Myricitrin Protects Cardiomyocytes from Hypoxia/Reoxygenation Injury: Involvement of Heat Shock Protein 90

Modulation of oxidative stress is therapeutically effective in ischemia/reperfusion (I/R) injury. Myricitrin, a naturally occurring phenolic compound, is a potent antioxidant. However, little is known about its effect on I/R injury to cardiac myocytes. The present study was performed to investigate the potential protective effect of myricitrin against hypoxia/reoxygenation (H/R)-induced H9c2 cardiomyocyte injury and its underlying mechanisms. Myricitrin pretreatment improved cardiomyocyte viability, inhibited ROS generation, maintained the mitochondrial membrane potential, reduced apoptotic cardiomyocytes, decreased the caspase-3 activity, upregulated antiapoptotic proteins and downregulated proapoptotic proteins during H/R injury. Moreover, the potential targets of myricitrin was predicted using Discovery Studio software, and heat shock protein 90 (Hsp90) was identified as the main disease-related target. Further mechanistic investigation revealed that 17-AAG, a pharmacologic inhibitor of Hsp90, significantly blocked the myricitrin-induced cardioprotective effect demonstrated by increased apoptosis and ROS generation. These results suggested that myricitrin provides protection to H9c2 cardiomyocytes against H/R-induced oxidative stress and apoptosis, most likely via increased expression of Hsp90.


INTRODUCTION
Myocardial ischemia/reperfusion (I/R) injury is the leading cause of morbidity and mortality after cardiac surgery and heart infarctions (Sharma et al., 2012). Accumulating evidence from in vivo and in vitro studies strongly suggests that apoptosis is one of the major mechanisms of cardiomyocyte injury following hypoxia and ischemia-reperfusion (Lam et al., 2013). Reactive oxygen species (ROS) overproduction plays a crucial role in myocyte apoptosis during I/R injury (Takano et al., 2003). Hypoxia/reoxygenation (H/R) initiates the mitochondrial apoptotic pathway by inducing massive ROS production and the loss of mitochondrial membrane potential, which stimulates cytochrome c release and caspase-9 activation, and subsequent caspase-3 activation, ultimately resulting in apoptosis (Ryter et al., 2007). Hence, the inhibition of intracellular ROS levels and prevention of cardiomyocyte apoptosis are effective strategies to ameliorate myocardial I/R injury.
Myricitrin, a naturally occurring flavonoid glycoside belonging to the flavonol subgroup, is abundantly distributed in the fruits, branches, bark, and leaves of Myrica rubra and other plants, such as Myrica esculenta, Ampelopsis grossedentata, Nymphaea lotus, and Chrysobalanus icaco (Huang et al., 2014). It has been shown that myricitrin possesses considerable antioxidant activity (Sun et al., 2013a;Domitrovic et al., 2015), antinociceptive action (Meotti et al., 2007), antiinflammatory activity (Shimosaki et al., 2011), and anti-fibrotic activity (Geng et al., 2016). We previously demonstrated that myricitrin is able to protect endothelia cells against ROS-induced apoptosis and prevent plaque formation at an early stage in an atherosclerotic mouse model (Sun et al., 2013a;Qin et al., 2015). We also reported that myricitrin inhibits high glucose-induced cardiomyocyte apoptosis to prevent diabetic cardiomyopathy (Zhang et al., 2016). These experiments have indicated that myricitrin can be used for the prevention and treatment of cardiovascular disease. However, there are few reports on the effects and mechanisms of myricitrin on cardiomyocyte apoptosis following I/R. Therefore, the objective of the present study was to investigate the effects of myricitrin on H9c2 cardiomyocytes subjected to H/R to simulate I/R. The underlying molecular targets of myricitrin against I/R injury were also elucidated using bioinformatics approaches.

Materials
Myricitrin was provided from the Institute of Medicinal Plant Development (Beijing, China) (Sun et al., 2013a). Cell culture products were purchased from Gibco BRL (Grand island, NY, United States). The fluorescent dye JC-1 was purchased from Sigma-Aldrich (St. Louis, MO, United States). The Annexin V/propidium iodide (PI) apoptosis detection kit was obtained from Invitrogen Corporation (Eugene, OR, United States). Caspase-3 fluorometric assay kits were acquired from BioVision (Milpitas, CA, United States). Primary antibodies against Bcl-2, Bax, Caspase-3, and β-actin were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, United States). Primary antibodies against Hsp90, Akt, and p-Akt were purchased from Abcam (Cambridge, United Kingdom). Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from CW Biotech (Beijing, China).

Cell Culture and Hypoxia/Reoxygenation
The H9c2 cardiomyocyte line was obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China) and cultured as previously described . Briefly, H9c2 cells were cultured in high glucose DMEM supplemented with 10% (v/v) fetal bovine serum, 1% penicillin/streptomycin (v/v), and 2 mM L-glutamine. The cells were maintained at 37 • C with 100% relative humidity in a CO 2 incubator containing 5% CO 2 .
The H/R procedures were modified from a previous study (Sun et al., 2013b;Wang et al., 2014). The H9c2 cardiomyocytes were cultured in an anaerobic glove box (Coy Laboratory, United States) under hypoxia for 6 h. Subsequently, the cells were removed from the anaerobic glove box and the medium was replaced with high glucose medium and maintained for 12 h in an incubator to mimic reperfusion. For all experiments, the cells were plated at an appropriate density according to the experimental design and grown for 24 h to reach 70 to 80% confluence prior to experimentation.

Experimental Protocols
The cultured H9c2 cardiomyocytes were randomly divided into different groups. In the control (Con) group, the H9c2 cardiomyocytes were incubated under normoxic conditions for equivalent durations with high glucose DMEM. The H/R group was conducted as described in the preceding section. In the myricitrin-treated group (H/R+Myr), the H9c2 cardiomyocytes were pre-treated with myricitrin for 12 h prior to H/R. The inhibitor-treated groups were processed the same as the H/R+Myr group, but the cells were pre-incubated with 2 µM 17-AAG for 1 h, then removed the medium and treated with myricitrin. The appropriate concentration of the selective HSP90 inhibitor, 17-AAG, was determined on the basis of data found in the literature (Wu et al., 2012) and our preliminary experiments (Supplementary Figure 1).

Measurement of LDH, CAT, GSH-Px, MDA Levels, and SOD Activity
The supernatant and cells were collected after the different treatments to measure the lactate dehydrogenase (LDH), catalase (CAT), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) levels as well as superoxide dismutase (SOD) activity using the corresponding detection kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions.

Detection of Intracellular ROS Production
The effect of myricitrin on the intracellular ROS levels was monitored using the total ROS detection kit according to the manufacturer's instructions (Enzo Life Sciences, Inc., Farmingdale, NY, United States). Briefly, the cells were preconditioned with myricitrin (40 µM) for 12 h, and further exposed to 6 h of hypoxia followed by 12 h reoxygenation. After treatment, the cells were harvested, placed into 5-mL roundbottom polystyrene tubes, and washed with 1× wash buffer. Subsequently, the cells were centrifuged for 5 min at 400 × g at room temperature and the supernatant was discarded. The cells were re-suspended in 500 µL of ROS detection solution, stained in the dark at 37 • C for 30 min, and analyzed using flow cytometry (Sun et al., 2011).

Determination of Mitochondrial Transmembrane Potential ( m)
We used 5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethylbenzimidazolylcarbocyanine iodide (JC-1) (Sigma-Aldrich, St. Louis, MO, United States) to analyze changes in the mitochondrial transmembrane potential as previously described (Sun et al., 2012). The cells were pre-incubated with 40 µM myricitrin for 12 followed by H/R procedure. After treatment, the cells were incubated with 2 µM JC-1 for 30 min in the dark and washed twice with phosphate-buffered saline (PBS). The cells labeled with JC-1 were analyzed by BD FACSCalibur flow cytometry using 488 nm excitation and green (525 nm) or orange-red (575 nm) emission wavelengths with CellQuest software. The JC-1 positive rate was expressed as the percentage of the red/green fluorescence intensity relative to that in the control group.

Flow Cytometric Detection of Apoptosis
The percentages of early apoptosis and necrosis were measured using an Annexin V-FITC/PI apoptosis kit for flow cytometry according to the manufacturer's instructions (Invitrogen). After treatment, the cells were harvested and washed twice with cold PBS and subsequently incubated with 5 µL of FITC-Annexin V and 1 µL of the PI working solution (100 µg/mL) in 100 µL of cell suspension for 15 min in the dark at room temperature. Cellular fluorescence was measured by flow cytometry analysis (FACS Calibur TM , BD Biosciences, San Jose, CA, United States).

Analysis of Caspase-9 Activity and Caspase-3 Activation
Caspase-9 activity (Sun et al., 2011) and caspase-3 activation (Sun et al., 2013a) were measured using the corresponding Fluorometric Assay Kits (BioVision, CA, United States) according to the manufacturer's instructions. To assess caspase-9 activity, the cells were resuspended in lysis buffer and kept on ice for 10 min. Then, 50 ml of 2× reaction buffer containing 10 mM dithiothreitol was added to each sample with 5 ml of 1 mM substrate LEHD-AFC for 1.5 h at 37 • C. The samples were determined using a microplate reader (TECAN Infinite M1000, Austria) at the excitation wavelength of 400 nm and the emission wavelength of 505 nm. For analysis of caspase-3 activation, the cells were harvested and incubated with 1 µL substrate FITC-DEVD-FMK for 1 h at 37 • C, and then centrifuged at 3000 rpm for 5 min; the supernate was removed. After washing twice with cold PBS, the cells were re-suspended in 100 µL of wash buffer and then transfer the cell suspension to each well in the black microtiter plate. The fluorescence intensity was detected using microplate reader with excitation wavelength of 485 nm and emission wavelength of 535 nm.

Western Blot Analysis
After treatment, H9c2 cells were harvested, washed with PBS, and lysed with cell lysis buffer containing 1% phenylmethylsulfonyl fluoride. Lysate preparation and Western blot analysis were performed as previously described (Sun et al., 2012). The immunoblots were developed using an electrochemiluminescence (ECL) kit.
Targets Predicted by Discovery Studio 4.5 The molecular targets of myricitrin were predicted using Discovery Studio (DS) 4.5 (BIOVIA Software, Inc., San Diego, CA, United States). Molecular structure of myricitrin was submitted to Monte Carlo based conformational analysis (Discovery Studio's FAST algorithm) allowing maximal 255 conformers with less than 20 kcal/mol above the energy minimum. PharmaDB is the only pharmacophore database implemented in DS 4.5. In the profiling with PharmaDB, all pharmacophore models with the shape of the binding pocket were selected for virtual screening using the default settings of the Ligand Profiler module of DS 4.5. The RIGID mode was used as the molecular mapping algorithm. The screening was conducted using the default settings and with a minimal inter-feature distance of 0.5 Å. After inputting all parameters, the job was run, and a result sheet was returned indicating which models were hits and how well myricitrin mapped into each model (fit value), respectively. A higher fit value represents a better fit (Rollinger et al., 2009;Yi et al., 2016).

Statistical Analyses
The results are expressed as the means ± standard deviation. Comparisons were performed using Student's t-test or one-way ANOVA followed by Tukey's multiple comparison test using Prism 5.00 software. Statistical significance was set at p < 0.05. All data are the result of at least three independent experiments.

Myricitrin Alleviated H/R Injury in H9c2 Cells
The protective effect of myricitrin against H/R-induced cell death was detected using an MTT assay. As shown in Figure 1A and Supplementary Figure 2, myricitrin pretreatment exhibited a strong protective effect after pretreatment for 12 h. The survival rate increased from 65.48 ± 6.42% (with H/R treatment alone) to 78.92 ± 5.41%, 83.56 ± 5.89%, and 87.48 ± 7.68% after treatment with 10, 20, and 40 µM myricitrin, respectively. LDH, which is secreted from cells after plasma membrane disruption, can be used as an indicator of cell injury. As shown in Figure 1B, myricitrin pretreatment decreased the LDH levels in the culture medium in a concentration-dependent manner (p < 0.05 or p < 0.01). This effect is consistent with its protective effects on cell viability, as assessed using an MTT assay.

Myricitrin Enhanced Anti-oxidant Enzyme Activity in H/R-Treated H9c2 Cells
Oxidant stress has long been associated with heart injury in response to I/R (Kalogeris et al., 2012). Membrane lipid oxidation is one of the primary events in oxidative damage, which can be assessed according to the degradation product MDA (Rodrigo et al., 2013). As shown in Figure 2A, the H/R group showed a significant increase in intracellular MDA levels, whereas a significant reduction in the levels of MDA was observed in the myricitrin pretreatment group compared with the H/R group.
To confirm the antioxidative effects of myricitrin, the activities of the endogenous antioxidative enzymes SOD, CAT, and GSH-Px were detected. Myricitrin pretreatment significantly enhanced the SOD, CAT, and GSH-Px activities compared to the H/R group (Figures 2B-D, p < 0.05 or p < 0.01).
Based on flow cytometry staining, we examined the effect of myricitrin treatment on the intracellular ROS levels. As shown in Figures 2E,F, H/R significantly increased the ROS levels by nearly threefold compared with the control, as demonstrated by a shift in fluorescence from left to right. However, myricitrin pretreatment (40 µM) partially blocked this effect, suggesting that myricitrin pretreatment decreased the ROS levels.

Myricitrin Decreased H/R-Induced Apoptosis in H9c2 Cells
Oxidative stress-induced apoptosis is associated with a loss of mitochondrial membrane potential ( m), an early marker of apoptosis (Sun et al., 2011). Mitochondrial depolarization was indicated as a decrease in the red/green fluorescence intensity ratio of JC-1 staining. The H/R group exhibited an increase in green fluorescence intensity, indicating ψm dissipation (Figures 3A,B), while pretreatment with myricitrin attenuated m depolarization, as indicated by the increased red fluorescence (p < 0.05). We further confirmed the anti-apoptotic effect of myricitrin through quantitative analysis of FITC-Annexin V/PI staining using flow cytometry. The percentage of apoptotic cells in the H/R group significantly increased from 1.23 ± 0.56% to 8.56 ± 0.97% compared with the control group ( Figures 3C,D). By contrast, pretreatment with myricitrin dramatically decreased the ratio of apoptotic cells to 3.89 ± 0.62%.

Myricitrin Modulated Apoptosis-Related Protein Activity and Expression in H/R-Treated H9c2 Cells
Caspase activity was also used to assess the level of apoptosis (Zhuang et al., 2000). As shown in Figures 4A,B, the caspase-9 activity and active caspase-3 levels markedly increased after H/R treatment. Myricitrin pretreatment significantly inhibited these effects (p < 0.05). To understand the mechanism of myricitrin treatment on the regulation of cell apoptosis, we measured the effects of myricitrin on the expression of apoptosis-related protein expression using Western blot analysis. As shown in Figures 4C,D, the H/R group showed a decrease in expression of the anti-apoptotic protein Bcl-2 and an increase in expression of the pro-apoptotic protein Bax. Pretreatment with myricitrin increased the Bcl-2/Bax expression FIGURE 5 | Profiling of the predicted protein targets of myricitrin via Discovery Studio. The Y -axis represents the compound myricitrin, the X-axis indicates the predicted pharmacophore models (pharmacological targets) of myricitrin. The color from blue to red represents a higher fit value and a better fit. ratio compared with the H/R group (1.28 ± 0.31 vs. 0.46 ± 0.12, respectively).

Target Prediction
The molecular targets of myricitrin were predicted using Discovery Studio software (Figure 5). The ranking according to fit score in descending order and the top 10 diseaserelated targets are shown in Table 1. Among these targets, we focused on heat shock protein 90 (Hsp90), a critical target in the protection of the myocardium from I/R injury.

Hsp90 Contributes to the Cytoprotection of Myricitrin against H/R Injury in H9c2 Cardiomyocytes
Accumulating evidence has shown that upregulation of Hsp90 expression protects cardiomyocytes from I/R-induced damage (Wu et al., 2012). Thus, we detected the expression levels of Hsp90 in myricitrin-treated H9c2 cardiomyocytes. As shown in Figure 6A, when H9c2 cells were treated with 40 µM myricitrin for 0-12 h, a significant increase in the expression level of Hsp90 was observed. Next, we detected the effects of myricitrin on the Hsp90 expression levels in H/R-treated cardiomyocytes. Figure 6B shows that myricitrin treatment significantly inhibited the downregulation of the H/R-induced Hsp90 expression.
To determine whether such elevated expression of Hsp90 is responsible for the cell protective effect of myricitrin, a selective inhibitor of Hsp90, 17-AAG, was utilized. As shown in Figure 7, the Hsp90 inhibitor 17-AAG reversed the protection against H/R injury by decreased cell viability (Figure 7A), phosphorylation levels of the prosurvival protein Akt (Figure 7B), and mitochondrial membrane potential (Figures 7C,D). In addition, in the presence of 17-AAG, the decreased mitochondrial ROS levels induced by myricitrin were also abolished (Figures 7E,F). These results suggest that Hsp90 is involved in the protective effect of myricitrin against H/R injury in H9c2 cells.

DISCUSSION
Oxidative stress has been implicated in injury associated with myocardial ischemia/reperfusion through induced ROS production (Kalogeris et al., 2012). Flavonoids, which contain a catechol group, have been described as scavengers and mimics of SOD and play an important role in oxidative stress (Meotti et al., 2006). Myricitrin, belonging to the flavonol subgroup, reportedly possesses effective antioxidant activity (Domitrovic et al., 2015), with stronger free radical scavenging activity than other flavonol rhamnosides or quercetin (Wu et al., 2008). Similarly, the results of the present study clearly showed that ROS production was well preserved as a result of myricitrin pretreatment during H/R; this effect was accompanied by significantly elevated activities of SOD, GSH-Px, and CAT and decreased levels of MDA, suggesting that the cardioprotective effects of myricitrin against I/R are largely associated with its anti-oxidative capacity.
In addition to producing oxidative injury, cardiomyocyte apoptosis is also one of the major contributors to the development of myocardial I/R injury (van Empel et al., 2005). Hypoxia and excess ROS alter the mitochondrial structure and trigger mitochondrial depolarization as well as dissipation of m (Sinha et al., 2013). Disruption of m induces the release of proapoptotic molecules from the mitochondria and subsequently activates caspases, resulting in apoptosis (Sinha et al., 2013). In the present study, the results indicated that myricitrin maintained the mitochondrial membrane potential, decreased caspase-9 activity and caspase-3 activation, and  Frontiers in Pharmacology | www.frontiersin.org reduced the apoptosis rate of the H9c2 cardiomyocytes against H/R. Caspase-3 activity can be induced by proapoptotic Bax and inhibited by anti-apoptotic Bcl-2 (Kaushal et al., 2004). Western blot analysis showed that myricitrin increased the Bcl-2/Bax ratio compared with the H/R group. These data demonstrated that myricitrin effectively inhibits H/R-induced apoptosis and provides further evidence supporting its anti-apoptotic role (Qin et al., 2015;Zhang et al., 2016). Myricitrin interacts with proteins, such as PI-3 kinase, nitric oxide synthase (NOS), PKCα, and PKCε (Pereira et al., 2011;Schwanke et al., 2013;Qin et al., 2015). In addition, previous studies have demonstrated that myricitrin potently inhibits calcium transport, likely through the interaction of the phenolic groups of this flavonoid with the Ca 2+ transporting protein (Thiyagarajah et al., 1991;Meotti et al., 2007). In the present study, we investigated the potential targets that may have significant relationships with the pharmacologic effects of myricitrin using bioinformatics approaches. Among the targets of myricitrin, the main protein involved in I/R injury is HSP90. Hsp90, as an abundant chaperone that regulates cell signaling and the functions of many proteins that bind to it, referred to as "client proteins" (Wang et al., 2009). According to previous studies, Hsp90 is a direct effector that enables the activation of the prosurvival PI3K/Akt pathway (Barksdale and Bijur, 2009). In recent years, studies have shown that Hsp90 mediates the mitochondrial import of PKCε (Budas et al., 2010;Zhong et al., 2014) and plays an integral role in endothelial NOS signaling (Amour et al., 2009), thereby efficiently protecting the myocardium against I/R injury (Kupatt et al., 2004;Zhu et al., 2016). Furthermore, Hsp90 facilitates the inhibitory effect of HAX-1 on SR calcium uptake, which is protective in I/R injury (Lam et al., 2013). Thus, we speculated that Hsp90 is one of the targets of myricitrin to relieve the H/R injury of cardiomyocytes.
Next, we further investigated the roles of Hsp90 in the cardioprotection of myricitrin against H/R injury. As expected, myricitrin pretreatment obviously blocked the inhibitory effect of H/R on the expression of Hsp90. Hsp90 acts as an upstream regulator of the serine/threonine protein kinase Akt survival pathway during cellular stress (Barksdale and Bijur, 2009). We previously showed that myricitrin mediates cardiovascular protection through activation of the Akt signaling pathway (Qin et al., 2015;Zhang et al., 2016). In the present study, the addition of the Hsp90 inhibitor 17-AAG substantially decreased Akt phosphorylation induced by myricitrin, thereby attenuating the prosurvival effects of the PI3-K/Akt pathway. In addition, inhibition of Hsp90 by 17-AAG markedly abolished myricitrininduced cardioprotection against H/R injury, as shown by the increased overproduction of mitochondrial ROS and loss of MMP. These findings are consistent with recent reports that Hsp90 is anti-apoptotic in cardiomyocytes through antioxidation and the preservation of mitochondrial function under stress conditions (Wu et al., 2012). Based on these results, we suggest that Hsp90 is involved in the cardio-protective effects of myricitrin and that the interaction between myricitrin and Hsp90 may promote the anti-apoptotic PI3-K/Akt signaling pathway in cardiomyocytes during H/R injury.

CONCLUSION
The results of the present study indicated that myricitrin, a dietary flavonoid compound, exerts a profound cardioprotective effect against H/R injury for H9c2 cardiomyocytes through suppression of oxidative stress and apoptosis and that Hsp90 contributes to the myricitrin-induced cardioprotective effect, at least in part, through the PI3-K/Akt pathway. Although more experiments are required to elucidate the precise mechanisms underlying this protection because HSP90 associates with a variety of other signaling molecules, these results suggest that myricitrin may be a promising therapeutic candidate for the treatment of myocardial I/R injury.

AUTHOR CONTRIBUTIONS
G-bS, J-xY, and X-bS conducted the study. MW designed the detailed experiments, performed the study, and collected and analyzed data. Y-yD, YT, PL, and X-sL took part in the cell experiments in this study. All Authors commented the study and approved the final manuscript.