The failing heart is subject to elevated metabolic demands, adverse remodeling, chronic apoptosis, and ventricular dysfunction. The interplay among such pathologic changes is largely unknown. Several laboratories have identified a unique posttranslational modification that may have significant effects on cardiovascular function. The Olinked β-N-acetylglucosamine (O-GlcNAc) posttranslational modification (O-GlcNAcylation) integrates glucose metabolism with intracellular protein activity and localization. Because O-GlcNAc is derived from glucose, we hypothesized that altered O-GlcNAcylation would occur during heart failure and figure prominently in its pathophysiology. After 5 d of coronary ligation in WT mice, cardiac O-GlcNAc transferase (OGT; which adds O-GlcNAc to proteins) and levels of OGlcNAcylation were significantly (P < 0.05) elevated in the surviving remote myocardium. We used inducible, cardiac myocyte-specific Cre recombinase transgenic mice crossed with loxP-flanked OGT mice to genetically delete cardiomyocyte OGT (cmOGT KO) and ascertain its role in the failing heart. After tamoxifen induction, cardiac OGlcNAcylation of proteins and OGT levels were significantly reduced compared with WT, but not in other tissues. WT and cardiomyocyte OGT KO mice underwent nonreperfused coronary ligation and were followed for 4 wk. Although OGT deletion caused no functional change in sham-operated mice, OGT deletion in infarcted mice significantly exacerbated cardiac dysfunction compared with WT. These data provide keen insights into the pathophysiology of the failing heart and illuminate a previously unrecognized point of integration between metabolism and cardiac function in the failing heart. heart failure | metabolism | O-GlcNAc | remodeling | infarct

O-linked β-N-acetylglucosamine (O-GlcNAc) is a dynamic, inducible, and reversible posttranslational modification of nuclear and cytoplasmic proteins on Ser/ Thr amino acid residues. In addition to its putative role as a nutrient sensor, we have recently shown pharmacologic elevation of O-GlcNAc levels positively affected myocyte survival during oxidant stress. However, no rigorous assessment of the contribution of O-GlcNAc transferase has been performed, particularly in the posthypoxic setting. Therefore, we hypothesized that pharmacological or genetic manipulation of OGlcNAc transferase (OGT), the enzyme that adds O-GlcNAc to proteins, would affect cardiac myocyte survival following hypoxia/reoxygenation (H/R). Adenoviral overexpression of OGT (AdOGT) in cardiac myocytes augmented O-GlcNAc levels and reduced post-hypoxic damage. Conversely, pharmacologic inhibition of OGT significantly attenuated O-GlcNAc levels, exacerbated post-hypoxic cardiac myocyte death, and sensitized myocytes to mitochondrial membrane potential collapse. Both genetic deletion of OGT using a cre-lox approach and translational silencing via RNAi also resulted in significant reductions in OGT protein and O-GlcNAc levels, and, exacerbated post-hypoxic cardiac myocyte death. Inhibition of OGT reduced O-GlcNAc levels on voltage dependent anion channel (VDAC) in isolated mitochondria and sensitized to calcium-induced mitochondrial permeability transition pore (mPTP) formation, indicating mPTP may be an important target of O-GlcNAc signaling and confirming the aforementioned mitochondrial membrane potential results. These data demonstrate that OGT exerts pro-survival actions during hypoxia-reoxygenation in cardiac myocytes, particularly at the level of mitochondria.

O-linked β-N-acetylglucosamine (O-GlcNAc) is an inducible, dynamically cycling and reversible post-translational modification of Ser/Thr residues of nucleocytoplasmic and mitochondrial proteins. We recently discovered that O-GlcNAcylation confers cytoprotection in the heart via attenuating the formation of mitochondrial permeability transition pore (mPTP) and the subsequent loss of mitochondrial membrane potential. Because Ca2+ overload and reactive oxygen species (ROS) generation are prominent features of post-ischemic injury and favor mPTP formation, we ascertained whether O-GlcNAcylation mitigates mPTP formation via its effects on Ca2+ overload and ROS generation. Subjecting neonatal rat cardiac myocytes (NRCMs, n>/=6/group) to hypoxia, or mice (n>/=4/group) to myocardial ischemia reduced O-GlcNAcylation, which later increased during reoxygenation/reperfusion. NRCMs (n>/=4/group) infected with an adenovirus carrying nothing (control), adenoviral O-GlcNAc transferase (adds O-GlcNAc to proteins, AdOGT), adenoviral O-GlcNAcase (removes O-GlcNAc to proteins, AdOGA), Vehicle, or PUGNAc (blocks OGA; increases O-GlcNAc levels), were subjected to hypoxia-reoxygenation or H2O2 and changes in Ca2+ levels (via Fluo-4AM and Rhod-2AM), ROS (via DCF), and mPTP formation (via calcein-MitoTracker Red colocalization) were assessed using time-lapse fluorescence microscopy. Both OGT and OGA overexpression did not significantly (p>0.05) alter baseline Ca2+ or ROS levels. However, AdOGT significantly (p<0.05) attenuated both hypoxia and oxidative stress-induced Ca2+ overload and ROS generation. Additionally, OGA inhibition mitigated both H2O2-induced Ca2+ overload and ROS generation. Although AdOGA exacerbated both hypoxia and H2O2-induced ROS generation, it had no effect on H2O2-induced Ca2+ overload. We conclude that inhibition of Ca2+ overload and ROS generation (inducers of mPTP) might be one mechanism through which O-GlcNAcylation reduces ischemia/hypoxia-mediated mPTP formation.

Key Words: myocardial ischemia Ⅲ glucose Ⅲ diabetes mellitus Ⅲ mitochondria M uch has been written about glycolysis, ␤-oxidation, and the other major metabolic pathways in cells. Yet, there are several underinvestigated accessory glycolytic pathways whose importance in the cardiovascular system is now beginning to be appreciated. Eukaryotic glycosylation represents a highly varied and complex collection of biological pathways, which are too broad for serious discussion here.This review focuses on one unique form of glycosylation, and the reader should refer to definitive sources 1 for information on other forms of glycosylation. The hexosamine biosynthetic pathway (HBP) exemplifies one such accessory pathway for glucose metabolism. Based on evidence from cell lines, 2 the HBP consumes a small fraction of glucose and involves a series of enzyme-catalyzed reactions ending with the formation Original

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Abstract-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 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 coworkers 5,6 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 def...

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The regulation of cardiomyocyte hypertrophy is a complex interplay among many known and unknown processes. One specific pathway involves the phosphatase calcineurin, which regulates nuclear translocation of the essential cardiac hypertrophy transcription factor, nuclear factor of activated T-cells (NFAT). Although metabolic dysregulation is frequently described during cardiac hypertrophy, limited insights exist regarding various accessory pathways. One metabolically derived signal, beta-O-linked N-acetylglucosamine (O-GlcNAc), has emerged as a highly dynamic posttranslational modification of serine and threonine residues regulating physiological and stress processes. Given the metabolic dysregulation during hypertrophy, we hypothesized that NFAT activation is dependent on O-GlcNAc signaling. Pressure overload-induced hypertrophy (via transverse aortic constriction) in mice or treatment of neonatal rat cardiac myocytes with phenylephrine significantly enhanced global O-GlcNAc signaling. NFAT-luciferase reporter activity revealed O-GlcNAc-dependent NFAT activation during hypertrophy. Reversal of enhanced OGlcNAc signaling blunted cardiomyocyte NFAT-induced changes during hypertrophy. Taken together, these results demonstrate a critical role of O-GlcNAc signaling in NFAT activation during hypertrophy and provide evidence that O-GlcNAc signaling is coordinated with the onset and progression of cardiac hypertrophy. This represents a potentially significant and novel mechanism of cardiac hypertrophy, which may be of particular interest in future in vivo studies of hypertrophy.nuclear factor of activated t cells; transverse aortic constriction CARDIAC HYPERTROPHY REFLECTS a compensatory response to pressure or volume overload, sarcomeric abnormalities, or loss of contractile ability through partial death of the myocardium (6, 14). The development of pathological pressure overloadinduced hypertrophy has deleterious consequences on the heart and often progresses to decompensation and overt heart failure, the leading cause of death in the industrialized world (30). The molecular mechanisms contributing to cardiac hypertrophy are common to several stressors (25), involve the activation of the nuclear factor of activated T-cells (NFAT), and induce the fetal gene program (17). Intracellular Ca 2ϩ participates as an obligatory signaling molecule during hypertrophy after an increase in cardiac workload or in response to myocyte stretch (2). Elevated Ca 2ϩ levels activate the phosphatase calcineurin culminating in NFAT dephosphorylation and nuclear translocation of NFAT (17). The final macromolecular response involves an increase in cell size and protein synthesis (25) and is accompanied by an eventual energetic defect (7,14,30). Although several groups have investigated the metabolic defects accompanying cardiac hypertrophy, little attention has been given to the contribution of accessory pathways of glucose metabolism, such as the hexosamine biosynthetic pathway (see Refs. 20, 21 for review). The hexosamine biosynthetic pathw...