HL-1 adult murine cardiomyocytes (kindly donated by Prof. W. Claycomb, Department of Biochemistry and Molecular Biology, LSUHSC, New Orleans, LA, USA) were cultured in Claycomb Medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM glutamine, and 0.1 mM norepinephrine in an atmosphere of 95% O2 and 5% CO2 at 37°C. The HL-1 cells were grown under fibronectin/gelatin, and were treated with CoCl2, carbobenzoxyl-leucinyl-leucinyl-leucinal-H (MG132), and MGO (Sigma-Aldrich, St. Louis, MO, USA).

The viability of the HL-1 cells was assessed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Briefly, after treatments, cells were incubated with MTT solution (0.5 mg/ml) for two hours at 37°C in a cell culture incubator. Subsequently, supernatants were removed and acidified isopropanol (0.04 N HCl in isopropanol) was added to dissolve the dark blue crystals of formazan produced by metabolically active cells. Formazan was quantified by measuring the absorbance of the samples using an ELISA automated microplate reader (Biotek, Winooski, VT, USA) at 570 nm, with a reference wavelength of 620 nm.

After the treatments, the cells were washed twice with phosphate-buffered saline (PBS) and denatured in a Laemmli buffer, sonicated and boiled at 95°C for 5 min. The denatured proteins were separated by polyacrylamide gel electrophoresis and electrophoretically transferred onto nitrocellulose membranes. The membranes were blocked for 1 h with 5% m/v of nonfat milk at room temperature and for 1 h with HIF-1α (1:500; SICGEN, Cantanhede, Portugal), β-actin (1:1000, Sigma Aldrich, St. Louis, MO, USA), GADPH (1:5000; SICGEN, Cantanhede, Portugal) and ubiquitin (1:1000, BioLegend, San Diego, CA, USA) primary antibodies at 4°C overnight, followed by incubation with the corresponding secondary antibodies at room temperature for 1 h. Specific protein bands were detected using an electrochemiluminescence detection system (Bio-Rad, USA). β-actin and GAPDH were used as loading control.

The HL-1 cells were washed with PBS, lysed with 50 mM Tris, pH 7.6, supplemented with 1 mM DTT and sonicated. After centrifugation (16000 g for 10 minutes at 4°C), the protein concentration was determined and 40 μg of protein was incubated with 100 μM Suc-LLVY-MCA (Biomol-Enzo Life Sciences, Farmingdale, NY, USA) in a 96-well plate. Chymotrypsin-like activity was monitored for 1 h at 37°C in 5 min periods (excitation wavelength 380 nm; emission wavelength 460 nm). Absorbance was measured on a Biotek Synergy HT spectrophotometer (Biotek, Winooski, VT, USA).

Cell lysates were prepared by solubilization in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.1% SDS, pH 7.6), containing protease and phosphatase inhibitors. The samples were centrifuged at 3200 rpm for 5 min, and the supernatants used for immunoprecipitation. Briefly, the supernatants were incubated with antibodies against HIF-1α. Non-specific (anti-GFP) antibodies were used for negative controls. Incubations proceeded overnight at 4°C followed by incubation with protein G-sepharose (Sigma-Aldrich, St. Louis, MO, USA) for 2 h at 4°C. The protein G-sepharose sediments were washed in RIPA buffer, eluted in Laemmli buffer, denatured at 95°C for 5 min, and analyzed by western blot.

The results are expressed as mean ± standard error of the mean of at least three independent experiments. The data were analyzed using one-way analysis of variance followed by the Tukey post-hoc test, using GraphPad Prism 5.0 software (San Diego, CA, USA). Differences between means were considered significant for values of p<0.05.

Hyperglycemia was previously shown to be involved in the loss of cell response to hypoxia. In this study, a cardiac cell line (HL-1) was used to investigate the role of MGO in the regulation of HIF-1α, a transcription factor involved in the cell response to low oxygen tension. The results presented in Figure 1A show that MGO induces a reduction in cell viability in a dose-dependent manner. We then assessed the effect of MGO in cells under hypoxia. To subject the cells to conditions that partially mimic hypoxia, HL-1 cells were incubated with CoCl2. HL-1 cells treated with CoCl2 for 6 h in the presence of 3 mM of MGO added to the culture in the final 3 h present a significant reduction in cell viability compared to cells exposed to hypoxia in the absence of MGO (Figure 1B). Importantly, incubation with CoCl2 alone did not affect cell viability.

MGO treatment in the absence (A) or presence of chemical hypoxia (B), and MTT assay was used to assess cell viability. The percentage of cytotoxicity was calculated relative to control (untreated cells). Data are mean ± standard error of the mean of three independent experiments performed in quadruplicate. * p<0.05, significantly different from control cells and MGO 1 mM/30 min; ** p<0.01, significantly different from control cells (one-way analysis of variance with Tukey's multiple comparison test). MGO: methylglyoxal.'> MGO treatment in the absence (A) or presence of chemical hypoxia (B), and MTT assay was used to assess cell viability. The percentage of cytotoxicity was calculated relative to control (untreated cells). Data are mean ± standard error of the mean of three independent experiments performed in quadruplicate. * p<0.05, significantly different from control cells and MGO 1 mM/30 min; ** p<0.01, significantly different from control cells (one-way analysis of variance with Tukey's multiple comparison test). MGO: methylglyoxal.' title='Cell viability under hypoxic conditions. HL-1 cells were subjected to MGO treatment in the absence (A) or presence of chemical hypoxia (B), and MTT assay was used to assess cell viability. The percentage of cytotoxicity was calculated relative to control (untreated cells). Data are mean ± standard error of the mean of three independent experiments performed in quadruplicate. * p<0.05, significantly different from control cells and MGO 1 mM/30 min; ** p<0.01, significantly different from control cells (one-way analysis of variance with Tukey's multiple comparison test). MGO: methylglyoxal.'/>

Figure 1.

Cell viability under hypoxic conditions. HL-1 cells were subjected to MGO treatment in the absence (A) or presence of chemical hypoxia (B), and MTT assay was used to assess cell viability. The percentage of cytotoxicity was calculated relative to control (untreated cells). Data are mean ± standard error of the mean of three independent experiments performed in quadruplicate. * p<0.05, significantly different from control cells and MGO 1 mM/30 min; ** p<0.01, significantly different from control cells (one-way analysis of variance with Tukey's multiple comparison test). MGO: methylglyoxal.

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Previous data from our group demonstrated that MGO induces the degradation of HIF-1α in retinal epithelial cells under hypoxia.2 However, the impact of MGO on HIF-1α in cardiac cells has never been addressed. We first investigated the effects of various MGO concentrations on HIF-1α levels in HL-1 cells under hypoxic conditions. After reaching confluence, the cells were exposed to CoCl2 to induce chemical hypoxia for 6 h and MGO (1 mM or 3 mM) was added in the last 30 min or 3 h of hypoxia. After the treatments, the cells were harvested and HIF-1α levels were determined by western blotting. As expected, under normoxic conditions HIF-1α was undetectable (Figure 2A, lane 1), but after 6 h of incubation with CoCl2 the amount of HIF-1α increased dramatically (Figure 2A, lane 2). However, when cells were simultaneously incubated with CoCl2 and MGO for 3 h, MGO impaired hypoxia-dependent accumulation of HIF-1α (Figure 2A; compare the 110 kDa band in lanes 4 and 6 with lane 2), suggesting that MGO induces degradation of HIF-1α accumulated in the course of hypoxia.

MGO on HIF-1α levels under hypoxia conditions. (A) HL-1 cells were exposed to CoCl2 (400 μM) for 6 h and then MGO (1 mM or 3 mM) was added in the last 30 min or 3 h. Whole-cell extracts were prepared and analyzed by western blotting using anti-HIF-1α or anti-β-actin antibodies (loading control); (B) HL-1 cells were exposed to CoCl2 (400 μM for 6 h) and MGO (3 mM for the last 3 h of treatment). HIF-1α was immunoprecipitated and immunoprecipitates were probed against HIF-1α and ubiquitin. MGO: methylglyoxal; WB: western blotting.'> MGO on HIF-1α levels under hypoxia conditions. (A) HL-1 cells were exposed to CoCl2 (400 μM) for 6 h and then MGO (1 mM or 3 mM) was added in the last 30 min or 3 h. Whole-cell extracts were prepared and analyzed by western blotting using anti-HIF-1α or anti-β-actin antibodies (loading control); (B) HL-1 cells were exposed to CoCl2 (400 μM for 6 h) and MGO (3 mM for the last 3 h of treatment). HIF-1α was immunoprecipitated and immunoprecipitates were probed against HIF-1α and ubiquitin. MGO: methylglyoxal; WB: western blotting.' title='Effect of MGO on HIF-1α levels under hypoxia conditions. (A) HL-1 cells were exposed to CoCl2 (400 μM) for 6 h and then MGO (1 mM or 3 mM) was added in the last 30 min or 3 h. Whole-cell extracts were prepared and analyzed by western blotting using anti-HIF-1α or anti-β-actin antibodies (loading control); (B) HL-1 cells were exposed to CoCl2 (400 μM for 6 h) and MGO (3 mM for the last 3 h of treatment). HIF-1α was immunoprecipitated and immunoprecipitates were probed against HIF-1α and ubiquitin. MGO: methylglyoxal; WB: western blotting.'/>

Figure 2.

Effect of MGO on HIF-1α levels under hypoxia conditions. (A) HL-1 cells were exposed to CoCl2 (400 μM) for 6 h and then MGO (1 mM or 3 mM) was added in the last 30 min or 3 h. Whole-cell extracts were prepared and analyzed by western blotting using anti-HIF-1α or anti-β-actin antibodies (loading control); (B) HL-1 cells were exposed to CoCl2 (400 μM for 6 h) and MGO (3 mM for the last 3 h of treatment). HIF-1α was immunoprecipitated and immunoprecipitates were probed against HIF-1α and ubiquitin. MGO: methylglyoxal; WB: western blotting.

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Interestingly, besides the presence of a 110 kDa band, under hypoxia HIF-1α accumulated as a slower migrating smear that most likely corresponded to ubiquitinated forms (Figure 2A). Strikingly, when HL-1 cells were simultaneously incubated with CoCl2 and MGO for 30 min, the intensity of the slower migrating bands was significantly increased, suggesting that ubiquitination was being enhanced (Figure 2A). However, for longer periods of incubation with MGO, reduction of both 110 kDa and high molecular weight bands of HIF-1α was observed (Figure 2A, lane 4 and lane 6 compared with lane 3 and lane 5), strongly suggesting that HIF-1α undergoes degradation in these conditions. To address whether MGO reduces the ubiquitination of HIF-1α, the protein was immunoprecipitated and probed with anti-ubiquitin antibodies. The results obtained show that levels of ubiquitinated HIF-1α increase in cells treated with MGO (Figure 2B, compare lane 1 with lane 2 of IP). We also assessed the effects on endogenous ubiquitin conjugates by western blot using polyclonal anti-ubiquitin antibodies (Figure 2B, Input). The results showed an increase in the endogenous conjugates of high molecular weight ubiquitin in cells subjected to hypoxia and in the presence of MGO.

Having established that MGO promotes degradation of HIF-1α under hypoxia, we proceeded to assess the involvement of UPP in the destabilization of HIF-1α. For this, HL-1 cells subjected to hypoxia in the presence of MGO were incubated with a proteasome inhibitor (MG132), after which HIF-1α levels were assessed. In these conditions an accumulation of HIF-1α was observed (Figure 3A, compare lane 3 with lane 5), suggesting that MGO induces HIF-1α degradation by the proteasome under hypoxia. Moreover, as expected, under normoxic conditions HIF-1α was undetectable. However, the results show an accumulation of HIF-1α when the cells were treated with a proteasome inhibitor in normoxia, confirming that HIF-1α is mainly degraded by the proteasome in normoxic conditions (Figure 3B, compare lane 1 with lane 2).

MGO on UPP activity. (A) HL-1 cells were treated with CoCl2 (400 μM; 6 h) with MGO (3 mM; 3 h) and MG132 (20 μM; 4 h). The cell extracts were used to measure chymotrypsin-like activity using the fluorogenic substrate Suc-LLVY-AMC. The degree of change in chymotrypsin-like activity was calculated relative to control (untreated) cells. The values in the graph correspond to measurements after 30 min of activity. Data are mean ± standard error of the mean of three independent experiments performed in quadruplicate; (B) HL-1 cells were exposed to CoCl2 (400 μM; 6 h), MGO (3 mM; 3 h) and MG132 (20 μM; 4 h). Whole-cell extracts were prepared and analyzed by western blotting using anti-HIF-1α or anti-β-actin antibodies (loading control). MGO: methylglyoxal; UPP: ubiquitin-proteasome pathway.'> MGO on UPP activity. (A) HL-1 cells were treated with CoCl2 (400 μM; 6 h) with MGO (3 mM; 3 h) and MG132 (20 μM; 4 h). The cell extracts were used to measure chymotrypsin-like activity using the fluorogenic substrate Suc-LLVY-AMC. The degree of change in chymotrypsin-like activity was calculated relative to control (untreated) cells. The values in the graph correspond to measurements after 30 min of activity. Data are mean ± standard error of the mean of three independent experiments performed in quadruplicate; (B) HL-1 cells were exposed to CoCl2 (400 μM; 6 h), MGO (3 mM; 3 h) and MG132 (20 μM; 4 h). Whole-cell extracts were prepared and analyzed by western blotting using anti-HIF-1α or anti-β-actin antibodies (loading control). MGO: methylglyoxal; UPP: ubiquitin-proteasome pathway.' title='Effect of MGO on UPP activity. (A) HL-1 cells were treated with CoCl2 (400 μM; 6 h) with MGO (3 mM; 3 h) and MG132 (20 μM; 4 h). The cell extracts were used to measure chymotrypsin-like activity using the fluorogenic substrate Suc-LLVY-AMC. The degree of change in chymotrypsin-like activity was calculated relative to control (untreated) cells. The values in the graph correspond to measurements after 30 min of activity. Data are mean ± standard error of the mean of three independent experiments performed in quadruplicate; (B) HL-1 cells were exposed to CoCl2 (400 μM; 6 h), MGO (3 mM; 3 h) and MG132 (20 μM; 4 h). Whole-cell extracts were prepared and analyzed by western blotting using anti-HIF-1α or anti-β-actin antibodies (loading control). MGO: methylglyoxal; UPP: ubiquitin-proteasome pathway.'/>

Figure 3.

Effect of MGO on UPP activity. (A) HL-1 cells were treated with CoCl2 (400 μM; 6 h) with MGO (3 mM; 3 h) and MG132 (20 μM; 4 h). The cell extracts were used to measure chymotrypsin-like activity using the fluorogenic substrate Suc-LLVY-AMC. The degree of change in chymotrypsin-like activity was calculated relative to control (untreated) cells. The values in the graph correspond to measurements after 30 min of activity. Data are mean ± standard error of the mean of three independent experiments performed in quadruplicate; (B) HL-1 cells were exposed to CoCl2 (400 μM; 6 h), MGO (3 mM; 3 h) and MG132 (20 μM; 4 h). Whole-cell extracts were prepared and analyzed by western blotting using anti-HIF-1α or anti-β-actin antibodies (loading control). MGO: methylglyoxal; UPP: ubiquitin-proteasome pathway.

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We also assessed the effect of hypoxia and MGO on chymotrypsin-like activity of the 20S proteasome by degradation of the Suc-LLVY-MCA fluorogenic peptide (Figure 3B). The cells were treated with chemical hypoxia for 6 h in the presence or absence of MGO added to the culture medium in the final 3 h of hypoxia. Incubation with MG132 was used as a positive control. The results presented in Figure 3B show that proteasome activity decreased by about 50% in cells subjected to hypoxia. Moreover, simultaneous incubation with MGO did not have additional effects on proteasome activity.

The authors declare that no experiments were performed on humans or animals for this study.

The authors declare that no patient data appear in this article.

The authors declare that no patient data appear in this article.