Diabetes and the Accompanying Hyperglycemia Impairs Cardiomyocyte Calcium Cycling through Increased Nuclear O-GlcNAcylation

Diabetic cardiomyopathy is characterized by impaired cardiac contractility leading to poor myocardial performance. We investigated the role that the hexosamine pathway, and especially altered nuclear O-Glc-NAcylation, plays in the development of diabetic cardiomyopathy. Incubating neonatal rat cardiomyocytes in high glucose (25 mm) resulted in prolonged calcium transients when compared with myocytes incubated in normal glucose (5.5 mm), which is consistent with delayed myocardial relaxation. High glucose-treated myocytes also exhibited reduced sarcoendoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) mRNA and protein expression, decreased SERCA2a promoter activity, and increased O-GlcNAcylation of nuclear proteins compared with myocytes treated with normal glucose. Exposure of myocytes to 8 mm glucosamine or an adenovirus expressing O-GlcNAc-transferase (OGT) resulted in prolonged calcium transient decays and significantly reduced SERCA2a protein levels, whereas treatment with an adenovirus encoding O-GlcNAcase (GCA) resulted in improved calcium transients and SERCA2a protein levels in myocytes exposed to high glucose. Effects of elevated glucose or altered O-GlcNAcylation were also observed on essential transcription factors involved in cardiomyocyte function. High glucose-treated myocytes (with or without OGT adenovirus) exhibited increased levels of O-GlcNAcylated specificity protein 1 compared with control myocytes, whereas infecting high glucose-treated myocytes with GCA adenovirus reduced the degree of specificity protein 1 Glc-NAcylation. Treatment of myocytes with 25 mm glucose, 8 mm glucosamine, or OGT adenovirus also significantly reduced levels of myocytes enhancer factor-2A protein compared with control myocytes, whereas infection with GCA adenovirus resulted in improved myocytes enhancer factor-2 expression. Our results suggest that the hexosamine pathway, and O-GlcNAcylation in particular, is important in impaired cardiac myocyte function and the development of diabetic cardiomyopathy. Diabetic cardiomyopathy is characterized by impaired cardiac contractility leading to poor myocardial performance. We investigated the role that the hexosamine pathway, and especially altered nuclear O-Glc-NAcylation, plays in the development of diabetic cardiomyopathy. Incubating neonatal rat cardiomyocytes in high glucose (25 mm) resulted in prolonged calcium transients when compared with myocytes incubated in normal glucose (5.5 mm), which is consistent with delayed myocardial relaxation. High glucose-treated myocytes also exhibited reduced sarcoendoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) mRNA and protein expression, decreased SERCA2a promoter activity, and increased O-GlcNAcylation of nuclear proteins compared with myocytes treated with normal glucose. Exposure of myocytes to 8 mm glucosamine or an adenovirus expressing O-GlcNAc-transferase (OGT) resulted in prolonged calcium transient decays and significantly reduced SERCA2a protein levels, whereas treatment with an adenovirus encoding O-GlcNAcase (GCA) resulted in improved calcium transients and SERCA2a protein levels in myocytes exposed to high glucose. Effects of elevated glucose or altered O-GlcNAcylation were also observed on essential transcription factors involved in cardiomyocyte function. High glucose-treated myocytes (with or without OGT adenovirus) exhibited increased levels of O-GlcNAcylated specificity protein 1 compared with control myocytes, whereas infecting high glucose-treated myocytes with GCA adenovirus reduced the degree of specificity protein 1 Glc-NAcylation. Treatment of myocytes with 25 mm glucose, 8 mm glucosamine, or OGT adenovirus also significantly reduced levels of myocytes enhancer factor-2A protein compared with control myocytes, whereas infection with GCA adenovirus resulted in improved myocytes enhancer factor-2 expression. Our results suggest that the hexosamine pathway, and O-GlcNAcylation in particular, is important in impaired cardiac myocyte function and the development of diabetic cardiomyopathy. The leading cause of mortality among diabetic patients in the United States is heart disease. Despite the numerous effects diabetes exerts on the cardiovascular system, there is substantial evidence indicating that a diabetes-specific cardiomyopathy occurs in the absence of coronary artery disease or hypertension (1Ahmed S.S. Jaferi G.A. Narang R.M. Regan T.J. Am. Heart J. 1975; 89: 153-158Crossref PubMed Scopus (203) Google Scholar, 2Galderisi M. Anderson K.M. Wilson P.W. Levy D. Am. J. Cardiol. 1991; 68: 85-89Abstract Full Text PDF PubMed Scopus (481) Google Scholar). Diabetic cardiomyopathy is characterized by impaired cardiac contractility and poor myocardial performance without an attendant vascular or valvular disease and can lead to congestive heart failure. Studies in diabetic human patients and animal models have demonstrated the early development of diastolic dysfunction prior to the alteration of systolic function (3Fein F.S. Kornstein L.B. Strobeck J.E. Capasso J.M. Sonnenblick E.H. Circ. Res. 1980; 47: 922-933Crossref PubMed Scopus (308) Google Scholar, 4Zarich S.W. Arbuckle B.E. Cohen L.R. Roberts M. Nesto R.W. J. Am. Coll. Cardiol. 1988; 12: 114-120Crossref PubMed Scopus (211) Google Scholar). Eventually, however, nearly all aspects of cardiac contractility appear to become impaired (5Trost S.U. Belke D.D. Bluhm W.F. Meyer M. Swanson E. Dillmann W.H. Diabetes. 2002; 51: 1166-1171Crossref PubMed Scopus (205) Google Scholar, 6Penpargkul S. Schaible T. Yipintsoi T. Scheuer J. Circ. Res. 1980; 47: 911-921Crossref PubMed Scopus (203) Google Scholar). Increasing evidence indicates that abnormalities in cardiac Ca2+ handling may be an important contributor to decreased contractile function in the diabetic heart. Ventricular contraction and relaxation are controlled largely by Ca2+ release from and uptake into the sarcoendoplasmic reticulum (SR) 1The abbreviations used are: SR, sarcoendoplasmic reticulum; SERCA2a, SR Ca2+ ATPase; Sp1, specificity protein 1; MEF, myocytes enhancer factor; PUGNAc, O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenyl carbamate; NG, normal (5.5 mm) glucose; HG, high (25 mm) glucose; AM, acetoxymethyl; OGT, UDP-N-acetylglucosamine:peptide N-acetylglucosaminyl transferase (O-GlcNAc-transferase); GCA, O-GlcNAcase; MEF, myocytes enhancer factor; m.o.i., multiplicity of infection; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; MES, 4-morpholineethanesulfonic acid; HPLC, high pressure liquid chromatography; STZ, streptozocin; Tricine, N-2-hydroxy-1,1-bis(hydroxymethyl)ethylglycine. (7Carafoli E. Annu. Rev. Biochem. 1987; 56: 395-433Crossref PubMed Scopus (1773) Google Scholar). All aspects of SR function in the diabetic heart are depressed as documented by decreased SR Ca2+ uptake, reduced SR Ca2+ content, and impaired SR Ca2+ release (8Penpargkul S. Fein F. Sonnenblick E.H. Scheuer J. J. Mol. Cell Cardiol. 1981; 13: 303-309Abstract Full Text PDF PubMed Scopus (191) Google Scholar, 9Bouchard R.A. Bose D. Am. J. Physiol. 1991; 260: H341-H354PubMed Google Scholar, 10Lagadic-Gossmann D. Buckler K.J. Le Prigent K. Feuvray D. Am. J. Physiol. 1996; 270: H1529-H1537PubMed Google Scholar). Impaired SR function appears to result from reduced activity and expression of the SR Ca2+ ATPase (SERCA2a) (5Trost S.U. Belke D.D. Bluhm W.F. Meyer M. Swanson E. Dillmann W.H. Diabetes. 2002; 51: 1166-1171Crossref PubMed Scopus (205) Google Scholar, 11Ganguly P.K. Pierce G.N. Dhalla K.S. Dhalla N.S. Am. J. Physiol. 1983; 244: E528-E535PubMed Google Scholar), which is responsible for sequestering Ca2+ and inducing diastolic relaxation. The underlying mechanisms leading to abnormal SR function and calcium flux in the diabetic heart are poorly understood. Diabetic hyperglycemia results in a number of pathophysiological changes in the vascular system, but investigations of its role in diabetic cardiomyopathy are limited. Recently, studies exposing cardiac myocytes to elevated extracellular glucose resulted in impaired cardiomyocytes contractility and calcium flux (12Ren J. Gintant G.A. Miller R.E. Davidoff A.J. Am. J. Physiol. 1997; 273: H2876-H2883PubMed Google Scholar) and increased Ca2+ i (13Gupta R.K. Wittenberg B.A. Biophys. J. 1993; 65: 2547-2558Abstract Full Text PDF PubMed Scopus (22) Google Scholar). The observation that the diastolic dysfunction observed in myocytes exposed to elevated extracellular glucose could be duplicated by incubation of cardiomyocytes with glucosamine, a precursor to cellular N- and O-linked glycosylation, suggests that the mechanism may involve increased flux of glucose into the hexosamine pathway (12Ren J. Gintant G.A. Miller R.E. Davidoff A.J. Am. J. Physiol. 1997; 273: H2876-H2883PubMed Google Scholar). Increased hexosamine flux is known to lead to insulin resistance in many tissues (14Marshall S. Bacote V. Traxinger R.R. J. Biol. Chem. 1991; 266: 4706-4712Abstract Full Text PDF PubMed Google Scholar), and recent studies (15Vosseller K. Wells L. Lane M.D. Hart G.W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5313-5318Crossref PubMed Scopus (393) Google Scholar, 16Parker G.J. Lund K.C. Taylor R.P. McClain D.A. J. Biol. Chem. 2003; 278: 10022-10027Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) indicate that dynamic O-GlcNAcylation (the dynamic addition and removal of a single O-linked N-acetylglucosamine residue) may prove to be an important player in diabetes. In this study, we investigated specifically whether the impaired myocardial calcium cycling observed in diabetic cardiomyopathy is linked to O-GlcNAcylation in a hyperglycemia-dependent manner. Using cultured neonatal rat cardiomyocytes, we demonstrate that elevated extracellular glucose impairs calcium cycling, that these changes appear specifically via increased cellular O-GlcNAcylation, and that the detrimental effect of increased cellular O-GlcNAcylation can be mitigated against through the use of adenovirally transfected O-GlcNAcase protein. Materials—Antibodies used in this study were as follows: anti-Sp1 (07–124; Upstate Biotechnology, Lake Placid, NY), anti-MEF-2 (predominantly MEF-2a, sc-313; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-O-GlcNAc monoclonal (CTD 110.6; provided as a gift by Dr. Gerald Hart, The Johns Hopkins University, Baltimore, MD) (17Comer F.I. Vosseller K. Wells L. Accavitti M.A. Hart G.W. Anal. Biochem. 2001; 293: 169-177Crossref PubMed Scopus (234) Google Scholar), and horseradish peroxidase-conjugated anti-rabbit IgG (Amersham Biosciences) and anti-mouse IgM (Sigma). Indo-1/AM and Pluronic were purchased from Molecular Probes (Eugene, OR). Pre-cast Tris/glycine SDS gels and all electrophoresis supplies were from Bio-Rad (Hercules, CA). O-(2-Acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenyl carbamate (PUGNAc) was purchased from Toronto Research Chemicals (North York, Ontario, Canada). All other chemicals were purchased from Sigma unless otherwise noted. Cardiomyocyte Isolation and Culture—Primary cultures of neonatal rat cardiomyocytes were prepared as described previously (18Hartong R. Villarreal F.J. Giordano F. Hilal-Dandan R. McDonough P.M. Dillmann W.H. J. Mol. Cell. Cardiol. 1996; 28: 2467-2477Abstract Full Text PDF PubMed Scopus (50) Google Scholar). Cells were plated onto gelatin-coated culture dishes or laminin-coated glass chamber slides. Plating medium consisted of and mm Cells were to to the for to culture medium and and with glucose (5.5 normal or elevated (25 high medium was with 8 mm glucosamine, or an control mm In were plated high the culture medium was the were of rat to the UDP-N-acetylglucosamine:peptide N-acetylglucosaminyl transferase which the responsible for the of single N-acetylglucosamine to M.A. Hart G.W. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and the human encoding the O-GlcNAcase the responsible for O-linked N-acetylglucosamine Wells L. F.I. G.J. Hart G.W. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), were provided by was into the of an described previously Meyer M. McDonough P.M. Hilal-Dandan R. Dillmann W.H. PubMed Scopus Google Scholar). adenovirus the or were by in in and single were and to high were and by were with a multiplicity of infection of 25 for all of of Ca2+ transients described previously Giordano F.J. Hilal-Dandan R. McDonough P.M. Bluhm W.F. Meyer M. Swanson E. Dillmann W.H. J. 1997; PubMed Scopus Google Scholar). were plated onto glass chamber and incubated for in medium as Cells were with culture medium (5.5 or 25 mm and incubated with the medium 1 and Pluronic for in a Cells were and incubated for in culture medium mm prior to to for of the were to and were and as described previously P.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). and systolic Ca2+ levels were as the and were from transients were and the of transient as a and the diastolic and systolic as SERCA2a protein levels, myocytes were with phosphate-buffered and with of mm mm mm 1 mm were incubated on for and cellular was for in the was by Bio-Rad protein of protein in and were without onto Tris/glycine proteins were to a Bio-Rad and were incubated with a SERCA2a (18Hartong R. Villarreal F.J. Giordano F. Hilal-Dandan R. McDonough P.M. Dillmann W.H. J. Mol. Cell. Cardiol. 1996; 28: 2467-2477Abstract Full Text PDF PubMed Scopus (50) Google Scholar) for 1 by a incubation with a of anti-rabbit (Amersham were by with and exposed to were from cellular O-GlcNAcylation and levels, nuclear were prepared from neonatal cardiomyocytes of the described by R.M. Res. 1983; PubMed Scopus Google Scholar). of were by on Tris/glycine gels and to were exposed to the (17Comer F.I. Vosseller K. Wells L. Accavitti M.A. Hart G.W. Anal. Biochem. 2001; 293: 169-177Crossref PubMed Scopus (234) Google Scholar) and exposed for 1 to anti-mouse were exposed to anti-MEF-2 for 1 by a to anti-rabbit and was as described were from neonatal cardiomyocytes the of R.M. Res. 1983; PubMed Scopus Google Scholar). was to with mm mm mm 1 mm and 1 mm and incubated with of anti-Sp1 Cruz on a for by incubation with of protein Cruz were in in of and for on Tris/glycine and to were incubated for 1 with the used to by to a anti-rabbit and were as described was from cultured myocytes the Anal. Biochem. 1987; PubMed Scopus Google Scholar) as described previously (5Trost S.U. Belke D.D. Bluhm W.F. Meyer M. Swanson E. Dillmann W.H. Diabetes. 2002; 51: 1166-1171Crossref PubMed Scopus (205) Google Scholar). The was on a and to a of the of a of the rat SERCA2a was used to a for was used as a were on and the and and SERCA2a of neonatal cardiomyocytes was the calcium Mol. Cell. Biol. 1987; PubMed Scopus Google Scholar). Cells were transfected in culture medium or with of a of the rat SERCA2a promoter to the of the R. Dillmann W.H. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar) or a control and of and with and into culture Cells to be treated with or were with an of 25 by were incubated for with PBS, and incubated for with in of mm and mm activity was of in a and of were diabetic described previously (5Trost S.U. Belke D.D. Bluhm W.F. Meyer M. Swanson E. Dillmann W.H. Diabetes. 2002; 51: 1166-1171Crossref PubMed Scopus (205) Google Scholar). diabetes was with a single of in to Diabetic were the degree of was via cardiac in and glucose was the Sigma glucose were from and by as described by and Anal. Biochem. 1983; PubMed Scopus Google Scholar). were in of and was for of were to the by for and for The was to a and were in and on a 1 The was as a and the was were in and and into and on a of the was by known of and the as and the for the control for and were on myocyte cultures from to control for among are as was of for calcium transients were a whether elevated extracellular glucose and increased O-GlcNAcylation an effect on calcium calcium transients were in neonatal rat the Ca2+ of are and that the to be we as the of observed and as a calcium we also transients to the diastolic and systolic and from these Treatment of myocytes with or 25 mm glucose resulted in for but the diastolic was significantly prolonged in myocytes exposed to high glucose. The to a in the calcium transient in normal and high glucose-treated myocytes was and and a in the myocytes to to prolonged the and a significantly high glucose systolic Treatment of myocytes with mm glucose with 8 mm glucosamine or increased to and which to a in diastolic calcium for to of myocytes with or effect on normal glucose-treated myocytes but exposed to 25 mm glucose exhibited prolonged that was to treatment with glucosamine and of high glucose-treated myocytes with resulted in a reduced that was to SERCA2a and of mRNA from myocytes demonstrated a substantial in SERCA2a mRNA with to 25 mm glucose compared with normal glucose-treated The in SERCA2a mRNA with high glucose treatment a in SERCA2a protein the role of myocytes were incubated in of of D. J. Biol. Chem. Full Text PDF PubMed Google Scholar), or 8 mm Cells exposed to mm glucose and 8 mm glucosamine exhibited a in SERCA2a protein compared with SERCA2a protein levels in myocytes exposed to 25 mm glucose and glucosamine were compared with myocytes exposed to mm glucose. effect was observed but we to SERCA2a protein levels of glucose we effect when were treated with mm glucose and a mm specifically the effect of increased O-GlcNAcylation on SERCA2a protein expression, we myocytes with adenovirus encoding OGT or GCA an of Treatment of myocytes with high glucose and resulted in a in SERCA2a protein expression compared with control with an adenovirus infection of high myocytes with resulted in improved SERCA2a protein levels The in SERCA2a mRNA and protein expression observed treatment of myocytes with high glucose to whether these effects were in by with the SERCA2a Cells were transfected with a of the rat SERCA2a promoter into the that myocytes exposed to 25 mm glucose or mm mm glucosamine exhibited a in SERCA2a promoter activity and compared with normal glucose of OGT effect on SERCA2a promoter activity in control but we reduced promoter activity in high glucose-treated with myocytes to and high glucose resulted in improved SERCA2a promoter activity, activity to control of myocytes with control effect on SERCA2a promoter activity to Effects of effects of 25 mm glucose and treatment on myocytes that increased O-GlcNAcylation was activity in the numerous transcription factors with the SERCA2a promoter and other of myocardial Ca2+ we whether the of O-GlcNAcylation was altered in nuclear from The monoclonal the of a single N-acetylglucosamine to (17Comer F.I. Vosseller K. Wells L. Accavitti M.A. Hart G.W. Anal. Biochem. 2001; 293: 169-177Crossref PubMed Scopus (234) Google Scholar) and a for the of nuclear Our results that levels of nuclear O-GlcNAcylation were increased in myocytes exposed to 25 mm glucose, mm glucose with 8 mm glucosamine or PUGNAc, or 25 mm glucose and with of high glucose-treated with resulted in a in cellular O-GlcNAcylation changes in O-GlcNAcylation were observed when myocytes were incubated with control Effects on and and The effects of high glucose treatment and elevated O-GlcNAcylation on SERCA2a expression and promoter activity to a mechanism increased to in SERCA2a expression. The transcription that are specifically by O-GlcNAcylation, and the SERCA2a promoter whether hyperglycemia could Sp1, we exposed myocytes to mm glucose, 25 mm glucose, or high glucose and and by Our results indicate that levels of protein were in myocytes exposed to 25 mm glucose, with or without compared with normal glucose-treated these increased levels of O-GlcNAcylation with control effect on expression or of the glucose of the medium the transcription is known to be to O-GlcNAcylation, is important for the expression of a of essential proteins in cardiomyocytes, SERCA2a R. Dillmann W.H. 1997; PubMed Scopus Google Scholar). whether elevated glucose could levels, we exposed myocytes to or 25 mm glucose and with of the 8 mm glucosamine, or from of nuclear that expression was decreased by that increased levels of nuclear treatment with 25 mm glucose, mm glucose with 8 mm glucosamine, or 25 mm glucose and with resulted in and in expression levels, compared with normal glucose of high myocytes with levels to with adenovirus effect of glucose We observed evidence of O-GlcNAcylation of protein. Effects of on for a mechanism diabetic hyperglycemia to flux through the hexosamine pathway, we of and in from diabetic by with exhibited hyperglycemia to Diabetic also exhibited elevated levels of and compared with control and in diabetic and in to heart which is in by early diastolic dysfunction M. S. B.E. 2002; PubMed Scopus Google Scholar). The in myocardial performance in diabetic cardiomyopathy is with of normal cardiomyocytes calcium flux G.N. Res. 1997; PubMed Scopus Google Scholar) and linked to reduced of calcium into the SR as a result of reduced SERCA2a activity and expression (5Trost S.U. Belke D.D. Bluhm W.F. Meyer M. Swanson E. Dillmann W.H. Diabetes. 2002; 51: 1166-1171Crossref PubMed Scopus (205) Google Scholar, 11Ganguly P.K. Pierce G.N. Dhalla K.S. Dhalla N.S. Am. J. Physiol. 1983; 244: E528-E535PubMed Google Scholar). diabetes is a the underlying mechanism for these in cardiomyocyte calcium In this study we demonstrate that of to high extracellular glucose can lead to impaired diastolic calcium and this appears to through a high in SERCA2a expression. we for a mechanism for these in contractile function and calcium with the that increased O-GlcNAcylation is to SERCA2a expression, in through effects on nuclear transcription Treatment of rat cardiomyocytes with elevated (12Ren J. Gintant G.A. Miller R.E. Davidoff A.J. Am. J. Physiol. 1997; 273: H2876-H2883PubMed Google Scholar) to result in prolonged impaired diastolic calcium uptake, and poor contractile performance. effects are to impaired cardiomyocyte function observed in myocytes from diabetic D. Buckler K.J. Le Prigent K. Feuvray D. Am. J. Physiol. 1996; 270: H1529-H1537PubMed Google Scholar). We and these by that neonatal rat cardiomyocytes exposed to elevated prolonged calcium transients and that the observed diastolic in calcium flux can be linked to reduced expression of the SR calcium release and as the of myocardial systolic contraction and diastolic The of calcium into the SR is controlled by SERCA2a, which also SR calcium M. S. J. Mol. Cell Cardiol. 2001; Full Text PDF PubMed Scopus Google Scholar). in the expression of SERCA2a result in a decreased of calcium and lead to prolonged calcium The results of with we (5Trost S.U. Belke D.D. Bluhm W.F. Meyer M. Swanson E. Dillmann W.H. Diabetes. 2002; 51: 1166-1171Crossref PubMed Scopus (205) Google Scholar) that from diabetic reduced levels of SERCA2a protein and that the contractile observed in these could be in expressing SERCA2a and Diabetic hyperglycemia can detrimental effects on the via but increased is on the that increased glucose flux through the hexosamine pathway plays an important role in the of diabetes (14Marshall S. Bacote V. Traxinger R.R. J. Biol. Chem. 1991; 266: 4706-4712Abstract Full Text PDF PubMed Google Scholar, D.A. Diabetes. 1996; PubMed Google Scholar, Diabetes. PubMed Scopus Google Scholar, M. R. M. L. J. 1997; 99: PubMed Scopus Google Scholar, Biochem. Biophys. PubMed Scopus Google Scholar). normal of glucose the cardiomyocyte is to the hexosamine pathway (14Marshall S. Bacote V. Traxinger R.R. J. Biol. Chem. 1991; 266: 4706-4712Abstract Full Text PDF PubMed Google Scholar). may that glucose may be for hexosamine the glucose of diabetic reduced insulin levels or insulin elevated may the myocyte via the glucose which is A. L. M. Meyer J. A. M. J. Am. J. Cardiol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). or insulin levels in the diabetic glucose flux through and glucose to as the hexosamine pathway, or for the of cellular glucose flux into hexosamine in cardiomyocytes from diabetic results demonstrate that from diabetic elevated of an important for N- and O-linked calcium cycling in diabetic may be in on an in levels, as by observation that infection effect on myocytes incubated in normal glucose OGT activity is by The observed in SERCA2a expression we observed in neonatal cardiomyocytes exposed to elevated appear to as a result of increased or result is in to the of contractile performance and calcium observed in rat myocytes exposed to elevated and treated with (12Ren J. Gintant G.A. Miller R.E. Davidoff A.J. Am. J. Physiol. 1997; 273: H2876-H2883PubMed Google Scholar). We are this results suggest that the abnormalities in cardiomyocyte calcium flux result from O-GlcNAcylation of cellular and in particular, an in the of N-acetylglucosamine on evidence indicates that hyperglycemia and elevated can expression in cardiac and E. A. Dillmann W.H. J. Mol. Cell Cardiol. 1988; Full Text PDF PubMed Scopus Google Scholar, W.H. Rev. 1988; PubMed Scopus Google Scholar, D.A. A.J. M.D. J.E. Proc. Natl. Acad. Sci. U. S. A. 89: PubMed Scopus Google Scholar). is known that dynamic O-GlcNAcylation the activity and function of and transcription factors G.W. Annu. Rev. Biochem. 1997; PubMed Scopus Google Scholar, L. Vosseller K. Hart G.W. 2001; PubMed Scopus Google Scholar). K. M.D. A.J. J.E. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar, F. J.E. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) demonstrate that the transcription is to dynamic O-GlcNAcylation and that this results in decreased activity of the protein. that the SERCA2a promoter that are important for expression V. T. M. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), we were by the of these cellular Our results evidence that the effects of hyperglycemia on cardiac and SERCA2a expression in particular, may be through effects on the transcription The in O-GlcNAcylation for a mechanism for the increased hexosamine and the of SERCA2a expression, with cardiomyocyte dysfunction in the diabetic hexosamine and control is by that to cellular O-GlcNAcylation also effects on the expression of transcription factors are known to be important for cardiomyocyte and function Annu. Rev. Cell Biol. PubMed Scopus Google Scholar), the mechanism changes in expression to cardiomyocyte calcium handling or contraction is studies in have demonstrated that is to effect on SERCA2a expression R. Dillmann W.H. 1997; PubMed Scopus Google Scholar). we are by the hyperglycemia and expression, and we to study this this study for the that the removal of is in to cardiomyocyte function to O-GlcNAcase was to diastolic calcium handling and to SERCA2a levels and promoter activity to normal levels in exposed to that otherwise result in increased cellular O-GlcNAcylation and a in function. a recent by G.J. Lund K.C. Taylor R.P. McClain D.A. J. Biol. Chem. 2003; 278: 10022-10027Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) demonstrated results in insulin in The of a O-GlcNAcase to the detrimental effects of elevated extracellular glucose may for a to cardiac dysfunction in diabetic We Dr. L. of of for with calcium