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The Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, Calif.
Abstract
To examine whether excessive protein O-GlcNAcylation plays a role in the dysfunction of the diabetic heart, we delivered adenovirus expressing O-GlcNAcase (Adv-GCA) into the myocardium of STZ-induced diabetic mice. Our results indicated that excessive cellular O-GlcNAcylation exists in the diabetic heart, and that in vivo GCA overexpression reduces overall cellular O-GlcNAcylation. Myocytes isolated from diabetic hearts receiving Adv-GCA exhibited improved calcium transients with a significantly shortened Tdecay (P<0.01) and increased sarcoplasmic reticulum Ca2+ load (P<0.01). These myocytes also demonstrated improved contractility including a significant increase in +dL/dt and eCdL/dt and greater fractional shortening as measured by edge detection (P<0.01). In isolated perfused hearts, developed pressure and eCdP/dt were significantly improved in diabetic hearts receiving Adv-GCA (P<0.05). These hearts also exhibited a 40% increase in SERCA2a expression. Phospholamban protein expression was reduced 50%, but the phosphorylated form was increased 2-fold in the diabetic hearts receiving Adv-GCA. We conclude that excess O-GlcNAcylation in the diabetic heart contributes to cardiac dysfunction, and reducing this excess cellular O-GlcNAcylation has beneficial effects on calcium handling and diabetic cardiac function.
Key Words: O-GlcNAcase adenovirus gene therapy diabetic cardiomyopathy Ca2+ handling
Introduction
Diabetic cardiomyopathy is characterized by impaired cardiac function independent of cardiovascular disease.1 The functional changes of diabetic cardiomyopathy are indicated by an early diastolic dysfunction, followed by systolic dysfunction.2eC4 Impaired sarcoplasmic reticulum calcium cycling, including decreased intracellular Ca2+ concentration, reduced SR Ca2+ content, decreased diastolic Ca2+ uptake and systolic Ca2+ release, and increased Ca2+ leak, have been well documented5eC7; however, the underlying mechanism has not been fully elucidated.
O-GlcNAcylation (O-linked -N-acetylglucosamine enzymatic glycosylation), which is defined as the covalent attachment of a single -N-acetylglucosamine (O-GlcNAc) molecule to serine or threonine residues, is a posttranslational modification of intracellular proteins. This modification affects transcription, translation, nuclear transport, and cell signaling.8eC12 Similar to phosphorylation, O-GlcNAc addition by O-linked GlcNAc transferase (OGT), or its removal by N-acetylglucosaminidase (O-GlcNAcase, GCA) is a highly dynamic and reversible process that is susceptible to perturbation under certain pathophysiological circumstances.13 Hyperglycemia, commonly seen in diabetes mellitus, increases glucose flux through the hexosamine pathway, resulting in increased production of UDP-GlcNAc (uridine diphosphatide GlcNAc, donor of O-GlcNAc), which serves as a substrate for O-GlcNAc modification.14,15 Excess O-linked glycosylation has been detected in the pancreas16 and corneas of diabetic rats17 as well as coronary endothelial cells and atherosclerotic plaques in diabetic patients.18 Furthermore, alteration of cellular O-GlcNAcylation levels in rat skeletal muscle or 3T3-L1 adipocytes has been linked to the development of insulin resistance in diabetes.14,19
The potential role of O-GlcNAcylation in diabetic hearts has been implicated by several studies. Exposing cardiac myocytes to high glucose or elevated glucosamine (a precursor of O-GlcNAc) results in a reduction in contractility and calcium flux in cardiac myocytes.20 Exposing isolated perfused hearts to glucosamine blunts the positive inotropy by phenylephrine.21 Moreover, increasing cellular O-GlcNAcylation by infecting neonatal cardiac myocytes with adenovirus expressing OGT or incubating with GCA inhibitor (PUGNAc) significantly impairs the calcium transient, whereas reducing cellular O-GlcNAcylation by GCA reverses the calcium transient dysfunction induced by incubation with high glucose.22 We hypothesize that excess O-GlcNAcylation in the diabetic heart plays a significant role in cardiac dysfunction. Using in vivo adenovirus-mediated gene delivery to the heart, we overexpressed GCA in STZ-induced diabetic mice and evaluated the effects of reducing cellular O-GlcNAcylation on diabetic cardiac dysfunction.
Materials and Methods
Preparation of Diabetic Mice
NIH Swiss mice (25 g) were made diabetic by a single IP injection with freshly prepared streptozotocin (STZ) solution (200 mg/kg body weight in citrate saline, pH 4.2) after overnight fasting.4 The diabetic status was assessed by measuring urine glucose (>22 mmol/L=diabetic) 3 days after STZ injection and was confirmed by blood glucose measurement at the time of euthanasia.
To rule out any potentially toxic effects of STZ on the heart, several STZ-injected mice were intensively treated with Ultralente human insulin (Eli Lilly and Company) with 50 U/g per day subcutaneously. The insulin treatment commenced on the third day after STZ injection and lasted for 2 weeks. The cellular O-GlcNAcylation in these hearts was compared with that of the noninsulin-treated diabetic hearts. In addition, several 20-week-old male polygenic diabetic NONcNZO10/LtJ mice (The Jackson Laboratory, Bar Harbor, Me)23,24 were used as a type II diabetic model to investigate the excess O-GlcNAcyaltion level in the diabetic heart independent of STZ administration. These mice developed hyperglycemia at 12 to 16 weeks. At the time of euthanasia, blood glucose levels were above 33.3 mmol/L. Animal procedures were performed in accordance with the guidelines established by the Committee on Animal Research at the University of California, San Diego.
In Vivo Adenoviral Gene Delivery
In vivo adenovirus gene delivery in diabetic mice was performed as previously described.7 Either adenovirus encoding human GCA (Adv-GCA) or adenovirus without encoding gene sequence (Adv-SReC) was directly injected into the left ventricular wall (5 sites, 10 e蘈 of 1010 pfu/mL each). In the experiments involving individual myocyte studies, a green fluorescent protein expressing adenovirus (Adv-GFP) was coinjected to help identify either Adv-GCAeC or Adv-SReCeCinfected myocytes after myocytes isolation.7 Another group of normal age-matched NIH Swiss mice receiving Adv-SReC was used as a control group in some series of experiments.
Measurement of Ca2+ Transients and Sarcoplasmic Reticulum Calcium Load
Single ventricular myocytes were enzymatically isolated and Ca2+ transients were measured as previously described.7,25 Only those myocytes infected with Adv-GCA or Adv-SReC (as indicated by GFP fluorescence) were studied. Ca2+ transients were recorded from at least 20 cells per heart and for at least 3 hearts per treatment. Diastolic and systolic intracellular Ca2+ levels were inferred from the basal and maximal indo-1 ratio per cycle, respectively. Diastolic decay time (Tdecay) was calculated from the normalized Ca2+ transient curve.
Sarcoplasmic reticulum calcium load was measured as described by Shannon et al.26 In brief, cells were superfused with normal Tyrode solution (1 mmol/L Ca2+) and paced at 0.3 Hz to steady state. The solution was then rapidly switched to a Na+- and Ca2+-free Tyrode solution with a rapid solution exchanger device. After 20 seconds, cells were rapidly exposed to 10 mmol/L caffeine (in Na+- and Ca2+-free Tyrode solution). The difference between the basal and peak Ca2+ transient induced by caffeine was used as an index of sarcoplasmic reticulum Ca2+ load.
Measurement of Myocyte Contractility by Edge Detection
The contractile properties of single myocytes were measured using edge detection as described.27 Myocyte fractional shortening, maximal shortening rate (+dL/dt), and relengthening rate (eCdL/dt) were analyzed with Felix32 software (Photon Technology International Inc). Again, only infected myocytes as indicated by GFP signal were studied. Data were collected from at least 10 cells per heart and 3 hearts per treatment.
Measurement of Ventricular Function by Isolated Perfused Hearts
Diabetic mice were randomly divided into 2 groups, with each receiving either Adv-GCA or Adv-SReC via in vivo adenovirus gene delivery. Five days after the procedure, hearts were isolated and Langendorff-perfused for functional analysis as previously described.7,28 The hearts were paced at 400 bpm, and the resulting pressure waves were analyzed for pressure derivatives [rate of contraction (+dP/dt), rate of relaxation (eCdP/dt)] and developed pressure. Another set of normal mice served as a nondiabetic control group and underwent the same procedure. At the end of the experiment, hearts were frozen in liquid N2 for Western blot analysis.
RNase Protection Assay
RNase protection assays were performed as previously described.29 The human GCA (hGCA) probe spans residues 1035 to 1143 in the published human cDNA sequence (NM_012215), and the mouse GCA (mGCA) probe spans residues 881 to 1020 in the published mouse sequence (AF132214). The mouse OGT probe spans residues 57 to 215 in the published sequence (AF363030), which yields a 159-bp signal for full-length OGT nucleocytoplasmic isoform (ncOGT) and a 150-bp signal for the OGT mitochondrial isoform (mitOGT).
Western Immunoblotting
Cytosolic and nuclear fractions were prepared by differential centrifugation. Cardiac tissues were homogenized with a Polytron homogenizer in a buffer containing 30 mmol/L Tris, 300 mmol/L sucrose, 50 mmol/L GlcNAc, and protease inhibitor cocktail (1:1,000, Sigma). First centrifugation was performed at 1500g for 15 minutes at 4°C to spin down the crude nuclear fraction. The supernatant was spun at 8000g for 15 minutes at 4°C and subsequently for 1 hour at 160 000g at 4°C. The final supernatant represented the cytosolic fraction. Whole heart tissue homogenate was prepared with 0.2 mL of lysis buffer (in mmol/L: 20 Tris, pH 7.4, 20 NaCl, 0.1 EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 1 dithiothreitol, 1 -glycerophosphate, 10 Na-pyrophosphate, 50 NaF, 1 Na-ortho-vanadate, 50 GlcNAc, proteinase inhibitor cocktail).
Twenty micrograms of protein was mixed with Laemmli sample buffer at room temperature for 10 minutes (samples for antieCO-GlcNAc antibody detection was heated at 70°C for 10 minutes) and then loaded onto 4% to 10% gradient Tris/glycine gels. Separated proteins were transferred to nitrocellular membranes and blocked overnight with 3% BSA at 4°C. Blots were incubated with a primary antibody (1:5000 CTD 110.6 antibody, a gift by Dr Gerald Hart, The Johns Hopkins University, Baltimore, Md; 1:1000 polyclonal SERCA2a antibody, Affinity Bioreagents, Inc; phospholamban antibody, phosphorylated phospholamban antibody, Upstate; -actin antibody, Sigma; 1:1000 anti-OGT antibody, a gift by Dr. John A. Hanover, National Institutes of Health, Boston, Mass) for 1 hour at room temperature, followed by a 1-hour incubation with a 1:5000 dilution of secondary antibody (anti-rabbit IgG-HRP conjugated, anti-rabbit IgG-HRP conjugated, anti-mouse IgM-HRP conjugated, anti-rabbit IgG-HRP conjugated; Sigma). Bands were visualized by reacting with chemiluminescent substrate (PerkinElmer Life Sciences) and exposed to film. Films were scanned and analyzed by Image-J software (NIH).
OGT Activity and GCA Activity Assay
The OGT activity assay was measured with the protein precipitated by 30% saturated ammonium sulfate from heart tissue extract as previously described,30,31 The peptide PGGSTPVSSANMM was used as a substrate. The OGT activity was expressed as dpm/e protein.
Cytosolic fractions prepared as described above were used for GCA activity measurement,32 except that 1 mmol/L PMSF was added to the homogenization buffer but not GlcNAc and proteinase inhibitor cocktail; 50 mmol/L GalNAc was added to inhibit lysosomal hexosaminidases. The activity was expressed as units/mg.
Statistic Analysis
All data are presented as mean±SEM. One-way ANOVA with appropriate post hoc or unpaired Student t test was used for comparison between two groups with SPSS v9.0 software package. P<0.05 was considered to be statistically significant.
Results
General Features of the Experimental Animals
All the diabetic mice used in this study had a blood glucose level >22 mmol/L at their time of euthanasia. As described previously,4 the diabetic mice studied here also had lower body weights (22.4±1.1 versus 29.1±0.5 g; P<0.01) and higher blood glucose levels after STZ injection (40.6±2.6 versus 9.5±0.5 mmol/L; P<0.05) than normal NIH Swiss mice. Diabetic mice were randomly divided into 2 groups: Dia+SReC group (mice receiving Adv-SReC) and Dia+GCA (mice receiving Adv-GCA). Before gene therapy, both body weights and blood glucose levels were comparable between these 2 groups (body weight: Dia+SReC, 22.2±1.3 g versus Dia+GCA, 22.6±1.0 g; blood glucose: Dia+SReC, 38.2±2.1 mmol/L versus Dia+GCA, 43.0±3.1 mmol/L; P>0.05), which indicated that mice in these 2 groups had a similar severity of diabetes. After gene therapy, no significant changes were observed in body weight or blood glucose level in both groups (P>0.05).
Excessive O-GlcNAcylation of cellular proteins in STZ-induced diabetic hearts was directly confirmed by Western blot analysis with antieCO-GlcNAc antibody. As shown in Figure 1, more O-GlcNAcylated proteins were detected in nuclear fractions (indicated by arrows) isolated from diabetic hearts than from normal hearts. Similarly, more O-GlcNAcylated proteins were also detected in cytosolic fractions (indicated by arrows) isolated from diabetic hearts than from normal hearts. Excess O-GlcNAcylation of cellular protein in diabetic hearts was prevented by insulin treatment. Additionally, as shown in Figure 2, similar excess cellular O-GlcNAcylation was also observed in the cytosolic and nuclear fraction from the polygenic type II diabetic hearts.
In order to further understand excess O-GlcNAcylation in the diabetic heart, the expression levels and enzymatic activities of OGT and GCA were also determined. As shown in Figure 3, the mRNA expression level of both ncOGT and mitOGT isoform was upregulated 20% to 30% in the diabetic heart. The protein expression levels of OGT were increased 30% in the diabetic heart. However, the OGT activity was not significantly changed in the diabetic heart relative to that in the normal heart (110.52±3.42 versus 96.51±9.29 dpm/e; P>0.05). Additionally, the mRNA expression level of GCA was also increased approximately 30% in the diabetic heart. Similarly to the OGT enzymatic activity, the GCA activity was not increased in the diabetic heart (0.36±0.03 versus 0.39±0.04 U/mg; P>0.05).
Confirming the Overexpression of GCA in Diabetic Hearts After Receiving In Vivo Adenoviral Gene Delivery
The overexpression of GCA mediated by adenovirus was confirmed by RNase protection assay with a human GCA (hGCA)eCspecific probe. As shown in Figure 4A, hGCA mRNA was well expressed in diabetic hearts 5 days after the Adv-GCA injection. The mRNA expression level of endogenous mouse GCA, detected by a second probe specific to mouse GCA sequence, was not affected by exogenous GCA overexpression. As shown in Figure 4B, the overall GCA activity was increased 50% in diabetic hearts receiving Adv-GCA gene therapy.
The reduction of cellular O-GlcNAcylation in diabetic hearts after GCA overexpression was further evaluated by Western blot using an O-GlcNAc antibody. Compared with diabetic hearts receiving Adv-SReC, O-GlcNAcylated proteins were less abundant in diabetic hearts receiving Adv-GCA treatment. As shown in Figure 5, a reduction in overall cellular O-GlcNAcylation was observed both in nuclear (indicated by arrows) and cytosolic fraction (indicated by arrows) from Dia+GCA group in comparison with those from Dia+SReC group. The overall reduction appeared more prominent in the nuclear fraction.
Effects of Overexpression of GCA on Ca2+ Transients in Diabetic Cardiac Myocytes
The effects of overexpression of GCA on Ca2+ transients were studied by directly coinjecting Adv-GCA and Adv-GFP into the hearts of diabetic mice 2 weeks after STZ induction. Cardiac myocytes were isolated after 5 days, and only GFP-positive cells were analyzed.
As shown in Figure 6A and 6B, cardiac myocytes from Dia+SReC group (n=80) exhibited significantly lower systolic and diastolic calcium concentration, as indicated by lower basal and peak indo-1 ratios (Rdia and Rsys) compared with cells isolated from Nor+SReC group (n=98) (Rdia: Dia+SReC, 0.67±0.01 versus Nor+SReC, 0.71±0.01; Rsys: Dia+SReC 0.78±0.01 versus Nor+SReC, 0.86±0.01; P<0.01). In addition, the diastolic Tdecay was prolonged 40% in myocytes from Dia+SReC in comparison with that in normal myocytes from Nor+SReC group (0.25±0.02 versus 0.15±0.01 seconds; P<0.01) (Figure 5C and 5D). These results indicate that calcium handling in diabetic cardiomyocytes was significantly impaired in STZ-induced diabetic hearts.
The beneficial effects of overexpressing GCA on Ca2+ transients in diabetic cardiac myocytes are also shown in Figure 6. Compared with myocytes from the Dia+SReC group, myocytes from the Dia+GCA group (n=104) had higher diastolic and systolic indo-1 ratio (Rdia, 0.71±0.01 versus 0.67±0.01; Rsys, 0.84±0.01 versus 0.78±0.01; P<0.01). The diastolic Ca2+ Tdecay in diabetic myocytes from Dia+GCA group was significantly shorter than that in those from Dia+SReC group (0.18±0.01 versus 0.25±0.02 seconds; P<0.0.1). All 3 parameters were altered toward a similar level as observed in myocytes from Nor+SReC group (P>0.05).
To further evaluate the effects of Adv-GCA on calcium handling, sarcoplasmic reticulum Ca2+ load was measured as caffeine-induced Ca2+ release in these myocytes. Sarcoplasmic reticulum Ca2+ load was 33% lower than normal in control diabetic myocytes from Dia+SReC group (0.14±0.01 versus 0.21±0.01; P<0.01). However, sarcoplasmic reticulum Ca2+ load was significantly increased 28% in cardiac myocytes from Dia+GCA group (0.18±0.02 versus 0.14±0.01; P<0.05).
We also examined the effects of Adv-GCA on Ca2+ transient in normal myocytes. Our measurements indicated that Rdia was higher (P<0.05) in myocytes isolated from normal hearts receiving Adv-GCA than those receiving Adv-SReC. Rsys and Tdecay were not changed in normal myocytes receiving Adv-GCA (data not shown).
Effects of Overexpression of GCA on Contractile Function in Diabetic Cardiac Myocytes
To evaluate the effects of overexpression of GCA on contractile function, isolated cardiac myocytes were analyzed with edge-detection technique. As shown in the Table, cardiac myocytes from Dia+SReC group manifested prominent contractile dysfunction, represented by a 48% reduction in fractional shortening (P<0.01) and a 53% reduction in +dL/dt (P<0.01) as well as 59% reduction in eCdL/dt (P<0.01), compared with myocytes from Nor+SReC group.
As shown in the Table, cardiac myocytes from Dia+GCA group manifested significantly improved contractile function. Compared with those from Dia+SReC group, myocytes from Dia+GCA group had a 74% increase in fractional shortening (P<0.01), a 79% increase in +dL/dt (P<0.01), and an 85% increase in eCdL/dt (P<0.01). Both fractional shortening and +dL/dt in myocytes from Dia+GCA group were recovered toward normal (P>0.05). However, there was still a 25% decrease in eCdL/dt of these myocytes, compared with those from Nor+SReC group (P<0.05).
Effects of Overexpression of GCA on Contractile Function in the Intact Diabetic Heart
We reported previously an 30% decrease in contractile function in isolated perfused hearts with STZ-induced diabetes.4 Because the enzymatic isolation of individual myocytes can be a selective process, involving the survival of only the healthiest myocytes, we sought to confirm our observations by examining contractile function in the whole heart. In this study, STZ-induced diabetic hearts receiving Adv-SReC (n=6) exhibited a 27% and a 21% reduction in eCdP/dt and developed pressure (DP), respectively, in comparison with normal control hearts (n=9) (eCdP/dt: 2296±120 versus 3138±259 mm Hg/S; DP: 90±5 versus 114±8 mm Hg; P<0.05). However, the diabetic hearts receiving Adv-GCA (n=7) had a significant 15% increase in eCdP/dt and DP relative to the diabetic hearts receiving Adv-SReC (eCdP/dt: 2629±63 versus 2296±120 mm Hg/S; DP: 104±2 versus 90±5 mm Hg; P<0.05). There was no significant difference in +dP/dt either between normal and the diabetic hearts or between diabetic hearts receiving Adv-SReC and diabetic hearts receiving Adv-GCA (P>0.05). These data demonstrated that overexpression of GCA in the diabetic heart had beneficial effects on its global contractility.
Effects of Overexpression of GCA on Sarcoplasmic Reticulum Ca2+ Regulating Protein
To understand the mechanism of the beneficial effects of GCA overexpression on the diabetic heart, protein expression levels of SERCA2a, phospholamban (PLB), and phosphorylated PLB (p-PLB) were examined. As shown in Figure 7, the expression levels of SERCA, PLB, and p-PLB were all significantly reduced in diabetic hearts. Compared with the expression in Dia+SReC group, SERCA2a expression was increased 40%, whereas PLB protein expression was reduced 50% in hearts from Dia+GCA group. Furthermore, the percentage of p-PLB from PLB was increased 2-fold in diabetic hearts from Dia+GCA group in comparison with Dia+SReC group.
Discussion
Our investigation demonstrates for the first time that excess protein O-GlcNAcylation occurs in vivo in the diabetic heart, and that a reduction of excessive O-GlcNAc modification by overexpressing an adenovirus-encoded O-GlcNAcase enzyme in the heart has beneficial effects on cardiac function in STZ-induced diabetes.
We observed, through protein analyses, that diabetic hearts exhibited excessive O-GlcNAcylation in both type I and type II diabetes. This is similar to what has been observed in other tissues in diabetic animals and diabetic patients.17,31 Although both mRNA and protein expression levels of OGT were upregulated in the STZ-induced diabetic heart, the enzymatic activity of OGT did not increase with protein amount. This finding is in agreement with previous observations,31,33 indicating that additional factors may regulate the activity of OGT.34 Additionally, unlike the increase in OGT activity observed in the pancreas from STZ-induced diabetic rats, there was no significant change in enzymatic activity in hearts from STZ-induced diabetic mice. Thus, it is suggested that OGT activity is modulated differentially in different types of tissue. Our previous studies have shown that intracellular UDP-GlcNAc levels are significantly elevated in the diabetic heart when compared with the normal heart.22 It is likely that excess substrate supply, derived from the enhanced hyperglycemia-driven hexosamine pathway flux, rather than altered enzymatic activity, leads to increased protein O-GlcNAc modification in the STZ-induced diabetic heart. O-GlcNAcase has been characterized as an enzyme specific for O-GlcNAc removal,35,36 and the adenovirus-mediated overexpression of GCA has been used successfully in our laboratory to reduce O-GlcNAcylation in high glucose- or glucosamine-treated neonatal cardiac myocytes.22 Although no significant GCA enzymatic change is detected in the diabetic heart, the present study demonstrates that overexpressing O-GlcNAcase in vivo is able to increase O-GlcNAcase activity 50% and is effective at reducing cellular O-GlcNAcylation levels in the heart. A reduction in cellular O-GlcNAcylation was observed both in cytosolic and nuclear fractions, suggesting that GCA overexpression may have diverse effects on cellular function.
The most interesting finding of our study is that overexpression of GCA has a beneficial effect on cardiac function in diabetes. The results presented here demonstrate that overexpression of GCA could dramatically improve calcium cycling in diabetic myocytes by enhancing the Ca2+ transient and sarcoplasmic reticulum Ca2+ loading. In the STZ-induced diabetic heart, decreased SERCA expression level and phosphorylation of PLB explain the cardiac dysfunction observed in these diabetic hearts. The alterations in sarcoplasmic reticulum protein expression observed after GCA overexpression further provide an explanation for these changes in calcium cycling. The higher SERCA2a expression and lower PLB expression in diabetic hearts after Adv-GCA delivery increased the SERCA2a/PLB ratio and SERCA2a activity, which mirrors the observed normalization of calcium transients and increases in sarcoplasmic reticulum calcium load. The improvement in calcium cycling can certainly contribute to the enhancement of contractility in diabetic myocytes overexpressing GCA, and further facilitates the global functional improvement observed in the diabetic heart. However, unlike the restoration of contractile function observed in diabetic hearts treated with insulin,37,38 contractile function increased only 15% in isolated perfused hearts overexpressing GCA. Other factors unrelated to O-GlcNAcylation, such as nonenzymatic glycation of SERCA2a,39 may also play a role in diabetic cardiac dysfunction.
There is compelling evidence indicating that O-GlcNAcylation involves regulation of gene transcription, protein synthesis, and degradation.10 A series of studies have established a role of O-GlcNAc in suppressing transcription through modifying RNA polymerase II,40 Sp1,41,42 and histone deacetylase.43 We have previously shown that SERCA2a promoter activity negatively corresponds with the amount of O-GlcNAcylation on Sp1 in cardiac myocytes.22 Therefore, it is likely that the increased SERCA2a expression observed in diabetic hearts receiving Adv-GCA results from a decrease in Sp1 O-GlcNAcylation via GCA overexpression. However, another series of studies suggest a positive role of O-GlcNAc in upregulating transcriptional events, including those specifically responsive to glucose.44,45 Additionally, O-GlcNAc modification has been demonstrated to inhibit the proteasome46eC48 and stabilize certain proteins. We postulate that different mechanisms may contribute to the down regulation of PLB observed in diabetic heart receiving Adv-GCA delivery.
Our study also demonstrated an increased phosphorylated PLB in diabetic heart overexpressing GCA. This provides another explanation for enhanced SERCA2a function after GCA gene delivery, as it has been shown that the phosphorylation of PLB promotes oligmerization of PLB and decreases its inhibitory effects on SERCA2a.49eC53 Reciprocal phosphorylation and O-GlcNAcylation occurring at the same amino acid site have been identified in several proteins, including Tau54 and RNA polymerase II.40 Moreover, recently, we determined that PLB could be immunoprecipitated with antieCO-GlcNAc antibody (CTD 110.6) from cardiac myocytes cultured with high glucosamine and adenovirus expressing OGT (data not shown). Therefore, we are currently investigating whether PLB phosphorylation is directly regulated by reciprocal PLB O-GlcNAcylation, or whether PLB phosphorylation was indirectly regulated by O-GlcNAcylation through altering protein kinase A or protein phosphatase activity.
In summary, our study demonstrates that excessive cellular O-GlcNAcylation exists in the diabetic heart, and that reducing excess O-GlcNAcylation by overexpressing GCA has beneficial effects on calcium handling and diabetic cardiac function. Overexpressing GCA mediated by adenovirus may provide a therapeutic means to ameliorate diabetic cardiomyopathy.
Acknowledgments
This research was supported by the NIH grants RO1 HL 66917 and RO1 HL 52946.
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