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Introduction

Saturated fatty acids, including palmitate (C16:0), but not monounsaturated oleate (C18:1), have been demonstrated to induce apoptosis in a number of cell types, including the cardiomyocyte element of the heart (1–3). Cardiac myocyte apoptosis is an important contributor to myocardial dysfunction and heart failure due to a systematic reduction in the number of cardiomyocytes (4). Abnormally high blood fatty acid levels in patients result in acute myocardial infarction and increase the extent and severity of myocardial injury (5–7). In vitro studies have indicated that palmitate-induced cell apoptosis in cardiomyocytes is associated with the mitochondria-dependent apoptotic pathway, including cytochrome c release, loss of the mitochondrial membrane potential and consequent caspase-3 activation (8,9).

A number of studies have hypothesized that increasing reactive oxygen species (ROS) production in cardiac cells and accompanying oxidative stress are major initiators of cardiovascular injury (10,11). The tumor suppressor protein, p53, activates transcription of proapoptotic genes in several cell types prior to the onset of apoptosis (12,13). The ratio of pro- and antiapoptotic Bcl-2 family members is crucial for cell apoptosis in response to lipotoxicity. p53 has been identified to regulate Bcl-2 family genes, leading to initiation of apoptosis (14). In addition, p53-transactivated genes have been revealed to increase ROS and general oxidative damage to all mitochondrial components, which disrupts mitochondrial oxidative phosphorylation, thereby contributing to ischemia/reperfusion injury (15–17). The extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway is important for cell proliferation, growth and cell death (18). Previous studies have demonstrated that activation of the ERK1/2 signaling pathway is involved in the apoptotic process (19,20). More recently, the ROS-activated ERK1/2 signaling pathway was found to be an important regulatory mechanism responsible for chemical hypoxia-induced cardiomyocyte injury (21). In addition, it has also been reported that excessive levels of glucose and free fatty acids induce tissue-specific cellular damage via increasing ROS (8,22,23). However, the underlying molecular mechanisms associated with cell apoptosis induced by palmitate remain unclear in cardiomyocytes.

The present study aimed to identify the mechanisms of lipotoxicity in H9c2 cells and to test the hypothesis that ROS production and the ERK1/2 signaling pathway are critical for palmitate-induced apoptosis in H9c2 cells.

Materials and methods

Chemicals and reagents

Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM) and penicillin/streptomycin were obtained from Hyclone Laboratories, Inc. (Logan, UT, USA). Antibodies against p-Akt, total-Akt, caspase-3, Bcl-2, Bax and poly (ADP-ribose) polymerase (PARP) and horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies against p-ERK1/2, total ERK1/2, p53 and β-actin were purchased from Millipore (Billerica, MA, USA). Enhanced chemiluminescence (ECL) reagent was purchased from Millipore. Palmitate and bovine serum albumin (BSA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of 5 mM PA/10% BSA were prepared as previously described (24) and stored at −20°C. Stock solutions were heated for 15 min at 55°C and then cooled to room temperature prior to use.

Cell culture

H9c2 cells (a subclone of an original clonal cell line derived from embryonic BD1X rat heart tissue exhibiting a number of skeletal muscle properties) were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China) and grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin in a humidified atmosphere of 95% air and 5% CO2 at 37°C. When cells reached ~80% confluence, various doses of palmitate were added to the complete medium and incubated for 12 h.

Evaluation of cell apoptosis by Hoechst 33342 staining

Cells were plated in 6-well chamber slides and allowed to adhere. Following 12 h of treatment, cells from each group were washed with phosphate-buffered saline (PBS) and fixed with 4% formalin for 10 min. Hoechst 33342 (10 μg/ml) was applied for 30 min under dark conditions to stain the nuclei of the fixed cells. Slides were then washed with PBS and mounted in a mounting medium (PBS:glycerol, 1:1). Cells were examined and images were captured using a fluorescence microscope. Apoptotic cells were identified as those with a nucleus exhibiting brightly stained condensed chromatin or unclear fragments. Normal nuclei exhibited blue chromatin with an organized structure. For each experimental condition, four separate cell populations were prepared. Apoptotic indices were determined by direct visualization and counting of a minimum of 500 cells/population (≥100 cells from five randomly selected fields). The apoptotic index was calculated using the following formula: (number of apoptotic cells/total cells counted) × 100.

Assessment of cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

Briefly, H9c2 cells were cultured in 96-well plates. Palmitate at concentrations of 0, 100 or 150 μM was added to the wells. Following treatment with palmitate for 12 h, the culture medium was replaced with 200 μl MTT solution (5 mg/ml stock solution in PBS, diluted with culture medium to a final concentration of 0.5 mg/ml). Following 4 h of incubation at 37°C, the solution was removed and the produced formazan was solubilized in 150 μl dimethyl sulfoxide. The absorbance was measured at 550 nM using an automated microplate reader.

Measurement of intracellular ROS levels

Intracellular ROS levels were determined using a peroxide-sensitive fluorescent probe, 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Beyotime Institute of Biotechnology, Shanghai, China). DCFH-DA is converted by intracellular esterases to DCFH, which is oxidized into the highly fluorescent dichlorofluorescein in the presence of a specific oxidant. Cells were plated in 6-well chamber slides and allowed to adhere. Following 12 h of treatment, cells were loaded with 1 mM DCFH-DA in DMEM in the dark for 30 min at 37°C. Following incubation, cells were washed with PBS three times and mounted with a coverslip. Slides were immediately analyzed and images were captured on an Olympus Inverted Fuorescence microscope. For every well, six fields were randomly selected to photograph and integrated optical densities of the images were determined. Positive cells were treated with 6 mM hydrogen peroxide for 1 h prior to addition of the fluorescent dye.

Western blot analysis

The cells were lysed in ice-cold RIPA buffer [50 mM Tris/HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% deoxycholate and 0.1% sodium dodecyl sulfate (SDS)] and protease inhibitor phenylmethanesulfonyl fluoride (PMSF). The protein concentration of the samples was determined using the bicinchoninic acid (BSA) protein assay reagent kit (Pierce Biotechnology, Inc., Rockford, IL, USA) with BSA as the standard. For western blot analysis, 40 μg protein was denatured by heating at 100°C for 10 min in SDS sample buffer, loaded onto and separated by 10 or 12% SDS polyacrylamide gels and then transferred to a polyvinylidene fluoride membrane. The membrane was blocked in 5% (w/v) nonfat milk with 0.05% Tween-20 Tris-buffered saline buffer for 1 h and then incubated overnight at 4°C with the following primary antibodies: monoclonal anti-Akt (1:1,000), anti-p-Akt (1:1,000), anti-caspase-3 (1:1,000), anti-PARP (1:1,000), anti-p-ERK1/2 (1:500), anti-ERK1/2 (1:500), anti-Bcl-2 (1:1,000), anti-p53 (1:500) and anti-Bax (1:1,000). Anti-β-actin (1:1,500) was used to determine equal loading of the protein. Following this, membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (1:3,000) for 1 h at 37°C, washed and visualized for immunoreactivity using an ECL system. Optical density was determined using FluorChem FC2 and readings were normalized to a control sample and presented in an arbitrary densitometry unit.

Statistical analysis

Quantitative data are presented as the mean ± SEM determined from at least three independent experiments. Statistical analysis was based on the Student’s t-test for comparison of two groups or one-way analysis of variance for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

Results

Palmitate-induced apoptosis of H9c2 cells

H9c2 cells were treated with various concentrations of palmitate (0, 100 and 150 μM) for 12 h. A marked increase in the number of apoptotic cells was observed in H9c2 cells by Hoechst 33342 staining (Fig. 1A and B) and decreased cell viability was measured by the MTT assay (Fig. 1C). Western blot analysis results revealed that expression of active caspase-3 (Fig. 1D and E) and cleaved PARP (Fig. 1D and F), two well-established hallmarks of apoptosis, was increased following treatment with palmitate (25,26). These results indicate that activation of caspase-3 and PARP is necessary for palmitate-induced apoptosis in H9c2 cells.

Figure 1 Palmitate-induced apoptosis of H9c2 cells. Cells were treated with palmitate (0, 100 and 150 μM) for 12 h. (A and B) Cell apoptosis was determined by Hoechst 33342 staining and (C) cell viability was measured using the MTT assay. Levels of (D and E) active caspase-3 and (D and F) cleaved PARP were measured by western blot analysis. Results are reported as mean ± SEM. *P<0.05, vs. control. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PARP, poly (ADP-ribose) polymerase; PA, palmitate.

ROS levels increased during palmitate treatment in H9c2 cells

Cellular ROS levels in H9c2 cells treated with various concentrations of palmitate (0, 100 and 150 μM) were examined. Following treatment with palmitate for 12 h, intracellular ROS levels were measured using a DCFH-DA assay. Intracellular ROS levels were identified to be significantly increased in H9c2 cells when exposed to 100 and 150 μM palmitate for 12 h compared with the control (Fig. 2). Intracellular ROS production was significantly deceased when exposed to 150 μM palmitate combined with the ERK1/2 inhibitor, U0126, compared with exposure to 150 μM palmitate alone (Fig. 2). These results indicate that palmitate induces H9c2 cell apoptosis and increases ROS generation simultaneously.

Figure 2 Effect of ROS generation during palmitate treatment in H9c2 cells. Cells were exposed to palmitate (0, 100 and 150 μM) or 150 μM palmitate combined with ERK1/2 inhibitor, U0126, for 12 h. Intracellular ROS was determined using DCFH-DA. Results are presented as mean ± SEM. *P<0.05, vs. control and *P<0.05, vs. 150 μM palmitate. ROS, reactive oxygen species; ERK1/2, extracellular signal-regulated kinase 1/2; DCFH-DA, 2′,7′-dichlorofluorescein diacetate; PA, palmitate.

Effect of Bax, Bcl-2 and p53 protein expression during palmitate treatment in H9c2 cells

To determine the correlation between palmitate-induced apoptosis and Bcl-2 family members, proapoptotic Bax and antiapoptotic Bcl-2 protein expression in H9c2 cells was determined by western blot analysis (Fig. 3A-C). Proapoptotic Bax expression was increased and antiapoptotic Bcl-2 levels were decreased by palmitate treatment. p53 protein expression was also found to be increased following palmitate-induced apoptosis in H9c2 cells (Fig. 3A and D). Results indicate that Bax and Bcl-2 are involved in H9c2 cell apoptosis via palmitate induction. In addition, p53 was also involved in palmitate-induced apoptosis in H9c2 cells.

Figure 3 Effect of palmitate on Bax, Bcl-2 and p53 protein expression in H9c2 cells. Cells were incubated with palmitate (0, 100 and 150 μM) for 12 h. Protein levels of (A and B) proapoptotic Bax, (A and C) antiapoptotic Bcl-2 and (A and D) p53 were measured by western blot analysis. Results are reported as mean ± SEM. *P<0.05, vs. control. PA, palmitate.

ERK1/2 and Akt signaling pathways were involved in palmitate-induced apoptosis of H9c2 cells

To investigate the apoptosis-related signaling pathways that are activated in H9c2 cells treated with palmitate for 12 h, levels of p-ERK1/2 and p-Akt were examined using western blot analayis following H9c2 cell exposure to various concentrations of palmitate (0, 100 and 150 μM). Levels of p-ERK1/2 were found to be significantly increased (Fig. 4A and B) and p-Akt was markedly decreased (Fig. 4A and C) following H9c2 cell treatment with palmitate. These observations demonstrate that the ERK1/2 and Akt signaling pathways were involved in palmitate-induced apoptosis in H9c2 cells. To further determine the role of ERK1/2 in palmitate-induced apoptosis of H9c2 cells, the ERK1/2 inhibitor, U0126 (10 μM), was used, as described previously (27). Levels of active caspase-3 and cleaved PARP were found to decrease significantly when treated with U0126 in the presence of 150 μM palmitate (Fig. 4D-F). Levels of ROS production were observed to be decreased significantly compared with 150 μM palmitate alone (Fig. 2). The results demonstrate that the ERK1/2 signaling pathway is involved in palmitate-induced apoptosis in H9c2 cells.

Figure 4 ERK1/2 and Akt signaling pathways are involved in palmitate-induced apoptosis in H9c2 cells. Cells were exposed to palmitate (0, 100 and 150 μM) or 150 μM palmitate combined the ERK1/2 inhibitor, U0126, for 12 h. Protein levels of (A and B) p-ERK1/2, (A and C) p-Akt, (D and E) cleaved PARP and (D and F) active caspase-3 were measured by western blot analysis. Results are presented as mean ± SEM. *P<0.05, vs. control and *P<0.05, vs. 150 μM palmitate. ERK1/2, extracellular signal-regulated kinase 1/2; PARP, poly (ADP-ribose) polymerase; PA, palmitate.

Discussion

Cardiac myocyte apoptosis is an important contributor to myocardial dysfunction and heart failure and serum fatty acid levels, including palmitate, have been observed at elevated levels in patients with acute myocardial infarction (4,6). A number of studies have reported that increased levels of ROS production are a contributing factor to apoptosis in cardiac myocytes (28–31). In the current study, ROS production was identified at increased levels following palmitate-induced apoptosis in H9c2 cells, consistent with previous studies (2,32). Increasing ROS production has been demonstrated to account for apoptosis in response to palmitate in numerous cell lines, including neutrophils and fibroblasts and endothelial, pancreatic β and hepatic cells (33–36). In addition, the results of the present study indicate that increased levels of ROS production may be one of the mechanisms by which palmitate induces apoptosis in H9c2 cells. By contrast, this observation is inconsistent with a previous study that demonstrated that palmitate-induced apoptosis in neonatal rat cardiomyocytes was not dependent on increased ROS production levels (37). The cause of this discrepancy is currently unknown. Nevertheless, present results support the hypothesis that increased ROS production promotes apoptosis in palmitate-induced in H9c2 cells.

The ratio of pro-and antiapoptotic Bcl-2 family members is crucial for cell apoptosis in response to lipotoxicity. The antiapoptotic protein Bcl-2 is considered an important cellular component and prevents cell apoptosis. Proapoptotic Bax promotes apoptosis. Previous studies have indicated that apoptosis is associated with upregulation of proapoptotic Bax and downregulation of antiapoptotic Bcl-2 (38,39). The results of the present study also demonstrated that levels of Bcl-2 were attenuated and Bax were increased in response to palmitate-induced apoptosis in H9c2 cells. These changes in levels of Bcl-2 family members may have a direct effect on mitochondrial membrane pore formation and consequent activation of caspase-3 and PARP activity, therefore leading to activation of the intrinsic apoptotic pathway. The tumor suppressor protein p53 is a transcription factor with a short half-life and is present at low levels in normal cells. p53 is known to activate transcription of proapoptotic genes in specific cell types prior to the onset of apoptosis (12,13). Bcl-2 family members may be directly transactivated by p53, which is a sensor of cellular stress, by transcriptionally regulating these genes to initiate intrinsic apoptosis (14). Previous studies have also demonstrated that p53 transactivates genes responsible for increased ROS production and general oxidative damage to all mitochondrial components. These events disrupt mitochondrial oxidative phosphorylation, thereby contributing to a number of human diseases, including ischemia/reperfusion injury (15–17). In the present study, results indicate that increased p53 levels were accompanied by increasing levels of ROS production, reducing the ratio of Bcl-2/Bax and resulting in palmitate-induced apoptosis in H9c2 cells. However, the mechanism by which palmitate induces apoptosis of H9c2 cells requires additional studies.

ERK1/2/mitogen-activated protein kinase is a well-known signal transduction cascade involved in the response to extracellular stimuli and is important for cell proliferation, growth and cell death (18,29). However, the mechanism by which the ERK1/2 signaling pathway is activated in apoptosis remains controversial. Previous studies have indicated that activation of the ERK1/2 signaling pathway mediates cell proliferation and survival (40,41). In addition, sustained activation of the ERK1/2 pathway has been associated with the apoptotic process (19,20,31). A recent study indicated that the ROS-activated ERK1/2 signaling pathway is an important regulatory mechanism responsible for chemical hypoxia-induced cardiomyocyte injury (21). In the present study, apoptosis induced by palmitate simultaneously increased ROS production and levels of p-ERK1/2 in H9c2 cells. The ERK1/2 inhibitor, U0126, decreased ROS production and attenuated palmitate-induced apoptosis in H9c2 cells through decreasing the activity of caspase-3 and PARP. In addition, the PI3K/Akt signaling pathway has been identified to play a major role in the prevention of apoptosis (42). Acute activation of this signaling pathway has been demonstrated to promote cardiomyocyte survival and function in vitro and in vivo(43). Current results indicate that p-Akt levels were decreased following palmitate-induced apoptosis in H9c2 cells.

The results of the present study indicate that palmitate induced apoptosis in H9c2 cells through activation of the ERK1/2 signaling pathway and increasing levels of intracellular ROS generation, resulting in activation of caspase-3 and PARP activity. These results are likely to provide insight into the mechanisms by which ROS mediate oxidative stress in response to palmitate, leading to lipotoxicity cardiomyopathy.

Acknowledgements

The current study was supported by the National Clinical Key Specialty Construction Projects and National Natural Science Foundation of China (no. 81100959).

References