ORIGINAL RESEARCH ARTICLE

Journal of

Bone Morphogenetic Protein-2 Antagonizes Bone Morphogenetic Protein-4 Induced Cardiomyocyte Hypertrophy and Apoptosis

Cellular Physiology

JING LU, BO SUN, RONG HUO, YU-CHUN WANG, DI YANG, YUE XING, XIAO-LIN XIAO, XIN XIE, AND DE-LI DONG* Department of Pharmacology (The State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), Harbin Medical University, Harbin 150086, P.R. China Our previous work showed that the expression of bone morphogenetic protein-4 (BMP4) was up-regulated in pathological cardiac hypertrophy models and BMP4 induced cardiomyocyte hypertrophy and apoptosis. Bone morphogenetic protein-2 (BMP2) and BMP4 share greater than 80% amino acid homology and there exists an interaction between BMP2 and BMP4, so the aim of the present study was to elucidate the changes of BMP2 in the cardiac hypertrophy models and the effects of BMP2 on BMP4-induced cardiomyocyte hypertrophy and apoptosis. The in vivo cardiac hypertrophy models were induced by pressure-overload and swimming exercise in mice. BMP2 mRNA and protein expressions increased in pressure-overload and swimming-exercise induced cardiac hypertrophy. BMP2 itself did not elicit cardiomyocyte hypertrophy and apoptosis, but antagonized BMP4-induced cardiomyocyte hypertrophy and apoptosis. BMP2 stimulated Akt in cardiomyocytes and Akt inhibitor prevented the antagonism of BMP2 on BMP4-induced cardiomyocyte apoptosis. Furthermore, BMP2 inhibited BMP4-induced JNK activation in cardiomyocytes. In conclusion, BMP2 antagonizes BMP4-induced cardiomyocyte hypertrophy and apoptosis. The anti-apoptotic effects of BMP2 on BMP4-induced cardiomyocyte apoptosis might be through activating Akt and inhibiting JNK activation. J. Cell. Physiol. 229: 1503–1510, 2014. © 2014 Wiley Periodicals, Inc.

Bone morphogenetic proteins (BMPs) are the largest subset in the transforming growth factor (TGF)-b superfamily. More than 30 members of BMP family have been described (Ducy and Karsenty, 2000). BMP family members have been associated with a number of pathologies, including kidney, bone as well as various cardiovascular diseases (Cai et al., 2012; Nishimura et al., 2012; Meng et al., 2013; Sun et al., 2013a). BMP2 and BMP4 belong to the same subclass of BMP family and share greater than 80% amino acid homology. There exists an interaction between BMP2 and BMP4 (Uchimura et al., 2009; Anderson et al., 2010). In the heart development, BMP2 and BMP4 function coordinately to direct normal lengthening of the outflow tract, proper positioning of the outflow vessels, and septation of the atria, ventricle and atrioventricular canal (Goldman et al., 2009). However, they appear to exert divergent effects in some diseases. In hypoxic pulmonary hypertension model, pulmonary BMP2 and BMP4 expressions (but not BMP5, 6, 7) are up-regulated (Frank et al., 2005), but BMP2 and BMP4 exert opposing roles. BMP2 protects against but BMP4 promotes hypoxic pulmonary hypertension (Frank et al., 2005; Anderson et al., 2010). Recent works show that BMP4 enhances, but BMP2 decreases, transient receptor potential channel (TRPC) expression, store-operated Ca2þ entry, and basal [Ca2þ]i in rat distal pulmonary arterial smooth muscle cells, which might contribute to the roles of BMP4 and BMP2 in pulmonary vascular remodeling during pulmonary arterial hypertension (Lu et al., 2010; Zhang et al., 2013). Pathological cardiac hypertrophy, characterized by cardiomyocyte hypertrophy, apoptosis and cardiac fibrosis, progressively leads to heart failure. Our previous works showed that the expression of BMP4 increased in pressureoverload and Ang II constant infusion-induced pathological cardiac hypertrophy, in turn, BMP4 induced cardiomyocyte hypertrophy, apoptosis and Kv4.3 Kþ channel remodeling (Sun et al., 2013a,b). We further found that BMP2 mRNA expression also increased in the pressure-overload induced heart hypertrophy model. Therefore, the aim of the present study was to elucidate the changes of BMP2 in the cardiac © 2 0 1 4 W I L E Y P E R I O D I C A L S , I N C .

hypertrophy models and the role of BMP2 in BMP4-induced cardiomyocyte hypertrophy and apoptosis. Materials and Methods Agents Anti-BMP-4, -BMP-2 antibodies were from Santa Cruz (Dallas); anti-GAPDH was from KangChen (Shanghai, China); anti-actin was from ZSGB-Bio (Beijing, China). Anti-sarcomeric a-actinin was from Sigma (Saint Louis, MO). Anti-p-JNK, -p-AKT, and anti-JNK, -AKT antibodies were from Cell Signaling Technology (Danvers, MA). Recombinant human BMP-4, BMP-2 and recombinant human noggin were purchased from R&D Systems (Minneapolis, MN). The real-time PCR primers were shown in Table I. Akt1/2 kinase inhibitor (Akti, 1,3-Dihydro-1-(1-((4-(6-phenyl-1H-imidazo[4,5-g]quinoxalin-7-yl) phenyl) methyl)-4-piperidinyl)-2H-benzimidazol-2one trifluoroacetate salt hydrate) was purchased from Sigma.

Conflict of interest: none. Jing Lu and Bo Sun contributed equally to this work. Contract grant sponsor: The National Basic Research Program of China; Contract grant number: 2012CB517803. Contract grant sponsor: The National Natural Science Foundation of China; Contract grant numbers: 81173049, 81121003, 81373406. *Correspondence to: De-Li Dong, Department of Pharmacology, Harbin Medical University, Baojian Road 157, Harbin 150086, Heilongjiang Province, P.R. China. E-mail: [email protected] Manuscript Received: 17 August 2013 Manuscript Accepted: 19 February 2014 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 23 February 2014. DOI: 10.1002/jcp.24592

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TABLE I. Sequences of qRT-PCR primers Gene name ANP BNP b-MHC BMP1 BMP2 BMP3 BMP4 GADPH

Accession no.

Species

NM_008725.2 NM_012612.2 NM_008726.4 NM_031545.1 NM_080728.2 NM_017240.1 NM_009755.3 NM_007553.2 NM_173404.3 NM_007554.2 NM_008084.2 NM_017008.3

Mouse Rat Mouse Rat Mouse Rat Mouse Mouse Mouse Mouse Mouse Rat

Forward primer(50 –30 )

Reverse primer (50 –30 )

CTCCGATAGATCTGCCCTCTTGAA

GGTACCGGAAGCTGTTGCAGCCTA

TGATTCTGCTCCTGCTTTTC

GTGGATTGTTCTGGAGACTG

CCAGAAGCCTCGAAATGTC

CTTTCTTTGCCTTGCCTTTGC

GACTCACGGCGGACTCTAAGC GACTCTGGTGAACTCTGTG GCACAGGGACAGAGACCAAACT CACCAGGGCCAGCACGTCAGAATC

GCGGCACTGACACTCGTAGC CTAACGACACCCGCAGCCCT CTGCTGCCGCTGTACCTGTCAT AGTGAATGGCGACGGCAGTTCT

AAGAAGGTGGTGAAGCAGGC

TCCACCACCCAGTTGCTGTA

Pressure overload-induced cardiac hypertrophy in vivo The pressure-overload heart hypertrophy model was obtained by subjecting the animals to transverse aortic constriction (TAC) as described in our previous works (Dong et al., 2010; Sun et al., 2013a). Adult Kunming male mice (22–26 g body weight) were anesthetized and performed endotracheal intubation. The chest was opened and the thoracic aorta was identified. A 7-0 silk suture was placed around the transverse aorta and tied around a 26-gauge blunt needle which was subsequently removed. The chest was closed and the animals were kept ventilated until recovery of autonomic breath. After 4 weeks, surviving animals were sacrificed and the heart was quickly excised and weighed in cold (4°C) buffer. The left ventricle and right ventricle were separated and weighted, and left ventricle tissue was then rapidly frozen in liquid nitrogen and stored at 80°C for subsequent Western blot or real-time PCR analysis. All procedures involving animals and their care were approved by the Institutional Animal Care and Use Committee of Harbin Medical University, P.R. China. Swimming exercise-induced physiological cardiac hypertrophy

dissolved in PBS solution and diluted in the culture media as the ratio 1.25:5,000. Protein/DNA ratio analysis Protein/DNA ratio analysis was performed as described in our previous work (Sun et al., 2013a). In brief, cells were rinsed in cold PBS twice and scraped with 100 ml of lysis buffer. The collected cells were immediately frozen and stored at 20°C. Then samples were thawed and vortexed, 1 ml sample was applied to determine the total protein content using BCA method. DNA concentrations were detected by using a DNA Quantitation Kit (Sigma-Aldrich, Saint Louis, MO). Real-time PCR analysis Detailed information was described in our previous work (Sun et al., 2013a). Transcript quantities were compared by using the relative quantitative method, where the amount of detected mRNA normalized to the amount of endogenous control (GAPDH). The relative value to the control sample is given by 2DDCT.

Swimming exercise was carried out as described in our previous work (Sun et al., 2013a). In brief, the swimming-trained mice were subjected to a 90-min swimming exercise session, twice a day, 5 days a week for 4 weeks. Water temperature was controlled at 30–32°C.

Western blot

Isolation and culture of cardiomyocyte

MTT assay

Cardiomyocyte cultures were prepared by dissociation of 1- to 3day-old neonatal rat (Wistar) hearts and were differentially plated to remove fibroblasts as described in our previous work (Dong et al., 2010). The purity of cardiomyocytes was increased by supplementing BrdU (5-Bromo-20 -deoxyuridine) to prevent noncardiomyocytes from developing. Culture medium was renewed after 48 h and cells were further cultured for 24 h. The cardiomyocytes were cultured in non-serum DMEM for 12 h before experiments. Cardiomyocytes were prepared for immunocytochemistry. Monoclonal antibody against sarcomeric a-actinin (Sigma) was added at dilutions of 1:200. Nuclear staining was performed with 1.3 mmol/L of bisbenzimide (Sigma). The cardiomyocyte surface was depicted by using Image-Pro Plus Version (5.0.1) software and the relative surface area was read with the arbitrary units (the number of pixels) for evaluating hypertrophy.

Viability of cells cultured in the 96-well culture plates was assessed by measuring mitochondrial dehydrogenase activity, using the colorimetric MTT assay (Sun et al., 2013a).

Drug treatment BMP4 (50 ng/ml), BMP2 (50 ng/ml), and noggin (100 ng/ml) were applied for 48 h or the time as indicated in the figures or figure legends. The culture media containing different drugs was renewed every 24 h. In the experiments, BMP4, BMP2, and noggin were JOURNAL OF CELLULAR PHYSIOLOGY

Detailed information was described in our previous works (Dong et al., 2010; Sun et al., 2013a). Western blot bands were quantified by using Odyssey infrared imaging system (LI-COR) and Odyssey v3.0 software.

TUNEL staining After three times PBS washing, treated myocytes were fixed by 4% paraformaldehyde, permeabilized in 0.1% Triton X-100 sodium citrate buffer. Then in situ cell death detection kits (Roche, Shanghai, China) were used to label apoptotic cells, and the nuclei were stained with DAPI. The numbers of total cells and TUNEL positive cells were automatically counted by Image-Pro plus version. The apoptosis rate was defined as ratio of apoptotic cells to total cells. Live- and dead-cell staining The LIVE/DEAD1 Viability/Cytotoxicity Assay Kit (Invitrogen, Beijing, China) was used to detect live and dead cells as described in our previous work (Li et al., 2012). Briefly, cells were grown on coverslips at a density of 3.75  104/ml and incubated overnight at 37°C in a humidified 5% CO2 incubator. The cells were washed with PBS and dyed according to the manufacturer’s instructions. The

BMP2 ANTAGONIZES BMP4

labeled cells were photographed under a fluorescence microscope. The live cells fluoresce green and dead cells fluoresce red.

Results BMP2 expression increases in pressure-overload and swimming exercise induced cardiac hypertrophy

Caspase-3 activity assay

Pressure-overload by transverse aorta constriction (TAC) for 4 weeks induced significant cardiac hypertrophy (Sun et al., 2013a). In this pathological cardiac hypertrophy model (Sun et al., 2013a), BMP2, 4 but not BMP1, 3 mRNA expression significantly increased (Fig. 1A). Western blot results showed that both precursor and mature forms of BMP2 protein also increased in this model (Fig. 1B). We further examined the expression of BMP2 in swimming exercise induced physiological cardiac hypertrophy model established previously by our group (Sun et al., 2013a). As shown in Figure 1C, BMP2 protein mature form significantly increased in physiological cardiac hypertrophy model. It was very intriguingly that both the precursor and mature forms of BMP2 were up-regulated in TAC model but only the mature form of BMP2 was upregulated in swimming model. BMPs are generally synthesized

Caspase-3 activity was determined by using Caspase-3 Activity Assay Kit (Beyotime Institute of Biotechnology, China) as described in our previous work (Sun et al., 2013a). Briefly, the cells were harvested and washed with cool PBS twice, then the cells were lysed with lysis buffer (100 ml per 2  106 cells) for 15 min on ice. The lysate was centrifuged at 13,500 rpm for 15 min at 4°C, then the supernatant was collected and protein concentration was determined by Bradford Protein Assay Kit (Beyotime Institute of Biotechnology, China). After incubating the mixture composed of 40 ml of cell lysate, 50 ml reaction buffer and 10 ml of 2 mM caspase3 substrate (Ac-DEVD-pNA) in 96-well plates at 37°C overnight, the absorbance of p-nitroanilide at 405 nm was determined by using a microtiter plate reader (Bio-TEK Epoch, BioTek Instrument, Winooski, VT). Caspase-3 activity was calculated as the ratio of p-nitroanilide content to total protein amount. The detail analysis procedure was described in the manufacturer’s protocol (Beyotime Institute of Biotechnology, China). Data analysis Data were presented as mean  SEM. Significance was determined by using Student t-test or one-way ANOVA, followed by Tukey post test. P < 0.05 was considered significant.

Fig. 1. Bone morphogenetic protein-2 (BMP2) expression increases in both pressure-overload and swimming exercise induced cardiac hypertrophy. A: BMP 2, 4 but not BMP1, 3 mRNA expression increased in pressure-overload induced pathological cardiac hypertrophy. Five hearts in each group (sham and TAC groups) were measured. **P < 0.01 versus sham. TAC, transverse aortic constriction. B: Both precursor and mature forms of BMP2 increased in protein level in pressure-overload induced pathological cardiac hypertrophy. Five hearts in each group (sham and TAC groups) were measured. **P < 0.01 versus sham. TAC, transverse aortic constriction. C: BMP2 expression increased in protein level in swimming exercise induced physiological cardiac hypertrophy. Five hearts in each group (control and swim groups) were measured. **P < 0.01 versus control. w, weeks.

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Fig. 2. Bone morphogenetic protein-2 (BMP2) antagonizes bone morphogenetic protein-4 (BMP4)-induced cardiomyocyte hypertrophy. A–C: BMP2 at 50 ng/ml did not induce significant cardiomyocyte hypertrophy, as demonstrated by the cell area, ANP, b-MHC expression and protein/DNA ratio, except that BMP2 treatment increased BNP mRNA expression. n ¼ 8 for ANP, BNP, and b-MHC analysis, n ¼ 5 for protein/DNA ratio analysis. The concentration of noggin was 100 ng/ml. **P < 0.01 versus control; # P < 0.05 versus BMP2. CTL, control. D–F: BMP2 (50 ng/ml) antagonized BMP4 (50 ng/ml) induced cardiomyocyte hypertrophy as demonstrated by the inhibited cell area, b-MHC expression and protein/DNA ratio. **P < 0.01 versus control; ##P < 0.01 versus BMP4. CTL, control.

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as inactive precursors which require intracellular proteolysis to become the activated mature forms. This difference might be due to the different proteolysis process and/or different regulation of gene expression in the TAC and swimming models. The above data suggested that BMP2 expression increased in both physiological and pathological cardiac hypertrophy models.

further examined the effects of BMP2 on BMP4-induced cardiomyocyte hypertrophy. As shown in Figure 2D–F, BMP2 (50 ng/ml) antagonized BMP4 (50 ng/ml)-induced cardiomyocyte hypertrophy as demonstrated by the inhibited cell area, b-MHC expression and protein/DNA ratio.

BMP2 antagonizes BMP4-induced cardiomyocyte hypertrophy

Our previous work showed that BMP4 could induce cardiomyocyte apoptosis (Sun et al., 2013a). The present results showed that BMP2 had no effect on cardiomyocyte apoptosis (Fig. 3A–C), but antagonized BMP4-induced cardiomyocyte apoptosis which was evaluated by TUNEL staining, caspase-3 activity and cell viability (Fig. 3A–C). We further examined the effects of BMP2 at 200 and 500 ng/ml concentrations on the cardiomyocyte viability. Results showed that BMP2 at 200 and 500 ng/ml concentrations had no significant effect on the cardiomyocyte viability (data not shown), indicating that the effect of BMP2 was distinct from that of BMP4 which reduced the cardiomyocyte viability even at 50 ng/ml concentration.

Since BMP2 expression increased in both physiological and pathological cardiac hypertrophy models, we examined whether BMP2 induced cardiomyocyte hypertrophy. BMP2 at 50 ng/ml did not induce cardiomyocyte hypertrophy, as demonstrated by the cell area, ANP, b-MHC expression and protein/DNA ratio, except that BMP2 treatment increased BNP mRNA expression which was inhibited by BMP inhibitor noggin (Fig. 2A–C). Our previous work showed that BMP4 could induce cardiomyocyte hypertrophy (Sun et al., 2013a). Here, we

BMP2 antagonizes BMP4-induced cardiomyocyte apoptosis

Fig. 3. Bone morphogenetic protein-2 (BMP2) antagonizes bone morphogenetic protein-4 (BMP4)-induced cardiomyocyte apoptosis as evaluated by TUNEL staining (A), caspase-3 activity (B) and cell viability assays (C). About 1,000 cells from two individual experiments were analyzed in each group for TUNEL staining in (A). **P < 0.01 vs control; ##P < 0.01 versus BMP4.

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BMP2 ANTAGONIZES BMP4

BMP2 activates Akt and inhibits BMP4-induced JNK activation in cardiomyocytes

BMP2 and BMP4 process the similar canonical Smad signal pathway, indicating that the antagonism of BMP2 on BMP4 might not be from the canonical Smad pathway. Therefore, we focused on the non-Smad signal pathways, Akt and JNK signalings. Akt plays an important role in regulating cardiomyocyte apoptosis. Adenoviral gene transfer of activated Akt1 protects cardiomyocytes from apoptosis in response to hypoxia in vitro (Matsui et al., 1999). Moreover, adenovirusmediated Akt1 gene transfer in the heart diminishes cardiomyocyte apoptosis and limits infarct size following ischaemia/reperfusion injury (Matsui et al., 2001). BMP2 was reported to activate Akt in chondrocytic cells, chondrosarcoma cells, and gastric cancer cells (Sugimori et al., 2005; Fong et al., 2008; Kang et al., 2010), therefore, firstly, we examined the effect of BMP2 on Akt activation in cardiomyocytes. BMP2 treatment for 30 min did not induce Akt activation (Fig. 4A), but after 12 h treatment, the phosphorylated Akt was increased and the increase was inhibited by co-treatment with noggin (Fig. 4B). Meanwhile, no effect of BMP4 on Akt activation after 12 and 48 h treatments was observed (Fig. 4C). The above data showed that BMP2 was up-regulated in heart hypertrophy and BMP2 activated Akt in cardiomyocytes, which was in agreement with the previous studies showing that Akt

was activated in in vivo pressure-overlaod induced cardiac hypertrophy models (Xie et al., 2004; Sbroggiò et al., 2011). Next, we examined whether the Akt activation contributed to the protection of BMP2 against BMP4-induced cardiomyocyte apoptosis. Firstly, the inhibitory effect of Akt1/2 kinase inhibitor (Akti,3-Dihydro-1-(1-((4-(6-phenyl-1Himidazo[4,5-g]quinoxalin-7-yl) phenyl) methyl)-4-piperidinyl)2H-benzimidazol-2-one trifluoroacetate salt hydrate) on BMP2-induced Akt activation was confirmed in cardiomyocytes (Fig. 5A). Akt inhibitor prevented BMP2induced protection against BMP4-induced decrease of cell viability measured by MTT assay (Fig. 5B). Furthermore, the effects of Akt inhibitor on BMP2-induced protection against BMP4-induced cardiomyocyte apoptosis were evaluated by the LIVE/DEAD1 viability, TUNEL staining, and caspase-3 activity measurements. As shown in Figure 5C–E, Akt inhibitor inhibited BMP2-induced protection against BMP4-induced cardiomyocyte apoptosis, indicating that BMP2-induced Akt activation contributed to the protection of BMP2 against BMP4-induced cardiomyocyte apoptosis. Akti is a specific Akt inhibitor which not only inhibits the activated Akt, but also inhibits the basal Akt activity. Therefore, we further examined the effect of Akti alone on cell viability. Results showed that Akti alone did not induce significant cardiomyocyte death, but it did reduce cell viability (Fig. 6A,B), indicating that akti alone inhibited cardiomyocyte growth. The present results were consistent to the fact that Akt plays important role in regulating cardiomyocyte proliferation and growth (Evans-Anderson et al., 2008). It was reported that BMP4 induced cardiomyocyte apoptosis through a JNK-dependent signaling pathway (Pachori et al., 2010). Our previous work also showed that BMP4 activated JNK in cardiomyocytes (Sun et al., 2013a). So we examined the effects of BMP2 on basal JNK and BMP4-induced JNK activation in cardiomyocytes. As shown in Figure 7A,B, BMP2 showed no effects on the basal JNK activity but inhibited BMP4-induced JNK activation. Finally, we examined the effects of BMP2 and BMP4 on the expressions of BMP2 and BMP4 themselves in cardiomyocytes. BMP4 treatment up-regulated BMP4, but did not affect BMP2 protein expression in cardiomyocytes (Fig. 7C), as evidenced in our previous work (Sun et al., 2013a). BMP2 treatment did not affect both BMP2 and BMP4 protein expressions in cardiomyocytes (Fig. 7C). Discussion

Fig. 4. Bone morphogenetic protein-2 (BMP2) but not bone morphogenetic protein-4 (BMP4) induces Akt activation in cardiomyocytes. A,B: BMP2 (50 ng/ml) increased p-Akt expression after 12 h but not 30 min treatment. **P < 0.01 versus control; # P < 0.05 versus BMP2. C: BMP4 (50 ng/ml) showed no effect on Akt activation after 12 and 48 h treatments.

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Although both BMP2 and BMP4 are critical for functional heart formation during embryogenesis and after birth, they exert opposing effects in some pathological conditions, such as pulmonary hypertension (Frank et al., 2005; Anderson et al., 2010). Our previous work showed that BMP4 expression was up-regulated in pathological cardiac hypertrophy models and BMP4 induced cardiomyocyte hypertrophy, apoptosis and Kv4.3 Kþ channel remodeling (Sun et al., 2013a,b). In the present study, we found that BMP2 antagonized BMP4-induced cardiomyocyte hypertrophy and apoptosis. Several lines of evidences show that BMP2 and BMP4 exert opposing effects in cardiovascular diseases, for example, BMP2 protects against but BMP4 promotes hypoxic pulmonary hypertension (Frank et al., 2005; Anderson et al., 2010). BMP2 and BMP4 also show opposing effects in cardiomyocytes. For example, BMP2 inhibits serum deprivation-induced apoptosis of neonatal cardiomyocytes (Izumi et al., 2001), limits infarct size after myocardial infarction and improves the contractility of cardiomyocytes in mice (Ebelt et al., 2013), while BMP4 mediates myocardial ischemic injury and induces cardiomyocyte apoptosis (Pachori et al., 2010). These results indicate that BMP2 is protective and BMP4 is deleterious in certain conditions. We proposed the mechanisms of roles of BMP2

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Fig. 5. Akt inhibitor (Akti) prevents BMP2-induced protection against BMP4-induced cell apoptosis. A: Akti inhibited BMP2-induced Akt activation. **P < 0.01 versus control; ##P < 0.01 versus BMP2. B: Akti prevented BMP2-induced protection against BMP4-induced decrease of cell viability. *P < 0.05 versus control; ##P < 0.01 versus BMP4; &&P < 0.01 versus BMP4 þ 2. C: Akti prevented BMP2-induced protection against BMP4-induced cell death. **P < 0.01 versus control; ##P < 0.01 versus BMP4; &&P < 0.01 versus BMP4 þ 2. n ¼ 10 section from different vision in each group. D: Quantification of the apoptotic cells by TUNEL staining. About 1,000 cells from two individual experiments were analyzed in each group. **P < 0.01 versus control; ##P < 0.01 versus BMP4; &&P < 0.01 versus BMP4 þ 2. E: Akti prevented BMP2-induced protection against BMP4-induced increase of caspase-3 activity. *P < 0.05 versus control; #P < 0.05 versus BMP4; &P < 0.05 versus BMP4 þ 2. Akti, Akt inhibitor (1,3-Dihydro-1-(1-((4-(6-phenyl-1H-imidazo[4,5-g]quinoxalin-7-yl) phenyl) methyl)-4-piperidinyl)-2H-benzimidazol-2-one trifluoroacetate salt hydrate). The concentrations of Akti, BMP2, and BMP4 were 0.5 mM, 50 ng/ml, and 50 ng/ml respectively.

and BMP4 in cardiomyocyte apoptosis in pathological cardiac hypertrophy in Figure 8: pathological stimuli increase both BMP2 and BMP4 expressions, BMP2 protects against cardiomyocyte apoptosis through activating Akt and inhibiting JNK, and BMP4 induced cardiomyocyte apoptosis through activating JNK. It was noteworthy that BMP2 antagonized BMP4-induced effects in vitro, but the in vivo heart hypertrophy model induced by pressure-overload still showed pathological phenotype. We speculated that the difference between the in vivo and in vitro data might be due to the relative quantities of BMP4 and BMP2 generated in cardiac tissues. We used the JOURNAL OF CELLULAR PHYSIOLOGY

same dose of BMP4 and BMP2 in in vitro experiments, but it was possible that the production of BMP2 was less than that of BMP4 in in vivo heart hypertrophy model since BMP4 induced own expression in a positive feedback manner, and the quantity of BMP2 was not enough to antagonize BMP4 effects in vivo. We could not accurately measure the protein quantities of BMP4 and BMP2 in cardiac tissues, so we did not provide the direct evidence to explain the difference of in vivo and in vitro results, which was a limitation of the present work. BMP2 and BMP4 diverge from a common ancestral gene, share >80% amino acid homology and similar signal

BMP2 ANTAGONIZES BMP4

Fig. 6. Akti alone inhibits cardiomyocyte viability. A: Akti (0.5 mM) alone reduced cardiomyocyte viability measured by MTT method. **P < 0.01 versus Control. n ¼ 3 individual experiments. B: Akti (0.5 mM) alone did not induce significant cell death evaluated by LIVE/DEAD1 viability assay. Akti, Akt inhibitor (1,3-Dihydro-1-(1((4-(6-phenyl-1H-imidazo[4,5-g]quinoxalin-7-yl) phenyl) methyl)-4piperidinyl)-2H-benzimidazol-2-one trifluoroacetate salt hydrate).

transduction pathways. The canonical BMPs signal transduction pathway has been summarized: upon binding by BMPs, BMP receptor type II phosphorylates type I receptors, and this complex, in turn, phosphorylates the receptor Smads 1/5/8. These form heteromeric complexes with Smad4, and this complex translocates to the nucleus and alters the expression of target genes (Zhang and Li, 2005; Anderson and Darshan, 2008). Since BMP2 and BMP4 process the similar canonical Smad signal pathway, so we focused on the non-Smad signal pathways, Akt and JNK, to investigate the mechanism of opposing effects of BMP2 and BMP4 on cardiomyocyte apoptosis. Akt activation initiates physiological heart hypertrophy and inhibits cardiomyocytes apoptosis in response to injury (Maillet et al., 2013). JNK activation induces cardiomyocytes apoptosis (Honsho et al., 2009; Chaanine et al., 2012). A recent work showed that both JNK and Akt activities increased with pressure overload, but JNK signaling dominated over Akt signaling for inducing mitochondrial apoptosis (Chaanine et al., 2012). These findings are highly in agreement with our present results that both BMP2 and BMP4 are up-regulated in pressure-overload cardiac hypertrophy, and BMP2 activates Akt and BMP4 activates JNK. In fact, BMPs signals are so complicated that the signal itself could exert opposite effects. de Pater et al. (2012) found that during cardiomyocyte differentiation, BMP signaling activity was required in the cardiac progenitor cells to induce cardiac JOURNAL OF CELLULAR PHYSIOLOGY

Fig. 7. Bone morphogenetic protein-2 (BMP 2) inhibits bone morphogenetic protein-4 (BMP4) induced JNK activation. A: BMP2 had no effect on JNK activation. B: BMP2 inhibited BMP4-induced JNK activation. C: BMP2 had no effect on BMP2 and BMP4 protein expression.

differentiation but dispensable and even deleterious once differentiation was initiated. Although our present results indicated that Akt activation and JNK inhibition might be involved in BMP2 protection against BMP4-induced cardiomyocyte apoptosis, we could not exclude the possibility that there were other signal pathways involved, such as BMP4induced CaMKII oxidation and BMP2-induced Smad1 activation (Izumi et al., 2001; Sun et al., 2013a). The interaction between Akt and JNK in cell survival and apoptosis has been well studied. Sunayama et al. (2005)

Fig. 8. Proposed mechanism underlying the roles of bone morphogenetic protein-2 (BMP2) and bone morphogenetic protein4 (BMP4) in cardiomyocyte apoptosis in pathological cardiac hypertrophy. (þ) indicated stimulation; () indicated inhibition. BMP4 pathway was presented in blue and BMP2 in black.

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reported that JNK antagonized Akt-mediated survival signals by phosphorylating 14-3-3. On the other hand, Akt exerts its anti-apoptotic effects through several downstream targets, including the pro-apoptotic Bc1–2 family member Bad, Forkhead transcription factors, and the cyclic AMP response element-binding protein (CREB). In terms of Bax, it was reported that Akt regulated cell survival and apoptosis by inhibiting Bax conformational change (Yamaguchi and Wang, 2001) and inhibited Bax translocation from cytoplasm to mitochondria (Tsuruta et al., 2002). In consideration of so many downstream targets of Akt in regulating cell apoptosis, we did not further investigate the downstream signals of Akt. Akt activation protects cardiomyocytes from apoptosis (Matsui et al., 1999, 2001; Dong et al., 2013), however, longterm Akt activation causes pathological hypertrophy and heart failure (Kim et al., 2003), indicating that the level of Akt activation determines the consequences, namely, moderate Akt activation is beneficial and excessive Akt activation is detrimental. We found that BMP2 moderately stimulated Akt activation, but did not induce cardiomyocyte hypertrophy (Fig. 2A–C), indicating that BMP2-induced Akt activation is beneficial. Izumi et al. (2001) previously had evaluated the effects of BMP2 on cardiomyocyte hypertrophy by measuring cell area, junB and BNP mRNA expression, and [3H] leucine incorporation in cultured rat neonatal cardiomyoctes. They found that, although BMP2 enhanced BNP mRNA expression, but did not induce significant cardiomyocyte hypertrophy. Their results are consistent with our present data. In the present work, we used Akt inhibitor to prevent BMP2induced protection against BMP4. Akt inhibitor also inhibited the basal Akt activity, which explained the phenomena that Akt inhibitor treatment strongly inhibited BMP2-induced protection against BMP4-induced decrease of cell viability (Fig. 5B). Whatever physiological and pathological cardiac hypertrophies, increases of cell size and protein synthesis are the common consequences. BMP2 expression increased in swimming exercise-induced physiological cardiac hypertrophy, but in vitro experiments did not evidence that BMP2 could induced cardiomyocyte hypertrophy (Fig. 2A–C), though BMP2 could increase phosphorylated Akt in cardiomyocytes (Fig. 4B). We did not clarify the mechanism that BMP2 antagonized BMP4-induced cardiomyocyte hypertrophy were through activating Akt, inhibiting JNK or other pathways, which was the limitation of the present study. In conclusion, both BMP2 and BMP4 are up-regulated in pathological cardiac hypertrophy, but BMP2 antagonizes BMP4-induced cardiomyocyte hypertrophy and apoptosis. BMP2 can be regarded as an endogenous BMP4 inhibitor and up-regulation of BMP2 would be an alternative strategy to prevent pathological cardiac hypertrophy. Literature Cited Anderson GJ, Darshan D. 2008. Small-molecule dissection of BMP signaling. Nat Chem Biol 4:15–16. Anderson L, Lowery JW, Frank DB, Novitskaya T, Jones M, Mortlock DP, Chandler RL, de Caestecker MP. 2010. Bmp2 and Bmp4 exert opposing effects in hypoxic pulmonary hypertension. Am J Physiol Regul Integr Comp Physiol 298:R833–R842. Cai J, Pardali E, Sanchez-Duffhues G, ten Dijke P. 2012. BMP signaling in vascular diseases. FEBS Lett 586:1993–2002. Chaanine AH, Jeong D, Liang L, Chemaly ER, Fish K, Gordon RE, Hajjar RJ. 2012. JNK modulates FOXO3a for the expression of the mitochondrial death and mitophagy marker BNIP3 in pathological hypertrophy and in heart failure. Cell Death Dis 3:265. de Pater E, Ciampricotti M, Priller F, Veerkamp J, Strate I, Smith K, Lagendijk AK, Schilling TF, Herzog W, Abdelilah-Seyfried S, Hammerschmidt M, Bakkers J. 2012. Bmp signaling exerts opposite effects on cardiac differentiation. Circ Res 110:578–587. Dong DL, Chen C, Huo R, Wang N, Li Z, Tu YJ, Hu JT, Chu X, Huang W, Yang BF. 2010. Reciprocal repression between microRNA-133 and calcineurin regulates cardiac

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Bone morphogenetic protein-2 antagonizes bone morphogenetic protein-4 induced cardiomyocyte hypertrophy and apoptosis.

Our previous work showed that the expression of bone morphogenetic protein-4 (BMP4) was up-regulated in pathological cardiac hypertrophy models and BM...
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