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Am J Physiol Heart Circ Physiol 292: H2227-H2236, 2007. First published January 5, 2007; doi:10.1152/ajpheart.01091.2006
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Glucosamine cardioprotection in perfused rat hearts associated with increased O-linked N-acetylglucosamine protein modification and altered p38 activation

Norbert Fülöp,1 Zhenghao Zhang,2 Richard B. Marchase,3 and John C. Chatham1,2,3

Departments of 1Medicine, 2Physiology, and 3Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama

Submitted 5 October 2006 ; accepted in final form 27 December 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
We have shown that, in the perfused heart, glucosamine improved functional recovery following ischemia and that this appeared to be mediated via an increase in O-linked N-acetylglucosamine (O-GlcNAc) levels on nucleocytoplasmic proteins. Several kinase pathways, specifically Akt and the mitogen-activated protein kinases (MAPKs) p38 and ERK1/2, which have been implicated in ischemic cardioprotection, have also been reported to be modified in response to increased O-GlcNAc levels. Therefore, the goals of this study were to determine the effect of ischemia on O-GlcNAc levels and to evaluate whether the cardioprotection resulting from glucosamine treatment could be attributed to changes in ERK1/2, Akt, and p38 phosphorylation. Isolated rat hearts were perfused with or without 5 mM glucosamine and were subjected to 5, 10, or 30 min of low-flow ischemia or 30 min of low-flow ischemia and 60 min of reperfusion. Glucosamine treatment attenuated ischemic contracture and improved functional recovery at the end of reperfusion. Glucosamine treatment increased flux through the hexosamine biosynthesis pathway and increased O-GlcNAc levels but had no effect on ATP levels. Glucosamine did not alter the response of either ERK1/2 or Akt to ischemia-reperfusion; however, it significantly attenuated the ischemia-induced increase in p38 phosphorylation and paradoxically increased p38 phosphorylation at the end of reperfusion. These data support the notion that O-GlcNAc may play an important role as an internal stress response and that glucosamine-induced cardioprotection may be mediated via the p38 MAPK pathway.

hexosamine biosynthesis; mitogen-activated protein kinase; Akt; ischemia

IT WAS RECENTLY SHOWN that in mammalian cells, a variety of different stress stimuli increased the level of O-linked N-acetylglucosamine (O-GlcNAc) on nuclear and cytoplasmic proteins. Inhibition of this response increased the sensitivity to stress, whereas augmentation of the O-GlcNAc levels increased tolerance to the same stress stimuli and improved cell survival (63). Sohn et al. (48) reported that in Chinese ovarian cancer cells, inhibition of glutamine:fructose-6-phosphate amidotransferase, which regulates the entry of glucose into the hexosamine biosynthesis pathway (HBP), decreased O-GlcNAc levels and reduced cell survival following heat stress (48). Taken together, these studies suggest that activation of metabolic pathways leading to increased levels of O-GlcNAc is an endogenous stress response of mammalian cells that could represent a potential target for the treatment of ischemic injury.

O-GlcNAc transferase (OGT) is the enzyme catalyzing protein O-GlcNAc modifications, and flux through this enzyme is very sensitive to the concentration of UDP-N-acetylglucosamine (UDP-GlcNAc), which is an end product of the HBP (26, 57). Tissue levels of UDP-GlcNAc are very sensitive to circulating glucose levels, and glutamine, the donor of the amine group, also increases HBP flux (57, 61). Glucosamine, which enters cells via the glucose transporter system and is phosphorylated to glucosamine-6-phosphate by hexokinase, also increased HBP metabolites, including UDP-GlcNAc (51). We have recently shown that in the heart, perfusion with glucosamine rapidly increased O-GlcNAc levels and decreased injury resulting from the Ca2+ paradox and improved recovery of function following zero-flow ischemia and reperfusion (28). Inhibition of OGT with alloxan abrogated this protection; however, the mechanisms underlying the protection associated with glucosamine have yet to be elucidated.

The response of the heart to external stress stimuli, including ischemia, is mediated, in part, by a number of signaling pathways, including extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) (36). The role of p38 in mediating the response of the heart to ischemic injury is somewhat controversial. In many studies ischemia increases p38 phosphorylation, and inhibition of p38 phosphorylation during sustained ischemia is reported to be cardioprotective (2, 3, 21, 31, 36, 39, 49). However, in contrast, brief activation of p38 before ischemia has been shown to contribute to the protection associated with ischemic preconditioning, although this protective effect is critically dependent on both timing and duration of activation (37). It is worth noting that p38 is frequently associated with activation of proapoptotic pathways such as caspase-3 or p53 (22, 34, 64). However, {alpha}B-crystallin and heat shock protein (HSP) 27, which are antiapoptotic, have been shown to play a role in ischemic protection (12, 18, 32, 41) and are both downstream of p38 (27, 44). Recently, several studies have demonstrated that ERK activation, especially during reperfusion, is important in mediating protection associated with insulin and ischemic preconditioning (2, 3, 15, 37). A number of studies have also reported that Akt activation protects against ischemia-reperfusion injury in the heart (11, 14, 17). Recently, Hausenloy et al. (15) described the reperfusion injury salvage kinase pathway in which activation of prosurvival kinases Akt and ERK1/2, particularly at the time of reperfusion, contribute to ischemic protection.

Interestingly, activation of HBP and increased O-GlcNAc levels have been reported to modify p38 phosphorylation and activity (6, 13, 19). We have recently shown that increasing O-GlcNAc levels in human neutrophils increased basal and agonist-stimulated phosphorylation of both p38 and ERK1/2 (23). Other studies have also demonstrated that increased HBP and/or O-GlcNAc levels also affect Akt phosphorylation (40, 52). Taken together, these studies suggest that key kinases implicated in ischemic cardioprotection can be modulated by changes in HBP flux and O-GlcNAc levels. Therefore, in light of recent studies showing that increased O-GlcNAc levels were associated with increased tolerance to stress, we investigated the effect of ischemia on the flux through the HBP and cardiac O-GlcNAc levels. We also examined the effect of glucosamine-mediated increase in protein O-GlcNAc levels on the response of p38, ERK1/2, and Akt phosphorylation to ischemia-reperfusion in the isolated perfused heart.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Animals. 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, Revised 1996). Nonfasted, male Sprague-Dawley rats (Charles Rivers Laboratories) weighing 351 ± 6 g were used.

Materials. All chemicals were purchased from Sigma-Aldrich unless otherwise stated.

Experimental groups. Hearts were divided into five perfusion groups: 1) normoxia, 2) 5 min of low-flow ischemia (LFI; 0.3 ml/min), 3) 10 min of LFI, 4) 30 min of LFI, and 5) 30 min of LFI and 60 min of reperfusion. In the normoxic group, hearts were perfused for a total of 60 min. In the LFI groups, hearts were allowed to stabilize for 30 min and then subjected to LFI for the indicated time period. In each perfusion group, hearts were perfused with or without glucosamine (5 mmol/l) for the duration of the experiment. There were four to six replicates in each group as indicated in the Table and figure legends.

Isolated heart perfusions. Animals were anesthetized with intraperitoneal ketamine hydrochloride injection (100 mg/kg, Lloyd Laboratories). Hearts were rapidly excised and perfused retrogradely as previously described (9), and coronary flow was adjusted to maintain a constant perfusion pressure of 75 mmHg. The perfusion buffer consisted of a Krebs-Henseleit buffer containing (in mmol/l) 1.0 lactate, 0.1 pyruvate, 0.32 palmitate, 0.5 glutamine, and 3% BSA (fatty acid free) (Serologicals Proteins) plus 50 µU/ml insulin (NovoNordisk). Cardiac function was monitored via a fluid-filled balloon placed into the left ventricle. End-diastolic pressure (EDP) was set to 5 mmHg by adjusting balloon volume. All hearts were paced at 320-beats/min rate during the whole experiment, except the LFI period and the first 5 min of reperfusion. Ventricular fibrillation was defined as fast mechanical activity with minimal left ventricular developed pressure during the unpaced, first 5 min of reperfusion. At the end of the experiment, hearts were freeze clamped with liquid nitrogen-cooled tongs.

Western blot analysis. Hearts were ground to a fine powder under liquid nitrogen; homogenized on ice in T-PER (Pierce) containing 5% protease inhibitor cocktail, 40 µmol/l O-(2-acetamido-2-deoxy-D-glucopyranosylidene)-amino-N-phenylcarbamate (Carbogen), 1 mmol/l sodium orthovanadate, and 20 mmol/l sodium fluoride; and centrifuged for 10 min at 15,000 g. The protein concentration of the supernatant was measured using Bio-Rad Protein Assay Kit. Whole heart lysates were separated on 10% SDS-PAGE and transferred to polyvinylidene difluoride membrane (Pall). Equal loading of protein was confirmed by Sypro Ruby staining (Bio-Rad) on the membranes. Blots were probed with the appropriate antibody in casein blocking buffer. Anti-O-GlcNAc antibody, CTD110.6 (Covance), total and phospho (Thr180/Tyr182)-p38 (Santa Cruz), total and phospho (Thr202/Tyr204)-ERK1/2 (Cell Signaling), and total and phospho (Ser473)-Akt (Cell Signaling) antibodies were used. Blots were visualized with enhanced chemiluminescent assay (Pierce), and the signal was detected with UVP BioChemi System (UVP). Densitometry was quantified using Labworks analysis software (UVP).

HPLC. Approximately 50 mg of frozen tissue powder were homogenized in 1 ml ice-cold 0.3 mol/l perchloric acid and centrifuged for 15 min at 15,000 g at 4°C. Perchloric acid was removed from the supernatant with two volumes of 1:4 trioctylamine:1,1,2-trichloro-trifluoroethane mixture. Samples were loaded on Partisil 10 SAX column (Beckman), nucleotide sugars were measured at 262 nm using 2 ml/min flow rate, and linear salt and pH gradient were from 5 to 750 mmol/l (NH4)H2PO4 and from pH 2.8 to 3.7 (42). 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); however, in the heart, the ratio of UDP-GlcNAc to UDP-GalNAc is ~3:1 (10).

Data analysis. Data are presented as means ± SE. Differences between experimental groups were evaluated with Student's t-test for unpaired data, two-way ANOVA, or one-way ANOVA tests as appropriate. Statistically significant differences between groups were defined as P < 0.05 and are indicated in the figure legends.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Glucosamine treatment improved functional recovery following ischemia. Before ischemia, there was no significant difference in contractile function between control and glucosamine groups; however, baseline coronary flow was significantly increased in the glucosamine group (Table 1). As previously reported in this model (54, 55), the onset of LFI resulted in a rapid decrease in contractile function and gradual increase in EDP (Fig. 1A). At the end of LFI, EDP was significantly attenuated in the glucosamine group (control 43.1 ± 3.1 vs. glucosamine 23.8 ± 3.0 mmHg; P < 0.05; Fig. 1B). After 30 min of LFI and 60 min of reperfusion, functional recovery was significantly higher in the glucosamine group compared with the control group (Fig. 1C); however, coronary flow was decreased by the same proportion in both groups compared with preischemic values (Fig. 1C). Interestingly, whereas four out of five control hearts (80%) fibrillated during early reperfusion, none of the glucosamine-treated hearts fibrillated (0%; P < 0.05; {chi}2-test).


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  		Table 1. Preischemic cardiac function in control and glucosamine-treated groups

 

Figure 1
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  		Fig. 1. A: Time course of changes in end-diastolic pressure (EDP; n = 11 replicates in each group). *P < 0.05 vs. control (two-way ANOVA with Bonferroni post hoc test). B: EDP at the end of 30 min of ischemia (n = 11 replicates in each group). *P < 0.05 vs. control (Student's t-test). C: percentage of recovery of function compared with baseline values (n = 5 replicates in each group). *P < 0.05 vs. control (Student's t-test). RPP: rate-pressure product (heart rate x left ventricular developed pressure); dP/dt, maximal rate of change in left ventricular pressure over time; GlcN, glucosamine.

 
Effect of ischemia-reperfusion and glucosamine on UDP-HexNAc, O-GlcNAc, and ATP. In the absence of glucosamine, 5 min of ischemia significantly increased UDP-HexNAc concentrations, and longer periods of ischemia (10 and 30 min) increased levels further (Fig. 2A). After 60 min of reperfusion, UDP-HexNAc levels returned to the normoxic levels. Under normoxic perfusion conditions, glucosamine increased UDP-HexNAc levels almost twofold (72 ± 7 vs. 126 ± 7 nmol/g tissue wet wt, P < 0.05; Fig. 2A). Similar to that in the control group, there was a significant increase in UDP-HexNAc levels in response to ischemia; however, after 30 min of ischemia, there was no significant difference in UDP-HexNAc levels between control and glucosamine-treated groups (Fig. 2A). At the end of reperfusion, UDP-HexNAc levels in the glucosamine-treated group also decreased compared with those at end ischemia but were still elevated compared with those in the control group.


Figure 2
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  		Fig. 2. A: UDP-HexNAc (i.e., UDP-N-acetylglucosamine plus UDP-N-acetylgalactosamine). B: ATP levels (n = 4 replicates in each group). O-GlcNAc, O-linked GlcNAc. *P < 0.05 vs. control (two-way ANOVA with Bonferroni post hoc test).

 
In the control group, protein O-GlcNAc levels increased in response to ischemia; after 10 min of ischemia, O-GlcNAc levels were ~1.5-fold higher than normoxic levels. However, in contrast to UDP-HexNAc levels, O-GlcNAc levels declined between 10 and 30 min of ischemia, and at the end of reperfusion, they were significantly lower than normoxic levels (Fig. 3A). In contrast to the control group, ischemia did not change O-GlcNAc levels in the glucosamine-treated group; however, at the end of reperfusion, O-GlcNAc levels were significantly increased compared with those in normoxia (Fig. 3B). In Fig. 4, comparisons of O-GlcNAc levels between control and glucosamine-treated groups are shown at each time point. It can be seen that glucosamine treatment significantly increased O-GlcNAc levels by at least 60% at all time points; the biggest difference in O-GlcNAc levels was at the end of reperfusion. This presumably reflects the fact that, in the control group at the end of reperfusion, O-GlcNAc levels were lower compared with those in normoxia, whereas in the glucosamine group, they were higher (Fig. 3, A and B).


Figure 3
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  		Fig. 3. Effect of ischemia-reperfusion (I/R) on O-GlcNAc. A: representative CTD110.6 immunoblots from control hearts (top) and CTD110.6 area densities relative to 0 min of ischemia (bottom) (n = 4 replicates in each group). B: representative CTD110.6 immunoblots from glucosamine-treated hearts (top) and CTD110.6 area densities relative to 0 min of ischemia (bottom) (n = 4 replicates in each group). *P < 0.05 vs. 0 min (one-way ANOVA with Dunnet post hoc test).

 

Figure 4
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  		Fig. 4. Effect of glucosamine on protein O-GlcNAc under normoxic conditions after 5, 10, and 30 min of ischemia and following I/R. Left: CTD110.6 immunoblots. Right: total area densities normalized to control group. *P < 0.05 vs. control (Student's t-test).

 
As expected, ATP levels decreased in both control and glucosamine-treated groups during ischemia and remained lower than normoxic perfusion at the end of ischemia-reperfusion (Fig. 2B). However, despite the attenuation of ischemic contracture and the improved functional recovery at the end of reperfusion, glucosamine treatment did not attenuate ATP loss during ischemia and did not increase ATP levels during reperfusion.

Effect of glucosamine on ERK, Akt, and p38 phosphorylation. Comparisons of total and phosphorylation levels of ERK, Akt, and p38 in control and glucosamine-treated groups at each time point are shown in Figs. 5, 6, and 7, respectively. The time courses of changes in phosphorylation in the two groups normalized to normoxic conditions are summarized in Fig. 8. Glucosamine treatment had no effect on ERK1/2 or Akt phosphorylation compared with that in the control group under any conditions (Figs. 5 and 6). Consistent with other studies (15), there was a marked decrease in the ratio of phospho-ERK1/2 to total ERK1/2 during ischemia and a significant increase after reperfusion compared with normoxic conditions in both groups (Fig. 8A). Akt phosphorylation increased following 5 min of ischemia; however, after a longer period of ischemia, Akt phosphorylation markedly decreased (Fig. 8B), similar to that seen with ERK (Fig. 8A). At the end of reperfusion, Akt phosphorylation returned to normoxic levels. The changes in Akt phosphorylation after 30 min of ischemia and reperfusion are consistent with previous studies (46).


Figure 5
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  		Fig. 5. Effect of glucosamine on ERK1/2 phosphorylation under normoxic conditions and following 5, 10, and 30 min of ischemia or I/R. Left: phospho- and total-ERK1/2 immunoblots. Right: phospho-/total area densities normalized to control group.

 

Figure 6
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  		Fig. 6. Effect of glucosamine on Akt phosphorylation under normoxic conditions and following 5, 10, and 30 min of ischemia or I/R. Left: phospho- and total-Akt immunoblots. Right: phospho-/total area densities normalized to control group.

 

Figure 7
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  		Fig. 7. Effect of glucosamine on p38 phosphorylation under normoxic conditions and following 5, 10, and 30 min of ischemia or I/R. Left: phospho- and total-p38 immunoblots. Right: phospho-/total area densities normalized to control group. *P < 0.05 vs. control (Student's t-test).

 

Figure 8
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  		Fig. 8. Effect of I/R and glucosamine on signaling pathways. A: ERK1/2 phosphorylation. B: Akt phosphorylation. C: p38 phosphorylation. *P < 0.05 (two-way ANOVA with Bonferroni post hoc test); n = 6 replicates in 0- and 30-min groups, n = 5 replicates in 90-min group, and n = 4 replicates in 5- and 10-min groups.

 
Under normoxic conditions and after 5 and 10 min of ischemia, p38 phosphorylation was unchanged by glucosamine treatment; however, at the end of ischemia, p38 phosphorylation was significantly attenuated in the glucosamine group (Fig. 7). In contrast, at the end of reperfusion, p38 phosphorylation was increased in the glucosamine group (Fig. 7). In Fig. 8C, it can be seen that, consistent with previous reports, ischemia increased phospho-p38 levels by more than threefold in the control group compared with normoxic levels (5) (Fig. 8C). However, in the glucosamine group, ischemia increased phospho-p38 by ~50%, consistent with the 30-min data in Fig. 7. At the end of reperfusion, phospho-p38 levels returned to normoxic levels in the control group, whereas in the glucosamine group, phospho-p38 levels were significantly elevated compared with both ischemic and normoxic conditions (Fig. 8C).

Interestingly, we found that there was a significant linear correlation between O-GlcNAc levels and p38 phosphorylation in the glucosamine-treated group (r2 = 0.31, P < 0.05, data not shown); however, there was no significant correlation in the control hearts (r2 = 0.01, P > 0.7).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 DISCLOSURES
 REFERENCES
 
In cell culture systems, previous studies have demonstrated that O-GlcNAc levels are increased in response to stress and that augmentation of this response increased tolerance to stress (63). We show here for the first time that, in the isolated, perfused hearts, 5–10 min of ischemia significantly increased O-GlcNAc levels, and this was associated with an increase in UDP-HexNAc levels consistent with an ischemia-induced increase in flux through the HBP. Surprisingly, however, after longer periods of ischemia, O-GlcNAc levels decreased, and, after 60 min of reperfusion, they were significantly lower than normoxia. Thus, whereas the increase in O-GlcNAc with ischemia is consistent with the notion that this stress response pathway is active in the heart, the subsequent decrease during reperfusion suggests that the regulation of this pathway may be more complex in the setting of ischemia-reperfusion in the intact heart than previously reported in isolated cell systems (63). Nevertheless, the changes in O-GlcNAc levels in response to ischemia and reperfusion reinforce the notion that this is a highly dynamic posttranslational modification that can be modulated in response to pathophysiological stimuli.

Consistent with our previous reports (28), glucosamine increased normoxic levels of both UDP-HexNAc and O-GlcNAc and improved functional recovery following ischemia-reperfusion. Interestingly, whereas the addition of glucosamine increased O-GlcNAc levels at all time points relative to controls, ischemia did not result in a further increase; however, glucosamine did prevent the decrease in O-GlcNAc that was seen at the end of ischemia and during reperfusion. Whereas the attachment of N-acetylgucosamine is important in the formation of proteoglycans in the Golgi and the endoplasmic reticulum, this is distinct from O-glycosylation, which is specific to the nucleus and the cytosol (47). We have shown that that inhibition of nucleocytoplasmic O-glycosylation with alloxan, an OGT inhibitor, resulted in a loss of protection of glucosamine treatment in isolated cardiomyocytes (8), which supports the notion that the cardioprotective effect of glucosamine is due to the increase in nucleocytoplasmic O-glycosylation. However, since glucosamine also increases the levels of UDP-GlcNAc, the substrate for N-glycosylation, we cannot entirely rule out a possible effect associated with increased levels N-glycosylation. We also found that glucosamine markedly attenuated ischemic contracture and also reduced the incidence of arrhythmias on reperfusion. In addition, glucosamine treatment significantly altered the response of p38 MAPK to ischemia-reperfusion without affecting the response of either ERK or Akt. These data provide further support for the idea that glucosamine cardioprotection is mediated via increased O-GlcNAc levels and show that this is associated with an altered response of p38 MAPK to ischemia-reperfusion.

It is increasingly apparent that protein O-GlcNAcylation is an important and widespread posttranslational modification implicated in regulating a diverse range of cellular processes. The number of proteins identified as being capable of posttranslational O-glycosylation is quickly growing and includes NF-{kappa}B, annexin, endothelial nitric oxide synthase, {alpha}B-crystallin, OGT, {alpha}-tubulin, c-myc, and HSP70 (53, 58). Increased levels of O-glycosylation have often been associated with adverse events such as insulin resistance (1), hyperglycemia-induced apoptosis (29), and impaired excitation-contraction coupling (10). Paradoxically, recent studies have shown that stress increased levels of protein O-GlcNAc in several different mammalian cell lines and that increasing the levels of protein O-GlcNAc enhanced cell survival (48, 63). Sohn et al. (48) also demonstrated that inhibition of the HBP increased hyperthermal sensitivity of cells, suggesting that activation of this pathway may be a component of endogenous cell survival pathway.

Zachara et al. (63) reported that O-GlcNAc levels increased in response to stress, possibly as a consequence of an increase in OGT activity. Here, in the intact heart, we found that after 5–10 min of ischemia, both UDP-HexNAc and O-GlcNAc levels were significantly increased. Since flux through OGT is very sensitive to UDP-GlcNAc levels (26, 57), the increase in UDP-HexNAc could explain the increase in O-GlcNAc levels seen here; however, we cannot rule out an increase in OGT activity. The increase in UDP-HexNAc during ischemia could be due to either an increase in flux through the hexosamine pathway and/or a consequence of decreased utilization of UDP-GlcNAc via other pathways. Sohn et al. (48) showed that inhibition of glutamine:fructose-6-phosphate amidotransferase, which regulates the entry of glucose into the HBP, prevented the stress-induced increase in O-GlcNAc and decreased hyperthermal tolerance. This would be consistent with increased flux through the HBP also increasing UDP-GlcNAc. However, UDP-GlcNAc is also required for multiple N-glycosylation reactions that are involved in protein synthesis; since ischemia is known to inhibit protein synthesis (60), it is possible that this could also contribute to the increase in UDP-GlcNAc seen here.

Interestingly, even though UDP-HexNAc continued to increase during ischemia, O-GlcNAc levels had decreased after 30 min of ischemia. The dissociation between UDP-HexNAc and O-GlcNAc suggests that OGT activity might be inhibited after prolonged ischemia. If so, this could also contribute to the further decline in O-GlcNAc levels seen during reperfusion. Regulation of OGT activity is complex and poorly understood; however, OGT is known to be subject to both phosphorylation and O-GlcNAc modification, and increased phosphorylation is believed to increase activity (25). Clearly, more studies are needed to understand the regulation of the HBP and O-GlcNAcylation in response to ischemia.

Given the wide range of proteins that are potential targets for O-GlcNAc modification, the specific mechanisms underlying the protection associated with glucosamine are likely complex. Increasing energy production or slowing the rate of energy utilization during ischemia has repeatedly been shown to improve functional recovery on reperfusion. However, we found here that glucosamine did not attenuate ATP hydrolysis during ischemia or increase ATP levels during reperfusion, suggesting that the protection was not mediated by alterations in energy metabolism. It should be noted that maintenance of ATP levels either during ischemia or reperfusion is not a prerequisite for cardioprotection. For example, although ischemic preconditioning significantly improves function recovery, it is not necessarily associated with increased ATP levels during ischemia or reperfusion (7, 24). Furthermore, Champattanachai et al. (8) recently demonstrated that glucosamine treatment significantly decreased both necrosis and apoptosis in isolated cardiomyocytes in response to hypoxia-reoxygenation without altering ATP levels. Previous studies have provided strong evidence linking the protective effect of glucosamine to changes in O-GlcNAc levels (8, 28). However, glucosamine could potentially be metabolized via glycolysis; thus, at this time, we cannot rule out the possibility that the mechanism of glucosamine cardioprotection could be due at least in part to the changes in substrate utilization. Clearly, further studies are needed to examine the effect of glucosamine on the regulation of substrate metabolism in the heart.

Activation of the MAPK pathway, particularly p38 and ERK1/2 MAPK, has been implicated in ischemic cardioprotection (2, 3, 15, 21, 31, 36, 37, 39, 49). Since we have shown in another cell system that agonist stimulation of the MAPK pathway was enhanced in response to increased O-GlcNAc levels (23), we examined the effect of glucosamine on the response of p38 and ERK1/2 MAPK to ischemia-reperfusion. Here we found that glucosamine significantly attenuated the ischemia-induced increase in p38 phosphorylation, which is consistent with reports that inhibition of p38 during sustained ischemia is protective (2, 3, 21, 31, 36, 39, 49). Surprisingly, at the end of reperfusion, p38 phosphorylation was increased in the glucosamine-treated group. There is relatively little information regarding the impact of reperfusion on p38 phosphorylation or on whether modulation of p38 activation during reperfusion impacts recovery. However, it has been shown that brief activation of p38 may contribute to cardioprotection seen with ischemic preconditioning. Activation of p38 can be proapoptotic, mediated via caspase-3 or p53 (21, 22, 34, 64); however, p38 has also been reported to activate prosurvival pathways. For example, {alpha}B-crystallin and HSP27 are downstream of p38, and both have been shown not only to play a role in ischemic protection but also to be subject to O-GlcNAc modification (43, 56, 58, 59). Furthermore, increased p38 phosphorylation has been associated with increased glucose transport (35), which might also be beneficial during reperfusion.

A number of studies have shown that increased ERK1/2 phosphorylation, especially during reperfusion, is cardioprotective (2, 3, 15, 37). However, whereas we saw the decrease in ERK1/2 phosphorylation during ischemia and an increase on reperfusion as recently reported by Hausenloy et al. (15), we found that glucosamine had no effect on ERK1/2 phosphorylation under any perfusion conditions. Akt has also been shown to have a protective effect in ischemia-reperfusion in the heart (14, 33). Recently, it has also been shown to mediate the effect of both ischemic pre- and postconditioning (15, 16, 50). We saw a brief increase in Akt phosphorylation after 5 min of ischemia; however, as described by Shao et al. (46), we saw a decrease in Akt phosphorylation at the end of ischemia and an increase during reperfusion. Despite the importance of Akt as a mediator of cardioprotection, we found no difference in the phosphorylation state of Akt between the control and the glucosamine group under any of the conditions. Thus these data suggest that glucosamine cardioprotection cannot be attributed to activation of either ERK1/2 or Akt prosurvival pathways.

It remains to be determined whether the changes in p38 phosphorylation seen with glucosamine are a cause or an effect of the improved recovery. For example, the severity of ischemic contracture is determined in part by diastolic Ca2+ levels (45); thus, since contracture was reduced, the decrease in p38 phosphorylation during ischemia could be a consequence of lower cytosolic Ca2+. This is consistent with reports that glucosamine decreases Ca2+ entry into cardiomyocytes (20, 38) and that increased cytosolic Ca2+ levels have been reported to activate p38 (30). Thus a decrease in cytosolic Ca2+ during ischemia could lead to a decrease in p38 phosphorylation; however, upon reperfusion, EDP is significantly reduced in the glucosamine group, suggesting lower diastolic Ca2+ levels, whereas p38 phosphorylation is increased. Consequently, the effects of glucosamine on p38 phosphorylation during ischemia and reperfusion are thus likely due to different mechanisms. It is also noteworthy that increased diastolic Ca2+ at the end of ischemia has been associated with an increased incidence of ventricular fibrillation on reperfusion (45) and that here we have found that glucosamine significantly reduced the incidence of ventricular fibrillation from 80% to 0% on reperfusion. However, ventricular fibrillation was evaluated only by qualtitive assessment of left ventricular developed pressure and heart rate; clearly, a more detailed evaluation of the electrophysiological effects of glucosamine during reperfusion is needed.

Since these studies were performed in an ex vivo, isolated, perfused heart model, it is premature to suggest that these results could be extrapolated to an in vivo environment. However, the substrates used here were designed to more closely mimic the in vivo metabolic milieu than in our earlier studies (28). It should also be noted that, previously, we demonstrated that glucosamine was protective against zero-flow ischemia, whereas here we used a model of very LFI, which may better reflect the conditions of myocardial infarction in vivo. Furthermore, in a rat model of trauma-hemorrhage and resuscitation, we found that O-GlcNAc levels in the heart were markedly lower at the end of resuscitation compared with those in sham-operated controls and that glucosamine treatment not only prevented the decrease in O-GlcNAc, it was also associated with improved cardiac function (62). These data are supportive of the idea that similar strategies might protect against myocardial ischemic injury in vivo. It should also be noted that glucosamine is a widely nutritional supplement used as a treatment of osteoarthritis; however, the serum glucosamine concentrations with commonly used oral dose (1,500 mg/day) (4) are well below the cytoprotective concentration (28, 62).

In conclusion, we have shown that short-term ischemia alone significantly increased UDP-GlcNAc levels in the heart and leads to increased cardiac O-glycosylation, which supports the notion that the HBP and O-glycosylation are endogenous stress-activated pathways (63). We also show that glucosamine attenuated contracture development during LFI and improved functional recovery following reperfusion. This protection was associated with increased UDP-HexNAc levels and increased protein O-GlcNAc levels but was not associated with increased ATP levels. We also found that glucosamine altered the response of p38 phosphorylation to ischemia and ischemia-reperfusion, and there was a significant correlation between phospho-p38 and O-GlcNAc levels in the glucosamine-treated group, whereas it had no effect on ERK1/2 or Akt. Further studies are clearly warranted to better understand the specific mechanisms underlying the protection seen with glucosamine. Nevertheless, these data support the concept that increasing protein O-GlcNAc levels are cardioprotective and suggest that one possible mechanism may be via altered p38 MAPK activation.


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This work was supported by National Heart, Lung, and Blood Institute Grants R01-HL-076175 (to R. B. Marchase), R01 HL-067464 and HL-079364 (to J. C. Chatham), and P50-HL-077100.


   DISCLOSURES
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R. B. Marchase and J. C. Chatham have intellectual property rights to disclose: a patent is pending that relates to the work presented in this article.


   ACKNOWLEDGMENTS
 
We thank Clarence Forrest and Charlye Brocks for technical assistance and Zachary T. Kneass and Tamas Nagy for insightful input.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. C. Chatham, Dept. of Medicine, Univ. of Alabama at Birmingham, MCLM 684, 1530 3rd Ave. S., Birmingham, AL 35294-0005 (e-mail: jchatham{at}uab.edu)

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.


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