Increased Enzymatic O-GlcNAcylation of Mitochondrial Proteins Impairs Mitochondrial Function in Cardiac Myocytes Exposed to High Glucose

Increased nuclear protein O-linked β-N-acetylglucosamine glycosylation (O-GlcNAcylation) mediated by high glucose treatment or the hyperglycemia of diabetes mellitus contributes to cardiac myocyte dysfunction. However, whether mitochondrial proteins in cardiac myocytes are also submitted to O-GlcNAcylation or excessive O-GlcNAcylation alters mitochondrial function is unknown. In this study, we determined if mitochondrial proteins are O-GlcNAcylated and explored if increased O-GlcNAcylation is linked to high glucose-induced mitochondrial dysfunction in neonatal rat cardiomyocytes. By immunoprecipitation, we found that several mitochondrial proteins, which are members of complexes of the respiratory chain, like subunit NDUFA9 of complex I, subunits core 1 and core 2 of complex III, and the mitochondrial DNA-encoded subunit I of complex IV (COX I) are O-GlcNAcylated. By mass spectrometry, we identified that serine 156 on NDUFA9 is O-GlcNAcylated. High glucose treatment (30 mm glucose) increases mitochondrial protein O-GlcNAcylation, including those of COX I and NDUFA9 which are reduced by expression of O-GlcNAcase (GCA). Increased mitochondrial O-GlcNAcylation is associated with impaired activity of complex I, III, and IV in addition to lower mitochondrial calcium and cellular ATP content. When the excessive O-GlcNAc modification is reduced by GCA expression, mitochondrial function improves; the activity of complex I, III, and IV increases to normal and mitochondrial calcium and cellular ATP content are returned to control levels. From these results we conclude that specific mitochondrial proteins of cardiac myocytes are O-GlcNAcylated and that exposure to high glucose increases mitochondrial protein O-GlcNAcylation, which in turn contributes to impaired mitochondrial function. Increased nuclear protein O-linked β-N-acetylglucosamine glycosylation (O-GlcNAcylation) mediated by high glucose treatment or the hyperglycemia of diabetes mellitus contributes to cardiac myocyte dysfunction. However, whether mitochondrial proteins in cardiac myocytes are also submitted to O-GlcNAcylation or excessive O-GlcNAcylation alters mitochondrial function is unknown. In this study, we determined if mitochondrial proteins are O-GlcNAcylated and explored if increased O-GlcNAcylation is linked to high glucose-induced mitochondrial dysfunction in neonatal rat cardiomyocytes. By immunoprecipitation, we found that several mitochondrial proteins, which are members of complexes of the respiratory chain, like subunit NDUFA9 of complex I, subunits core 1 and core 2 of complex III, and the mitochondrial DNA-encoded subunit I of complex IV (COX I) are O-GlcNAcylated. By mass spectrometry, we identified that serine 156 on NDUFA9 is O-GlcNAcylated. High glucose treatment (30 mm glucose) increases mitochondrial protein O-GlcNAcylation, including those of COX I and NDUFA9 which are reduced by expression of O-GlcNAcase (GCA). Increased mitochondrial O-GlcNAcylation is associated with impaired activity of complex I, III, and IV in addition to lower mitochondrial calcium and cellular ATP content. When the excessive O-GlcNAc modification is reduced by GCA expression, mitochondrial function improves; the activity of complex I, III, and IV increases to normal and mitochondrial calcium and cellular ATP content are returned to control levels. From these results we conclude that specific mitochondrial proteins of cardiac myocytes are O-GlcNAcylated and that exposure to high glucose increases mitochondrial protein O-GlcNAcylation, which in turn contributes to impaired mitochondrial function. Previous reports have demonstrated that exposure to high glucose impairs calcium flux in cardiac myocytes (1Davidoff A.J. Ren J. Am. J. Physiol. 1997; 272: H159-H167PubMed Google Scholar, 2Clark R.J. McDonough P.M. Swanson E. Trost S.U. Suzuki M. Fukuda M. Dillmann W.H. J. Biol. Chem. 2003; 278: 44230-44237Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar) and that this change is mediated through increased O-linked β-N-acetylglucosamine glycosylation (O-GlcNAcylation) of the nuclear transcription factor Sp1 resulting in decreased sarco (endo) plasmic reticulum Ca2+-ATPase (SERCA2a) gene expression (2Clark R.J. McDonough P.M. Swanson E. Trost S.U. Suzuki M. Fukuda M. Dillmann W.H. J. Biol. Chem. 2003; 278: 44230-44237Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Furthermore, we found that decreasing excessive protein O-GlcNAcylation in the diabetic mouse heart by expression of a transgene encoding β-N-acetyl-d-glucosaminidase (O-GlcNAcase or GCA, (3Dong D.L. Hart G.W. J. Biol. Chem. 1994; 269: 19321-19330Abstract Full Text PDF PubMed Google Scholar, 4Gao Y. Wells L. Comer F.I. Parker G.J. Hart G.W. J. Biol. Chem. 2001; 276: 9838-9845Abstract Full Text Full Text PDF PubMed Scopus (525) Google Scholar)) improved calcium handling and contractile function (5Hu Y. Belke D. Suarez J. Swanson E. Clark R. Hoshijima M. Dillmann W.H. Circ. Res. 2005; 96: 1006-1013Crossref PubMed Scopus (185) Google Scholar). O-GlcNAcylation is an abundant post-translational protein modification occurring on serine and threonine residues (6Vosseller K. Sakabe K. Wells L. Hart G.W. Curr. Opin. Chem. Biol. 2002; 6: 851-857Crossref PubMed Scopus (106) Google Scholar, 7Wells L. Hart G.W. FEBS Lett. 2003; 546: 154-158Crossref PubMed Scopus (172) Google Scholar, 8Zachara N.E. Hart G.W. Biochim. Biophys. Acta. 2006; 1761: 599-617Crossref PubMed Scopus (324) Google Scholar). O-GlcNAcylation and phosphorylation are reversible and highly dynamic processes (9Khidekel N. Ficarro S.B. Clark P.M. Bryan M.C. Swaney D.L. Rexach J.E. Sun Y.E. Coon J.J. Peters E.C. Hsieh-Wilson L.C. Nat. Chem. 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Chem. 1986; 261: 8049-8057Abstract Full Text PDF PubMed Google Scholar). In contrast to enzymatic glycosylation of nuclear and cytosolic proteins, O-GlcNAcylation of mitochondrial proteins has only been explored in a limited fashion. In a recent report a shorter splice variant of O-GlcNAc transferase (OGT) 2The abbreviations used are: OGT, O-GlcNAc transferase; NCM, neonatal rat cardiac myocytes; GCA, O-GlcNAcase; pNP-O-GlcNAc, p-nitrophenyl-N-acetyl-β-d-glucosaminide; HG, high glucose; NG, normal glucose; DTT, dithiothreitol; WGA, wheat germ agglutinin. has been identified which exhibits preferred mitochondrial localization (16Love D.C. Kochan J. Cathey R.L. Shin S.H. Hanover J.A. J. Cell Sci. 2003; 116: 647-654Crossref PubMed Scopus (155) Google Scholar). Recombinant mOGT has O-GlcNAc transferase activity and substrate specificity, which is different from that of OGT (17Lazarus B.D. Love D.C. Hanover J.A. Glycobiology. 2006; 16: 415-421Crossref PubMed Scopus (102) Google Scholar). Despite these facts, it is unclear if mitochondrial proteins in cardiac myocytes are modified by O-GlcNAcylation, if high glucose exposure of cells modifies the extent of O-GlcNAcylation or if increased mitochondrial protein O-GlcNAcylation alters mitochondrial function. Further exploring the occurrence of O-GlcNAcylation on mitochondrial proteins in cardiac myocytes and determining its influence on mitochondrial function has relevance for diabetes induced cardiovascular disease. Cardiovascular complications are the leading cause of diabetes-related morbidity and mortality (18Comer F.I. Vosseller K. Wells L. Accavitti M.A. Hart G.W. Anal. Biochem. 2001; 293: 169-177Crossref PubMed Scopus (234) Google Scholar) and include a diabetes mediated cardiomyopathy which occurs in the absence of coronary artery disease or hypertension (19Holt G. Snow C. Senior A. Haltiwanger R. Gerace L. Hart G. J. Cell Biol. 1987; 104: 1157-1164Crossref PubMed Scopus (316) Google Scholar, 20Walgren J.L. Vincent T.S. Schey K.L. Buse M.G. Am. J. Physiol. Endocrinol Metab. 2003; 284: E424-E434Crossref PubMed Scopus (136) Google Scholar). Several factors have been implicated in the development of diabetic cardiomyopathy, including metabolic, biochemical, and ultrastructural changes within the cardiac myocyte (21Belke D.D. Larsen T.S. Gibbs E.M. Severson D.L. Am. J. Physiol. Endocrinol Metab. 2000; 279: E1104-E1113Crossref PubMed Google Scholar, 22Bhimji S. Godin D.V. McNeill J.H. Acta Anat (Basel). 1986; 125: 195-200Crossref PubMed Scopus (27) Google Scholar, 23Rodrigues B. Cam M.C. McNeill J.H. Mol. Cell Biochem. 1998; 180: 53-57Crossref PubMed Scopus (241) Google Scholar). In particular, because of the primary role of mitochondria in ATP production, impairment of mitochondrial respiratory function is a key contributing factor to decreased contractile function of the diabetic heart. The mechanisms, which contribute to the diabetes-induced decrease in mitochondrial function, have been incompletely explored. Our results show for the first time that specific mitochondrial proteins of cardiac myocytes are O-GlcNAcylated and that exposure to high glucose further enhances the O-GlcNAc modification. A mitochondrial DNA-encoded protein representing subunit I of complex IV is also O-GlcNAcylated, making it likely that O-GlcNAcylation is a modification occurring in mitochondria. In addition, we found that the high glucose induced excessive mitochondrial protein O-GlcNAcylation impairs mitochondrial respiratory complex function, which can be markedly improved by diminishing O-GlcNAcylation through O-GlcNAcase expression. This report demonstrates therefore that the repertoire of O-GlcNAcylation modified proteins includes mitochondrial proteins from cardiac myocytes and that excessive mitochondrial protein O-GlcNAcylation has significant negative effects on mitochondrial function. Isolation and Culture of Neonatal Rat Cardiac Myocytes (NCM)—Primary cultures of neonatal rat cardiomyocytes were prepared as described previously (24Hartong 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 (3 × 106 per 10-cm plate) were plated onto gelatin-coated culture dishes. Plating medium consisted of 4.25:1 Dulbecco’s modified Eagle’s medium: M199, 10% horse serum, 5% fetal bovine serum, 1% penicillin/streptomycin/fungizone, and 5.5 mmol of l,d-glucose. Cells were allowed to adhere to the plates for at least 24 h before different treatments. Cells were cultured in maintenance medium (4.5:1 Dulbecco’s modified Eagle’s medium: M199, 2% fetal bovine serum, 1% penicillin/streptomycin/fungizone) supplemented with either normal glucose (5.5 mm plus 25 mm mannitol) or high glucose (30 mm) concentrations, referred to as NG and HG, respectively. Cells exposed to high glucose were also treated with adenovirus (Adv) encoding human O-GlcNAcase (Adv-GCA) or vector control (Adv-CTR) (2Clark R.J. McDonough P.M. Swanson E. Trost S.U. Suzuki M. Fukuda M. Dillmann W.H. J. Biol. Chem. 2003; 278: 44230-44237Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar), and are referred to as HGGCA or HGCTR. Cells were infected at a multiplicity of infection of 20/cell for both viruses. The culture medium for 2 the cells were and mouse were and in of and bovine with a at medium for at × for the and the with the of the and were at × for and the as the cytosolic protein The as the mitochondrial were further with a as described previously R. K. B. Biochim. Biophys. Acta. PubMed Scopus Google Scholar, F. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). mitochondrial in and in of 1 25 bovine and at × for 1 h in a The lower a and of and at × for with the mitochondria used for and a described before R. K. B. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). protein from treated NCM, the cells were and with a at for in mitochondria 2 and at × for at and the the nuclear and cellular The at × for The as cytosolic protein The mitochondria with mitochondria and at × for The The in 1% at × for the for were determined by and or mitochondrial were on proteins were to The were in 5% used to O-GlcNAc modification a from Hart of and IV subunit I I, were also used as primary and were used as mitochondrial from were in 1% subunit I of complex of mitochondrial with I on a for h at of protein were and for 2 h at the were and with of by for were and of were on for In addition, 25 of mitochondria protein used as and 25 of mitochondrial also of subunits and core 2 of protein the used to and used to for subunits and were from the O-GlcNAc modification on COX I, were treated in NG, HG, and HGGCA as described for 2 the cells were in and COX I with I were by the and with I with I for COX I were of O-GlcNAcylated with were used to the O-GlcNAcylated mitochondrial proteins with the described FEBS Lett. 2006; PubMed Scopus Google Scholar). of cell proteins in of as described were used in with wheat germ the were at for and the The and proteins with and with of for and by The of cell and proteins in the were also in the The with and treatments. O-GlcNAc on with by with and mitochondrial protein NDUFA9 from mouse by and treated by for h at The were treated with and were with as described previously Hart G.W. 2006; PubMed Scopus Google Scholar, L. Vosseller K. Cole Hart G.W. Mol. Cell 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) The were by to mass the were in a The were with in The and as the The at 5% and is to The is to increased to for and for to to 5% The were on the mass a at 2 at the The used in by on the abundant the of the dynamic and the of the of on serine with at the and to I were treated with different glucose and or the complex I activity to the described D. M. S. A. A. Biochem. Biophys. Res. 2003; PubMed Scopus Google Scholar). to and IV activity of complex and complex IV of the mitochondrial to a previously described G. G. 1996; PubMed Google Scholar, Dillmann W.H. Biol. 2003; PubMed Scopus Google Scholar) with modification. treatment described were and by with in medium A for in with of protein were used for complex function in a in a at and complex IV activity were as the of in the of the specific and for complex III, and and for complex for complex III, and for complex IV were used as ATP cellular ATP by cell to the with were with ATP by addition of of and at for with the were and used for ATP The cells in plates were in as and at for the and protein to cellular ATP content. mitochondrial as described before by D.D. Swanson E. Suarez J. Dillmann W.H. Am. J. Physiol. Circ. Physiol. 2007; PubMed Scopus Google Scholar). GCA activity as described by and Hart (3Dong D.L. Hart G.W. J. Biol. Chem. 1994; 269: 19321-19330Abstract Full Text PDF PubMed Google Scholar) as were in of mm 2 mm with of bovine at for The were with addition of of and at activity is as the of in and on neonatal rat myocytes were from at least different and are as or as with with to be if O-GlcNAc modification occurs on mitochondrial proteins mitochondrial and cytosolic proteins were from mouse and submitted to with the O-GlcNAc O-GlcNAc modification on several cytosolic and mitochondrial with cytosolic proteins, mitochondrial proteins have O-GlcNAc modification. mitochondrial proteins show O-GlcNAc like the protein in the that the proteins are from mitochondria and from by proteins from the cytosolic were and with an in is in mitochondrial In the mitochondrial protein COX I has a and in the protein O-GlcNAc in Cardiac Myocytes by High and O-GlcNAcase are exposed to normal glucose mm glucose plus 25 mm to or high glucose (30 In exposed to normal glucose mitochondrial protein O-GlcNAcylation to that in mitochondrial proteins from mouse of to high glucose with control adenovirus increased O-GlcNAcylation A and Our have that expression of an GCA transgene in markedly decreased O-GlcNAcylation of nuclear proteins (2Clark R.J. McDonough P.M. Swanson E. Trost S.U. Suzuki M. Fukuda M. Dillmann W.H. J. Biol. Chem. 2003; 278: 44230-44237Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). we if GCA protein also the excessive mitochondrial protein O-GlcNAcylation occurring in exposed to high The results show that mitochondrial O-GlcNAc modification is markedly by GCA expression. The were and with of plus 1 A. Hart G.W. Mol. Cell 2007; 6: Full Text Full Text PDF PubMed Scopus Google Scholar), the the of to GCA expression in the mitochondria and increased exposure to and were to the protein mitochondrial protein O-GlcNAcylation from with The O-GlcNAcylation with of O-GlcNAcylation by O-GlcNAcase by and by High if increased mitochondrial protein O-GlcNAcylation by high glucose exposure mitochondrial function, we determined mitochondrial respiratory complex function. The mitochondrial respiratory of complexes which and determined the of complex I, III, and in exposed to normal and high The activity of complex I is a D. M. S. A. A. Biochem. Biophys. Res. 2003; PubMed Scopus Google Scholar). in high glucose treatment complex I activity by in to normal glucose treatment GCA expression in high cells returned complex I activity to the control with a control adenovirus transgene on complex I activity results were found for complex and IV activity by a G. G. 1996; PubMed Google Scholar, Dillmann W.H. Biol. 2003; PubMed Scopus Google Scholar). in complex III, high glucose exposure of the activity by with normal glucose control High glucose exposure decrease the activity of complex IV by with normal glucose control GCA expression in exposed to high glucose increased complex and IV control the activity of complex increased by with normal glucose control and the activity of complex IV increased by with normal glucose with a control on complex or IV activity complex III, and for complex activity by a change with high glucose exposure of O-GlcNAcase ATP high glucose treatment induced a decrease in mitochondrial complex and is linked to cellular ATP reduced cellular ATP content by with normal 1 of O-GlcNAcase increased cellular ATP in high cells with the control change cellular ATP treatment with GCA increased ATP content than control treatment by High and by O-GlcNAcase were in mm or 25 mm glucose for h by infection with the mitochondria calcium A. A. Sci. S. A. 2001; PubMed Scopus Google Scholar) and GCA or a transgene expression for an the of this time mitochondrial were High glucose treatment decreased mitochondrial by of O-GlcNAcylation by O-GlcNAcase expression, in treated with high increased mitochondrial calcium control of O-GlcNAcylated in protein members in mitochondrial specific protein members are specific at by and with the The are and with to the of the in complex IV subunit I (COX I) identified as an O-GlcNAcylated protein in the COX I also as a in protein prepared from a of mitochondria and protein from the mitochondrial before COX I is by mitochondrial and in the mitochondrial proteins by as subunit NDUFA9 of complex I, core and core 2 of complex are also O-GlcNAcylated with 1 of the In addition to the with to mitochondrial proteins and we of the O-GlcNAc on mitochondrial respiratory protein NDUFA9 with treatment and as described of of O-GlcNAc modification for by mass spectrometry, treatment this are with which are A mass of to or of the O-GlcNAc modification on this we identified on NDUFA9 and in the also explored if high glucose treatment of increases O-GlcNAcylation of COX I, which we found to be the In addition, we determined if increased O-GlcNAc modification of COX I induced by high glucose treatment can be by GCA expression. these COX I protein is by with the of COX I on with or I O-GlcNAc modified COX I is for COX I protein The that of O-GlcNAc modified COX I increased by in high with control The increased O-GlcNAcylation of COX I is returned to normal by O-GlcNAcase expression. adenovirus has to residues and to proteins with high which has been used to proteins FEBS Lett. 2006; PubMed Scopus Google Scholar, Hart G.W. 1994; PubMed Scopus Google Scholar). protein from treated with normal or high glucose or with to the O-GlcNAc modified mitochondrial were used to the of O-GlcNAc modified or in and that the of or NDUFA9 proteins different in HG, proteins are O-GlcNAcylated and to and proteins and in GCA treatment reduced the proteins to which O-GlcNAcylation on or of COX I or NDUFA9 on and are of O-GlcNAcylated in and O-GlcNAcylated NDUFA9 in NDUFA9 are significant and NG and and HGGCA The and were from and show O-GlcNAcase in that mitochondrial OGT has been (16Love D.C. Kochan J. Cathey R.L. Shin S.H. Hanover J.A. J. Cell Sci. 2003; 116: 647-654Crossref PubMed Scopus (155) Google Scholar) and mitochondrial GCA expression we if is O-GlcNAcase activity within mitochondria. we the O-GlcNAcase activity of mitochondria from mouse heart. The results show that is O-GlcNAcase activity in mitochondria In this report we that in cardiac myocytes mitochondrial proteins are modified by O-GlcNAcylation and that the O-GlcNAcylation mitochondrial function. show that mitochondrial proteins are O-GlcNAcylated including mitochondrial proteins of the respiratory complexes like NDUFA9 of complex I, core and core 2 of complex III, and subunit I of complex IV (COX the first we found the O-GlcNAc modification on mitochondrial of NDUFA9 and which that O-GlcNAc modification occurs in mitochondrial In addition to these identified protein members of mitochondrial mitochondrial proteins significant O-GlcNAcylation 1 and Several protein with O-GlcNAcylation are on of mitochondrial proteins with a specific at O-linked also be that the of O-GlcNAc modification of cytosolic proteins is than that of mitochondrial proteins The O-GlcNAcylation of specific mitochondrial proteins to an enzymatic occurring mitochondria. The COX I subunit of complex IV is by mitochondrial making it highly likely that its O-GlcNAcylation occurs in the mitochondrial also be that a mitochondria shorter splice variant of OGT has been which has different substrate from that of In addition, we O-GlcNAcase expression and activity in the mitochondria. results the that O-GlcNAcylation in the mitochondria as in cell have the occurrence of mitochondrial protein O-GlcNAcylation, including on C. B. J. Y. Mol. Cell 2004; 3: Full Text Full Text PDF PubMed Scopus Google Scholar). we also identified that mitochondrial is O-GlcNAc modified at serine from a mitochondrial protein O-GlcNAcylation and its is In this mass we found that NDUFA9 can be modified at serine This a and for O-GlcNAc modification on mitochondrial phosphorylation mass are to O-GlcNAc because the glycosylation and is treatment to O-GlcNAc and it with a which can be by The increases or mass by and it is to O-GlcNAc on proteins L. Vosseller K. Cole Hart G.W. Mol. Cell 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, A. Hart G.W. Mol. Cell 2007; 6: Full Text Full Text PDF PubMed Scopus Google Scholar). reports have that exposure of cells to high glucose results in increased O-GlcNAcylation of nuclear and cytosolic proteins (2Clark R.J. McDonough P.M. Swanson E. Trost S.U. Suzuki M. Fukuda M. Dillmann W.H. J. Biol. Chem. 2003; 278: 44230-44237Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, Y. Belke D. Suarez J. Swanson E. Clark R. Hoshijima M. Dillmann W.H. Circ. Res. 2005; 96: 1006-1013Crossref PubMed Scopus (185) Google Scholar) its on O-GlcNAcylation of mitochondrial proteins has been explored. that high glucose exposure of cardiac myocytes results in increased O-GlcNAcylation of mitochondrial In particular, we the mitochondrial COX I protein of complex IV and nuclear NDUFA9 protein of complex found that these proteins increased O-GlcNAcylation treatment with high The high in O-GlcNAcylation of mitochondrial proteins by decreased mitochondrial complex function in addition to cellular ATP and decreased mitochondrial calcium levels. of a O-GlcNAcase transgene in cardiac myocytes exposed to high glucose markedly mitochondrial protein of mitochondrial protein O-GlcNAcylation by increases in complex ATP and of mitochondrial calcium exposure to high that excessive O-GlcNAcylation of mitochondrial proteins has effects on mitochondrial function. the by which protein O-GlcNAcylation protein function are explored (10Hart G.W. Housley M.P. Slawson C. Nature. 2007; 446: 1017-1022Crossref PubMed Scopus (1099) Google Scholar). O-GlcNAcylation of proteins is a dynamic and it is to protein on of serine and threonine by phosphorylation and O-GlcNAcylation can have effects on protein function. include modification of the by phosphorylation or O-GlcNAc the phosphorylation induced modification and its on protein function. In addition, and modification of as as (10Hart G.W. Housley M.P. Slawson C. Nature. 2007; 446: 1017-1022Crossref PubMed Scopus (1099) Google Scholar). modification of protein function by O-GlcNAc from the protein modification by phosphorylation and Our show a increased O-GlcNAcylation of specific proteins, for COX I protein of complex IV and decreased activity of complex The and a O-GlcNAcylation induced of specific phosphorylation is a of diabetes mellitus and a diabetes mediated for cardiovascular including the development of heart is S. 2007; PubMed Scopus Google Scholar). mitochondrial function in the diabetic cardiac myocyte is also Y. N. Res. PubMed Scopus Google Scholar) the have been explored. on of increased O-GlcNAcylation of mitochondrial proteins in cardiac myocytes exposed to high that excessive O-GlcNAcylation of mitochondrial proteins also occurs in the diabetic heart. In demonstrated that O-GlcNAc modification occurs on mitochondrial proteins of cardiac myocytes and that excessive O-GlcNAc modification by high glucose contributes to impaired mitochondrial function.