High glucose induces mitochondrial dysfunction independently of protein O-GlcNAcylation.

Diabetes is characterized by hyperglycaemia and perturbations in intermediary metabolism. In particular, diabetes can augment flux through accessory pathways of glucose metabolism, such as the hexosamine biosynthetic pathway (HBP), which produces the sugar donor for the ß-O-linked-N-acetylglucosamine (O-GlcNAc) post-translational modification of proteins. Diabetes also promotes mitochondrial dysfunction. Nevertheless, the relationships among diabetes, hyperglycaemia, mitochondrial dysfunction and O-GlcNAc modifications remain unclear. In the present study, we tested whether high-glucose-induced increases in O-GlcNAc modifications directly regulate mitochondrial function in isolated cardiomyocytes. Augmentation of O-GlcNAcylation with high glucose (33 mM) was associated with diminished basal and maximal cardiomyocyte respiration, a decreased mitochondrial reserve capacity and lower Complex II-dependent respiration (P<0.05); however, pharmacological or genetic modulation of O-GlcNAc modifications under normal or high glucose conditions showed few significant effects on mitochondrial respiration, suggesting that O-GlcNAc does not play a major role in regulating cardiomyocyte mitochondrial function. Furthermore, an osmotic control recapitulated high-glucose-induced changes to mitochondrial metabolism (P<0.05) without increasing O-GlcNAcylation. Thus, increased O-GlcNAcylation is neither sufficient nor necessary for high-glucose-induced suppression of mitochondrial metabolism in isolated cardiomyocytes.

HASF is a stem cell paracrine factor that activates PKC epsilon mediated cytoprotection.

Despite advances in the treatment of acute tissue ischemia significant challenges remain in effective cytoprotection from ischemic cell death. It has been documented that injected stem cells, such as mesenchymal stem cells (MSCs), can confer protection to ischemic tissue through the release of paracrine factors. The study of these factors is essential for understanding tissue repair and the development of new therapeutic approaches for regenerative medicine. We have recently shown that a novel factor secreted by MSCs, which we called HASF (Hypoxia and Akt induced Stem cell Factor), promotes cardiomyocyte proliferation. In this study we show that HASF has a cytoprotective effect on ischemia induced cardiomyocyte death. We assessed whether HASF could potentially be used as a therapeutic agent to prevent the damage associated with myocardial infarction. In vitro treatment of cardiomyocytes with HASF protein resulted in decreased apoptosis; TUNEL positive nuclei were fewer in number, and caspase activation and mitochondrial pore opening were inhibited. Purified HASF protein was injected into the heart immediately following myocardial infarction. Heart function was found to be comparable to sham operated animals one month following injury and fibrosis was significantly reduced. In vivo and in vitro HASF activated protein kinase C ε (PKCε). Inhibition of PKCε blocked the HASF effect on apoptosis. Furthermore, the beneficial effects of HASF were lost in mice lacking PKCε. Collectively these results identify HASF as a protein of significant therapeutic potential, acting in part through PKCε.

O-linked ß-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 ß-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.

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.

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 beta-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.

Augmented O-GlcNAc signaling attenuates oxidative stress and calcium overload in cardiomyocytes.

O-linked ß-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 Ca(2+) 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 Ca(2+) overload and ROS generation. Subjecting neonatal rat cardiac myocytes (NRCMs, n ≥ 6 per group) to hypoxia, or mice (n ≥ 4 per group) to myocardial ischemia reduced O-GlcNAcylation, which later increased during reoxygenation/reperfusion. NRCMs (n ≥ 4 per 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 H(2)O(2), and changes in Ca(2+) 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 Ca(2+) or ROS levels. However, AdOGT significantly (P < 0.05) attenuated both hypoxia and oxidative stress-induced Ca(2+) overload and ROS generation. Additionally, OGA inhibition mitigated both H(2)O(2)-induced Ca(2+) overload and ROS generation. Although AdOGA exacerbated both hypoxia and H(2)O(2)-induced ROS generation, it had no effect on H(2)O(2)-induced Ca(2+) overload. We conclude that inhibition of Ca(2+) overload and ROS generation (inducers of mPTP) might be one mechanism through which O-GlcNAcylation reduces ischemia/hypoxia-mediated mPTP formation.

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.

Cardiac hypertrophy drives PGC-1α suppression associated with enhanced O-glycosylation.

The peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) regulates metabolism and is essential for normal cardiac function. Its activity is suppressed during pressure overload induced cardiac hypertrophy and such suppression at least partially contributes to the associated morbidity. The O-linked ß-N-acetylglucosamine post-translational modification (O-GlcNAc) of proteins is a glucose-derived metabolic signal. The relationship between O-GlcNAc, and PGC-1α activity in cardiac hypertrophy is unknown. We hypothesized that hypertrophy-induced suppression of PGC-1α was at least partially regulated by O-GlcNAc signaling. Treatment of neonatal rat cardiac myocytes with phenylephrine (an inducer of cardiomyocyte hypertrophy) significantly enhanced global O-GlcNAc signaling. Quantitative real-time PCR analysis revealed a downregulation of PGC-1α with concomitant suppression of fatty acid oxidation/mitochondrial genes. Transverse aortic constriction in mice decreased the basal expression of PGC-1α and its downstream genes. Reduction of O-GlcNAc signaling alleviated suppression of PGC-1α and most of its downstream genes. Interestingly, augmentation of O-GlcNAc signaling with glucosamine or PUGNAC (a O-GlcNAcase inhibitor) reduced glucose starvation-induced PGC-1α upregulation even in the absence of hypertrophy. Finally, we found that PGC-1α itself is O-GlcNAcylated. Together, these results reveal the recruitment of O-GlcNAc signaling as a potentially novel regulator of PGC-1α activity during cardiac hypertrophy. Furthermore, O-GlcNAc signaling may mediate constitutive suppression of PGC-1α activity in the heart. Such findings illuminate new possibilities regarding the inter-regulation of O-GlcNAc signaling and also may have some implications for metabolic dysregulation during cardiac diseases.

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.

Calorie restriction attenuates hypertrophy-induced redox imbalance and mitochondrial ATP-sensitive K+ channel repression.

Oxidative stress has been implicated in the pathogenesis of cardiac hypertrophy and associated heart failure. Cardiac tissue grows in response to pressure or volume overload, leading to wall thickening or chamber enlargement. If sustained, this condition will lead to a dysfunctional cardiac tissue and oxidative stress. Calorie restriction (CR) is a powerful intervention to improve health and delay aging. Here, we investigated whether calorie restriction in mice prevented isoproterenol-induced cardiac hypertrophy in vivo by avoiding reactive oxygen species (ROS) production and maintaining antioxidant enzymatic activity. Additionally, we investigated the involvement of mitochondrial ATP-sensitive K+ channels (mitoKATP) in cardiac hypertrophy. CR was induced by 40% reduction in daily calorie ingestion. After 3 weeks on CR or ad libitum (Control) feeding, Swiss mice were treated intraperitoneally with isoproterenol (30 mg/kg per day) for 8 days to induce hypertrophy. Isoproterenol-treated mice had elevated heart weight/tibia length ratios and cardiac protein levels. These gross hypertrophic markers were significantly reduced in CR mice. Cardiac tissue from isoproterenol-treated CR mice also produced less H2O2 and had lower protein sulfydryl oxidation. Additionally, calorie restriction blocked hypertrophic-induced antioxidant enzyme (catalase, superoxide dismutase and glutathione peroxidase) activity repression during cardiac hypertrophy. MitoKATP opening was repressed in isolated mitochondria from hypertrophic hearts, in a manner sensitive to calorie restriction. Finally, mitoKATP inhibition significantly blocked the protective effects of calorie restriction. Altogether, our results suggest that CR improves intracellular redox balance during cardiac hypertrophy and prevents this process in a mechanism involving mitoKATP activation.

Tissue protection mediated by mitochondrial K+ channels.

Two distinct K+ uniporters have been described in mitochondria, ATP-sensitive and Ca2+-activated. Both are capable of protecting tissues against ischemia and other forms of injury when active. These findings indicate a central role for mitochondrial K+ uptake in tissue protection. This review describes the characteristics of mitochondrial K+ uniport, physiological consequences of this transport, forms of tissue damage in which K+ channels are implicated and possible mechanisms through which protection occurs.

Mitochondrial ATP-sensitive K+ channels are redox-sensitive pathways that control reactive oxygen species production.

Pharmacological mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)) opening protects against ischemic damage and mimics ischemic preconditioning. However, physiological and pathological signaling events that open this channel are still not fully understood. We found that catalase, which removes H(2)O(2), is capable of reversing the beneficial effects of ischemic preconditioning but not of mitoK(ATP) agonist diazoxide. On the other hand, 2-mercaptopropionylglycine prevented cardioprotection in both cases, suggesting that this compound may present effects other than scavenging of reactive oxygen species. Indeed, 2-mercaptopropionylglycine and a second thiol-reducing agent, dithiothreitol, impair diazoxide-mediated activation of mitoK(ATP) in isolated heart mitochondria. This demonstrates that mitoK(ATP) activity is regulated by thiol redox status. Furthermore, stimulating the generation of endogenous mitochondrial reactive oxygen species or treating samples with H(2)O(2) strongly enhances mitoK(ATP) activity, in a manner probably dependent on redox sensors located in the channel's sulfonylurea receptor. We also demonstrate that mitoK(ATP) channel activity effectively prevents mitochondrial reactive oxygen release. Collectively, our results suggest that mitoK(ATP) acts as a reactive oxygen sensor that decreases mitochondrial free radical generation in response to enhanced local levels of oxidants. As a result, these channels regulate mitochondrial redox state under physiological conditions and prevent oxidative stress under pathological conditions such as ischemia/reperfusion.

Diazoxide prevents reactive oxygen species and mitochondrial damage, leading to anti-hypertrophic effects.

Pathological cardiac hypertrophy is characterized by wall thickening or chamber enlargement of the heart in response to pressure or volume overload, respectively. This condition will, initially, improve the organ contractile function, but if sustained will render dysfunctional mitochondria and oxidative stress. Mitochondrial ATP-sensitive K+ channels (mitoKATP) modulate the redox status of the cell and protect against several cardiac insults. Here, we tested the hypothesis that mitoKATP opening (using diazoxide) will avoid isoproterenol-induced cardiac hypertrophy in vivo by decreasing reactive oxygen species (ROS) production and mitochondrial Ca2+-induced swelling. To induce cardiac hypertrophy, Swiss mice were treated intraperitoneally with isoproterenol (30 mg/kg/day) for 8 days. Diazoxide (5 mg/kg/day) was used to open mitoKATP and 5-hydroxydecanoate (5 mg/kg/day) was administrated as a mitoKATP blocker. Isoproterenol-treated mice had elevated heart weight/tibia length ratios and increased myocyte cross-sectional areas. Additionally, hypertrophic hearts produced higher levels of H2O2 and had lower glutathione peroxidase activity. In contrast, mitoKATP opening with diazoxide blocked all isoproterenol effects in a manner reversed by 5-hydroxydecanoate. Isolated mitochondria from Isoproterenol-induced hypertrophic hearts had increased susceptibility to Ca2+-induced swelling secondary to mitochondrial permeability transition pore opening. MitokATP opening was accompanied by lower Ca2+-induced mitochondrial swelling, an effect blocked by 5-hydroxydecanoate. Our results suggest that mitoKATP opening negatively regulates cardiac hypertrophy by avoiding oxidative impairment and mitochondrial damage.

Ischemic preconditioning requires increases in reactive oxygen release independent of mitochondrial K+ channel activity.

Mitochondrial ATP-sensitive K+ channels (mitoKATP) mediate ischemic preconditioning, a cardioprotective procedure. MitoKATP activity has been proposed to either enhance or prevent the release of reactive oxygen species. This study tested the redox effects of mitoKATP in order to clarify the role of these channels during preconditioning. We found no evidence that mitoKATP channels increase mitochondrial reactive oxygen species release directly. In addition, neither ischemic preconditioning nor the mitoKATP agonist diazoxide increased antioxidant defenses. Furthermore, increases in reactive oxygen species observed during ischemic preconditioning were not inhibited by mitoKATP antagonists, suggesting that they occur upstream of channel activity. Antioxidants were tested to verify if diazoxide-promoted ischemic protection was dependent on reactive oxygen species. N-Acetylcysteine proved to be an inadequate antioxidant for these tests since it directly interfered with the ability of diazoxide to activate mitoKATP. Catalase reversed the beneficial effect of preconditioning, but not of diazoxide, indicating that reactive oxygen species mediating preconditioning occur upstream of mitoKATP. Taken together, these results demonstrate that ischemic preconditioning increases reactive oxygen release independently of mitoKATP and suggest that the activity of this channel prevents oxidative reperfusion damage by decreasing reactive oxygen species production.

Mitochondrial ATP-sensitive K+ channels prevent oxidative stress, permeability transition and cell death.

Ischemia followed by reperfusion results in impairment of cellular and mitochondrial functionality due to opening of mitochondrial permeability transition pores. On the other hand, activation of mitochondrial ATP-sensitive K(+) channels (mitoK(ATP)) protects the heart against ischemic damage. This study examined the effects of mitoK(ATP) and mitochondrial permeability transition on isolated rat heart mitochondria and cardiac cells submitted to simulated ischemia and reperfusion (cyanide/aglycemia). Both mitoK(ATP) opening, using diazoxide, and the prevention of mitochondrial permeability transition, using cyclosporin A, protected against cellular damage, without additive effects. MitoK(ATP) opening in isolated rat heart mitochondria slightly decreased Ca(2+) uptake and prevented mitochondrial reactive oxygen species production, most notably in the presence of added Ca(2+). In ischemic cells, diazoxide decreased ROS generation during cyanide/aglycemia while cyclosporin A prevented oxidative stress only during simulated reperfusion. Collectively, these studies indicate that opening mitoK(ATP) prevents cellular death under conditions of ischemia/reperfusion by decreasing mitochondrial reactive oxygen species release secondary to Ca(2+) uptake, inhibiting mitochondrial permeability transition.