Published online before print November 20, 2008, doi: 10.1161/CIRCRESAHA.108.189431
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© 2009 American Heart Association, Inc.
Molecular Medicine |
Unique Hexosaminidase Reduces Metabolic Survival Signal and Sensitizes Cardiac Myocytes to Hypoxia/Reoxygenation Injury
From the Institute of Molecular Cardiology (G.A.N., H.T.F., T.H., S.P.J.) and Department of Physiology and Biophysics (G.A.N., S.P.J.), University of Louisville, Ky; Division of Endocrinology and Metabolism (W.D.), Department of Medicine, University of California; and Department of Biological Chemistry (N.E.Z.), Johns Hopkins University School of Medicine, Baltimore, Md.
Correspondence to Steven P. Jones, PhD, Institute of Molecular Cardiology, Department of Medicine, 580 S Preston St, Baxter II–404C, Louisville, KY 40202. E-mail Steven.P.Jones{at}Louisville.edu
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Abstract |
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Metabolic signaling through the posttranslational linkage of N-acetylglucosamine (O-GlcNAc) to cellular proteins represents a unique signaling paradigm operative during lethal cellular stress and a pathway that we and others have recently shown to exert cytoprotective effects in vitro and in vivo. Accordingly, the present work addresses the contribution of the hexosaminidase responsible for removing O-GlcNAc (ie, O-GlcNAcase) from proteins. We used pharmacological inhibition, viral overexpression, and RNA interference of O-GlcNAcase in isolated cardiac myocytes to establish its role during acute hypoxia/reoxygenation. Elevated O-GlcNAcase expression significantly reduced O-GlcNAc levels and augmented posthypoxic cell death. Conversely, short interfering RNA directed against, or pharmacological inhibition of, O-GlcNAcase significantly augmented O-GlcNAc levels and reduced posthypoxic cell death. On the mechanistic front, we evaluated posthypoxic mitochondrial membrane potential and found that repression of O-GlcNAcase activity improves, whereas augmentation impairs, mitochondrial membrane potential recovery. Similar beneficial effects on posthypoxic calcium overload were also evident. Such changes were evident without significant alteration in expression of the major putative components of the mitochondrial permeability transition pore (ie, voltage-dependent anion channel, adenine nucleotide translocase, cyclophilin D). The present results provide definitive evidence that O-GlcNAcase antagonizes posthypoxic cardiac myocyte survival. Moreover, such results support a renewed approach to the contribution of metabolism and metabolic signaling to the determination of cell fate.
Key Words: O-GlcNAc • mitochondria • hypoxia • cell death • posttranslational modification
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Introduction |
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O-linked β-N-acetylglucosamine (O-GlcNAc) is a metabolic posttranslational modification of nucleocytoplasmic proteins. Following its discovery in 1984,1 numerous proteins have been identified as being O-GlcNAc–modified. Such targets are diverse and include transcription factors, RNA-binding proteins, cytoskeletal proteins, nuclear pore proteins, phosphatases, and kinases.2,3 Unlike traditional N-linked protein glycosylation, O-GlcNAcylation of proteins involves the addition of one GlcNAc molecule to the Ser/Thr amino acid residues with no further elongation into more complex oligosaccharides. The GlcNAc moiety is added to serine and threonine amino acid residues by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase. O-GlcNAcylation is highly inducible, dynamic, and reversible, and the cycle of turnover of the sugar moiety exceeds the turnover of the protein itself.4 O-GlcNAc differs from phosphorylation in that the enzymes involved, OGT and O-GlcNAcase, are coded for by single genes, contrary to the numerous genes controlling protein phosphorylation/dephosphorylation.
Zachara and coworkers5,6 showed that O-GlcNAc levels change in response to stress and that augmentation of O-GlcNAc levels attenuated cell injury following lethal stress. We recently showed that enhanced O-GlcNAc levels attenuated injury following myocardial infarction, oxidative stress, and hypoxia.7,8 In the present study, we address the role of O-GlcNAcase in cardiac myocyte survival following hypoxic stress. Here, we evaluate whether manipulation of O-GlcNAcase to alter O-GlcNAc levels affects sensitivity to in vitro hypoxia/reoxygenation. Our findings definitively implicate O-GlcNAcase in the pathogenesis of posthypoxic cardiomyocyte death. In addition, this study emphasizes the significant contribution of metabolic signaling to posthypoxic damage.
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Materials and Methods |
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Neonatal Rat Cardiac Myocyte Isolation and Culture
Neonatal rat cardiac myocytes (NRCMs) were isolated from 1- to 2-day-old Sprague–Dawley rats and cultured according to a well-characterized protocol.7–12 The first 4 days of culture medium contained the antimitotic 5-bromodeoxyuridine (0.1 mmol/L) to inhibit fibroblast growth in addition to 5% FBS, penicillin/streptomycin, and vitamin B12. Twenty-four hours before experimentation, medium was changed to serum-free DMEM. All animals were used in compliance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health. The experimental protocol for the present study was reviewed and approved by the Institutional Animal Care and Use Committee of the University of Louisville.
Gene Transfer
NRCMs were infected with replication-deficient adenoviruses carrying O-GlcNAcase gene (AdO-GlcNAcase, 48 hours)8,13 or green fluorescent protein (AdGFP, Vector Biolabs), as described previously.11 Doses used include 0 and 100 multiplicities of infection (mois) of AdO-GlcNAcase or AdGFP. Twenty-four hours before experimentation, medium was changed to serum-free DMEM. Functional expression was confirmed by immunoblot analysis.
Enzyme Inhibition
NRCMs were treated with O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbamate14,15 (ie, PUGNAc: 200 µmol/L), an inhibitor of O-GlcNAcase, or vehicle (0.1% ethanol) overnight and subjected to hypoxia/reoxygenation or total protein isolated. Sample size is equal to 6 per group per treatment.
RNA Interference
Cultured NRCMs7,8 were transfected with 60 nmol/L short interfering (si)RNA directed against O-GlcNAcase (siRNA ID no. 190811; with sense strand 5'-GCAACUUAUGACUCCAUCtt-3' and antisense strand 3'-GAUGAGAGUCAUAAGUUGCtc-5') or scrambled sequence as a nonsilencing control (Ambion). Myocytes were transfected with SiPORT NeoFX transfection reagent according to the instructions of the manufacturer (Ambion). Seventy-two hours following transfection, total protein was isolated and immunoblotted for O-GlcNAcase protein and O-GlcNAc levels. Similarly treated myocytes were subjected to hypoxia/reoxygenation and posthypoxic media harvested to assess cell injury.
Protein Expression
Total cellular proteins were isolated from NRCMs as described previously.7,8 NRCMs were washed with PBS, harvested in ice-cold lysis buffer (containing 68 mmol/L sucrose, 200 mmol/L mannitol, 50 mmol/L KCl, 1mmol/L EGTA, 1mmol/L DTA, and 5 mmol/L HEPES) with freshly added 0.2 µmol/L DTT, 0.1% (vol/vol) protease inhibitor stock, 0.4% (vol/vol) NP-40, 0.4% (vol/vol) Triton-X, and posttranslational modification inhibitor stock. Extracts were sonicated and the resulting lysates centrifuged at 15 000g for 5 minutes at 4°C to remove cell debris. Forty micrograms of protein (according to Bradford assay) was applied to each lane of a 6% SDS-PAGE electroblotted onto poly(vinylidene fluoride) membranes for O-GlcNAcase protein, 10% Bis-Tris gel and electroblotted onto poly(vinylidene fluoride) membranes for cyclophilin D (CypD), adenine nucleotide translocase (ANT), and voltage-dependent anion channel (VDAC), whereas 50 µg of protein was applied to each lane of a 4% to 12% gradient NuPAGE Bis-Tris gel (Invitrogen) and electroblotted onto nitrocellulose membranes (Invitrogen) for O-GlcNAc levels. Reagent-grade nonfat milk (Bio-Rad) 5% (wt/vol) in Tris-buffered saline was used for blocking. Blots were incubated with anti–O-GlcNAcase (1:200) anti–O-GlcNAc antibodies (1:1000 CTD 110.6; Covance or 1:2000 RL2; Affinity BioReagents) anti-CypD (1:1000, Affinity BioReagents), anti-ANT (1:1000, Santa Cruz Biotechnology), anti-VDAC (1:1000, Santa Cruz), or 
-tubulin (1:1000, Santa Cruz), as primary antibodies overnight at 4°C. Blots were then incubated for one hour with 0.1 µg/mL of secondary antibody (goat anti-chicken IgG–HRP-conjugated, goat anti-mouse IgM–HRP-conjugated, donkey anti-goat IgG–HRP-conjugated, or goat anti-mouse IgG–HRP-conjugated) and detected with an enhanced chemiluminescent detection system (Pierce).
Densitometry
Densitometry was performed using nonsaturated chemiluminescent membranes exposed and quantitated using Fuji LAS-3000 Bio Imaging analyzer. Multiple exposures from every experiment were used to confirm that the signal was within the linear range. Densitometric analysis of O-GlcNAc levels using CTD 110.6 and RL2 antibodies were performed on the entire lane. O-GlcNAc levels were normalized to the appropriate control (Ponceau stain) and then expressed as a percentage of control (set at 100%).
In Vitro Hypoxia/Reoxygenation Injury in Cardiac Myocytes
Cardiac myocytes were subjected to hypoxia using 1x Esumi lethal ischemic media, pH 6.2 (containing 117 mmol/L NaCl, 12 mmol/L KCl, 0.9 mmol/L CaCl2, 0.49 mmol/L MgCl2, 4 mmol/L HEPES, 20 mmol/L sodium lactate, and 5.6 mmol/L L-glucose) by sealing the myocytes in humidified hypoxic chambers (Billups-Rothenberg Inc),16 flushing each chamber with a gas mixture consisting of 5% CO2 and 95% N2 for 15 minutes, and incubating the hypoxic chamber in a modular incubator at 37°C for 3 hours. Following hypoxia, the media were changed to 1x Esumi control media (pH 7.4, 137 mmol/L NaCl, 3.8 mmol/L KCl, 0.9 mmol/L CaCl2, 0.49 mmol/L MgCl2, 4 mmol/L HEPES, and 5.6 mmol/L D-glucose) and culture dishes were reoxygenated for 1 or 6 hours in the modular incubator or on the fluorescent microscope during imaging, as appropriate. Similarly treated NRCMs were subjected to 4 or 9 hours of normoxia in 1x Esumi control media to serve as normoxic/aerobic controls.
Cell Death
Cell death was assessed for NRCMs as previously described.8 Normoxic or posthypoxic LDH release was spectrophotometrically determined using a commercially available kit (Sigma) following hypoxia/reoxygenation, and the results were expressed as LDH release relative to total LDH in the cells and normalized to the appropriate controls (1 hour of reoxygenation data) or normoxic untreated control (for 6 hours of reoxygenation data). Similarly treated NRCMs were stained with the fluorescent DNA-binding dyes Hoechst 33342 (5 µg/mL) and propidium iodide (PI) (5 µg/mL) (Invitrogen) during the last 30 minutes of reoxygenation.8,17 The stained nuclei were then visualized using a x20 objective on a Nikon-TE2000E2 fluorescence microscope, Xcite light source; 350/50 nm excitation and 470/40 nm emission filter for Hoechst and 560/40 nm excitation and 630/60 nm emission filter for PI. Four fields per treatment in triplicate were counted and data were expressed as percentage PI-positive nuclei/total nuclei. Because the nuclear stain Hoechst 33342 is membrane-permeable, it was used to determine total cells in each field and not as an index of apoptosis.
Assessment of Mitochondrial Membrane Potential
Using time-lapse fluorescence microscopy,7–12,18 detection of mitochondrial membrane potential changes was performed by following changes in tetramethylrhodamine methyl ester (TMRM) fluorescence treated with AdGFP, AdO-GlcNAcase, vehicle, PUGNAc, scrambled (Scr) RNAi or O-GlcNAcase RNAi as described previously.8
Assessment of Calcium Overload
Calcium levels were assessed in NRCMs treated with AdGFP, AdO-GlcNAcase, vehicle, or PUGNAc and subjected to 3 hours of hypoxia using time-lapse fluorescent microscopy by following the changes in Rhod-2AM fluorescence. Cardiac myocytes were plated on 35 mm glass bottom culture dishes and loaded with 2 µmol/L Rhod-2AM before hypoxia/reoxygenation. Imaging was initiated at reoxygenation in isolated myocytes by exciting Rhod-2AM with an Xcite light source through a 546/11 nm bandpass filter and emission assessed through a 567/15 nm bandpass filter. Fluorescence intensity was monitored throughout the protocol every 90 seconds. All experimental groups were repeated in at least 4 separate isolations.
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Results |
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O-GlcNAcase (AdO-GlcNAcase) Overexpression Exacerbates Posthypoxic Cardiac Myocyte Death
Forty-eight hours following AdO-GlcNAcase infection of isolated NRCMs (n
5 per group), total cellular proteins were isolated for O-GlcNAcase protein and O-GlcNAc levels (via western blot analysis). Adenoviral overexpression of O-GlcNAcase significantly (P<0.05) augmented O-GlcNAcase protein levels (Figure 1A). Such elevation corresponded with a significant (P<0.05) reduction in O-GlcNAc levels (48±7% of 0 moi AdO-GlcNAcase) (Figure 1B). Similar findings were seen with another O-GlcNAc antibody, RL2 (65±8% of 0 moi AdO-GlcNAcase; supplemental Figure V, A). Immunoblots for O-GlcNAc levels show multiple immunopositive bands because O-GlcNAc is a posttranslational modification, not a single protein (Figure 1C and supplemental Figure V, A). Previous studies have demonstrated similar findings in various cell lines5 and NRCMs.7,8 Equal protein loading was confirmed by densitometric analysis of Ponceau-stained membranes (see supplemental Figure VII).
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To evaluate the effects of O-GlcNAcase overexpression on posthypoxic cardiac myocyte survival, similarly treated cardiac myocytes were subjected to hypoxia (3 hours) and reoxygenation (1 or 6 hours). Posthypoxic media was harvested for LDH release, and additional posthypoxic NRCMs were stained with PI to assess cell death. Myocytes infected with AdO-GlcNAcase were more sensitive to hypoxia-induced injury by the first hour of reoxygenation according to LDH release (150±23% of 0 moi AdO-GlcNAcase, P<0.05; supplemental Figure III, A) and PI positivity (17±2% versus 10±1% for AdGFP; supplemental Figure III, D) compared to 0 moi AdO-GlcNAcase or AdGFP, respectively.
To determine whether the detrimental effect of AdO-GlcNAcase on posthypoxic cardiac myocytes was short-lived, similarly treated NRCMs were reoxygenated for 6 hours after 3 hours of hypoxia. The longer duration of reoxygenation (6 hours) still showed exacerbated cellular injury in AdO-GlcNAcase NRCMs, according to LDH release (179±14% versus 136±8% for AdGFP, P<0.05; Figure 1D) and PI positivity (34±3% versus 24±2% for AdGFP, P<0.05; Figure 1E) compared to AdGFP.
Cell damage was not significantly different among AdO-GlcNAcase, AdGFP, or uninfected NRCMs under normoxia according to LDH release (109±3% of control versus 93±3% of control, P>0.05; supplemental Figure I, A) and PI positivity (14±1% for AdO-GlcNAcase versus 13±1% for AdGFP versus 12±1% for control, P>0.05; supplemental Figure II, A). Such results confirm that viral infection does not affect cell survival or O-GlcNAc levels in this system.
O-GlcNAcase Inhibition Attenuates Posthypoxic Cardiac Myocyte Injury
NRCMs (n=6 per group) were treated with PUGNAc (O-GlcNAcase inhibitor) overnight before protein harvest, then immunoblotted for O-GlcNAc levels. PUGNAc significantly increased O-GlcNAc levels (878±215% of control, P<0.05) compared to vehicle (Figure 2A and 2B). Supplemental Figure V, B, contains results with an additional O-GlcNAc antibody (RL2).
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Additional NRCMs (n=6 per group) were treated with PUGNAc, subjected to hypoxia/reoxygenation, and media harvested to measure LDH release. Inhibition of O-GlcNAcase (with PUGNAc) significantly attenuated posthypoxic LDH release 70±10% of control, P<0.05; supplemental Figure III, B) and PI positivity (9±1% versus 13±1% for vehicle, P<0.05; supplemental Figure III, E) at the end of the first hour of reoxygenation.
The protective effect of augmented O-GlcNAc levels (with PUGNAc) was still seen at 6 hours of reoxygenation with LDH release (133±11% for PUGNAc, P<0.05 versus 177±14% for vehicle; Figure 2C) and PI positivity (21±2% versus 36±2% of vehicle; Figure 2D). PUGNAc or vehicle treatment did not significantly alter normoxic/aerobic cellular viability compared with untreated NRCMs under normoxia according to LDH release (P>0.05, 90±8% of control versus 104±8% of control; supplemental Figure I, B) and PI positivity (P>0.05, 12±1% for PUGNAc versus 13±1% for vehicle versus 13±1% for control; supplemental Figure II, B).
Knockdown of O-GlcNAcase Reduces Posthypoxic Cardiac Myocyte Injury
NRCMs (n=6 per group) were treated with 60 nmol/L O-GlcNAcase RNAi or Scr RNAi for 72 hours to knockdown O-GlcNAcase expression. O-GlcNAcase knockdown significantly (P<0.05) reduced O-GlcNAcase protein levels compared to Scr RNAi, despite no change in -tubulin levels (Figure 3A). O-GlcNAcase RNAi significantly (P<0.05) augmented O-GlcNAc levels (132±12% of control, P<0.05) compared to Scr RNAi (Figure 3B and 3C). Additional NRCMs were treated with O-GlcNAcase or Scr RNAi, subjected to hypoxia/reoxygenation, and media harvested to measure LDH release. O-GlcNAcase RNAi significantly (P<0.05) reduced posthypoxic LDH release after 1 hour (72±10% of Scr RNAi; supplemental Figure III, C) and 6 hours (130±4% for O-GlcNAcase RNAi versus 170±26% for Scr RNAi; Figure 3D) of reoxygenation compared to Scr RNAi. In addition, O-GlcNAcase RNAi significantly diminished PI positivity at 6 hours (22±2% for O-GlcNAcase RNAi versus 33±2% for Scr RNAi, P<0.05; Figure 3E) compared with Scr RNAi. O-GlcNAcase or Scr RNAi treatment did not cause significant cell damage compared with untreated NRCMs under normoxia/aerobic conditions (supplemental Figures I, C, and II, C).
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Effect of O-GlcNAcase Manipulation on Mitochondrial Membrane Potential Preservation
Based on our recent findings linking O-GlcNAc–mediated cardioprotection to mitochondria,7,8 we assessed the effects of altered O-GlcNAcase activity on posthypoxic mitochondrial membrane potential. NRCMs were treated with either AdGFP (48 hours, 100 mois), AdO-GlcNAcase (48 hours, 100 mois), vehicle (overnight), PUGNAc (before hypoxia/reoxygenation, 200 µmol/L), Scr RNAi (72 hours, 60 nmol/L), or O-GlcNAcase RNAi (72 hours, 60 nmol/L) and loaded with the mitochondrial membrane potential indicator TMRM (50 nmol/L). Cardiac myocytes were then subjected to either 4 hours of normoxia, or, 3 hours of hypoxia and 1 hour of reoxygenation. During the reoxygenation period, myocytes were evaluated for changes in mitochondrial membrane potential using time-lapse fluorescence microscopy. Dissipation of mitochondrial membrane potential is reflected by a loss of TMRM fluorescence. AdGFP-infected NRCMs loaded with TMRM maintained mitochondrial membrane potential over time under normoxia and showed no significant difference in mitochondrial membrane potential over time (supplemental Figure IV). Hypoxia/reoxygenation induced mitochondrial membrane potential loss. O-GlcNAcase overexpression aggravated the loss of mitochondrial membrane potential on reoxygenation, shown by impaired recovery of TMRM fluorescence (Figure 4A and 4B). Conversely, inhibition of O-GlcNAcase (via PUGNAc or O-GlcNAcase RNAi) attenuated the loss of mitochondrial membrane potential compared to vehicle or Scr RNAi, as shown by the significant restoration of TMRM fluorescence toward that of the normoxic control, and protected cells from posthypoxic mitochondrial dysfunction (Figure 4C through 4F).
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Effect of O-GlcNAcase Manipulation on mPTP Component Expression
To test the hypothesis that O-GlcNAc–mediated protection involved alteration in expression of the putative major components of mPTP, we treated cells with the various interventions and harvested proteins. Lysates were immunoblotted for the likely mPTP components VDAC, ANT, and CypD. None of the interventions significantly altered the expression of VDAC, ANT, or CypD (Figure 5).
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Effect of O-GlcNAcase Manipulation on Calcium Levels
Cardiac myocytes were treated with either AdGFP (48 hours, 100 mois), AdO-GlcNAcase (48 hours, 100 mois), vehicle (overnight), or PUGNAc (overnight, 200 µmol/L), loaded with the mitochondrial calcium indicator Rhod-2AM (2 µmol/L) and subjected to 3 hours of hypoxia and one of reoxygenation. Time-lapse fluorescence microscopy was initiated at the beginning of reoxygenation for changes in mitochondrial calcium levels. Augmentation of calcium levels is reflected by an increase in Rhod-2AM fluorescence. Hypoxia sensitized myocytes to calcium overload. O-GlcNAcase overexpression (AdO-GlcNAcase) exacerbated the hypoxia-induced calcium overload on reoxygenation reflected by a significant increase in Rhod-2AM fluorescence (Figure 6A and 6B), whereas O-GlcNAcase inhibition (PUGNAc) attenuated hypoxia-induced calcium overload compared to vehicle (Figure 6C and 6D).
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Discussion |
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O-GlcNAc signaling has emerged as an integral element in the armamentarium of the cell against lethal stress. Previous work from this laboratory and others suggests that pharmacologically elevated O-GlcNAc levels are protective in cell models following hypoxia/oxidative stress8,19–22 and, in vivo, following myocardial ischemia/reperfusion.7 However, such studies were never able to achieve the standard of genetic gain- and loss-of-function insights on O-GlcNAcase, as are available in the present work. Here, overexpression of O-GlcNAcase reduces O-GlcNAc levels, exacerbates the loss of mitochondrial membrane potential following hypoxia, and sensitizes myocytes to posthypoxic cell death. The present results also demonstrate that inhibition of O-GlcNAcase (via siRNA or PUGNAc) augments O-GlcNAc levels, enhances the recovery of mitochondrial membrane potential following hypoxia, and attenuates posthypoxic cell death.
Recently, Taylor et al identified a unique response of cells to O-GlcNAc levels, namely that O-GlcNAc is not a simple "readout" of glucose availability.23 These results were further confirmed by Cheung et al in Neuro-2a cells.24 Indeed, such insights are consistent with ongoing work from this laboratory that O-GlcNAc signaling represents a stress responsive program, not simply a fuel gauge. Such insights are particularly true in light of the data of Taylor et al23 and Cheung and Hart24 indicating that complete removal of glucose significantly augments O-GlcNAc levels. Because the hexosamine biosynthetic pathway produces the monosaccharide donor for the O-GlcNAcylation of proteins, several groups have manipulated this pathway as a means of better understanding the role of O-GlcNAc modification of proteins and have shown that enhanced hexosamine biosynthetic pathway flux is cytoprotective following acute stress.19,21,22,25
Zachara et al first showed that enhanced O-GlcNAc levels improved cell survival following stress.5 Since then, we and others have reported on the protective role of enhanced O-GlcNAc levels in different systems.7,8,19–22,25–27 In the heart, Champattanachai et al showed that inhibition of O-GlcNAcase with PUGNAc in NRCMs improved cardiac myocyte viability following hypoxia/reoxygenation.25 In several studies of the isolated perfused heart, Liu and colleagues showed that enhanced O-GlcNAc levels protected the heart against injury resulting from calcium paradox and hypoxia/reoxygenation.20–22 We have also shown that pretreatment of mice with PUGNAc (an O-GlcNAcase inhibitor) reduced infarct size in vivo and that ischemic preconditioning augmented O-GlcNAc levels.7 Moreover, we recently showed that augmenting O-GlcNAc levels by overexpressing OGT attenuated posthypoxic injury, whereas inhibition of OGT (pharmacologically or genetically) in cardiomyocytes exacerbated posthypoxic injury at the mitochondrial level. Such findings are supported by the study by Champattanachai et al19 showing that overexpression of OGT attenuated loss of mitochondrial membrane potential induced by H2O2 and increased mitochondrial Bcl-2. Here, we show that manipulation of O-GlcNAcase to alter O-GlcNAc levels significantly affects cardiac myocyte survival following hypoxia, although we found no evidence for differences in apoptosis at 6 hours reoxygenation (see supplemental Figure VII).
From a molecular vantage point, we have identified VDAC,7,8 a putative member of the mitochondrial permeability transition pore, to be O-GlcNAc modified and also showed that enhanced O-GlcNAc levels attenuated calcium-induced mitochondrial permeability transition pore (mPTP) formation in adult cardiac mitochondria. The present study supports mitochondrial involvement as a potential mechanism in O-GlcNAc–mediated cardioprotection in that following hypoxia, reduction of O-GlcNAc levels (by O-GlcNAcase overexpression) diminished the recovery of mitochondrial membrane potential, whereas augmented O-GlcNAc levels using PUGNAc enhanced the recovery of mitochondrial membrane potential during reoxygenation. The calcium overload data further support potential involvement of mitochondria in O-GlcNAc signaling induced alterations in cell survival. Yet, there were no significant changes in total protein expression of the prominent, although putative, constituents of the mPTP. Whether the physical alteration of mPTP components by O-GlcNAc explains the protective effects observed remains the subject of ongoing investigation.
O-GlcNAcase structurally has both hexosaminidase and histone acetyltransferase domains and is functionally involved with the removal of O-GlcNAc from proteins and acetylation of free histones.28–30 Because O-GlcNAcase has been shown to be cleaved by caspase 3,30 an executioner caspase in apoptosis, into N-terminal hexosaminidase domain and an C-terminal HAT domain, we hypothesize that cleavage of O-GlcNAcase might result in the loss of internal regulation of its hexosaminidase activity, thereby increasing the rate of removal of O-GlcNAc from proteins and hence the severity of posthypoxic injury. However, significant additional efforts should be directed toward understanding the molecular regulation of O-GlcNAc signaling.
The reductionist approach used in the present study allows clear focus on the role of O-GlcNAcase in hypoxia/reoxygenation injury. It is becoming increasingly evident that O-GlcNAc signaling exerts its influence based on context and such potentially differing effects deserve investigation.31,32 Ongoing pursuits will continue to identify the protein targets and attempt to elucidate how O-GlcNAc signaling might be altered in diabetes and aging, in contrast to acute events such as myocardial ischemia. Clearly, O-GlcNAc signaling warrants such continued and intensive attention in the cardiovascular system.
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Acknowledgments |
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We acknowledge the expert technical assistance of Linda Harrison (University of Louisville).
Sources of Funding
This work is supported by NIH grant R01 083320, American Heart Association National Center Scientist Development Grant 0535270N, and Kentucky Science and Engineering Foundation grant KSEF-1677-RDE-011 (to S.P.J.). G.A.N. is an American Heart Association Predoctoral Fellow, Great Rivers Affiliate (0815502D). H.T.F. is an American Heart Association Postdoctoral Fellow, Great Rivers Affiliate (0825643D).
Disclosures
None.
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Footnotes |
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Original received May 6, 2008; first resubmission received August 14, 2008; second resubmission received October 15, 2008; accepted November 6, 2008.
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References |
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