Novel Therapeutic Effects of Pterosin B on Ang II-Induced Cardiomyocyte Hypertrophy

Pathological cardiac hypertrophy is characterized by an abnormal increase in cardiac muscle mass in the left ventricle, resulting in cardiac dysfunction. Although various therapeutic approaches are being continuously developed for heart failure, several studies have suggested natural compounds as novel potential strategies. Considering relevant compounds, we investigated a new role for Pterosin B for which the potential life-affecting biological and therapeutic effects on cardiomyocyte hypertrophy are not fully known. Thus, we investigated whether Pterosin B can regulate cardiomyocyte hypertrophy induced by angiotensin II (Ang II) using H9c2 cells. The antihypertrophic effect of Pterosin B was evaluated, and the results showed that it reduced hypertrophy-related gene expression, cell size, and protein synthesis. In addition, upon Ang II stimulation, Pterosin B attenuated the activation and expression of major receptors, Ang II type 1 receptor and a receptor for advanced glycation end products, by inhibiting the phosphorylation of PKC-ERK-NF-κB pathway signaling molecules. In addition, Pterosin B showed the ability to reduce excessive intracellular reactive oxygen species, critical mediators for cardiac hypertrophy upon Ang II exposure, by regulating the expression levels of NAD(P)H oxidase 2/4. Our results demonstrate the protective role of Pterosin B in cardiomyocyte hypertrophy, suggesting it is a potential therapeutic candidate.


Introduction
Pathological cardiac hypertrophy is characterized by the thickening of cardiac muscle, which leads to irreversible morphological and functional changes and is mainly observed in patients with end-stage heart disease. The pathological hypertrophic heart is closely associated with the development of heart failure, arrhythmias, cerebrovascular disease, sudden death, etc [1]. Pressure overload, such as hypertension, is one of the major causes of pathological cardiac hypertrophy, in which angiotensin II (Ang II) has been reported to play a pivotal role [2,3]. Ang II, a vasoconstrictor peptide of the renin-angiotensin system (RAS), plays an important role in the regulation of blood

Anti-Hypertrophic Effect of Pterosin B on Ang II-Treated Cardiomyocytes
Ang II is known as one of the major factors inducing cardiac hypertrophy [6]. Thus, we induced cardiomyocyte hypertrophy by treatment with 300 nM Ang II using H9c2 cells, an embryonic rat heart-derived cell line [11]. As Pterosin B has rarely been studied in hypertrophic cells, we wondered whether it has an effect on cardiomyocyte hypertrophy. First, we measured the viability and cytotoxicity of cell induced by Ang II with or without Pterosin B (10-100 µM) treatment. The results showed that a 300 nM Ang II treatment did not affect cell viability ( Figure 1A) or cytotoxicity ( Figure 1B). Pterosin B at concentrations as high as 50 µM had no effects on Ang II-treated cells, but cell viability was decreased by approximately 30% in 100 µM Pterosin B-treated cells. However, no significant differences in induced cytotoxicity were detected with concentrations as high as 100 µM. Considering these results, we treated cells with concentrations of Pterosin B up to 50 µM to investigate its anti-hypertrophic effect. We then analyzed hypertrophy-related genes, namely, ANP, BNP, and NFATc1, in Ang II-treated cells treated with or without Pterosin B (Figure 2A). The mRNA expression levels of ANP and BNP increased approximately 1.5-fold upon 300 nM Ang II stimulation, and these increases were attenuated by Pterosin B treatment in a dose-dependent manner ( Figure 2B,C). The expression of NFATc1 was increased approximately 2.5-fold, and it was also decreased by Pterosin B treatment ( Figure 2D). Although Pterosin B showed a tendency to decrease all three hypertrophy-related genes, 50 µM Pterosin B administered after Ang II treatment had a particularly strong effect, significantly reducing all three genes. Therefore, we used 50 µM Pterosin B for further experiments to confirm its effects on cardiomyocyte hypertrophy. As the most representative characteristic of myocardial hypertrophy, the size of myocardial cells was measured through immunofluorescence staining ( Figure 2E). The results showed that the cell surface area of the cardiomyocytes was increased by approximately 250% by Ang II, and the surface area of the cells in the Pterosin B-treated group was maintained at the level of the control cells ( Figure 2F). Additionally, there was no significant change in cell surface area in the group treated with Pterosin B alone. Then, we analyzed the synthesis of new intracellular proteins, which is one of the characteristics of cardiomyocyte hypertrophy ( Figure 2G). As a result of visualizing the newly synthesized protein through fluorescence staining, we confirmed that the synthesis of new protein was inhibited in the Pterosin B treatment group. Considering these results, we suggest that Pterosin B can affect Ang II-induced cardiomyocyte hypertrophy. characteristic of myocardial hypertrophy, the size of myocardial cells was measured through immunofluorescence staining ( Figure 2E). The results showed that the cell surface area of the cardiomyocytes was increased by approximately 250% by Ang II, and the surface area of the cells in the Pterosin B-treated group was maintained at the level of the control cells ( Figure 2F). Additionally, there was no significant change in cell surface area in the group treated with Pterosin B alone. Then, we analyzed the synthesis of new intracellular proteins, which is one of the characteristics of cardiomyocyte hypertrophy ( Figure 2G). As a result of visualizing the newly synthesized protein through fluorescence staining, we confirmed that the synthesis of new protein was inhibited in the Pterosin B treatment group. Considering these results, we suggest that Pterosin B can affect Ang IIinduced cardiomyocyte hypertrophy.

Pterosin B Attenuates AT1R-Mediated HMGB1 Secretion in Ang II-Induced Hypertrophic Cardiomyocytes
It has been reported that AT1R-mediated signaling pathways are mainly involved in Ang II-induced hypertrophic effects in the heart [27,28]. In addition, we previously demonstrated the importance of the AT1R-HMGB1-RAGE axis in Ang II-induced cardiomyocyte hypertrophy [11]. Therefore, we examined the protein expression levels of AT1R and RAGE in Ang II-treated cardiomyocytes by immunoblot analysis ( Figure 3A). The protein expression levels of AT1R and RAGE in the Ang II-treated cells were increased approximately 1.5-fold compared to those of the control, showing that Pterosin B significantly attenuated these receptors ( Figure 3B,C). Next, we determined the protein level of intracellular HMGB1 in Ang II-treated cell lysates and the amount of secreted HMGB1 in Ang II-treated culture medium ( Figure 3D). The protein levels of HMGB1 were increased in the Ang II-treated cell lysates and culture media. The expression and secretion of HMGB1 were decreased in the Pterosin B-treated cells compared with the Ang II-treated cells ( Figure 3E,F). In parallel, we investigated the protein levels of TLR2 and TLR4, which are activated by HMGB1 [17], in Ang II-induced hypertrophic cardiomyocytes. The results showed that the expression levels of TLR2 and TLR4 were increased approximately 1.5-fold in the Ang II-treated cells ( Figure 3F), and the levels of TLR2 and TLR4 were decreased in the Pterosin B-treated cells. In particular, both TLR2 and TLR4 showed a tendency to respond to Ang II and Pterosin B, but only the effect on TLR2 was significant ( Figure 3G,H). In addition, we examined the protein expression of MyD88, which is a as a downstream adaptor protein critical for the TLR signaling pathway [29], and the results showed that MyD88 expression was also increased by Ang II treatment and was subsequently reduced by Pterosin B treatment ( Figure 3F,I). These results suggested that Pterosin B may regulate the AT1R-HMGB1-RAGE axis as well as the TLR2-MyD88 pathway in Ang II-treated cardiomyocytes.

Pterosin B Attenuates Ang II-Mediated Signaling Pathways
Next, we investigated the effect of Pterosin B on the AT1R downstream signaling pathway in Ang II-induced cardiomyocyte hypertrophy. The results showed that Ang II increased the PKC phosphorylation by approximately 1.7-fold compared to the level of the control group. The level of phospho-PKC was decreased and maintained at the control level in Pterosin B-treated cells. In addition, the increased levels of phosphorylated ERK in the Ang II-treated cells were decreased in the Pterosin B-treated cells ( Figure 4A,B). We further investigated the phosphorylation of NF-κB, a

Pterosin B Attenuates Ang II-Mediated Signaling Pathways
Next, we investigated the effect of Pterosin B on the AT1R downstream signaling pathway in Ang II-induced cardiomyocyte hypertrophy. The results showed that Ang II increased the PKC phosphorylation by approximately 1.7-fold compared to the level of the control group. The level of phospho-PKC was decreased and maintained at the control level in Pterosin B-treated cells. In addition, the increased levels of phosphorylated ERK in the Ang II-treated cells were decreased in the Pterosin B-treated cells ( Figure 4A,B). We further investigated the phosphorylation of NF-κB, a major transcription factor in myocardial hypertrophy. The phosphorylation level of NF-κB was increased approximately 1.5-fold in the Ang II-treated cardiomyocytes, whereas Pterosin B reduced the phosphorylation level of NF-κB to a level similar to that of the control group ( Figure 4C,D). These results suggested that Pterosin B can modulate myocardial hypertrophy by regulating the PKC-ERK-NF-κB signaling pathway in Ang II-stimulated hypertrophic cardiomyocytes.
Molecules 2019, 24, x FOR PEER REVIEW 7 of 15 major transcription factor in myocardial hypertrophy. The phosphorylation level of NF-κB was increased approximately 1.5-fold in the Ang II-treated cardiomyocytes, whereas Pterosin B reduced the phosphorylation level of NF-κB to a level similar to that of the control group ( Figure 4C,D). These results suggested that Pterosin B can modulate myocardial hypertrophy by regulating the PKC-ERK-NF-κB signaling pathway in Ang II-stimulated hypertrophic cardiomyocytes.

Pterosin B Attenuates Ang II-Induced Intracellular ROS
In the cardiovascular system, the AT1R-NOX axis generates cytosolic ROS upon Ang II stimulation, and these ROS activate various hypertrophic signaling factors, such as MAP kinase and NF-kB [30][31][32]. Therefore, we investigated whether Pterosin B can control cytosolic ROS production in Ang II-stimulated cardiomyocytes. The cells treated with Ang II with or without Pterosin B were incubated with the ROS indicator H2DCFDA, and intracellular ROS levels were assessed through confocal microscopy ( Figure 5A). The cytosolic ROS level of Pterosin B-pretreated cells after Ang II stimulation for 2 h was maintained to a level similar to that of the control group, although the

Pterosin B Attenuates Ang II-Induced Intracellular ROS
In the cardiovascular system, the AT1R-NOX axis generates cytosolic ROS upon Ang II stimulation, and these ROS activate various hypertrophic signaling factors, such as MAP kinase and NF-kB [30][31][32]. Therefore, we investigated whether Pterosin B can control cytosolic ROS production in Ang II-stimulated cardiomyocytes. The cells treated with Ang II with or without Pterosin B were incubated with the ROS indicator H 2 DCFDA, and intracellular ROS levels were assessed through confocal microscopy ( Figure 5A). The cytosolic ROS level of Pterosin B-pretreated cells after Ang II stimulation for 2 h was maintained to a level similar to that of the control group, although the cytosolic ROS level was increased by approximately 15% in the Ang II-treated cardiomyocytes ( Figure 5A,B). In addition, the expression levels of NOX2 and NOX4, which are the major isoforms in cardiomyocytes [33], were evaluated by immunoblotting ( Figure 5C). The expression levels of NOX2 and NOX4 were increased in the Ang II-induced cardiomyocytes by approximately 35% and 30%, respectively ( Figure 5D,E). The expression of NOX2 and NOX4 was significantly decreased in the Pterosin B-treated cells. Based on these results, we suggest that Pterosin B regulates intracellular ROS levels by regulating NOX2 and NOX4 expression. cytosolic ROS level was increased by approximately 15% in the Ang II-treated cardiomyocytes ( Figure 5A,B). In addition, the expression levels of NOX2 and NOX4, which are the major isoforms in cardiomyocytes [33], were evaluated by immunoblotting ( Figure 5C). The expression levels of NOX2 and NOX4 were increased in the Ang II-induced cardiomyocytes by approximately 35% and 30%, respectively ( Figure 5D,E). The expression of NOX2 and NOX4 was significantly decreased in the Pterosin B-treated cells. Based on these results, we suggest that Pterosin B regulates intracellular ROS levels by regulating NOX2 and NOX4 expression.

Discussion
Our data showed that Pterosin B attenuates AT1R-mediated signaling pathways upon Ang IIstimulation of hypertrophic cardiomyocytes, in which Pterosin B inhibits the increase in cytosolic HMGB1 protein levels and secretion into the extracellular environment that leads to the activation of RAGE and its downstream signaling. It has been suggested that Ang II increases the protein levels of RAGE, TLR2, TLR4, HMGB1, and even AT1R itself through NF-κB activation, resulting in a positive feedback loop [34,35]. In these experiments, Pterosin B blocked the positive feedback loop by inhibiting PKC-ERK-NF-κB activation. An in vitro assay suggested that Pterosin B (50-500 μM) may not be able to bind RAGE directly ( Figure S1). We also confirmed that Pterosin B suppresses Ang IIinduced excessive production of ROS, which are critical mediators of signaling pathways that lead to cardiac hypertrophy ( Figure 6). It has been reported that ROS production is mediated through PKC and NOX activation upon Ang II stimulation. Similar to our findings on NOX expression levels, various studies have supported the association of NOX activity and expression levels with ROS

Discussion
Our data showed that Pterosin B attenuates AT1R-mediated signaling pathways upon Ang II-stimulation of hypertrophic cardiomyocytes, in which Pterosin B inhibits the increase in cytosolic HMGB1 protein levels and secretion into the extracellular environment that leads to the activation of RAGE and its downstream signaling. It has been suggested that Ang II increases the protein levels of RAGE, TLR2, TLR4, HMGB1, and even AT1R itself through NF-κB activation, resulting in a positive feedback loop [34,35]. In these experiments, Pterosin B blocked the positive feedback loop by inhibiting PKC-ERK-NF-κB activation. An in vitro assay suggested that Pterosin B (50-500 µM) may not be able to bind RAGE directly ( Figure S1). We also confirmed that Pterosin B suppresses Ang II-induced excessive production of ROS, which are critical mediators of signaling pathways that lead to cardiac hypertrophy ( Figure 6). It has been reported that ROS production is mediated through PKC and NOX activation upon Ang II stimulation. Similar to our findings on NOX expression levels, various studies have supported the association of NOX activity and expression levels with ROS production. Dai et al. emphasized the roles of NOX2 and NOX4 to produce intracellular ROS by Ang II-stimulated AT1R, depending on their intracellular location [36,37]. Zhang et al. demonstrated that NOX 4 expression levels also affect ROS production through the use of silencing and overexpression experiments [38], and using an overexpression in vivo model, Dai et al. also evaluated the regulatory effects of NOX2 expression levels on vascular remodeling and hypertension [39]. However, additional validation such as NADH Oxidase activity is needed to elucidate the ability of Pterosin B against intracellular ROS. In addition, a recent study revealed a new role for ROS, showing that, under hypoxic conditions and ultraviolet radiation, they can induce the secretion of HMGB1 from the nucleus into the extracellular area [40,41].
Molecules 2019, 24, x FOR PEER REVIEW 9 of 15 production. Dai et al. emphasized the roles of NOX2 and NOX4 to produce intracellular ROS by Ang II-stimulated AT1R, depending on their intracellular location [36,37]. Zhang et al. demonstrated that NOX 4 expression levels also affect ROS production through the use of silencing and overexpression experiments [38], and using an overexpression in vivo model, Dai et al. also evaluated the regulatory effects of NOX2 expression levels on vascular remodeling and hypertension [39]. However, additional validation such as NADH Oxidase activity is needed to elucidate the ability of Pterosin B against intracellular ROS. In addition, a recent study revealed a new role for ROS, showing that, under hypoxic conditions and ultraviolet radiation, they can induce the secretion of HMGB1 from the nucleus into the extracellular area [40,41]. Considering the findings from our study, we suggest that extracellular HMGB1, as a downstream signal of AT1R stimulation, is one of the major mediators of RAGE activation. A ubiquitous nuclear protein, HMGB1 acts as a damage-associated molecular pattern (DAMP) upon passive or active secretion from damaged or stressed cells [42]. Various studies have shown the critical roles of extracellular HMGB1 in cardiovascular diseases, including myocarditis and cardiomyopathies [43,44]. Our previous studies also showed that an anti-HMGB1 antibody attenuated RAGE expression and its downstream effector NF-κB activation in Ang II-induced endothelial dysfunction and cardiomyocyte hypertrophy [11,34]. Although the mechanism of extracellular HMGB1 secretion upon Ang II-induced cardiomyocyte hypertrophy is poorly understood, Oh et al. demonstrated that classical PKC, not ERK or NF-κB, can phosphorylate HMGB1, resulting in extracellular HMGB1 secretion from LPS-stimulated Raw264.7 cells, and An et al. showed PKC-induced phosphorylation and secretion of HMGB1 using a bone cancer pain model [45,46]. Regarding HMGB1 secretion in an Ang II-induced environment, Zhou et al. recently provided Considering the findings from our study, we suggest that extracellular HMGB1, as a downstream signal of AT1R stimulation, is one of the major mediators of RAGE activation. A ubiquitous nuclear protein, HMGB1 acts as a damage-associated molecular pattern (DAMP) upon passive or active secretion from damaged or stressed cells [42]. Various studies have shown the critical roles of extracellular HMGB1 in cardiovascular diseases, including myocarditis and cardiomyopathies [43,44]. Our previous studies also showed that an anti-HMGB1 antibody attenuated RAGE expression and its downstream effector NF-κB activation in Ang II-induced endothelial dysfunction and cardiomyocyte hypertrophy [11,34]. Although the mechanism of extracellular HMGB1 secretion upon Ang II-induced cardiomyocyte hypertrophy is poorly understood, Oh et al. demonstrated that classical PKC, not ERK or NF-κB, can phosphorylate HMGB1, resulting in extracellular HMGB1 secretion from LPS-stimulated Raw264.7 cells, and An et al. showed PKC-induced phosphorylation and secretion of HMGB1 using a bone cancer pain model [45,46]. Regarding HMGB1 secretion in an Ang II-induced environment, Zhou et al. recently provided another novel mechanism, in which Ang II can induce HMGB1 release by increasing the hyperacetylation of HMGB1, by separation from a complex of HMGB1 and SIRT1 in Raw264.7 cells [47]. To address the possible mechanisms controlling HMGB1 release in Ang II-induced cardiomyocyte hypertrophy, we need further experiments using a PKC inhibitor or an anti-SIRT1 antibody.
Finally, we further assessed hERG assay to predict the possible use of Pterosin B as a pharmaceutical compound. Pterosin B at 50 µM showed the hERG inhibition by approximately 6.7% ( Figure S2). As a cardiac repolarizing potassium channel, the hERG channel has been reported to be associated with long QT syndrome due to unintended blocking [48]. As diverse chemicals can bind and block the hERG channel, several agents, such as terfenadine and astemizole, have been withdrawn from the market because of the potential occurrence of sudden cardiac death [49,50]. Therefore, further study using the new synthesis of Pterosin B derivatives is necessary to overcome this safety issue.

Reagent
The Pterosin B was purchased from Santa Cruz Biotechnology (catalog number: sc-476730, Dallas, TX, USA) and dissolved in DMSO before use.

Induction of Cardiomyocyte Hypertrophy In Vitro
H9c2 cells (Korean Cell Line Bank, Seoul, Korea) were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplied with 10% Fetal Bovine Serum (FBS; Atlas Biologicals, Fort Collins, CO, USA) and 1% Penicillin-Streptomycin (P/S; Gibo, Thermo Fisher Scientific, Waltham, MA, USA) at 37 • C under humidified conditions (5% CO 2 atmosphere). The cells were cultured for 24 h in DMEM supplied with 0.5% FBS to expose the cells to starvation. To induce hypertrophy, 300 nM Ang II is treated in the cells for 48 h then, the hypertrophy was examined.

Cell Viability/Cytotoxicity Assay
For analyze the cell viability / cytotoxicity, 5 × 10 3 cells per well were seeded in 96-well plates and incubated for 24 h at 37 • C under humidified conditions (5% CO 2 atmosphere). Then, cells were incubated for 24 h in a DMEM containing 0.5% FBS. The cells were treated with Pterosin B at concentrations of 10, 25, and 50 µM for 1 h, and Ang II was then treated for 48 h.

Cell Viability
Then, EZ-Cytox Kit (DoGen, Seoul, Korea) is an assay kit based on water soluble tetrazolium salt (WST) assay. The assay reagent was added to each well at final concentration of 0.5 mg/mL and the cells were incubated for 2 h at 37 • C under humidified conditions (5% CO 2 atmosphere). To check cell viability, the absorbance was measured at 450 nm by a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

Cytotoxicity Assay
The experiment was conducted using Lactate dehydrogenase (LDH) Cytotoxicity Detection Kit (Takara Bio Inc, Kusatsu, Japan), according to a manufacturer's protocol. Briefly, the Pterosin B-treated cells were incubated for 48 h then and 100 µL of cell-culture supernatant was transferred into optically clear 96-well flat bottom microtiter plate. To determine the LDH activity, 100 µL of reaction mixture was added and incubated for 30 min at room temperature. The absorbance at 490 nm was measured using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The absorbance value of assay medium as background control was subtracted from other values of test samples.

Immunocytochemistry and Cardiomyocyte Cell Size Measurement
2 × 10 4 cells per well were seeded in 4-well chamber slide and induced cardiomyocyte hypertrophy by Ang II treatment. 48 h after the incubation, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature, it was then washed twice with PBS and was permeabilized with 0.2% Triton X-100 for 10 min. Next, it was washed with PBS, was blocked with blocking solution (1% bovine serum albumin) for 1 h, and then was stained with Texas Red™-X Phalloidin (Thermo Fisher Scientific, Waltham, MA, USA). Immunofluorescence was detected by confocal microscopy (LSM 700; Carl Zeiss, Wetzlar, Germany). More than 50 cells in 10 randomly selected regions per experiment were measured in six independent experiments. The cell size was measured using ImageJ software, version 1.48v (National Institutes of Health, Bethesda, MD, USA).

Quantification of Protein Synthesis
To measure protein synthesis, we used Click-iT™ HPG Alexa Fluor™ 594 Protein Synthesis Assay Kit (Thermo Fisher, Waltham, MA, USA). Cells were incubated in L-methionine-free DMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) for 30 min prior to the addition of 50 µM methionine analog L-homopropargylglycine (HPG) for 30 min. After fixation and permeabilization, the Click-iT reaction using Alexa-488 was performed according to the manufacture's protocol. Immunofluorescence was detected via confocal microscopy (LSM700; Carl Zeiss, Jena, Germany) Then, immune-fluorescence intensities were measured and analyzed using Zen software (Carl Zeiss, Jena, Germany), in which around 5-10 randomly selected regions per experiment were measured in three independent experiments and fluorescence intensities of each group were normalized by number of 4 ,6-diamidino-2-phenylindole (DAPI)-positive cells.

Measuring Cytosolic ROS Levels
Cytosolic ROS level was measured in 300 nM Ang II with or without treatment of 50 µM Pterosin B treated cells. The cells were plated in 24-well plates. Next day, 50 µM Pterosin B was treated for 1 h followed by 2 h treatment of 300 nM Ang II. Next, the cells were washed with PBS twice, and were resuspended in pre-warmed PBS containing 10 µM CM-H2DCFDA dye (C6827, Thermo Fisher Scientific, Waltham, MA, USA) for ROS detection. The cells were then incubated at 37 • C for 10 min to render the dye responsive to oxidation. Fluorescence images were obtained from 6 independent experiments (10-15 randomly selected regions per each experiment). Fluorescence intensities were measured using CELL IMAGING CELENA ® X high content imaging system (CX 30000, Logos Biosystems, Anyang, Korea).

Statistical Analysis
The data was expressed as the mean ± SEM. The significance of differences between two groups was compared using Student's t-test. Comparisons of more than two groups were performed via one-way ANOVA with Bonferroni correction. P values were indicated in figure legends.

Conclusions
In the present study, we investigated the effects of Pterosin B on Ang II-induced cardiomyocyte hypertrophy, in which Pterosin B attenuated activation of major hypertrophic signaling pathways and also can regulate excessive cytosolic ROS production in the presence of Ang II. However, additional in depth study to elucidate the underlying mechanisms in details and to validate its therapeutic efficacy using in vitro and in vivo models.