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CELLULAR METABOLISM
1Department of Cell Biology and 2Department of Medicine, Division of Cardiovascular Disease, University of Alabama at Birmingham, Birmingham, Alabama
Submitted 7 April 2006 ; accepted in final form 30 July 2006
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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hexosamine biosynthesis; calcium; protein O-glycosylation
The O-GlcNAc modification of proteins is unlike other glycosylation events in that it occurs through an enzyme-catalyzed reaction in the cytosol and the nucleus rather than in the Golgi apparatus or the endoplasmic reticulum. Posttranslational modification by O-GlcNAc is a dynamic and reversible process, regulated by the activities of two key enzymes, O-GlcNAc transferase (OGT) (18, 21) and N-acetylglucosaminidase (O-GlcNAcase) (11). In some cases, O-GlcNAcylation is reciprocal with phosphorylation (5, 7), and proteins that have been identified as being modified by O-GlcNAcylation include kinases, phosphatases, cytoskeletal proteins, nuclear hormone receptors, nuclear pore proteins, signal transduction molecules, and actin regulatory proteins (38). Furthermore, O-GlcNAcylation has also been reported to influence protein transcription and translation, nuclear targeting and transport, and protein degradation (7, 13, 29). Consequently, alterations in O-GlcNAc levels have important and wide-ranging effects on cell function.
Schaffer et al. (30) reported that hyperglycemia significantly reduced hypoxia-induced apoptosis and necrosis in isolated cardiomyocytes and showed that this was associated with decreased Ca2+ overload. Our laboratory (25) recently reported that increased levels of O-GlcNAc in cardiomyocytes decreased angiotensin II-mediated increase in cytosolic Ca2+. The facts that O-GlcNAc levels are increased by hyperglycemia and that elevated O-GlcNAc levels increase the tolerance of cells to stress raise the possibility that the protection associated with increased glucose utilization could be mediated, at least in part, via this pathway. Therefore, the goal of this study was to determine in isolated neonatal cardiomyocytes whether an increase in protein O-GlcNAcylation resulted in improved survival following ischemia-reperfusion and if so, whether protection conferred by hyperglycemia was also mediated via the same pathway.
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METHODS |
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Neonatal rat ventricular myocyte primary cultures. Animal experiments were approved by the University of Alabama Institutional Animal Care and Use Committee and conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85–23, 1996). Primary cultures of neonatal rat ventricular myocytes (NRVMs) were obtained from 2- to 3-day-old neonatal Sprague-Dawley rats and cultured as described previously (15). Within 1–2 days of isolation, a confluent monolayer of spontaneously beating NRVMs had formed, and cells were used as described below.
Simulated ischemia and reperfusion. Ischemia and reperfusion were induced based on the method described by Brar et al. (3). After 1–2 days in culture, NRVMs were exposed to ischemia by addition of a fresh Esumi modified ischemic medium (137 mM NaCl, 12 mM KCl, 0.49 mM MgCl2, 0.9 mM CaCl2·2H2O, 4 mM HEPES, and 20 mM sodium lactate, pH 6.2) and then incubated in a chamber with an atmosphere of 95% argon and 5% CO2 for 4 h. After 4 h of ischemia, cells were returned to the maintenance growth medium [serum-free 4:1 (vol/vol) Dulbecco's modified Eagle's medium/medium 199 with Hanks' salts, supplemented with 2% Nutridoma and 1% penicillin/streptomycin] and then incubated in an atmosphere of 21% O2 and 5% CO2 for 16 h. In control normoxia experiments, cells were incubated with fresh maintenance growth medium in an atmosphere of 21% O2 and 5% CO2 for 20 h.
Measurement of cell viability, necrosis, and apoptosis. Cell viability was measured by Trypan blue exclusion. We mixed 10 µl of cell suspension with 90 µl of 0.04% Trypan blue in PBS. A total of at least 200 cells were counted using a hemocytometer.
Necrosis was assessed by determining the release of lactate dehydrogenase (LDH) in culture medium and the LDH in remaining attached cells using an LDH assay kit (Sigma). The percent LDH release was calculated as the ratio of the LDH released into the medium to the total LDH (release plus cellular content).
Apoptosis was assessed using the in situ cell death detection kit (Roche). Permeabilized cells were exposed to the TdT-mediated dUTP nick end labeling reaction mixture and were counterstained with 0.1 mg/ml propidium iodide solution. In each treatment, a total of at least 300 cells were counted through a x40 objective with excitation wavelength at 495 nm.
Measurement of UDP-GlcNAc and ATP.
UDP-N-acetylglucosamine (UDP-GlcNAc) and ATP were analyzed in acid extracts by performing HPLC as described previously (26, 28). Nucleotide sugars were monitored by UV detector at 262 nm, and known standard sugars were used for calibration. This method cannot separate UDP-GlcNAc from UDP-N-acetylgalactosamine (UDP-GalNAc), so the results are presented as the sum of UDP-GlcNAc and UDP-GalNAc (UDP-HexNAc) (28); however, in cardiomyocytes the ratio of UDP-GlcNAc to UDP-GalNAc is 
3:1 (6).
Measurement of O-GlcNAc levels. O-GlcNAc levels were determined by Western blot analysis using the anti-O-GlcNAc antibody CTD110.6 (Covance) as previously described (20). The specificity of CTD110.6 for O-GlcNAc was previously reported by Comer et al. (8).
Cells were lysed with 1x RIPA [50 mM Tris·HCl, pH 8.0, 150 mM NaCl, 1% (vol/vol) Nonidet P-40, 0.5% (wt/vol) sodium deoxycholate, 1 mM EDTA, and 0.1% SDS] containing 2% protease inhibitor cocktail (Sigma) on ice for 30 min. Lysed proteins were harvested and assayed for protein concentration using the Bio-Rad protein assay kit. Proteins (10 µg) were separated on 7.5% SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Millipore). The blots were then soaked in 100% methanol, dried, and probed with the anti-O-GlcNAc antibody CTD110.6 (Covance) diluted 1:5,000 times in casein blocking buffer (Pierce) containing 0.01% Tween 20 for 2 h at room temperature. After being washed three times with PBS, the membrane was then incubated with a 1:5,000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgM (Calbiochem) in 1% casein/PBS with 0.01% Tween 20 for 1 h. After further washing in PBS, the immunoblots were developed with enhanced chemiluminescence (SuperSignal West Pico; Pierce), and visualization was performed using the BioImaging system of UVP (Upland, CA).
Previous studies have reported that stress resulted in a global increase in O-GlcNAc levels (40). Therefore, densitometric analysis of CTD110.6 immunoblots was performed on the entire lane of each sample with the use of LabWorks analysis software (UVP), and the mean intensity was normalized to the control group value. Duplicate gels were stained with Coomassie brilliant blue R-250 for assessment of equal protein loading.
Assessment of ischemia and ischemia-reperfusion on cytosolic Ca2+ levels and translocation of nuclear factor of activated T cells. A direct measure of Ca2+ levels at the end of ischemia was performed using the fluorescent Ca2+ indicator fura-2 AM (Molecular Probes). NRVMs were loaded with fura-2 AM as previously described, and cytosolic Ca2+ levels were determined in cells at the end of 4 h by using alternating 340- and 380-nm excitation wavelengths and emission at 510 nm (12).
Hunton et al. (15) previously showed that a sustained increase in cytosolic Ca2+ levels resulted in the nuclear translocation of a nuclear factor of activated T cells (NFAT) transcription factor, which is mediated by activation of the Ca2+/calmodulin-dependent protein phosphatase calcineurin. Since calcineurin activation has been implicated in mediating cardiomyocyte apoptosis (24, 27), we examined NFAT translocation at the end of ischemia-reperfusion as an indirect assessment of Ca2+ influx and a surrogate marker for calcineurin activation. One day after cell isolation, NRVMs were infected with an adenovirus (100 multiplicity of infection) encoding enhanced green fluorescent protein linked to the nuclear localization region of NFAT (GFP-NFAT). The day after infection, cells were subjected to ischemia-reperfusion experiments as described above. This chimeric GFP-NFAT protein remains in the cytoplasm in resting cells and translocates into the nuclear when cytoplasmic Ca2+ increases sufficiently to activate the Ca2+-dependent phosphatase calcineurin. At the end of the experiment, cells were fixed and imaged through a x40 objective with excitation wavelength at 495 nm; 200 cells were counted for each condition.
Statistics. All data are presented as means ± SE. Unpaired t-tests and one-way and repeated-measures ANOVA were used where appropriate, followed by Bonferroni's multiple comparison test using Prism 4.0c (GraphPad Software, San Diego, CA). Statistically significant differences between groups were defined as P < 0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-actin levels (data not shown). In the control group, UDP-HexNAc levels were significantly increased at the end of ischemia compared with normoxic conditions (2.3 ± 0.2 vs. 3.0 ± 0.2 µmol/g; P < 0.05).
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During ischemia, O-GlcNAc levels increased 45% in the control group (P = 0.07) and more than twofold in the glucosamine-treated group (P < 0.05) (Fig. 2, A and B). During the first hour of reperfusion, O-GlcNAc levels increased in both untreated and glucosamine-treated groups, but this response was markedly enhanced in the glucosamine-treated group (Fig. 2B). In contrast, in the absence of glucose there was no increase in O-GlcNAc levels on reperfusion (Fig. 2, A and B). In untreated cells, O-GlcNAc levels returned to baseline after 8 h of reperfusion and remained relatively constant for the remainder of the reperfusion period. However, in the glucosamine-treated group, O-GlcNAc levels remained elevated for 8 h of reperfusion, returning close to baseline levels by 12 h. In the absence of glucose, there was a gradual decrease in O-GlcNAc levels throughout the reperfusion period. The ability to increase O-GlcNAc levels during the first few hours of reperfusion was associated with improved viability (Fig. 2C), and there was a significant correlation between O-GlcNAc levels and cell viability during both early (2 h) and late (16 h) reperfusion (Fig. 2D).
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50% increase in O-GlcNAc in the glucosamine-treated group, this did not reach statistical significance. We believe that the apparently attenuated response of O-GlcNAc to glucosamine treatment in these experiments is most likely a consequence of the short exposure time required for visualizing the immunoblot due to the very high levels of O-GlcNAc seen with PUGNAc treatment.
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Increased O-GlcNAc attenuates Ca2+-mediated nuclear translocation of NFAT during ischemia and ischemia-reperfusion. Cytosolic Ca2+ levels were measured using fura-2 in untreated and glucosamine-treated NRVMs under normoxic conditions and at the end of 4 h of ischemia. In untreated cells, cytosolic Ca2+ levels increased from 110 ± 12 nM under normoxic conditions to 150 ± 5 nM at the end of ischemia (P < 0.05). In glucosamine-treated cells, normoxic cytosolic Ca2+ levels were not significantly different from those in untreated cells (114 ± 8 nM); however, at the end of ischemia, cytosolic Ca2+ levels were significantly lower than in untreated cells (106 ± 7 nM; P < 0.05).
To determine whether the reduction in Ca2+ during ischemia in the glucosamine-treated group might contribute to the reduction in injury shown in Figs. 1 and 2, we assessed nuclear translocation of GFP-NFAT as an indicator of Ca2+-induced calcineurin activation as previously described (15). Calcineurin has been implicated in mediating cardiomyocyte apoptosis (24, 27). As shown in Fig. 5, under normoxic conditions GFP-NFAT was restricted to the cytoplasm in >90% of the transfected cells; however, at the end of ischemia-reperfusion there was marked increase in nuclear translocation (36 ± 2%) in untreated cells. Glucosamine treatment significantly reduced NFAT translocation compared with untreated controls (18 ± 2%; P < 0.05), whereas alloxan markedly increased NFAT nuclear translocation (87 ± 3%; P < 0.05). Similarly to glucosamine treatment, PUGNAc treatment attenuated NFAT nuclear translocation compared with controls (13 ± 1%; P < 0.05). The impact of glucosamine, alloxan, and PUGNAc on NFAT translocation was remarkably similar to their effects on viability shown in Fig. 4A.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Zachara et al. (40) demonstrated that in COS-7 cells, O-GlcNAc levels increased almost twofold within 3 h following heat stress, returning to baseline levels between 24–48 h, and they showed that this was due, at least in part, to an increase in OGT activity. In the present study, we found that ischemia-reperfusion stress in untreated NRVMs increased O-GlcNAc levels even more quickly with a maximum level, almost threefold higher than baseline, and that this was completely ablated when cells were reperfused in the absence of glucose (Fig. 2B). This finding suggests that increased metabolism of glucose via the HBP is required for the increase in O-GlcNAc in response to ischemia and also may contribute to the increase in O-GlcNAc reported by Zachara et al. (40). This is supported by the fact that increasing glucose concentrations also increased cell survival via a mechanism consistent with increased HBP flux and O-GlcNAc levels (Fig. 3). Furthermore, the strong correlation between cell viability and O-GlcNAc levels during reperfusion provides additional support for the notion that the level of O-GlcNAc in cells is an important determinant of cell viability following a stress such as ischemia-reperfusion (Fig. 2D).
Zachara et al. (40) found that increased O-GlcNAc levels were associated with faster induction of heat shock protein 70 (HSP70) expression, suggesting a possible mechanism underlying the cytoprotection resulting from increased flux through OGT. However, we found no increase in HSP70 expression in response to glucosamine treatment in NRVMs (data not shown), which is consistent with a study by Sohn et al. (32), who showed that overexpression of OGT increased O-GlcNAc levels and cell survival in Chinese hamster ovary cells within 60 min without any change in HSP70 expression during this time frame. The dissociation between changes in cell survival and HSP70 expression suggests that other mechanism(s) operating over a shorter time frame also may contribute to the protection associated with increased O-GlcNAc levels.
Increasing glycolytic ATP production, especially during ischemia, has been shown to decrease injury and improve recovery on reperfusion (9). Since glucosamine could increase glycolytic flux by metabolism via glucosamine-6-phosphate isomerase or deaminase (2, 41), this might represent an alternative protective mechanism independent of alterations in O-GlcNAc levels. We therefore assessed ATP levels in control and glucosamine-treated groups under normoxic conditions, at the end of ischemia, and at the end of ischemia-reperfusion. If the addition of glucosamine increased glycolytic ATP synthesis, we would have expected to see increased levels of ATP, especially at the end of ischemia. However, although ATP levels were slightly increased at the end of ischemia and decreased at the end of reperfusion, these differences did not reach statistical significance (Table 1). Thus these data suggest that the protection seen with glucosamine was not due to increased glycolytic ATP synthesis during ischemia. It is important to note that although maintenance of ATP levels during either ischemia or reperfusion is often associated with improved functional recovery and decreased injury, this is not a prerequisite for ischemic protection. For example, although ischemic preconditioning significantly improves function recovery compared with control hearts following ischemia and reperfusion, ATP hydrolysis during ischemia has been reported to be increased (16), and following reperfusion, ATP levels were not increased in a preconditioned group (4).
Stimulation of myocardial glucose utilization repeatedly has been shown to afford protection against ischemic injury (33); however, there is little consensus regarding the mechanism(s) underlying this protection. In the present study we found that hyperglycemia was protective against ischemia-reperfusion in NRVMs (Fig. 3), that this protection was associated with an increase in O-GlcNAc levels, and that when glucose entry into the HBP was inhibited with azaserine, both the protection and the increase in O-GlcNAc were attenuated (Fig. 3). Thus, although we cannot exclude changes in energy metabolism playing a role in hyperglycemic protection, these data suggest this protection may be mediated, at least in part, by increased HBP flux and O-GlcNAc levels. The fact that the increase in O-GlcNAc seen with hyperglycemia was less pronounced than that seen with glucosamine is not surprising, since glucosamine enters the HBP directly and essentially unregulated, whereas glucose entry into the HBP is regulated by GFAT. However, it is noteworthy that in the hyperglycemia experiments, densitometric analysis showed differential response of individual bands in the CTD110 immunoblot, suggesting that the protection might be mediated via specific proteins rather than a global increase in O-GlcNAc levels.
Work in our laboratory has previously shown that glucosamine inhibited angiotensin II induced, calcineurin-mediated NFAT translocation (15) and have subsequent studies have shown that this was due to attenuation of Ca2+ influx (25). Since calcineurin activation has been implicated in mediating cardiomyocyte apoptosis (24, 27), we asked whether protection resulting from increased O-GlcNAc levels may also be associated with reduced calcineurin activation. We found that at the end of ischemia-reperfusion, there was marked increase in nuclear localization of GFP-NFAT consistent with an increase in cytosolic Ca2+ (Fig. 5). Importantly, both glucosamine and PUGNAc significantly attenuated the nuclear translocation of GFP-NFAT, whereas alloxan significantly increased GFP-NFAT nuclear localization. Furthermore, the effects of the different interventions on GFP-NFAT nuclear localization are remarkably similar to their impacts on cell viability (Fig. 4). It should be noted that these experiments do not address whether glucosamine attenuates severe Ca2+ overload that is also associated with ischemia-reperfusion injury. However, our laboratory has recently reported that in the whole heart, glucosamine markedly reduced tissue injury due to severe Ca2+ overload induced by the Ca2+ paradox (20). Thus the data shown in the present study combined with our group's earlier study (20) suggests that the protection associated with increased O-GlcNAc levels may be due, at least in part, to inhibition of Ca2+ entry into NRVMs and subsequent attenuation of Ca2+-mediated necrosis and apoptosis.
The specific proteins affected by O-GlcNAcylation that might be mediating the protection against ischemia-reperfusion injury remain to be identified. However, our previous studies have shown that the effects of glucosamine on cardiomyocyte Ca2+ homeostasis were specific for a capacitative Ca2+ entry (CCE) pathway rather than other sarcolemmal Ca2+ channels (15). The transient receptor potential (TRP) channel protein family are prime candidates for mediating CCE (10, 23), and analysis of the protein sequences for both TRP1 and TRP4 indicates that both proteins contain serine residues with a potentially high affinity for O-GlcNAc modification (www.cbs.dtu.dk/services/YinOYang/). Alternatively, kinases such as Akt, ERK1/2, p38, and PKC that have been shown to play a role in mediating ischemic cardioprotection (1, 14) also have been shown to be targets for O-GlcNAcylation or for their activity to be modulated by changes in O-GlcNAc levels (38). Clearly, further studies are warranted not only to identify cardiac proteins that are targets for O-GlcNAcylation but also to determine how these proteins are affected by ischemic stress and how changes in the levels of O-GlcNAc alter the response to stress.
We have shown in the present study that alloxan, a putative inhibitor of OGT (17), markedly reduced O-GlcNAc levels at the end of ischemia-reperfusion, and this was associated with increased cellular injury. We also have shown that the absence of glucose during reperfusion also attenuated the increase in O-GlcNAc levels and reduced cell viability. These data support the notion that metabolism of glucose via the HBP and the formation of O-GlcNAc is a normal stress response and that inhibition of these pathways decreases the tolerance to ischemia-reperfusion stress. However, it is important to note that alloxan is a uracil analog, and thus we cannot rule out potential nonspecific effects of alloxan (34). Nevertheless, in the absence of ischemia-reperfusion, alloxan had minimal effects on viability and no effects on necrosis or apoptosis. Since ablation of the OGT gene is embryonically lethal (31), more specific demonstration of the role of OGT in mediating glucosamine cardioprotection will require the development of tissue-specific, conditional knockout mice or the use of small interfering RNA approaches to reduce OGT expression in cardiomyocytes.
There was a significant correlation between O-GlcNAc levels and cell viability in untreated cells or when glucosamine was used to increase O-GlcNAc (Fig. 2); however, PUGNAc did not lead to greater protection despite the fact that O-GlcNAc levels were markedly higher that in the glucosamine-treated group (Fig. 4). Indeed, if anything, PUGNAc appeared to be somewhat less protective than glucosamine. It has been reported that acceptor protein specificity changes with hyperglycemia or glucosamine treatment (19, 36), and we found that glucosamine and PUGNAc treatment led to increased O-GlcNAc levels in different protein bands. Thus the fact that PUGNAc was not as protective as glucosamine despite the higher overall level of O-GlcNAc may be due to different proteins being O-GlcNAcylated in response to the different interventions. Furthermore, Zachara and Hart (39) proposed that high as well as low levels of O-GlcNAc might trigger apoptosis. Consequently, the dose and duration of PUGNAc treatment used in the present study may have resulted in an excessive level of O-GlcNAc that was sustained for the entire reperfusion period. In contrast, although glucosamine treatment increased O-GlcNAc levels seven- to eightfold during reperfusion (Fig. 2), this was a transient response, and at the end of reperfusion O-GlcNAc levels were only approximately twofold higher than in the untreated group. This suggests that protection may be a result not only of the effect of increased O-GlcNAc levels on specific proteins but also the duration of the increase in O-GlcNAc levels. Cleary, further studies are warranted not only to identify the specific proteins involved in O-GlcNAc-mediated protection but also to better understand the dynamics of O-GlcNAcylation in response to stress.
In conclusion, we have shown that increasing O-GlcNAc levels with glucosamine, high glucose, or PUGNAc are all associated with increased cell survival following ischemia and reperfusion. We also found that ischemia-reperfusion increased O-GlcNAc levels in untreated cells, which was inhibited by reperfusion in the absence of glucose, and that this was associated with lower viability. Furthermore, the improved cell survival in glucosamine- and PUGNAc-treated cells was associated with reduced cytosolic Ca2+ levels during ischemia and ischemia-reperfusion. These data support the concept that activation of metabolic pathways leading to an increase in O-GlcNAc levels is an endogenous stress-activated response and that augmentation of this response improves cell survival. Thus strategies designed to activate these pathways may represent novel interventions for inducing cardioprotection.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* R. B. Marchase and J. C. Chatham contributed equally to this work. 
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REFERENCES |
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P < 0.05 vs. normoxia.
