Published online before print February 19, 2008, doi: 10.1161/CIRCULATIONAHA.107.730515
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(Circulation. 2008;117:1172-1182.)
© 2008 American Heart Association, Inc.
Molecular Cardiology |
Cardioprotection by N-Acetylglucosamine Linkage to Cellular Proteins
From the Institute of Molecular Cardiology, University of Louisville School of Medicine, Louisville, Ky (S.P.J., G.A.N., B.G.H., A.B.); and Department of Biological Chemistry (N.E.Z., G.W.H.), Institute of Molecular Cardiobiology (Y.T., E.M.), and Johns Hopkins University National Heart, Lung, and Blood Institute Proteomics Center (G.W.H., E.M.), Johns Hopkins University School of Medicine, Baltimore, Md.
Correspondence to Steven P. Jones, PhD, Institute of Molecular Cardiology, 580 S Preston St, Baxter II, 404C, Louisville, KY 40202. E-mail Steven.P.Jones{at}Louisville.edu
Received July 26, 2007; accepted December 14, 2007.
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Abstract |
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Background— The modification of proteins with O-linked β-N-acetylglucosamine (O-GlcNAc) represents a key posttranslational modification that modulates cellular function. Previous data suggest that O-GlcNAc may act as an intracellular metabolic or stress sensor, linking glucose metabolism to cellular function. Considering this, we hypothesized that augmentation of O-GlcNAc levels represents an endogenously recruitable mechanism of cardioprotection.
Methods and Results— In mouse hearts subjected to in vivo ischemic preconditioning, O-GlcNAc levels were significantly elevated. Pharmacological augmentation of O-GlcNAc levels in vivo was sufficient to reduce myocardial infarct size. We investigated the influence of O-GlcNAc levels on cardiac injury at the cellular level. Lethal oxidant stress of cardiac myocytes produced a time-dependent loss of cellular O-GlcNAc levels. This pathological response was largely reversible by pharmacological augmentation of O-GlcNAc levels and was associated with improved cardiac myocyte survival. The diminution of O-GlcNAc levels occurred synchronously with the loss of mitochondrial membrane potential in isolated cardiac myocytes. Pharmacological enhancement of O-GlcNAc levels attenuated the loss of mitochondrial membrane potential. Proteomic analysis identified voltage-dependent anion channel as a potential target of O-GlcNAc modification. Mitochondria isolated from adult mouse hearts with elevated O-GlcNAc levels had more O-GlcNAc–modified voltage-dependent anion channel and were more resistant to calcium-induced swelling than cardiac mitochondria from vehicle mice.
Conclusions— O-GlcNAc signaling represents a unique endogenously recruitable mechanism of cardioprotection that may involve direct modification of mitochondrial proteins critical for survival such as voltage-dependent anion channel.
Key Words: infarction • ischemia • mitochondria • myocardial infarction • acetylglucosamine
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Introduction |
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The modification of nuclear and cytoplasmic proteins with monosaccharides of O-linked β-N-acetylglucosamine (O-GlcNAc) is an essential posttranslational modification of metazoans. The addition of O-GlcNAc to the protein backbone is analogous to protein phosphorylation. O-GlcNAc levels are dynamic and change in response to extracellular stimuli, morphogens, cell cycle, development, and cellular stress.1,2 The addition of O-GlcNAc to serine and threonine residues is catalyzed by uridine diphospho-N-acetylglucosamine:polypeptide β-N-acetylglucosaminyltransferase (OGT),3,4 whereas removal of O-GlcNAc is catalyzed by a β-hexosaminidase (O-GlcNAcase).5 Ultimate confirmation of the key role of O-GlcNAc in regulating processes within the cell is reflected by lethality consequent to deletion of OGT at the single-cell level.6,7
Clinical Perspective p 1182
Up to 5% of glucose imported into the cell is converted to UDP-GlcNAc through the hexosamine biosynthetic pathway. UDP-GlcNAc serves as a donor for the synthesis of other sugar nucleotides, glycolipids, glycosylphosphatidylinositol anchors, N-linked glycosylation, Golgi-mediated O-linked glycosylation, and O-GlcNAc. Levels of O-GlcNAc within the cell are sensitive to changes in extracellular glucose concentrations.1,2 Accordingly, extensive efforts have focused on the idea that O-GlcNAc is a metabolic sensor or signal.8 Zachara and coworkers9 demonstrated recently that cell lines respond to various stressors by augmenting O-GlcNAc levels and that this may by a critical maneuver for cell survival. This finding suggests an endogenous autoprotective mechanism that motivated us to investigate whether transient cellular stress may trigger such a recruitable process in the myocardium. We initially addressed the cardioprotective process known as ischemic preconditioning, wherein brief periods of ischemia render the heart resistant to subsequent lethal ischemia. In this report we describe changes in O-GlcNAc levels during cardiac myocyte injury and specifically evaluate whether augmentation of O-GlcNAc levels is sufficient to confer cardioprotection in vitro and in vivo. Our findings implicate O-GlcNAc modification during ischemic preconditioning and represent the first reversible posttranslational modification other than phosphorylation to figure prominently in determining cardiac myocyte fate during lethal stress. Furthermore, our data indicate that at least 1 potential target for the O-GlcNAc modification is voltage-dependent anion channel (VDAC), a member of the mitochondrial permeability transition pore (mPTP), thereby providing unique insights into the cardioprotective mechanism.
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Methods |
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See the online-only Data Supplement for complete experimental methods.
Murine In Vivo Ischemia/Reperfusion and Infarct Size Determination
Ligation of the left coronary artery and infarct size determination were performed as described previously.10–22
Cardiac Myocyte Isolation, Culture, and Fluorescence
Cardiac myocytes were isolated from 1- or 2-day–old Sprague-Dawley rats as described.23 Fluorescence-activated cell sorting and confocal microscopy were performed as described previously.20,24,25
Identification of O-GlcNAc–Modified Proteins From Adult Mouse Hearts
Cells were lysed in buffer containing 25 mmol/L HEPES, pH 7.0, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% NP-40, 0.1% SDS, 1% protease inhibitor cocktail (Sigma, St Louis, Mo), 1% phosphatase inhibitor cocktail (Pierce), and 1 µmol/L O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) (O-GlcNAcase inhibitor). Proteins modified by O-linked GlcNAc were immunoprecipitated with anti-O-GlcNAc antibodies (Covance) with the use of a protein immunoprecipitation kit (Sigma). Proteins were released from the beads by boiling in Laemmli buffer containing 50 mmol/L dithiothreitol and separated by 12% SDS-PAGE. Separated proteins were subjected to Western blot analysis with the use of anti-VDAC antibodies (Sigma).
Adult Mouse Heart Mitochondrial Isolation and Swelling Assay
Mitochondria were isolated from whole mouse hearts as described previously.26,27 The mitochondrial swelling assay was performed as described in previous reports.28
Statistical Analyses
Data were analyzed by unpaired t test or ANOVA with post hoc analysis (Bonferroni) with the use of StatView (SAS Institute) software. For box plots, data in the upper border indicate the 75th percentile, and data in the lower border indicate the 25th percentile; the median (50th percentile) is between the 2. For bar and line graphs, data are reported as mean±SEM. Differences were accepted as significant when P<0.05.
All authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Results |
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O-GlcNAc Levels After Ischemic Preconditioning In Vivo
Mice were subjected to either acute or delayed ischemic preconditioning (or respective sham). At the appropriate time (acute, immediately; delayed, 24 hours later), the hearts were removed, total proteins were isolated as described in Methods, and samples were subjected to Western blotting with the O-GlcNAc–specific antibody (CTD110.6).29 As shown in Figure 1, cardiac O-GlcNAc levels were significantly augmented after acute (135±7%) or delayed (140±4%) ischemic preconditioning compared with the respective sham group (104±3% and 95±4%). The appearance of numerous immunopositive bands was expected and has been reported previously.9,29 The antibody (CTD110.6) used here is specific for the posttranslational modification (O-GlcNAc), not for a single protein.29
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Reduction of Myocardial Infarct Size by PUGNAc In Vivo
We intraperitoneally injected mice with 50 mg/kg of PUGNAc (O-GlcNAcase inhibitor30), 8 hours before surgery, to ascertain whether augmentation of O-GlcNAc levels is sufficient to reduce infarct size in vivo. Several mice not undergoing surgery were also euthanized to determine whether the dosing regimen of PUGNAc was sufficient to increase cardiac O-GlcNAc levels. As shown in Figure 1D, such treatment significantly augmented myocardial O-GlcNAc levels. Additional mice were treated similarly and subjected to 40 minutes of left coronary artery ischemia and 24 hours of reperfusion. At the end of reperfusion, Evans blue dye and 2,3,5-triphenyltetrazolium chloride were used to define the area at risk and infarct size, respectively (Figure 1E). PUGNAc significantly reduced infarct size compared with vehicle (Figure 1C). The ischemic preconditioning group is presented to give a better idea of the ability to observe protective effects in our model and demonstrate the endogenous capacity to augment O-GlcNAc levels and reduce infarct size.
O-GlcNAc Levels in Cardiac Myocytes
O-GlcNAc levels were assessed via Western blot analysis with the use of an O-GlcNAc–specific antibody in isolated cardiac myocytes after exposure to various durations of hydrogen peroxide in serum-free media (Figure 2). PUGNAc was used to test the hypothesis that reversal of such decline in O-GlcNAc levels could attenuate the extent of cardiomyocyte death. As shown in Figure 2B, PUGNAc significantly augmented O-GlcNAc levels in cardiac myocytes compared with vehicle in isolated cardiac myocytes. Additional myocytes were treated with vehicle or PUGNAc and exposed to varying durations of hydrogen peroxide exposure. Cells were then harvested and examined for changes in O-GlcNAc levels. Myocytes treated with PUGNAc showed markedly higher levels of O-GlcNAc throughout the time course compared with myocytes treated with vehicle. Furthermore, the decrement in O-GlcNAc levels was minimized and retarded in the PUGNAc- compared with the vehicle-treated group. From the time course data, cardiac myocytes experience a significant decrement in O-GlcNAc levels at 
40 minutes (Figure 2B and 2C). The decrement in cellular O-GlcNAc levels may correspond to important catastrophic events culminating in cardiomyocyte death, as addressed below. We next tested the hypothesis that reversal of the decrement in O-GlcNAc levels could attenuate cell death.
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O-GlcNAc and Cardiomyocyte Survival
Cardiac myocytes were treated with PUGNAc or vehicle, exposed to hydrogen peroxide (0.1 mmol/L), and coincubated with propidium iodide and annexin V to assess cell death (Figure 3). At the end of 150 minutes of hydrogen peroxide exposure, the percentage of propidium iodide–positive cells was significantly augmented in vehicle compared with PUGNAc and control groups. Within 150 minutes, exposure to hydrogen peroxide also induced a significant increase in annexin V fluorescence positivity in vehicle compared with PUGNAc or control groups. Cardiomyocyte death was significantly attenuated in cells treated with PUGNAc compared with cells treated with vehicle. Saponin permeabilization was used at the end of the experimental protocol to confirm equal numbers of cells per field (Figure 3B). Additional groups of myocytes were treated with vehicle or PUGNAc, then challenged with peroxide, and cells were harvested for total caspase activity after 16 hours (Figure 3E). This indicates evidence of persistent protective effects, potentially related to apoptosis.
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Preservation of Mitochondrial Membrane Potential
To evaluate the mechanistic implications of the protective effects of enhanced O-GlcNAc protein modification, we focused on early events governing cell death. Specifically motivated by the importance of maintaining mitochondrial integrity to enhance cell survival, we ascertained whether mitochondrial membrane potential was affected by alterations in O-GlcNAc levels. Such an avenue is particularly attractive given the early decline in O-GlcNAc levels shown in Figure 2B and 2C, which mirrors the early loss of mitochondrial membrane potential we have reported previously.20,23–25,31–33 Figure 2 clarifies that treatment of cardiac myocytes with the O-GlcNAcase inhibitor PUGNAc augments O-GlcNAc levels, and Figure 3 indicates that PUGNAc treatment attenuates the extent of cardiomyocyte death. In Figure 4, we treated cardiac myocytes with PUGNAc or vehicle, loaded them with the mitochondrial membrane potential indicator tetramethylrhodamine ethyl ester (TMRE), and exposed the cells to oxidant stress (hydrogen peroxide). Vehicle cardiac myocytes experience a catastrophic loss of mitochondrial membrane potential after exposure to hydrogen peroxide for 1 hour. PUGNAc, which augments O-GlcNAc levels, significantly attenuated the loss of mitochondrial membrane potential according to confocal microscopy (Figure 4A and 4B) and flow cytometry (Figure 4C and 4D) in a dose-dependent manner. These data provide important mechanistic implications for the protective effect of O-GlcNAc, which will be discussed below.
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PUGNAc-Mediated Protection Is Glibenclamide Sensitive
Glibenclamide blocks numerous forms of "preconditioning," both ischemic and pharmacological. In Figure 5, we show that PUGNAc-mediated cytoprotection in neonatal rat cardiomyocytes is largely blocked by the KATP channel antagonist glibenclamide. These data support the notion that some of the protective mechanisms required by augmented O-GlcNAc levels are shared with ischemic preconditioning (ie, KATP activation). Although glibenclamide-sensitive pathways are apparently required for PUGNAc-mediated protection, it is unclear whether such pathways are the only mechanism of PUGNAc-mediated protection. To evaluate other potential changes that might occur after PUGNAc treatment, we performed Western blots for the cytoprotectant heat shock protein (HSP)70 in neonatal rat cardiomyocytes (Figure 5E). PUGNAc treatment significantly augmented HSP70 levels compared with vehicle treatment. Whether augmentation of HSP70 levels is instrumental for PUGNAc-mediated protection remains to be tested.
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Protein Modification by O-GlcNAc
Numerous studies have documented an ever-growing list of proteins modified by O-GlcNAc. However, sparse information exists on specific cardiac proteins modified by O-GlcNAc. According to the immunoblotting data in Figures 1 and 2
, it appears that several proteins are modified by O-GlcNAc in cardiac myocytes. In Figure 6, we attempted to specifically identify some of these as potential candidates for cardioprotection. Proteins isolated from cultured cardiac myocytes treated with vehicle or PUGNAc were harvested as described in the online-only Data Supplement. Two-dimensional gel electrophoresis revealed at least 13 spots on which the O-GlcNAc modification (as evidenced by CTD antibody positivity) was augmented after PUGNAc treatment. Analysis of these 13 spots (Table) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry revealed several proteins involved in metabolism and 1 (VDAC) that piqued our interest because of its participation in the mPTP. Next, we performed an immunoprecipitation experiment to confirm that VDAC was modified by pulling down the O-GlcNAc–modified proteins and performing an immunoblot against VDAC. Indeed, the band corresponding to 30 kDa was also augmented in the PUGNAc sample compared with vehicle (Figure 6C).
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Our focus on VDAC is based on its identity as a central element in formation of the mPTP, which represents a significant and proximal event in the commitment to cell death. Although this serves only as an initial foray into the likely numerous proteomic changes associated with O-GlcNAc in this system, this singular finding is intriguing and consistent with the in vitro evidence for O-GlcNAc–mediated cardioprotection. It is important to note that lower-molecular-weight proteins are not shown in the 1-dimensional gels earlier in this study because the Western blots were not optimized for lower-molecular-weight ranges. Nevertheless, the appearance of the lower-molecular-weight bands appeared after the saturation of some of the higher-molecular-weight bands (by which time the exposure was stopped for the analyses shown in the earlier figures).
On the basis of the preliminary findings in Figure 6A through 6C (and the Table), adult wild-type mice were treated with PUGNAc (50 mg/kg IP) or isovolumic vehicle (as in Figure 1D), and cardiac mitochondrial proteins were evaluated to ascertain whether such changes in VDAC modification suggested by the 2-dimensional gels (Figure 6A through 6C) also occurred in the adult myocardium. Fractionation of hearts from vehicle- and PUGNAc-treated mice yielded clean, largely intact mitochondria (Figure 6D). Further examination of the mitochondrial fraction revealed that more VDAC was O-GlcNAc modified after PUGNAc treatment compared with vehicle and that such change occurred without a difference in total VDAC (Figure 6E).
O-GlcNAc Modification and mPTP Formation
To test the potential link between O-GlcNAc modification and a functional biochemical assessment of mitochondrial function within the mechanistic context of cell survival, cardiac mitochondria isolated from PUGNAc- and vehicle-treated adult mouse hearts (see above) were subjected to calcium-induced mitochondrial swelling (Figure 7). Mitochondria isolated from vehicle- and PUGNAc-treated mice were intact (Figure 6D), were free of cellular debris (Figure 7A), and maintained stable calcium-free absorbance levels throughout the assay (Figure 7B). The formation of mPTP occurred in vehicle-treated mitochondria, as evidenced by the decrease in spectrophotometric absorbance (520 nm) after the addition of 0.1 mmol/L calcium chloride (Figure 7C). Conversely, cardiac mitochondria isolated from PUGNAc-treated mice revealed resistance to the induction of mPTP. These data provide a novel molecular link between the identity of an O-GlcNAc–modified protein and a potential direct mechanism of cytoprotection.
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Discussion |
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Innumerable studies have focused on phosphorylation, the most widely described posttranslational modification in mammalian cells. The goal of this study was the primary establishment of the relationship between cardiac myocyte pathophysiology and the less studied posttranslational modification O-GlcNAc. The salient findings of the present study are as follows: (1) ischemic preconditioning enhances O-GlcNAc levels in vivo; (2) elevation of O-GlcNAc levels is sufficient to reduce infarct size after in vivo myocardial ischemia/reperfusion injury; (3) cardiac myocytes experience a decrement in O-GlcNAc levels during lethal cell injury; (4) the decrement in O-GlcNAc levels occurs early in cardiomyocyte injury in conjunction with the loss of mitochondrial membrane potential; (5) augmentation of O-GlcNAc levels attenuates the loss of mitochondrial membrane potential and cell death; (6) PUGNAc-mediated protection apparently shares some mechanisms with ischemic preconditioning; (7) at least several proteins are O-GlcNAc modified; and (8) augmenting O-GlcNAc levels apparently reduces sensitivity to mPTP formation. The present study establishes a new mechanistic paradigm in experimental cardioprotection and provides a previously unrecognized potential mechanism of ischemic preconditioning.
Others have shown that glucose transport activity is significantly enhanced after ischemic preconditioning.34 This could increase glucose flux and ultimately contribute to enlargement of the UDP-GlcNAc pool. Theoretically, increasing the size of the UDP-GlcNAc pool could increase O-GlcNAc levels (because UDP-GlcNAc is the sugar donor), as demonstrated previously in isolated cells9 and supported by the present findings from in vivo ischemic preconditioning. However, the approximate equivalence of PUGNAc and ischemic preconditioning in the extent of infarct size reduction does not conclusively demonstrate dependence of ischemic preconditioning on the O-GlcNAc modification, which is the subject of several ongoing investigations.
The current list of O-GlcNAc targets is sizable and growing, a fact not surprising when it is considered that O-GlcNAc is a posttranslational modification involving serine and threonine residues. As indicated by the present data, enhanced O-GlcNAc protein modification may act (at least partially) on the mitochondria to effect protection. Active pursuit of potential mitochondrial targets should continue, especially in light of the importance of mitochondria in cell survival. Additional emerging evidence suggests that O-GlcNAc protein modification of the proteasome could be responsible for the protective effects of enhanced O-GlcNAcylation.35 Marchase et al36 recently found that hyperglycemia, via enhanced flux through the hexosamine biosynthetic pathway, attenuated capacitative calcium entry in a manner similar to that of the in vitro model system. Thus, the present protective effects may at least partially be explained by attenuation of calcium overload, which is a known contributor to cardiomyocyte death. Subsequently, the same group found that elevating flux through the hexosamine biosynthetic pathway (thereby increasing O-GlcNAc levels) reduced cardiac myocyte damage in vitro.37,38 Such findings are consistent with those of the present study.
The formation of the mPTP is a harbinger of cell death.39–41 Although a consensus has not been reached on the obligatory presence of all putative members, it is widely accepted that VDAC (also known as porin), adenine nucleotide translocator, and cyclophilin-D constitute the core components of mPTP. Within the context of the present study, we are tempted to speculate that the O-GlcNAc modification of VDAC represents a heretofore unappreciated cardioprotective posttranslational modification. Furthermore, such modification may, at least in part, offer an explanation for the mechanism of cardioprotection.
The opening of the mPTP produces unregulated ingress and egress of molecules <1.5 kDa in the mitochondria. Such pathological disturbance uncouples electron transport from the production of ATP by destroying the electrochemical gradient across the inner mitochondrial membrane (ie, the mitochondrial membrane potential, indicated by TMRE) and releases proapoptotic proteins. Thus, the correlation of augmented levels of O-GlcNAc–modified VDAC with cytoprotection warrants attention as a potential mechanism. One might conjecture that the modification of VDAC by O-GlcNAc interferes with the formation of mPTP and thus protects the myocytes. Indeed, our swelling data from adult cardiac mitochondria support this precise notion, leading to our hypothetical scheme (Figure I in the online-only Data Supplement). At a minimum, these findings provide a molecular link between O-GlcNAc modification and cardioprotection, although definitive evaluation of such an exciting possibility and the complete characterization of O-GlcNAc–modified proteins in the heart will undoubtedly be the subject of numerous future studies. A recent report also suggests that mPTP can occur in murine embryonic fibroblasts in the genetic absence of VDAC,42 although this was not shown in cardiac mitochondria. Although this may be true in fibroblasts that have no VDAC, the present mitochondria all had abundant expression of VDAC, and the former report42 does not indicate that constitutive VDAC does not participate significantly in the formation of mPTP. Thus, it is difficult to make direct comparisons between the 2 studies. It is important to emphasize that many proteins are apparently O-GlcNAc modified and that VDAC is a likely mechanistic possibility; however, our data do not exclude the contribution of other protein targets in this system, such as adenine nucleotide translocator, cyclophilin-D, and other unidentified proteins. Indeed, it is likely that other O-GlcNAc–modified proteins contribute to this process, or the de novo production of cardioprotective proteins may also be involved (Figure 5).
Although the performance of in vivo myocardial ischemia/reperfusion studies lends credence to the idea that this could be a future therapeutic avenue in humans, the present data serve merely as proof of principle on which additional investigations will be based. The present in vivo infarct model has several obvious limitations. Healthy adult mice are likely unrepresentative of diseased human patient populations, and animal models with known risk factors should yield further insight into the potential clinical applicability of such findings.
The present data demonstrate a previously unrecognized endogenously recruitable mechanism of cardioprotection involving enhancement of the posttranslational modification O-GlcNAc. As our understanding of this area matures, we can elucidate the identity of specific protein targets of O-GlcNAc and develop a more integrated understanding of this process. This area of study may yield viable therapeutic options to combat postischemic myocardial injury, and, in a broader sense, we can begin to establish the biological role of O-GlcNAc in both the healthy and diseased myocardium.43
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Acknowledgments |
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Sources of Funding
This study was funded by National Institutes of Health grants to Dr Jones (R01 HL083320), Dr Bhatnagar (R01 HL55477 and P01 ES11860), Dr Hart (N01-HV-28180, R01 DK61671, R01 HD13563, and CA42486), and Dr Marbán (P01 HL081427). Dr Jones is also supported by a Scientist Development Grant from the American Heart Association (SDG 053270N). Dr Hill (0415165B, Ohio Valley) and Gladys Ngoh (0715493B, Great Rivers) are American Heart Association Predoctoral Fellows.
Disclosures
Under a licensing agreement between Covance Research Products and Johns Hopkins University, Dr Hart receives a share of royalty received by the university on sales of the CTD110.6 antibody. The terms of this arrangement are being managed by Johns Hopkins University in accordance with its conflict-of-interest policies. The other authors report no conflicts of interest.
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References |
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1. Wells L, Vosseller K, Hart GW. Glycosylation of nucleocytoplasmic proteins: signal transduction and O-GlcNAc. Science. 2001; 291: 2376–2378.
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The online-only Data Supplement, consisting of expanded Methods and figures, is available with this article at http://circ.ahajournals.org/cgi/content/ full/CIRCULATIONAHA.107.730515/DC1.
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Circulation 2008 117: 1121-1123.
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