Original articleGlutamine-induced protection of isolated rat heart from ischemia/reperfusion injury is mediated via the hexosamine biosynthesis pathway and increased protein O-GlcNAc levels
Introduction
Despite significant advances in treatment, coronary heart disease still accounts for 1 in 5 deaths in the United States [1]; furthermore, deaths from congestive heart failure have also increased [2], in large part due to increased survival following myocardial infarction. Thus, despite improvements in the management of patients with coronary heart disease, there is a growing need for the development of new treatments for use not only in response to an acute myocardial infarction, but also prior to surgery. Glutamine has been shown to preserve cardiomyocyte viability and enhance functional recovery from acute ischemia in isolated perfused rat heart [3], [4]. In vivo administration of glutamine has also been shown to improve cardiac function following ischemia/reperfusion [5]. However, the mechanisms underlying this protection have yet to be determined. Khogali et al. found that glutamine increased myocardial ATP/ADP and GSH/GSSG ratios following ischemia/reperfusion and suggested that the protection could be attributed to increased flux through the TCA cycle [3]. However, Wischmeyer et al. found that improved cardiomyocyte survival with glutamine treatment was associated with increased HSP-72 expression [5].
Recently, we reported that acutely increasing flux through the hexosamine biosynthesis pathway (HBP) with glucosamine significantly improved the tolerance of the isolated perfused heart to ischemia/reperfusion and appeared to be mediated by increased protein O-GlcNAc levels [6]. This was consistent with the report by Zachara et al., who showed that increased O-GlcNAc levels were an endogenous stress response and that augmentation of this response was cytoprotective [7]. Interestingly, glutamine:fructose-6-phosphate amidotransferase (GFAT) regulates the metabolism of glucose via the HBP and glutamine is the essential amino donor for the formation of glucosamine-6-phosphate, which is subsequently metabolized to UDP-N-acetylglucosamine (UDP-GlcNAc). Increasing glutamine concentrations dramatically increases UDP-GlcNAc, especially if glucose is also elevated [8]. Importantly, UDP-GlcNAc is the obligatory substrate for the transfer of O-GlcNAc to proteins by O-GlcNAc transferase (OGT) and flux through OGT is sensitive to changes in UDP-GlcNAc levels [9].
The importance of glutamine in regulating HBP flux combined with recent evidence demonstrating that increased HBP flux is cardioprotective led us to postulate that the ischemic protection seen with glutamine was mediated via the same pathway. We report here that, in the isolated rat heart, ischemic protection afforded by glutamine treatment was associated with a significant increase in the levels of UDP-GlcNAc and protein O-GlcNAc. Furthermore, inhibition of either GFAT or OGT blocked the glutamine-induced increase in O-GlcNAc and reversed the cardioprotection. These data provide further evidence supporting the role of the HBP and O-GlcNAc in mediating cardioprotection and provide new insights into the protective mechanisms associated with glutamine treatment.
Section snippets
Materials
All chemicals were purchased from Sigma-Aldrich unless otherwise stated.
Animals
All 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 National Institute of Health (NIH publication no. 85-23, 1996). Non-fasted, male Sprague–Dawley rats weighing 250–300 g were used throughout.
Isolated heart perfusion
Hearts were isolated and perfused as previously described [6], [10]. Briefly, rats were
Inhibition of HBP and OGT reverses protection associated with glutamine treatment
Cardiac function prior to ischemia in all four groups is summarized in Table 1. Although LVDP was significantly lower in the glutamine + azaserine group compared to untreated controls, heart rate, RPP and ± dP/dt were not significantly different between any of the groups. In time-control normoxic perfusions, there were no significant differences in any functional parameters after 105 min perfusion with either azaserine or alloxan compared to untreated control hearts (Table 2). Although RPP in both
Discussion
Increasing glutamine levels above the normal plasma level of 0.4 mM has been shown to enhance recovery from a variety of experimental and clinical challenges including burns, sepsis, trauma, hyperthermia and myocardial ischemia [3], [4], [5], [15], [16], [17]. However, the mechanism underlying the protection afforded by glutamine is unclear. We demonstrate here for the first time that ischemic protection resulting from glutamine treatment is mediated, at least in part, via increased flux
Acknowledgments
This work was supported by grants from the NHLBI HL-076165 (RBM); HL-67464 and HL079364 (JCC); and SCCOR grant HL-077100.
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