Heberty T. Facundo

O-GlcNAc Signaling in the Cardiovascular System

Cardiovascular function is regulated at multiple levels. Some of the most important aspects of such regulation involve alterations in an ever-growing list of posttranslational modifications. One such modification orchestrates input from numerous metabolic cues to modify proteins and alter their localization and/or function. Known as the &bgr;-O-linkage of N-acetylglucosamine (ie, O-GlcNAc) to cellular proteins, this unique monosaccharide is involved in a diverse array of physiological and pathological functions. This review introduces readers to the general concepts related to O-GlcNAc, the regulation of this modification, and its role in primary pathophysiology. Much of the existing literature regarding the role of O-GlcNAcylation in disease addresses the protracted elevations in O-GlcNAcylation observed during diabetes. In this review, we focus on the emerging evidence of its involvement in the cardiovascular system. In particular, we highlight evidence of protein O-GlcNAcylation as an autoprotective alarm or stress response. We discuss recent literature supporting the idea that promoting O-GlcNAcylation improves cell survival during acute stress (eg, hypoxia, ischemia, oxidative stress), whereas limiting O-GlcNAcylation exacerbates cell damage in similar models. In addition to addressing the potential mechanisms of O-GlcNAc–mediated cardioprotection, we discuss technical issues related to studying protein O-GlcNAcylation in biological systems. The reader should gain an understanding of what protein O-GlcNAcylation is and that its roles in the acute and chronic disease settings appear distinct.

O-linked β-N-acetylglucosamine transferase is indispensable in the failing heart

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 O-linked 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 O-GlcNAcylation 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 O-GlcNAcylation 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.

Non-canonical glycosyltransferase modulates post-hypoxic cardiac myocyte death and mitochondrial permeability transition

O-linked beta-N-acetylglucosamine (O-GlcNAc) is a dynamic, inducible, and reversible post-translational 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 post-hypoxic setting. Therefore, we hypothesized that pharmacological or genetic manipulation of O-GlcNAc 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 that 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.

Unique Hexosaminidase Reduces Metabolic Survival Signal and Sensitizes Cardiac Myocytes to Hypoxia/Reoxygenation Injury

Metabolic signaling through the posttranslational linkage of N-acetylglucosamine (O-GlcNAc) to cellular proteins represents a unique signaling paradigm operative during lethal cellular stress and a pathway that we and others have recently shown to exert cytoprotective effects in vitro and in vivo. Accordingly, the present work addresses the contribution of the hexosaminidase responsible for removing O-GlcNAc (ie, O-GlcNAcase) from proteins. We used pharmacological inhibition, viral overexpression, and RNA interference of O-GlcNAcase in isolated cardiac myocytes to establish its role during acute hypoxia/reoxygenation. Elevated O-GlcNAcase expression significantly reduced O-GlcNAc levels and augmented posthypoxic cell death. Conversely, short interfering RNA directed against, or pharmacological inhibition of, O-GlcNAcase significantly augmented O-GlcNAc levels and reduced posthypoxic cell death. On the mechanistic front, we evaluated posthypoxic mitochondrial membrane potential and found that repression of O-GlcNAcase activity improves, whereas augmentation impairs, mitochondrial membrane potential recovery. Similar beneficial effects on posthypoxic calcium overload were also evident. Such changes were evident without significant alteration in expression of the major putative components of the mitochondrial permeability transition pore (ie, voltage-dependent anion channel, adenine nucleotide translocase, cyclophilin D). The present results provide definitive evidence that O-GlcNAcase antagonizes posthypoxic cardiac myocyte survival. Moreover, such results support a renewed approach to the contribution of metabolism and metabolic signaling to the determination of cell fate.

O-GlcNAc signaling is essential for NFAT-mediated transcriptional reprogramming during cardiomyocyte hypertrophy

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 O-GlcNAc 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.

Mitochondria and Cardiac Hypertrophy

Cardiac tissue responds to long-term hemodynamic load through initiation of a hypertrophic remodeling program. Importantly, if not counteracted this response will eventually lead to organ failure. Cardiac hypertrophic adaptations are complex, and involve multiple cellular events and the mechanisms underlying the development of cardiac hypertrophy are not well understood. Mitochondrial dysfunction has been indicated as a potential and important player in the development of cardiac hypertrophy. Additionally, substantial evidence shows that a significant portion of mitochondrial processes, necessary for normal cardiomyocyte physiology, are impacted by these hypertrophic changes. In this chapter, we will present and discuss the adaptations and changes in the mitochondrial electron transport system, mitochondrial metabolism, mitochondrial biogenesis, oxidative stress, the opening of the mitochondrial permeability transition pore following hypertrophic stimuli, as well as, review the various drugs (targeting mitochondria) that can be used in treatment of cardiac hypertrophy.

AMPK Activators: Not Just for Diabetes?

See related articles, pages 403–411 A patent inability to properly use glycolytic and fatty acid substrates and progressive insulin resistance characterize non–insulin dependent diabetes mellitus. Curiously, the failing myocardium may also share these unfavorable characteristics. Insights from the clinical arena indicate a significant relationship between insulin resistance and New York Heart Association heart failure classification.1 In fact, insulin resistance may beget heart failure and heart failure may beget insulin resistance.2 Fueled by a recent resurgence in studies of the metabolic derangements contributing to cardiovascular disease, we are beginning to comprehend the complexities of metabolism or at least appreciate the bounds of our ignorance. Our understanding of such issues surrounding diabetes and heart failure is predicated on identifying the proximal regulators of substrate availability, sensing, and utilization. One potentially satisfying candidate appears to be AMP-dependent protein kinase (AMPK)3,4 and is the focus of the study by Gundewar et al in this issue of Circulation Research .5 AMPK has emerged as a principal figure in the story of metabolic regulation and dysregulation in diabetes, exercise, and, to a lesser extent, myocardial ischemia. AMPK activation appears to be beneficial in the context of myocardial ischemia and reperfusion, at least in terms of infarct size reduction. Such findings are confirmed by the present5 and previous studies.6 However, the intriguing element of the present study5 is the significant effect of chronic, postischemic treatment with the AMPK-activating biguanide metformin in the context of the failing heart (Figure). Despite a prior contraindication for heart failure because of concerns about the generation of lactic acidosis, interest has been renewed (although it actually never waned for some determined investigators) for the potential use of the insulin “sensitizer” metformin in diabetics with heart failure. Such interest is based not simply on the …

AMP-Dependent Protein Kinase Activators Not Just for Diabetes?

See related articles, pages 403–411 A patent inability to properly use glycolytic and fatty acid substrates and progressive insulin resistance characterize non–insulin dependent diabetes mellitus. Curiously, the failing myocardium may also share these unfavorable characteristics. Insights from the clinical arena indicate a significant relationship between insulin resistance and New York Heart Association heart failure classification.1 In fact, insulin resistance may beget heart failure and heart failure may beget insulin resistance.2 Fueled by a recent resurgence in studies of the metabolic derangements contributing to cardiovascular disease, we are beginning to comprehend the complexities of metabolism or at least appreciate the bounds of our ignorance. Our understanding of such issues surrounding diabetes and heart failure is predicated on identifying the proximal regulators of substrate availability, sensing, and utilization. One potentially satisfying candidate appears to be AMP-dependent protein kinase (AMPK)3,4 and is the focus of the study by Gundewar et al in this issue of Circulation Research .5 AMPK has emerged as a principal figure in the story of metabolic regulation and dysregulation in diabetes, exercise, and, to a lesser extent, myocardial ischemia. AMPK activation appears to be beneficial in the context of myocardial ischemia and reperfusion, at least in terms of infarct size reduction. Such findings are confirmed by the present5 and previous studies.6 However, the intriguing element of the present study5 is the significant effect of chronic, postischemic treatment with the AMPK-activating biguanide metformin in the context of the failing heart (Figure). Despite a prior contraindication for heart failure because of concerns about the generation of lactic acidosis, interest has been renewed (although it actually never waned for some determined investigators) for the potential use of the insulin “sensitizer” metformin in diabetics with heart failure. Such interest is based not simply on the …

AMP-Dependent Protein Kinase Activators

See related articles, pages 403–411 A patent inability to properly use glycolytic and fatty acid substrates and progressive insulin resistance characterize non–insulin dependent diabetes mellitus. Curiously, the failing myocardium may also share these unfavorable characteristics. Insights from the clinical arena indicate a significant relationship between insulin resistance and New York Heart Association heart failure classification.1 In fact, insulin resistance may beget heart failure and heart failure may beget insulin resistance.2 Fueled by a recent resurgence in studies of the metabolic derangements contributing to cardiovascular disease, we are beginning to comprehend the complexities of metabolism or at least appreciate the bounds of our ignorance. Our understanding of such issues surrounding diabetes and heart failure is predicated on identifying the proximal regulators of substrate availability, sensing, and utilization. One potentially satisfying candidate appears to be AMP-dependent protein kinase (AMPK)3,4 and is the focus of the study by Gundewar et al in this issue of Circulation Research .5 AMPK has emerged as a principal figure in the story of metabolic regulation and dysregulation in diabetes, exercise, and, to a lesser extent, myocardial ischemia. AMPK activation appears to be beneficial in the context of myocardial ischemia and reperfusion, at least in terms of infarct size reduction. Such findings are confirmed by the present5 and previous studies.6 However, the intriguing element of the present study5 is the significant effect of chronic, postischemic treatment with the AMPK-activating biguanide metformin in the context of the failing heart (Figure). Despite a prior contraindication for heart failure because of concerns about the generation of lactic acidosis, interest has been renewed (although it actually never waned for some determined investigators) for the potential use of the insulin “sensitizer” metformin in diabetics with heart failure. Such interest is based not simply on the …