfunction (Nakamura et al., 2008). In addition, AT1R-deficient mice exhibited heart atrophy and lower blood pressure, indicating that AT1R was involved in the development of cardiovascular systems (Van Esch et al., 2010). Several studies have confirmed the

0301-4681/$ - see front matter & 2013 International Society of Differentiation. Published by Elsevier B.V. All rights reserved. Join the International Society for Differentiation (www.isdifferentiation.org) http://dx.doi.org/10.1016/j.diff.2013.06.007

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involvement of Ang II in the differentiation of stem/progenitor cells (Zambidis et al., 2008; Kim et al., 2008). Zambidis et al. showed that the differentiation of human hemangioblasts into either hematopoietic or endothelial progenitor cells may be modulated by angiotensin receptors. In addition, Kim et al. showed that AT1R was involved in the Ang II-induced differentiation of human adipose tissue-derived mesenchymal stem cells to contractile smooth muscle-like cells. A recent study showed that Ang II had an effect on proliferation and differentiation of mouse induced pluripotent stem cells into mesodermal progenitor cells through AT1R (Ishizuka et al., 2012). However, the effect of AT1R on the cardiac differentiation is still unclear. In the present study, we investigated the effect of the AT1R on the cardiac differentiation of mouse ESCs and the underlying molecular mechanisms.

2. Materials and methods 2.1. Chemicals and reagents Angiotensin II (Ang II), Losartan, β-mercaptoethanol, vitamin C, Hoechst33342 and anti-AGTR1 antibody produced in rabbit and anti-mouse α-actinin monoclonal antibody were purchased from Sigma (St. Louis, MO, USA). TRIzol reagent, PD98059, SB203580 and SP600125 were purchased from Promega (Madison, WI, USA). Leukemia inhibitory factor (LIF) was purchased from Chemicon (Temecula, CA, USA). Dulbeccos's modified Eagle's medium (DMEM) and fetal bovine serum were purchased from Hyclone (Logan, UT, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies (goat anti-rabbit and goat anti-mouse), TRITCconjugated secondary anti-body, GATA4 polyclonal antibody were purchased from Santa Cruz (CA, USA). Cardiac troponin-T antibody was purchased from Abcam (Cambridge, UK). Phosphor-ERK1/2 antibody, ERK1/2 antibody, phosphor-p38 antibody and p38 antibody, phosphor-JNK/SAPK antibody and JNK/SAPK antibody were purchased from Cell Signaling Technology (Dancers, MA, USA). 2.2. ESC culture and differentiation Mouse R1 ES cell line was a gift from Prof. Yang. The cells were cultured in high glucose (4.5 g/L) DMEM, supplemented with 20% batch-tested fetal bovine serum, 2 mmol/L L-glutamine, 1% nonessential amino acids, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.1 mmol/L β-mercaptoethanol and 500 U/mL LIF (Wobus et al., 2002). To induce embryoid body (EB) formation, the ESC colonies were suspended in differentiation culture medium, which consisted of high glucose (4.5 g/L) DMEM, 20% fetal bovine serum, 0.1 mmol/L nonessential amino acid, 100 U/mL penicillin, 100 μg/ mL streptomycin, 10 4 mol/L vitamin C, and then seeded into domestic Petri dishes (Qingdao Alpha Company, China). After 5 days the EBs were plated onto gelatin-coated culture dishes and were incubated in differentiation culture medium to differentiate further and examined daily. The medium was changed every other day. The time that ESCMs began to beat spontaneously, the numbers of beating EBs were recorded.

Relative changes of mRNA amount were calculated using the DDCT method, using mouse 18S RNA as an internal control as previously described (Liu et al., 2009). PCR amplification parameters are as follows: initial denaturation at 95 1C for 12 min; 45 cycles of 1 s at 95 1C for denaturation, 5 s at 60 1C for annealing and 10 s at 72 1C for extension. The primers used for qRT-PCR are: AT1R: 5′-TGTCCACCCGATGAAGTCTC-3′ and 5′-AGCGCAAACAGTGATATTGGT-3′; α-MHC: 5′-GCCCAGTACCTCCGAAAGTC-3′ and 5′-GCCTTAACATACTCCTCCTTGTC-3′; β-MHC: 5′-ACCCCTACGATTATGCG-3′ and 5′-GTGACGTACTCGTTGCC-3′; troponin-T: 5′-GGCAGAACCGCCTGGCTGAA-3′ and 5′-CTGCCACAGCTCCTTGGCCT-3′; GATA4: 5′-CCCTACCCAGCCTACATGG-3′ and 5′-ACATATCGAGATTGGGGTGTCT-3′. 2.4. Western blot analysis Total cellular protein was prepared using a standard method. Briefly, embryoid bodies were collected using a cell scraper (Nunc, USA) and washed once in phosphate-buffered saline and lysed in modified Radio Immunoprecipitation Assay (RIPA) lysis buffer (Sigma-Aldrich, St. Louis, MO, USA). The protein concentration was measured by the bicinchoninic acid (BCA) protein assay. A total of 50 μg protein was subjected to electrophoresis on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Proteins were transferred onto polyvinylidene fluoride membrane by electrophoresis at 100 V for 2 h. Membranes were blocked in 5% non-fat milk or 5% bovine serum albumin blocking buffer and then incubated overnight with primary antibodies at 4 1C. The membranes were washed and incubated for 1 h with HRP-labeled secondary antibody at room temperature, and the labeled proteins were detected using enhanced chemiluminescence reagents (Santa Cruz, CA, USA). 2.5. Immunofluorescence and confocal microscopy The EBs were washed three times with phosphate-buffered saline (PBS), fixed with 4% polyoxymethylene for 15 min at room temperature and permeabilized with PBST (phosphate buffered saline with 0.5% Triton X-100). The samples were blocked for 1 h with PBS containing 5% bovine serum albumin (BSA) at room temperature and then incubated with cardiac α-actinin antibody (1:1000 dilution) or GATA4 antibody (1:100 dilution) overnight at 4 1C. After washing, cells were incubated with TRITC-conjugated secondary antibodies (mouse IgG antibody-TRITC, or goat IgG antibody-TRITC, 1:200 dilution) for 1 h. The nuclei were counterstained with Hoechst33342 (Sigma-Aldrich, St. Louis, MO, USA) for 5 min and visualized microscopically with an Olympus FV 1000 confocal laser scanning microscope (Olympus Corporation, Tokyo, Japan). 2.6. Statistic analysis Data represent the mean7SEM from at least three independent experiments. These results were analyzed by one-way ANOVA followed by a post hoc comparison using Tukey's test. Differences were considered to be significant when P-value o0.05.

2.3. RNA extraction and quantitative real-time RT-PCR

3. Results

Total RNA was extracted from ESCs with TRIzol reagent according to the manufacture's instruction (Promega, Madison, WI, USA). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed using the SYBR Green real-time PCR system following the manufacture's protocol (Applied Biosystems, Carlsbad, CA, USA) with the RNA samples obtained from four independent experiments and each sample were tested in triplicate.

3.1. ES cells differentiated into cardiomyocytes after induction ES cells were propagated for 2 days. Cardiac differentiation was initiated by inducing EB formation from undifferentiated ES cells. After 5 days suspension culture, EBs were allowed to differentiate further in adherent cultures. Spontaneously contracting EBs appeared at differentiation day 7. The number of beating EBs in

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6-cm-plates was recorded at different time points. Spontaneously contracting cells appeared as clusters and were identified approximately 12% of the individual EBs at differentiation day 9, increased to 58% of the EBs at day 14 and then began to decline. By day 18, the percentage of beating EBs was 40% (Fig. 1A). GATA4 is a marker of cardiomyocytes. We detected the expression of GATA4 with Western blotting during the differentiation from day 0 to day 18. We found that the expression of GATA4 at the protein level gradually increased and reached a maximum 14 days after induction (Fig. 1B). 3.2. AT1R was expressed in ESC-CMs The expression of AT1R in the cardiomyocytes derived from mouse embryonic stem cells (ESCs) was analyzed by qRT-PCR and Western blotting analysis. As shown in Fig. 2A, the mRNA expression of AT1R increased during ESCs differentiation. Accordingly, the expression of AT1R at the protein level also gradually increased and reached a maximum 14 days after induction (Fig. 2B). 3.3. AT1R agonist promoted cardiac differentiation of embryonic stem cells To evaluate the effect of AT1R agonist on the cardiac differentiation of ESCs, Ang II was applied at different concentrations (0.01, 0.1, 1, 10 μmol/L) and for different periods (7–14, 5–14, 3–14, 0–14 days). The differentiation efficiency was evaluated by calculating the percentage of beating EBs 14 days after induction. As shown in Fig. 2, Ang II promoted cardiac differentiation in a doseand time-dependent manner. Compared to 51% of the control group (without Ang II treatment), the percentage of beating EBs

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was increased to 58%, 72%, 85% and 63% in the ESCs treated with Ang II ranging from 0.01 to 10 μmol/L for 14 days, respectively (Fig. 3A). Both 0.1 and 1 μmol/L Ang II groups showed significant differences with the control group, with 1 μmol/L Ang II the most efficient dose. The expression of troponin-T and GATA4 was also up-regulated by 1 μmol/L Ang II treatment (Fig. 3B). In order to evaluate the effects of AT1R activation at different period of cardiac differentiation, the ESCs were treated with 1 μmol/L Ang II for 7–14 days, 5–14 days, 3–14 days, 0–14 days, respectively, and the percentage of beating EBs was increased to 60%, 65%, 73% and 85% in different periods, compared with 51% in control group (Fig. 3C). The result indicates that the whole course treatment (0– 14 days) of 1 μmol/L Ang II is the most efficient for cardiac differentiation of ESCs. The Ang II treatment was also upregulated the expression of troponin-T and GATA4 (Fig. 3D). The period of 0–14 days with 1 μmol/L Ang II was chosen as the optimum time and dose for the rest of the study. To further confirm the effect of AT1R on the cardiac differentiation of ESCs, cells were pre-incubated for 1 h with AT1R selective antagonist Losartan (1 μmol/L) before the treatment of 1 μmol/L Ang II from day 0 to day 14. The concentration of Losartan was optimized according to published literature (Kim et al., 2008; Ishizuka et al., 2012). The percentage of beating EBs was counted 9–14 days after induction. As shown in Fig. 4A, Ang II significantly increased the percentage of beating EBs (83%), while Losartan reduced the percentage of beating EBs (53%) to the level seen in the control group (51%). There was no statistical difference between Losartan alone (48%) and the control groups. This result was also reflected by the changes in cardiac gene expression (Fig. 4B). Ang II increased the expression of α-MHC, β-MHC and troponin-T at the mRNA level (α-MHC: 17.1 fold, P o0.05; β-MHC:

Fig. 1. Identification of cardiomyocytes derived from embryonic stem cells. (A) Differentiation efficiency was evaluated by calculating the percentage of beating embryoid bodies (EBs). Data were obtained from three independent experiments. (B) Expression of GATA4 at different times was examined by Western Blotting analysis. GAPDH was used as an internal control. A representative figure from three independent experiments is shown.

Fig. 2. The expression of Angiotensin II receptor type 1 (AT1R) during the cardiac differentiation of embryonic stem cells (ESCs). (A) The expression of AT1R mRNA in differentiating ESCs was evaluated by real-time PCR. The expression level at day 0 was set 1, and the fold changes at indicated time points were calculated relative to day 0. Values are mean 7 SD. *Po 0.05 vs. day 0. (B) The expression of AT1R proteins in differentiating ESCs was detected by Western Blotting analysis. GAPDH was used as an internal control. A representative figure from three independent experiments is shown.

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Fig. 3. Dose- and time-dependent effects of Ang II on the cardiac differentiation of embryonic stem cells (ESCs). (A) Differentiation efficiency was evaluated by calculating the percentage of beating embryoid bodies (EBs). EBs were treated with AngII at the concentration of 0.01, 0.1, 1, 10 μmol/L from day 0 to day 14. The medium was changed every other day. A total of 100 EBs were counted at day 14 for different groups. Percentages of beating EBs were calculated. *P o0.05 vs. the control (EBs without AngII treatment). (B) Cells were treated with or without 0.01, 0.1, 1, 10 μmol/L Ang II and the expression of GATA4 and troponin-T was assessed by Western Blotting at day 14. GAPDH was used as an internal control. A representative figure from three independent experiments is shown. (C) Differentiation efficiency was evaluated by calculating the percentage of beating embryoid bodies (EBs). EBs were treated with 1 μmol/L AngII for 7–14, 5–14, 3–14 and 0–14 days, respectively. A total of 100 EBs were counted at day 14 for different groups. Percentages of beating EBs were calculated. *P o0.05 vs. the control (EBs without AngII treatment). (D) Cells were treated with 1 μmol/L Ang II for 7–14, 5–14, 3–14 and 0–14 days and the expression of GATA4 and troponin-T was assessed by Western Blotting at day 14. GAPDH was used as an internal control. Representatives of three independent experiments are shown.

15.8 fold, P o0.05; troponin-T: 12.8 fold, Po 0.05), suggesting that cardiac differentiation was enhanced by Ang II. However, preincubation of ESCs with Losartan abolished this enhancement. At the protein level, Ang II also increased troponin-T and α-actinin expression while pre-exposure to Losartan reversed this effect significantly (P o0.05, Fig. 4D and F). No statistical difference was observed between Losartan and the control group. In addition to the structural proteins, we also observed that Ang II significantly increased the expression of a cardiac specific transcription factor, GATA4 (9.9 fold, P o0.05; Fig. 4B), while preincubation with Losartan abolished this effect. Furthermore, Western blotting analysis and immunofluorescence microscopy also revealed the same change in protein expression (Fig. 4C and E). No statistical significance was observed between Losartan and the control group. 3.4. AT1R agonist promoted the cardiac differentiation of ESCs via ERK, p38 and JNK activation Previous studies (Wang, 2007) have suggested that mitogenactivated protein kinase (MAPK) is involved in cardiac development. Furthermore, AT1R signaling has been shown to be essential for the activation of MAPK. We therefore tested whether the activation of ERK, JNK and p38 MAPK led to AT1R-dependent cardiac development. Western blotting analysis showed that Ang II significantly increased ERK, JNK and p38 MAPK activation, as assessed using phosphorylation-specific antibodies that detect the activated kinases. When the cells were pre-exposed to Losartan, the phosphorylation of ERK, JNK and p38 MAPK was inhibited (Fig. 5A–C), suggesting that AT1R is required for the activation of the MAPK pathway during the cardiac differentiation of ESCs. However, when the cells were treated with Losartan alone, the phosphorylation of ERK, JNK and p38 MAPK was not changed significantly compared with the control group.

To further investigate the impact of MAPK activity on Ang IIinduced cardiac differentiation, cells were treated with Ang II alone or pre-incubated with PD98059 (20 μmol/L, a specific inhibitor of ERK), SB203580 (20 μmol/L, a specific inhibitor of p38 MAPK) or SP600125 (20 μmol/L, a specific inhibitor of JNK) for 1 h before treatment with 1 μmol/L Ang II for 14 days. To exclude possible toxic effects induced by PD98059, SB203580 and SP600125, we performed cell proliferation assay with WST-1 Cell Proliferation and Cytotoxicity Assay Kit. The results demonstrated that the concentration of 20 μmol/L PD98059, SB203580 or SP600125 had no toxic effects (data not shown). Cardiac differentiation was assessed by counting the percentage of beating EBs at day 9–day 14. As shown in Fig. 5D–F, PD98059 alone did not significantly reduce the percentage of beating EBs compared to the control group (without Ang II treatment), but the increase of beating EBs following Ang II treatment was significantly inhibited by PD98059 (P o0.05). In contrast, SB203580 alone did significantly inhibit cardiac differentiation (P o0.05). Co-administration of SB203580 with Ang II also inhibited cardiac differentiation compared to both the control group and the group treated with Ang II alone (Po 0.05). Similarly, SP600125 had the similar effect as SB203580. SP600125 alone did significantly inhibit cardiac differentiation (P o0.05). Co-administration of SP600125 with Ang II also inhibited cardiac differentiation compared to both the control group and the group treated with Ang II alone (P o0.05). In addition, cardiac differentiation was also assessed by detecting the expression of cardiac markers at the protein level on day 14. As shown in Fig. 5G, PD98059 alone had little effect on the expression of cardiac markers, but the up-regulated expression of these markers following Ang II treatment was reduced in the presence of PD98059. As shown in Fig. 5H, SB203580 alone inhibited the expression of the cardiac markers. Co-administration SB203580 and Ang II could abolish the up-regulation effect of Ang II on the expression of the cardiac markers. As shown in Fig. 5I, SP600125

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Fig. 4. Ang II induces differentiation embryonic stem cell to cardiomyocytes through angiotensin type 1 receptor. (A) The effect of AT1R on the differentiation efficiency was evaluated by calculating the percentage of beating EBs. Embryoid bodies (EBs) were pre-incubated for 1 h with 1 μmol/L AT1R antagonist Losartan, and then treated with 1 μmol/L Ang II for 14 days. The medium was changed every other day. Percentages of beating EBs were calculated from day 9 to day 14. *Po 0.05 vs. the control (without Ang II or Losartan treatment). #Po 0.05 vs. Ang II treated group. (B) The expression of α-MHC, β-MHC, troponin-T and GATA4 mRNA in ESCs treated with Ang II alone or after pretreatment with Losartan was detected by real-time PCR at day 14. The expression level of the control group (without Ang II or Losartan treatment) was set 1, and the fold changes in other groups were calculated relative to the control. Values are mean 7SD of three independent experiments. *P o 0.05 vs. the control. #Po 0.05 vs. Ang II treated group. (C and D) The expression of troponin-T and GATA4 in ESCs treated with Ang II alone or after pre-treatment with Losartan was detected by Western Blotting at day 14. The densitometric analysis was performed with images of three independent experiments and the results are shown at the top of the representative images. *Po 0.05 vs. the control (without Ang II or Losartan treatment). #Po 0.05 vs. Ang II treated group. (E and F) The expression of GATA4 and α-actinin in ESCs treated with Ang II alone or after pre-treatment with Losartan was detected by confocal microscopy at day 14. Representatives of three independent experiments are shown.

alone inhibited the expression of the cardiac markers. Coadministration SP600125 and Ang II could abolish the upregulation effect of Ang II on the expression of the cardiac markers.

4. Discussion A major finding of this study is that AT1R agonist Ang II promotes cardiac differentiation of ESCs by a mechanism that involves ERK, P38 and JNK signaling pathways. Our results suggest that AT1R plays a vital role in cardiac differentiation. Recent studies reported that ESC-derived cariomyocytes exhibit fully functional AT1R (Oshra et al., 2008; Lagerqvist et al., 2011). We also detected the presence of AT1R in ESC differentiated cardiomyocytes in both our previous (Cui et al., 2010) and present studies. Moreover, the expression pattern of AT1R during the

differentiation of ESC suggests that AT1R may play important roles in the late stage of cardiac differentiation. Previous studies have demonstrated the role of AT1R in the differentiation of bone marrow mesenchymal stem cells and pancreatic progenitor cells (Klei et al., 2013; Leung et al., 2012) and in the development of cardiac system (Nakamura et al., 2008; Van Esch et al., 2010; Everett et al., 1997). Recently, Xing et al. (2012) reported that the combination of Ang II and 5-azacytidine promoted cardiomyocyte differentiation of rat bone marrow mesenchymal stem cells, which indicates that Ang II is involved in cardiac differentiation. However, whether AT1R had effect on the cardiac differentiation of ESCs remained unclear. ESCs have tremendous capacity for expansion and cardiac differentiation potential, and represent an attractive candidate cell source for obtaining cardiomyocytes to be used in cell replacement therapy for patients suffered from myocardial infarction. While AT1R antagonist

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Fig. 5. Mitogen-activated protein kinases (MAPKs) pathways involved in Ang II-dependent cardiac differentiation. (A–C) The phosphorylation of ERK, p38 and JNK was detected in embryonic stem cells treated with Ang II alone or after pre-treatment with Losartan from day 0 to day 7 using the phosphorylation-specific antibodies at day 7. Representatives of three independent experiments are shown. (D–F) Embryoid bodies (EBs) were treated with Ang II alone or after pre-treatment with PD98059 (20 μM, a specific inhibitor of ERK), SB203580 (20 μM, a specific inhibitor of p38 MAPK) or SP600125 (20 μM, a specific inhibitor of JNK) for 1 h from day 0 to day 14. Percentages of beating EBs were calculated from day 9 to day 14. *Po 0.05 vs. the control (without Ang II treatment). #Po 0.05 vs. Ang II treated group. (G–I) EBs were treated with Ang II alone or after pre-treatment with PD98059 (20 μM), SB203580 (20 μM) or SP600125 (20 μM) for 1 h from day 0 to day 14. The expression GATA4 and troponin-T in ESCs were detected by Western Blotting at day 14. GAPDH was used as an internal control. Representatives of three independent experiments are shown.

is widely used for myocardial infarction treatment. Thus, it is of considerable importance to understand the effect of AT1R on cardiac differentiation of ESCs. From our results, we suggest that activation of AT1R may be useful for increasing production of cardiomyocytes from ESCs. Moreover, AT1R inhibitor alone did not have significant effect on cardiac differentiation, which suggests that AT1R may not be a major contributor to cardiac differentiation in the absence of exogenous Ang II. Since Losartan alone has no negative effect on cardiac differentiation of ESCs, we conclude that patients who transplanted ESCs can use Losartan. It has been reported that MAPK signaling pathways are involved in cardiac differentiation (Eriksson and Leppa, 2002; Aouadi et al., 2006). It is also well known that MAPK signaling pathways have crosslink with Ang II in different physiological or biological processes (Hayashida et al., 2001; Kang et al., 2006). However, whether MAPK pathways are involved in Ang II induced cardiac differentiation is not clear. In our study, the levels of ERK, p38 and JNK phosphorylation were increased following Ang II treatment, suggesting that ERK, p38 and JNK pathways are involved in Ang II-dependent cardiac differentiation. Further investigation indicated that the ERK inhibitor PD98059 alone had no obvious effect on cardiac differentiation, but the effect of Ang II on promoting cardiac differentiation was abolished by PD98059. In contrast, the p38 inhibitor SB203580 alone or the JNK inhibitor SP600125 alone could inhibited cardiac differentiation, which suggest that p38 and JNK play more important roles in Ang IIindependent cardiac differentiation compared with ERK. This is consistent with previous studies (Eriksson and Leppa, 2002) that

inhibition of p38 pathway completely prevented the formation of beating cardiomyocytes, whereas inhibition of the ERK only partially prevented the differentiation of P19 cells. In addition, recent studies showed that ERK, p38 and JNK were involved in cell differentiation and embryonic development. Wang (2007) reported that MAPK signaling pathways are involved in cardiac development. Chye et al. (2012) demonstrated that activation of p38 and JNK involved in apoptosis induced by paraphenylenediamine in chang liver cells. Wu et al. (2010) and Barruet et al. (2011) showed that p38 is crucial during the differentiation of mouse ES cell into mesodermal lineages. Moreover, many differentiation processes require p38 activation, including adipocytic differentiation (Engelman et al., 1998), myogenic differentiation (Cuenda and Cohen, 1999; Galbiati et al., 1999; Zetser et al., 1999), neuronal differentiation (Morooka and Nishida, 1998; Iwasaki et al., 1999), erythroid differentiation (Nagata et al.,1998) and growth/ differentiation factor 5-induced chondrogenesis of ATDC5 cells (Nakamura et al.,1999). Behrens et al. (1999) reported that c-Jun was essential for embryonic development, as fetuses lacking Jun die at mid-gestation with impaired hepatogenesis and primary Jun-/- fibroblasts have a severe proliferation defect and undergo premature senescence in vitro, whereas Kanzawa et al. (2006) showed that inhibition of JNK improved neuronal differentiation. The discrepancies in these studies may be due to the stage and duration in which agonists or antagonists were administered. In addition, the concentration and cell type may also contribute to different results. The roles of MAPK pathways in cardiac differentiation need to be investigated further.

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In conclusion, the present results suggest that the AT1R agonist Ang II is able to promote cardiac differentiation and that the MAPK signaling pathway is one of the underlying molecular mechanisms. Inhibition of AT1R partially abolished the stimulation effect of Ang II on cardiac differentiation of ESCs. This study has shown for the first time that AT1R is involved in cardiac differentiation of ESCs. Meanwhile, the finding on that AT1R inhibitor alone in the absence of exogenous Ang II has no negative effect on cardiac differentiation may provide useful information for the application of ESCs in clinics. Acknowledgments We are grateful to Prof. Huangtian Yang, Shanghai Jiao Tong University, for providing us the mouse R1 ES cell line. We thank Prof. Youyi Zhang and Dr. Yao Song, Institute of Vascular Medicine, Peking University Third Hospital, for their helpful discussion. This work was supported by the National Natural Sciences Foundation of China (30570711, 30871253, 90919022), the 111 Project of China, Leading Academic Discipline Project of Beijing Education Bureau and Beijing Natural Sciences Foundation (7122200). References Aouadi, M., Bost, F., Caron, L., Laurent, K., Le Marchand Brustel, Y., Binétruy, B., 2006. p38 mitogen-activated protein kinase activity commits embryonic stem cells to either neurogenesis or cardiomyogenesis. Stem Cells 24, 1399–1406. Barruet, E., Hadadeh, Q., Peireti, F., Renault, V.W., Hadial, Y., Bernot, D., Toumaire, R., Negre, D., Juhan, V.I., Alessi, M.C., Binetruy, B., 2011. P38 mitogen activated protein kinase controls two-successive-steps during the early mesodermal commitment of embryonic stem cells. Stem Cells and Development 20, 1233–1246. Behrens, A., Sibilia, M., Wagner, E.F., 1999. Amino-terminal phosphorylation of cJun regulates stress-induced apoptosis and cellular proliferation. Nature Genetics 21, 326–329. Cai, C.L., Liang, X., Shi, Y., Chu, P.H., Pfaff, S.L., Chen, J., Evans, S., 2003. Isl1 identifies a cardiac progenitor population that proliferates prior to differentiation and contributes a majority of cells to the heart. Developmental Cell 5, 877–889. Chau, M.D., Tuft, R., Fogarty, K., Bao, Z.Z., 2006. Notch signaling plays a key role in cardiac cell differentiation. Mechanisms of Development 123, 626–640. Chye, S.M., Tiong, Y.L., Yip, W.K., Koh, R.Y., Len, Y.W., Seow, H.F., Ng, K.Y., Ranjit, D.A., Chen, S.C., 2012. Apoptosis induced by para-phenylenediamine involves formation of ROS and activation of p38 and JNK in chang liver cells. Environmental Toxicology , http://dx.doi.org/10.1002/tox.21828 (Epub ahead of print). Cuenda, A., Cohen, P., 1999. Stress-activated protein kinase-2/p38 and a rapamycinsensitive pathway are required for C2C12 myogenesis. Journal of Biological Chemistry 274, 4341–4346. Cui, J.J., Yang, H.T., Jia, Z.Q., Yan, L.H., Zhang, C.G., Yang, H.T., Zhou, C.Y., Zhang, Y.Z., 2010. Expression of angiotensin II type 1 and type 2 receptors in mouse embryonic stem cell-derived cardiomyocytes. Chinese Journal of Biochemistry and Molecular Biology 26, 643–650. Engelman, J.A., Lisanti, M.P., Scherer, P.E., 1998. Specific inhibitors of p38 mitogenactivated protein kinase block 3T3-L1 adipogenesis. Journal of Biological Chemistry 273, 32111–32120. Eriksson, M., Leppa, S., 2002. Mitogen-activated protein kinases and activator protein 1 are required for proliferation and cardiomyocyte differentiation of P19 embryonal carcinoma cells. Journal of Biological Chemistry 277, 15992–16001. Everett, A.D., Fisher, A., Tufro, M.A., Harris, M., 1997. Developmental regulation of angiotensin type 1 and 2 receptor gene expression and heart growth. Journal of Molecular and Cellular Cardiology 29, 141–148. Galbiati, F., Volonte, D., Engelman, J.A., Scherer, P.E., Lisanti, M.P., 1999. Targeted downregulation of caveolin-3 is sufficient to inhibit myotube formation in differentiating C2C12 myoblasts. Journal of Biological Chemistry 274, 30315–30321. Hayashida, W., Kihara, Y., Yasaka, A., Inagaki, K., Iwanaga, Y., Sasayama, S., 2001. Stage-specific differential activation of mitogen-activated protein kinases in hypertrophied and failing rat hearts. Journal of Molecular and Cellular Cardiology 33, 733–744. Ishizuka, T., Goshima, H., Ozawa, A., Watanabe, Y., 2012. Effect of angiotensin II on proliferation and differentiation of mouse induced pluripotent stem cells into mesodermal progenitor cells. Biochemical and Biophysical Research Communications 420, 148–155. Iwasaki, S., Iguchi, M., Watanabe, K., Hoshino, R., Tsujimoto, M., Kohno, M., 1999. Specific activation of the p38 mitogen-activated protein kinase signaling pathway and induction of neurite outgrowth in PC12 cells by bone morphogenetic protein-2. Journal of Biological Chemistry 274, 26503–26510.

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