https://orcid.org/0000-0002-8405-0536
Abstract
Oxidative stress significantly contributes to heart disease, and thus might be a promising target for ameliorating heart failure. Mounting evidence suggests that selenium has chemotherapeutic potential for treating heart disease due to its regulation of selenoproteins, which play antioxidant regulatory roles. Oxidative stress-induced cardiomyocyte cell cycle arrest contributes to the loss of cardiomyocytes during heart failure. The protective effects and mechanism of selenium against oxidative stress-induced cell cycle arrest in cardiomyocytes warrant further study. H9c2 rat cardiomyoblast cells were treated with hydrogen peroxide in the presence or absence of selenium supplementation. Na2SeO3 pretreatment alleviated H2O2-induced oxidative stress, increased thioredoxin reductase (TXNRD) activity and glutathione peroxidase (GPx) activity and counteracted the H2O2-induced cell cycle arrest at the S phase. These effects were accompanied by attenuation of the H2O2-induced strengthening of the G2/M-phase inhibitory system, including increased mRNA and protein levels of cyclin-dependent kinase 1 (CDK1) and decreased p21 mRNA levels. Notably, Na2SeO3 pretreatment activated the PI3K/AKT signaling pathway, and inhibition of PI3K counteracted the protective effects of selenium on H2O2-induced cell cycle arrest. We corroborated our findings in vivo by inducing oxidative stress in pig heart by feeding a selenium deficient diet, which decreased the TXNRD activity, inactivated PI3K/AKT signaling and strengthened the G2/M-phase inhibitory system. We concluded that the cardioprotective effects of selenium supplementation against oxidative stress-induced cell cycle arrest in cardiomyocytes might be mediated by the selenoprotein-associated (GPx and TXNRD) antioxidant capacity, thereby activating redox status-associated PI3K/AKT pathways, which promote cell cycle progression by targeting the G2/M phase inhibitory system. This study provides new insight into the underlying mechanisms of cardioprotection effects of selenium at the cellular level.
Selenium alleviates oxidative stress-induced cell cycle arrest in cardiomyocytes mediated by antioxidant capacity of selenoproteins, thus activating PI3K/AKT pathways, which promote cell cycle progression by targeting the G2/M phase inhibitory system.
Selenium is an essential trace element that could decrease inflammation, prevent platelets from aggregating, and decrease the risk of heart failure through its incorporation into selenoproteins. Severe deficiency of selenium causes myocardial dysfunction in humans and animals. This work shows that selenium supplementation attenuates oxidative stress-induced cell cycle arrest in cardiomyocytes in vitro and in vivo, and the cardioprotective effects are mediated by the selenoprotein-associated antioxidant capacity, thereby activating the redox status-associated PI3K/AKT pathway, which promotes cell cycle progression by targeting the G2/M phase inhibitory system. This study provides new insight into the underlying mechanisms of the cardioprotection effects of selenium at the cellular level.
Introduction
Heart failure is a major public health issue affecting more than 23 million people worldwide. The risk increases with age, thus posing a considerable burden to the health-care system, owing to substantial morbidity and mortality.1 Heart failure is also emerging as a major challenge in animal production in the presence of environmental and nutritional deficiency stresses.2 The loss of cardiomyocytes is a major contributor to the occurrence of heart failure.3 The adult human heart can lose as many as 1 billion cardiomyocytes in response to myocardial infarction,4 and these cells cannot be replenished, owing to cell cycle arrest and ceased proliferation of cardiomyocytes in adults.5 Numerous cardiovascular studies have identified oxidative stress as an important pathophysiological factor in the progression of heart failure that contributes to cardiomyocyte cell cycle arrest,6–8 H2O2 induces cell cycle arrest at the G1 and G2 phases in cardiomyoblast H9c2 cells,9 and hypoxia has been reported to stimulate cardiomyocyte proliferation through decreasing the production of reactive oxygen species (ROS).10 These findings suggest that oxidative stress may be a promising target for switching on the arrested cell cycle of cardiomyocytes in the failing heart of humans and animals.
Selenium is a metalloid trace element that prevents the oxidative modification of lipids and proteins, thereby decreasing inflammation, preventing platelets from aggregating, and decreasing the risk of heart failure or death associated with heart failure through its incorporation into selenoproteins, thus contributing to the alteration of redox signaling.11–13 Severe deficiency in selenium can cause myocardial dysfunction in humans and animals, including Keshan disease in humans and mulberry heart disease in pigs, both of which are typically accompanied by congestive heart failure.14–16 It was also demonstrated that ROS accumulation and heart injury were features of selenium deficiency in mice.17 Thus, it was suggested that reduced antioxidant capacity was strongly correlated with selenium deficiency-associated cardiomyopathies.11 Although the underlying mechanism of heart failure induced by selenium deficiency at the cellular level is not fully clear, previous studies have uncovered several beneficial effects of selenium on the inhibition of cell death (including autophagy, apoptosis14 and ferroptosis18) and a lack of selenoprotein synthesis increases intracellular ROS production and inhibits cell cycle progression in H9c2 cardiomyocytes.19 However, the potential effects of selenium on oxidative stress-associated changes in cell cycle progression in cardiomyocytes and the underlying mechanism warrant further study.
The inhibition of the cell cycle in cardiomyocytes is mainly regulated by two inhibitory systems: the G1-phase inhibitory system, through repression of cyclin D1 (CCND1) expression and promotion of p21 and p27, and the G2/M-phase inhibitory system, through inhibition of CDK1 activation and promotion of p21, which inhibits the G2/M-phase entry of cardiomyocytes.20 Mounting evidence indicates that the PI3K/AKT signaling pathway acts as a central effector in cardiomyocyte cell cycle entry or arrest, which targets cyclins and CDK inhibitors (p21, 27, 57), thus regulating cell cycle phase progression.21,22 Particularly in neonatal and adult cardiomyocytes, the activation of PI3K/AKT is concomitant with an increase in the cell proliferation ratio.23 Notably, the PI3K/AKT signaling pathway is regulated by selenoprotein-mediated alteration of the redox status, which influences cell cycle progression in cardiomyocytes.19 Therefore, exploration of the effects of selenium on PI3K/AKT signaling and its involvement in oxidative stress-induced cell cycle arrest is of great significance for future applications of selenium in treating heart disease.
The aim of this study was to elucidate the protective effects and mechanisms of selenium supplementation against oxidative stress-induced cell cycle arrest in cardiomyocytes. In vitro, we established an H2O2-induced oxidative stress model in H9c2 and determined the protective effects of selenium on oxidative stress-induced cell cycle arrest. In our previous study, we established a selenium-deficiency pig model via feeding a selenium deficient diet, this selenium-deficiency pig model exhibits oxidative stress in organs,24,25 accompanied by decreased selenium content, and GPx and TXNRD activity. These results were consistent with Lei and colleagues’ previous study in pigs.26 Because of the similarities in organ size, anatomy, immunology and physiology between pigs and humans with respect to the cardiovascular system, selenium deficiency induced-oxidative stress heart is the most attractive model for studies of oxidative stress-related heart disease. Thus, selenium deficiency was used to induce oxidative stress in pig hearts in vivo, and the protective effects of selenium on cardiomyocytes were investigated. Additionally, inhibition of the PI3K/AKT signaling pathway was performed to reveal the potential protective mechanism of selenium supplementation against heart failure in humans and animals.
Materials and methods
Reagents and antibodies
Sodium selenite (Na2SeO3), H2O2 and LY294002 were purchased from Sigma-Aldrich (Sigma-Aldrich Co., St. Louis, MO, USA). High-glucose Dulbecco's modified Eagle's medium (DMEM-H), fetal bovine serum (FBS), glutamine and antibiotics were procured from Gibco (Grand Island, NY, USA). The primary antibodies against PI3K, AKT1, phospho-AKT1-S473, GSK3β, phospho-GSK3β-S9 and CDK1 were purchased from ABclonal Technology (Wuhan, China). Primary antibodies against pigs AKT1 and phospho-AKT1-S473, PTEN and GAPDH were purchased from Cell Signaling Tech. (Danvers, MA, USA). Detailed primary antibody information is listed in Table S1 (ESI†).
Cell culture and treatment
H9c2 cells were obtained from the National Infrastructure of Cell Line Resource (Beijing, China). H9c2 cells were maintained in DMEM-H supplemented with 10% FBS, penicillin (100 IU mL−1), streptomycin (100 μg mL−1) and 2 mM glutamine, and were cultured in a humidified incubator containing 5% CO2 at 37 °C. To induce selenium deficiency, H9c2 cells were grown in a starvation medium containing only 5% FBS for 24 h. This determined a reduction of the selenium content in the medium to about 0.85 ng mL−1 (10 nM L−1). Detection of the selenium content in FBS was performed according to our previously reported protocol,27 the sample pretreatment is according to the Chinese national standard method (GB 5009.93-2017), the extraction method was modified from that described by Bakırdere28 and total selenium analysis was performed using an Agilent 7900 inductively coupled plasma mass spectrometry (ICP-MS) system (Agilent Technologies, Santa Clara, CA, USA). To evaluate the protective effects of selenium on H9c2 cells under oxidative stress, we supplemented the starvation medium with a final concentration of 0, 100, 250, 500, 1000 or 2000 nM Na2SeO3 and/or H2O2. Cells cultured in DMEM without supplementation with Na2SeO3 and H2O2 were used as controls.
Animals and experimental diets
Pig heart samples were obtained from our previous experiment. The experimental pigs were tested in full sibling pairs.24 The Se-Def basal diet (BD) was composed of corn and soybeans (Table S2, ESI†) produced in the Se-deficient area of HeiLongJiang, and contained 0.007 mg Se kg−1. The diet was formulated according to the nutritional requirements described in NY/T 65-2004, to achieve the National Research Council (2012) recommended levels. A total of 24 weaned pigs (from 12 full-siblings, 45 days of age) were divided into two equal groups (n = 12) and fed the BD supplemented with 0 or 0.3 mg Se kg−1 selenomethionine (Se-Met), the trial lasted 16 weeks. All procedures used in this study were approved by the Animal Care and Use Committee of the Institute of Animal Science, Chinese Academy of Agricultural Sciences. Pigs were individually housed in cages and given ad libitum access to fresh water and feed. The 12 pigs in each group were slaughtered through electric shock and exsanguinated. The hearts were rapidly removed, washed with ice-cold isotonic saline, snap-frozen in liquid nitrogen and stored at −80 °C until use.
MTT assays
To evaluate the cell viability, we seeded H9c2 cells into 96-well plates at 1.0 × 104 per well and cultured them for 24 h. Then, the H9c2 cells were pretreated with 0, 100, 250, 500, 1000 or 2000 nM Na2SeO3 from 24 to 96 h, and 0, 250, 500, 750, 1000 or 2000 μM H2O2 was added for the last 0 to 6 h. Operations were strictly carried out according to the MTT kit (Bio Friend Ltd, Beijing, China) instructions, 10 μL MTT solution was added and incubated for 4 h at 37 °C. The absorbance was measured at 490 nm with an Infinite F50 plate reader (TECAN, Switzerland). The percentage of cell survival in the treated cells was expressed as the absorbance normalized to that of the control cells.
Cell cycle analysis
For cell cycle analysis, H9c2 cells (1 × 105) were seeded in six-well cell culture plates. After a 72 h period of culture in starvation medium with the addition of 500 nM Na2SeO3, and/or 500 μM H2O2 treatment was performed for the last 4 h. The cells were then harvested and fixed with 75% cold ethanol. After fixation, the cells were resuspended in 0.4 mL staining reagent containing 50 mg mL−1 propidium iodide and 100 mg mL−1 RNase and incubated for 30 min at 37 °C in the dark. The samples were analyzed with a BD LSR Fortessa flow cytometer (BD Biosciences) with a 488 nm excitation laser. The cell cycle phases were determined with modfit software (Verity Software House, Topsham, ME, USA).
Measurement of intracellular ROS levels
Intracellular ROS levels were measured on the basis of H2O2-sensitive DCFH-DA fluorescence. After Na2SeO3 and/or H2O2 treatment, H9c2 cells were washed with phosphate buffered saline (PBS) and stained with 10 μM DCFH-DA for 30 min at 37 °C in the dark. The H9c2 cells were pre-warmed in growth medium for 30 min to recover the cells, and the coverslips were observed by laser scanning confocal microscopy (Leica TCS SP8, Germany). Images were quantified with ImageJ (NIH, Bethesda, MD, USA).
Determination of antioxidant activity
H9c2 cell and porcine heart extracts were prepared with sonication (Sonics VCX105, USA) in ice-cold PBS and centrifuged at 12 000g for 15 min to remove debris. The SOD activity was measured spectrophotometrically (550 nm) on the basis of the reaction with TMB, the MDA levels were determined spectrophotometrically (450 nm) on the basis of the reaction with thiobarbituric acid. The total antioxidant capacity (T-AOC) was determined spectrophotometrically (520 nm) through the reaction with Fe3+ and phenanthrolines. The MDA level, SOD activity and T-AOC were determined with commercially available kits (Nanjing Jiancheng Biology Engineering, Nanjing, China) according to the manufacturer's instructions. The total protein concentration was determined by using the BCA Protein Assay kit (Thermo Fisher Scientific Inc., Waltham, MA, USA).
RNA extraction, cDNA synthesis and RT-qPCR
H9c2 cells and porcine hearts were harvested in Trizol, and 1 μg total RNA was used to synthesize cDNA with PrimeScript™ RT 1st Master Mix (TaKaRa, Japan) according to the manufacturer's instructions. RT-qPCR was performed with TB Green® Premix Ex TaqTM II (Tli RNaseH Plus) and an ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The primers specific for p21, CDK1, CDK2, CDK4, CCNB1 and GAPDH used in this experiment are listed in Table S3 (ESI†). Relative gene expression levels were calculated with the 2−ΔΔCt method.29GAPDH and β-actin were used as endogenous controls,26,30 in brief, GAPDH was used as the reference gene and its reliability was confirmed by the perfect parallelism of its Ct values in all the 18 cell samples with those of β-actin (Fig. S1, ESI†).
GPx and TXNRD activity
The GPX activity was measured by the GR-coupled standard test with a total glutathione peroxidase assay kit with NADPH (Beyotime Biotechnology, Shanghai, China). The NADPH-dependent TXNRD activity was detected on the basis of the catalytic reduction of 5,5-dithio-bis-(2-nitrobenzoic acid) to 5-thio-2-nitrobenzoate measured at 412 nm.
Western blot analysis
H9c2 cells and porcine hearts were harvested and resuspended in RIPA lysis buffer supplemented with a protease inhibitor cocktail and phosphatase inhibitor cocktail for protein extraction, and the protein concentrations of all samples were determined with a BCA Protein Assay Kit. Aliquots of the lysates containing 30 μg protein were separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto PVDF membranes. The membranes were blocked in 5% bovine serum albumin in Tris-buffered saline for 1 h at room temperature and then incubated with the indicated primary antibodies overnight at 4 °C. The dilution details for all antibodies are listed in Table S1 (ESI†). After three washes, the membrane was then incubated with anti-rabbit HRP-conjugated IgG as a secondary antibody for 1 h at room temperature, and the immunoreactive bands were developed with an enhanced chemiluminescent substrate (Thermo Fisher Scientific Inc.). Images of blotting were analyzed using Bio-Rad QuantityOne® software (Bio-Rad Laboratories, Richmond, CA, USA).
Statistical analysis
Data are expressed as mean ± standard errors (S.E.M.) from all independent experiments. The statistical analysis was performed with Student's t-test in SPSS 17.0 (SPSS Inc., Chicago, IL, USA), and statistically significant differences were assumed at a P-value < 0.05. Densitometric analyses of western blot bands were performed using Quantity One 1-D analysis software (Bio-Rad, USA).
Results
Selenium supplementation decreases H2O2-induced cytotoxic effects in H9c2 cells
To determine the cytotoxic effects of H2O2 in H9c2 cells, we evaluated the viability of H9c2 cells exposed to increasing concentrations of H2O2 (0, 250, 500, 750, 1000 and 2000 μM) for 0.25, 0.5, 1, 2, 4 and 6 h by using MTT assays. As shown in Fig. 1a, with up to 250 μM H2O2 no significant changes were detected in cell viability, whereas the cell viability was markedly inhibited after treatment with 500, 750, 1000 and 2000 μM H2O2 for 4 h, showing approximately 25% lower cell viability than that of the control at a concentration of 500 μM, whereas >50% lower cell viability was observed at concentrations of 750, 1000 and 2000 μM. On the basis of the above results, H2O2 was used at a concentration of 500 μM for 4 h in subsequent experiments. To confirm the safe concentration range of Na2SeO3 for H9c2 cells, we incubated cells with different concentrations of Na2SeO3 (0, 100, 250, 500, 1000 and 2000 nM) for 24 h, 48 h and 72 h, after which the cell viability was measured. Fig. 1b shows that no cytotoxicity was observed in the presence of different concentrations of Na2SeO3 for 24 h incubation, whereas growth-retarding effects were observed over time in response to a high concentration of Na2SeO3 (1000 to 2000 nM Na2SeO3 incubation for 72 h).
Selenium supplementation attenuates H2O2-induced cytotoxicity in H9c2 cells. Cell viability was detected with MTT assays in H9c2 cells exposed to (a) increasing concentrations of H2O2 (from 0 to 2000 μM) from 0 to 6 h, (b) different concentrations of Na2SeO3 (0, 100, 250, 500, 1000 and 2000 nM) for 24 h, 48 h and 72 h, (c) and different concentrations of Na2SeO3 (0, 100, 250, 500, 1000 and 2000 nM) for 72 h in the presence of 500 μM H2O2 for 4 h. Data are expressed as means ± S.E.M. (n = 3) of three independent experiments. Con represents cells were treated with neither Na2SeO3 nor H2O2. *P < 0.05 vs. control cells.
We evaluated the potential selenium cardioprotective properties in response to H2O2 treatment. According to previous findings, incubating cells with Na2SeO3 for 72 h can effectively decrease the intracellular ROS levels and increase the T-AOC and GPX activity.31 Thus, H9c2 cells were pretreated with different concentrations of Na2SeO3 (0, 100, 250, 500, 1000 and 2000 nM) for 72 h, and then exposed to 500 μM H2O2 for an additional 4 h. Up to 250 nM Na2SeO3 significantly decreased the cell viability, as compared with that of the control group, whereas 500 nM Na2SeO3 pretreatment for 72 h resulted in the recovery of the H2O2-induced cell viability, which returned to normal levels; Na2SeO3 in the range of 1000–2000 nM attenuated the decrease in the H2O2-induced cell viability due to the growth-retarding effects of high concentrations of Na2SeO3 (Fig. 1c). On the basis of these above results, 500 nM Na2SeO3 pretreatment for 72 h was chosen as a working treatment for all subsequent experiments. These results demonstrated that Na2SeO3 pretreatment rescues the cytotoxic effects induced by H2O2.
Selenium supplementation rescues H2O2-induced cell cycle arrest in the S phase in H9c2 cells
To investigate the effects of Na2SeO3 on the cell cycle progression under oxidative stress in H9c2 cells, we pretreated H9c2 cells with 500 nM Na2SeO3 for 72 h and then optionally exposed the cells to H2O2 for 4 h, we then analyzed the cell cycle distribution by flow cytometry. H2O2 incubation significantly increased the population of cells in the S phase from 25.17 ± 0.88% to 39.59 ± 0.82%, but decreased the population in the G2/M phase from 12.30 ± 0.88% to 5.21 ± 0.77% and the G1 phase from 62.54 ± 0.22% to 55.29 ± 0.05%, as compared with the control (Fig. 2a and b). As shown in Fig. 2b, compared with H2O2 incubation, Na2SeO3 pretreatment for 72 h significantly counteracted the H2O2-induced increase in the population of cells in the S phase from 39.59 ± 0.82% to 35.06 ± 0.41%, but counteracted the H2O2-induced decrease in the population of cells in the G2/M phase from 5.21 ± 0.77% to 8.63 ± 0.23% (Fig. 2a and b) and that of cells in the G1 phase from 55.29 ± 0.05% to 56.61 ± 0.08%. The G2/M-phase inhibitory system (CDK1 and p21) regulates cardiomyocyte switching from the S phase to the G2/M phase.20,32 Cyclin B1 (CCNB1) binds CDK1 and forms a complex that participates in regulating cell cycle progression.33 Accordingly, the mRNA expression levels of CDK1 and CCNB1 significantly decreased under H2O2 incubation, as compared with the control levels, whereas that of p21 significantly increased (Fig. 2c). Compared with H2O2 incubation, Na2SeO3 incubation not only reversed the H2O2-induced decrease in CDK1 and CCNB1 but also reversed the H2O2-induced increase in p21 (Fig. 2c). These results indicated that incubation with 500 μM H2O2 inhibited H9c2 cell entry into the G2/M phase and arrested H9c2 cells in the S phase. Moreover, Na2SeO3 pretreatment rescued the H2O2-induced cell cycle arrest at the S phase in H9c2 cells, possibly by reversing the H2O2 induced promotion of the G2/M phase inhibitory system (CDK1 and p21).
Effects of selenium on H2O2-induced cell cycle arrest in H9c2 cells. H9c2 cells were cultured for 72 h in 5% FBS-containing medium supplemented with 0 or 500 nM Na2SeO3, and then the cells were cultured in 0 or 500 μM H2O2. (a) Representative flow cytometry diagrams of H9c2 cells. (b) Quantification of the results in (a). (c) CDK1, CDK2, CDK4, CCNB1 and p27 mRNA expression levels in H9c2 cells. Data are expressed as means ± S.E.M. (n = 3). *P < 0.05 vs. untreated cells; #P < 0.05 vs. H2O2 alone.
Selenium supplementation ameliorates H2O2-induced oxidative stress and increases both GPx and TXNRD activity in H9c2 cells with H2O2 treatment
To evaluate the effects of selenium supplementation on H2O2-induced oxidative stress, we cultured H9c2 cells in the presence of Na2SeO3 for 72 h and then optionally exposed the cells to H2O2 for 4 h, and oxidative parameters were then measured. Intracellular ROS levels were measured with DCFH-DA fluorescence assays. The green fluorescence distinctly increased after treatment with H2O2, thus indicating that the levels of ROS clearly increased to levels approximately two-fold higher than those in the control cells after treatment with 500 μM H2O2 (Fig. 3a and b). Selenium pretreatment significantly reversed the H2O2-induced increase in green fluorescence, thus indicating that the levels of ROS clearly decreased with pretreatment with 500 μM Na2SeO3 under H2O2 incubation. H2O2 effectively promoted the production of ROS in H9c2 cells, as compared with the control cells, yet selenium counteracted the H2O2-induced ROS production. The levels of MDA significantly increased and the SOD activity significantly decreased in cells treated with 500 μM H2O2 compared with the control cells (Fig. 3c and d). However, selenium pretreatment not only reversed the H2O2-induced increase in MDA but also reversed the H2O2-induced decrease in SOD activity in H9c2 cells (Fig. 3c and d). In addition, there was no significant difference in T-AOC among the control, H2O2, Na2SeO3, and Na2SeO3 + H2O2 treated cells (Fig. 3e). Together, these results indicated that selenium pretreatment counteracted H2O2-induced oxidative stress.
Effects of selenium supplementation on oxidative stress and selenium-containing enzymes induced by H2O2 in H9c2. H9c2 cells were cultured for 72 h in 5% FBS-containing medium supplemented with 0 or 500 nM Na2SeO3; the cells were then cultured in 0 or 500 μM H2O2. (a) ROS fluorescence images visualized by fluorescence microscopy (magnification, ×200; scale bar, 10 μm). (b) Quantitative analysis of the ROS positive cells of (a). The (c) MDA content, (d) SOD relative level, (e) T-AOC, (f) GPx activity and (g) TXNRD activity were detected with commercial reagent kits. Data are expressed as means ± S.E.M. (n = 3). *P < 0.05 vs. untreated cells, #P < 0.05 vs. H2O2 alone.
Selenium participates in antioxidant defense mainly through incorporation into different selenoproteins.34 H9c2 cells were cultured in the presence of Na2SeO3 for 72 h and then optionally exposed to H2O2 for 4 h. The enzymatic activity of two major selenoproteins (Gpx and TXNRD) was subsequently detected. H2O2 incubation significantly increased the enzymatic activity of GPx and decreased that of TXNRD, relative to the control levels (Fig. 3f and g), whereas selenium supplementation significantly increased the enzymatic activity of both GPx and TXNRD in H9c2 cells under H2O2 incubation. These results indicated that selenium supplementation ameliorated the H2O2-induced decrease in TXNRD enzymatic activity, and also increased the GPx activity in H9c2 cells with H2O2 treatment.
Selenium supplementation ameliorates H2O2-induced inactivation of the PI3K/AKT signaling pathway in H9c2 cells
PI3K/AKT signaling is a downstream target of ROS production that is extensively involved in the cell cycle and apoptosis.35 The PI3K/AKT signaling pathway has been reported to be involved in oxidative stress-induced cardiotoxicity.36 We used western blot analysis to evaluate the PI3K/AKT activation in H9c2 cells cultured in the presence of Na2SeO3 for 72 h and then optionally exposed to H2O2 for 4 h, to elucidate the mechanisms underlying the protective effects of selenium against H2O2-induced cell cycle arrest. The protein levels of PI3K, the phosphorylation of downstream protein AKT (Ser-473) and the level of upstream protein PTEN were analyzed by western blotting. H2O2 incubation significantly decreased the protein content of PI3K to levels below those of the control cells, whereas the decrease recovered with Na2SeO3 pretreatment, however, PTEN showed no significant difference among the control, H2O2, Na2SeO3, and Na2SeO3 + H2O2 treated cells (Fig. 4a–c). Moreover, H2O2 incubation decreased AKT phosphorylation relative to the control levels, and Na2SeO3 pretreatment counteracted the effect of H2O2 treatment (Fig. 4a and d). The results indicated that H2O2 induced inactivation of the PI3K/AKT signaling pathway, whereas selenium supplementation ameliorated this adverse effect in H9c2 cells.
Selenium supplementation ameliorates the H2O2-induced inactivation of the PI3K/AKT signaling pathway in H9c2 cells. H9c2 cells were cultured for 72 h in 5% FBS-containing medium supplemented with 0 or 500 nM Na2SeO3, and then the cells were cultured in 0 or 500 μM H2O2. (a) Western blot analysis of phosphorylation of AKT (Ser-473) and upstream protein expression levels of PTEN and PI3K. Semi-quantitative analyses of the protein levels of (b) PTEN, (c) PI3K, and (d) phosphorylation of AKT. Data are expressed as means ± S. E. M. (n = 3). *P < 0.05 vs. untreated cells, #P < 0.05 vs. H2O2 alone.
Selenium supplementation ameliorates H2O2-induced cell cycle arrest at the S phase through the PI3K/AKT signaling pathway in H9c2 cells
To validate whether the PI3K/AKT pathway might contribute to the cardioprotective effect of selenium, we pretreated H9c2 cells with LY294002 (10 μM), a PI3K inhibitor, for 1 h before H2O2 treatment. LY294002 reversed the protective effects of selenium supplementation on H2O2-induced cell cycle arrest, as determined by the cell phase distribution and cell cycle regulatory systems. In H9c2 cells, LY294002 incubation weakened the protective effects of selenium on H2O2-induced cell cycle arrest at the S phase by increasing the population of cells in the S phase from 30.61 ± 0.56% to 32.66 ± 0.69% and decreasing the population of cells in the G2/M phase from 7.65 ± 0.20% to 6.84 ± 0.10% (Fig. 5a and b). In addition, the mRNA and protein levels of CDK1 and the mRNA level of CCNB1 decreased, whereas the mRNA level of p21 increased (Fig. 5c, d and f). The results indicated that inhibition of the PI3K/AKT signaling pathway by LY294002 antagonized the protective effect of selenium against H2O2-induced H9c2 cell cycle arrest in the S phase by targeting the G2/M phase inhibitory system (CDK1 and p21). AKT inhibits the activity of GSK3β by phosphorylating serine-9 in the N-terminal tail of GSK3β.37 The efficacy of PI3K inhibition was confirmed through the phosphorylation of GSK3β, and LY294002 antagonized the effect of Na2SeO3 on the dephosphorylation of GSK3β in H2O2-induced H9c2 cells (Fig. 5e). Therefore, these data suggest that the protective effect of Na2SeO3 in H2O2-induced cell cycle arrest at the S phase in H9c2 cells is mediated at least in part through PI3K/AKT/GSK3β signaling.
The alleviation of H2O2-induced cell cycle arrest by selenium supplementation depends on the PI3K/AKT signaling pathway in H9c2 cells. H9c2 cells were treated with 0 or 500 nM Na2SeO3 for 72 h, and then pre-cultured in serum-free medium in the presence or absence of 10 μM LY294002, an inhibitor of PI3K, and stimulated further with 500 μM H2O2 for an additional 4 h. (a) Representative flow cytometry diagrams of H9c2 cells. (b) Quantitative analysis of the results of (a). (c) CDK1, CDK2, CDK4, CCNB1 and p21 mRNA expression levels in H9c2 cells. (d) After the cells were lysed, the proteins were separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detected by western blotting. Semi-quantitative analyses of phosphorylation of GSK3β (e) and CDK1 (f). GAPDH was used for normalization. Data are expressed as means ± S.E.M. (n = 3). *P < 0.05 vs. untreated cells, #P < 0.05 vs. H2O2 alone, δP < 0.05 vs. H2O2 and Na2SeO3.
Selenium deficiency induced-oxidative stress inactivates PI3K/AKT signaling and promotes the G2/M-phase inhibitory system
In the Se-Def group compared with the Se-Met group, the MDA content in the porcine heart was significantly higher (Fig. 6a). The T-AOC content in the porcine heart was lower in the Se-Def group than the Se-Met group, however, the SOD activity showed no significant difference between the Se-Def and the Se-Met group (Fig. 6b and c). Meanwhile, the TXNRD activity was significantly lower in the Se-Def group than the Se-Met group (Fig. 6d). A marked decrease in the protein content of PI3K and the phosphorylation of downstream protein AKT (Ser-473) was detected in the Se-Def group compared with the Se-Met group (Fig. 6e and f). Additionally, the mRNA and protein content of CDK1 were significantly lower in the Se-Def group than the Se-Met group (Fig. 6e–g), the mRNA expression level of CCNB1 decreased, whereas the mRNA level of p21 increased (Fig. 6i and j). These results indicated that selenium deficiency-induced oxidative stress in the porcine heart inactivated the PI3K/AKT signaling pathway, thus promoting the G2/M-phase inhibitory system.
Selenium deficiency induced oxidative stress and inactivated the PI3K/AKT signaling pathway in the porcine heart. The (a) MDA content, (b) relative SOD levels, (c) T-AOC and (d) TXNRD activity were detected with commercial reagent kits (n = 4). (e) After the porcine heart tissue was lysed, the proteins were separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detected by western blotting. (f) Densitometry scanning analysis of the expression of PI3K and the cell cycle regulatory protein CDK1 (n = 3). (g) Semi-quantitative analyses of phosphorylation of AKT (n = 3). (h) CDK1, CDK2, CDK4, CCNB1 and p21 mRNA expression levels in pig hearts (n = 6). GAPDH was used for normalization. Data are expressed as means ± S.E.M. *P < 0.05.
Discussion
Selenium has cardioprotective effects due to its regulation of selenoproteins, which have antioxidant capacity, and thus has attracted the attention of researchers. However, the protective effects and mechanisms of selenium against oxidative stress-induced cardiomyocyte cell cycle arrest are unclear. We demonstrated that selenium supplementation markedly attenuates oxidative stress-induced cell cycle arrest at the S phase in H9c2 cells and counteracts the selenium-deficiency induced promotion of the G2/M phase inhibitory system in pig hearts. Importantly, we verified that the cardioprotective effects of selenium on oxidative stress-induced cell cycle arrest might be mediated by the selenoprotein-associated antioxidant capacity, which probably affects redox status-related pathways, such as the activation of the PI3K/AKT pathway, that promote cell cycle progression by targeting the G2/M phase inhibitory system, including CDK1 and p21 (Fig. 7). These results provide new insights into the molecular mechanisms underlying the protective effects of selenium on oxidative stress-induced cardiomyopathy in clinical trials and in the animal production industry.
The overall mechanism through which selenium supplementation alleviates oxidative stress-induced cell cycle arrest in cardiomyocytes. Oxidative stress was induced in H9c2 cells by H2O2 and in pig hearts by selenium deficiency. Oxidative stress inactivates the PI3K/AKT signaling pathway, thus activating the G2/M phase inhibitory system through directly or indirectly (GSK3β-mediated) activating p21, and then inhibits the expression of CDK1, which contributes to the inhibition of cardiomyocyte entry into the G2/M phase. Selenium supplementation reverses the effects of oxidative stress on cell cycle arrest, an effect possibly mediated by the antioxidant capacity of the selenoproteins (GPx and TXNRD).
Numerous studies have used the H9c2 rat cardiomyoblast cell line as an animal-free alternative model,9,19,38,39 because of growing concern regarding the need to reduce the number of animals used in research (National Centre for the Replacement, Refinement and Reduction of Animals in Research, London, UK; NIH Office of Laboratory Animal Welfare, Bethesda, MD). Additionally, H9c2 cells show many similarities to primary cardiomyocytes, including the membrane morphology, G-protein signaling and electrophysiological properties, although H9c2 cells are unable to beat.40–42 Importantly, H9c2 cells are a proliferating cell line, which provides an excellent alternative in vitro model for cardiomyocyte cell cycle experimental studies. In this study, we established an H2O2 induced-oxidative stress model in H9c2 cells in vitro, in which the cell cycle was arrested at the S phase (Fig. 2). A previous study has demonstrated that H2O2-induced K562 cells are arrested at the S phase,43 whereas the cell cycle is arrested at G2/M in H9c2 cells incubated with 250 mM H2O2 for 4 h.9 The potential reasons for the above discrepancy are multifactorial and may be due to differences in the sensitivity of cell phase-specific checkpoint proteins to H2O2 in different cell cycle phases, and to large variations in the H2O2 concentration. In this study, H2O2 promoted the G2/M phase inhibitory system by decreasing CDK1 and increasing p21 at the mRNA level in H9c2 cells (Fig. 2c). Selenium has been reported to promote G2/M phase progression by targeting CDK1, CCND1, p21 and p27.44,45 Additionally, in this study, selenium supplementation attenuated the H2O2-induced promotion of the G2/M inhibitory system and inhibited the S phase inhibitory system (Fig. 2). Therefore, we conclude that selenium supplementation alleviates H2O2-induced cell cycle arrest at the S phase through targeting G2/M-phase inhibitory systems in H9c2 cells.
Studies reported that the medium is typically deficient in selenium in cell culture studies, and the level of basal selenium appears not adequate to get the maximum possible activity of GPx with different cells in different culture conditions.46–48 The results reported in this study indicated that at least 72 h and 500 nM Na2SeO3 pretreatment were necessary to exhibit the protective effect against oxidative stress-induced cell cycle arrest (Fig. 1 and 2). Toxicity effects were observed over time in response to a too high concentration (>500 nM Na2SeO3 incubation for 72 h) of Na2SeO3 (Fig. 1b), while short-term (<72 h) supplementation with 500 nM Na2SeO3 shows no protective effect against oxidative stress-induced toxicity in H9c2 cells (Fig. 1c). This might suggest that a high concentration of selenium for a long period is required to protect cardiomyocytes from oxidative stress-induced cell cycle arrest, and these data were consistent with a previous report that high doses of selenium for long periods are required to protect thyroid follicular cells from damage-induced mortality.49
Emerging evidence suggests that oxidative stress is responsible for the development and progression of heart failure.7,50 Deficiency in trace elements participating in antioxidant defense is present in patients with heart disease.51 Selenium has been suggested as a potential co-treatment for heart disease, owing to its participation in antioxidant defense due to its regulation of selenoproteins, which mainly play antioxidant regulatory roles,34 especially the GPx and TXNRD family, which play central roles in the heart's antioxidant system, and selenium deficiency is involved in heart disease.34,52–55 Herein, in H9c2 cells selenium supplementation ameliorated the H2O2-induced oxidative stress and significantly increased the activity of both GPx and TXNRD in H2O2-induced oxidative stress (Fig. 3). H2O2 is widely accepted to decrease antioxidant enzyme activity, while the inhibition of enzyme activity by H2O2 is time- and concentration-dependent, repeated low doses of H2O2 have been reported to enhance the activity of antioxidant enzymes.56,57 In the present study, under H2O2-induced oxidative stress, the enzymatic activity of GPx increased, whereas that of TXNRD decreased without selenium supplementation, possibly because the GPx and TXNRD systems differ in their responses to oxidative stress.58 The early response of the GSH-dependent system to alleviate the H2O2-induced oxidative stress might account for the more selenium incorporated into GPx and thus elevated GPx enzymatic activity under H2O2 induction. In addition, selenium supplementation ameliorated the H2O2-induced oxidative stress and significantly increased the activity of both GPx and TXNRD in H2O2-induced oxidative stress in H9c2 cells (Fig. 3), since supplementation of 500 nM Na2SeO3 is adequate for GPx to maximize its activity, and enough selenium was left for the synthesis of TXNRD, thus inducing the increased TXNRD activity. Therefore, the increased GPx and TXNRD enzyme activity in the presence of selenium supplementation in H2O2-induced H9c2 cells contributed to improving oxidative stress, thus alleviating oxidative stress-induced cell cycle arrest in H9c2 cells. Accordingly, the TXNRD activity was significantly decreased in hearts (Fig. 6d), kidneys25 and livers24 of pigs fed selenium deficient diets, and the GPx activity also significantly decreased in selenium deficient pigs24,25,30 and chickens.59 On the basis of the above results, GPx and TXNRD might contribute to the protective effects of selenium supplementation on oxidative stress-induced cell cycle arrest in cardiomyocytes. It warrants further study to explore other selenoproteins that might be involved in the process.
The PI3K/AKT pathway is involved in the response to H2O2-induced oxidative stress in cardiomyocytes.60,61 In particular, the PI3K/AKT pathway is an intracellular signaling pathway that plays a crucial role in cardiomyocyte cell cycle processes.22 Activation of PI3K phosphorylates and activates AKT, which has a number of downstream effects such as inhibiting p27.62 Previous evidence has indicated that PI3K/AKT promotes cell cycle progression in cardiomyocytes.23 Activation of the PI3K/AKT pathway phosphorylates the N terminal serine of GSK3β, thus inhibiting GSK3β activity and increasing the expression of the downstream protein cyclin D1, thereby resulting in cell cycle progression in skeletal muscle.22 Additionally, activation of PI3K/AKT increases CDK2 expression and decreases p27 expression, thus facilitating cell cycle progression.22,62 Given these considerations, we found that activation of the PI3K/AKT pathway appears to be responsible for the protective effect of selenium in the cell cycle arrest induced by H2O2, because LY294002, an inhibitor of PI3K, abolished the cardioprotective effects of selenium (Fig. 5). In addition, selenium supplementation resulted in the recovery of the H2O2-induced decrease in phospho-AKT and an increase in phospho-GSK3β (Fig. 4d and 5e). Consistent with previous results indicating that activation of PI3K/AKT increases cyclin-dependent kinase and decreases CDK inhibitor expression,22 selenium supplementation-mediated activation of the PI3K/AKT pathway may account for the increased expression of CDK1 and CCNB1 and the decreased expression of p21 at the mRNA level after H2O2 incubation (Fig. 2). Furthermore, in vivo study, selenium deficiency induced oxidative stress and inactivated PI3K/AKT signaling in the pig heart (Fig. 6) and chicken heart,63 and subsequently inhibited CDK1 and CCNB1, and increased p21 in the porcine heart (Fig. 6). Therefore, the selenium supplementation-mediated activation of PI3K may aid in alleviating oxidative stress-induced cell cycle arrest through inhibition of cell entry into the G2/M phase by targeting the G2/M inhibitory system (CDK1 and p21).
The protective effect of selenium on oxidative stress-induced cell cycle arrest through PI3K/AKT signaling was demonstrated in the present study. The relationship between H2O2-induced oxidative stress and the inactivation of the PI3K/AKT signaling pathway has been confirmed in H9c2 cells in previous studies.64 The adverse effects of oxidative stress occur mainly through the promotion of oxidation-induced inactivation of phosphatases, notably the effects of oxidation and phosphorylation of PTEN, which regulates cell cycle progression.65–67 Additionally, PTEN is phosphorylated and undergoes disulfide bond formation between Cys-297 and Cys-311 under H2O2-induced oxidative stress in H9c2 cells.68 In this study, the total PTEN levels showed no significant alterations under H2O2-induced oxidative stress (Fig. 4b), although the oxidation and phosphorylation of PTEN might have been altered under this condition. We speculate that multiple factors, including oxidation and phosphorylation of PTEN or other factors, contribute to altering the phosphorylation of PI3K/AKT signaling under H2O2-induced oxidative conditions in H9c2 cells. Whether selenium supplementation directly affects PTEN activity altered by oxidative stress, thus regulating PI3K/AKT activity, warrants further study.
Conclusions
In summary, we conclude that selenium supplementation protects cardiomyocytes against oxidative stress-induced cell cycle arrest, potentially via a process involving selenoprotein-associated (GPx and TXNRD) alleviation of oxidative stress, thereby activating the redox status-associated PI3K/AKT pathway, which promotes cell cycle progression by targeting the G2/M phase inhibitory system (CDK1 and p21). These findings provide new insight into the underlying mechanisms of the cardioprotection effects of selenium at the cellular level.
Funding
This research was funded by the National Key Research and Development Program of China, grant/award number: 2018YFD050040001-02/03; National Natural Science Foundation of China, grant/award number: 31802073; The Chinese Academy of Agricultural Science and Technology Innovation Program, grant/award number: ASTIP-IAS-12.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgements
This research was funded by the National Key Research and Development Program of China, grant/award number: 2018YFD050040001-02/03; National Natural Science Foundation of China, grant/award number: 31802073; The Chinese Academy of Agricultural Science and Technology Innovation Program, grant/award number: ASTIP-IAS-12.
References
Footnotes
Electronic supplementary information (ESI) available. See DOI: 10.1039/d0mt00225a
