Protein O-GlcNAcylation and Cardiovascular (Patho)physiology*

Our understanding of the role of protein O-GlcNAcylation in the regulation of the cardiovascular system has increased rapidly in recent years. Studies have linked increased O-GlcNAc levels to glucose toxicity and diabetic complications; conversely, acute activation of O-GlcNAcylation has been shown to be cardioprotective. However, it is also increasingly evident that O-GlcNAc turnover plays a central role in the delicate regulation of the cardiovascular system. Therefore, the goals of this minireview are to summarize our current understanding of how changes in O-GlcNAcylation influence cardiovascular pathophysiology and to highlight the evidence that O-GlcNAc cycling is critical for normal function of the cardiovascular system.

titative analysis of glucose flux via the HBP in the heart has yet to be performed. Although glucose availability is an important factor in O-GlcNAc synthesis, glutamine is critical as the amine donor for glucosamine 6-phosphate, whereas fatty acid metabolism is likely the primary source for the acetyl moiety. Thus, multiple nutrients contribute to both UDP-GlcNAc and protein O-GlcNAc synthesis. In addition to its synthesis, the levels of protein O-GlcNAc are also regulated by the activity of ␤-Nacetylhexosaminidase (O-GlcNAcase (OGA)), which catalyzes removal of this post-translational modification.
A little over a decade following the identification of O-GlcNAc protein modification by Torres and Hart (3), the small heat shock protein ␣B-crystallin was shown to be an O-GlcNAc target in rat heart (4). Using vascular smooth muscle cells, Han and Kudlow (5) demonstrated in 1997 that O-GlcNAcylation of the transcription factor Sp1 modulated its susceptibility to proteasomal degradation, concluding that this may provide a link between nutritional status and transcriptional regulation. In the same year, Yki-Järvinen et al. (6) reported that OGT activity was significantly higher in rat heart compared with liver, fat, and other types of striated muscle; they also hypothesized that O-GlcNAcylation could be involved in mediating glucose toxicity in insulin-responsive tissues. To our knowledge, these were the first reports of O-GlcNAcylated proteins in cardiac or vascular tissues, as well as the first to suggest that protein O-GlcNAcylation may contribute to the adverse effects of insulin resistance and diabetes in the cardiovascular system.
Studies in cardiovascular tissues can be broadly divided into two categories: 1) the adverse effects of chronically elevated O-GlcNAc levels typically associated with cardiometabolic diseases, such as diabetes, and 2) the protective effects of increased O-GlcNAc levels observed in the setting of acute injury models ( Fig. 1). Although we will summarize these findings, there have been a number of recent reviews that have covered these areas in greater detail (7)(8)(9)(10)(11)(12)(13). Consequently, our goal here is to focus on some emerging areas, including the role of O-GlcNAcylation in the regulation of vascular function, cardiac hypertrophy, Ca 2ϩ signaling, and epigenetics, and we will highlight evidence that O-GlcNAc cycling is critical for normal function of the cardiovascular system (Fig. 2).

O-GlcNAcylation and the Heart
As glucose is a substrate for UDP-GlcNAc synthesis via the HBP, glucose availability is a key mediator of O-GlcNAcylation. Consequently, chronically elevated protein O-GlcNAcylation in hearts from diabetic animals has been implicated in glucose toxicity and linked to multiple facets of cardiomyocyte dysfunction in diabetes including impaired contractility, mitochondrial dysfunction, and metabolic dysfunction (14 -22). Moreover, many transcription factors that regulate cell survival are also targets for O-GlcNAcylation (23) and could potentially contribute to an impaired stress response of diabetic cardiomyocytes. Increased HBP flux has also been linked to insulin resistance, and many proteins that play a key role in metabolic regulation, such as AMPK (AMP-activated protein kinase), IRS1/2, Akt, and GLUT4, have been shown to be O-GlcNAc-modified, resulting in blunted activity (24,25). Although these findings have yet to be confirmed in the heart, they illustrate the potential mechanisms by which O-GlcNAcylation could contribute to metabolic dysfunction characteristic of the heart in the setting of metabolic disease. It is of note that increasing HBP flux and O-GlcNAcylation in the isolated perfused heart using glucosamine resulted in an increase in fatty acid oxidation, which was associated with O-GlcNAcylation of the fatty acid transporter FAT (fatty acid translocase)/CD36 (26). This suggests that elevated O-GlcNAc levels could contribute not only to cardiac glucotoxicity, but also to cardiac lipotoxicity. Increased O-GlcNAcylation may also be detrimental to cardiomyocytes by attenuating responsiveness to hypertrophic and autophagic stimuli (17,18), dysregulation of the cardiac circadian clock (27), and impaired cardiac cell differentiation (28).
Collectively, these reports support the widely held belief that increased O-GlcNAc levels are detrimental to the heart. However, in 2004, Zachara et al. (29) showed for the first time that acute increases in cellular O-GlcNAc levels were cytoprotective. Subsequently, there have been numerous reports demonstrating that increased O-GlcNAcylation, either by augmenting synthesis with glucosamine or by attenuating degradation by inhibition of OGA, is cardioprotective in the setting of acute injury, such as oxidative stress, calcium overload, endoplasmic reticulum stress, and ischemia/reperfusion ( Fig. 1) (30 -37). Conversely, decreasing O-GlcNAc levels through overexpression of OGA decreased tolerance of cardiomyocytes to oxidative stress (38). These in vitro observations also translated to in vivo models, in which administration of the OGA inhibitor O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino N-phenylcarbamate (PUGNAc) decreased infarct size similar to that seen with ischemic preconditioning (34), as well as improved cardiac function following trauma/hemorrhage (39,40).
Of particular note, in both the trauma/hemorrhage studies and the in vitro ischemia/reperfusion models, increasing the O-GlcNAc levels during resuscitation/reperfusion was found to be particularly effective in improving survival and recovery of cardiac function, respectively (35, 39 -41). Interestingly, ischemic preconditioning has been shown to increase cardiac O-GlcNAc levels (34,42); however, whether this contributes to the cardioprotection following this intervention is not known. Remote ischemic preconditioning also appears to influence myocardial O-GlcNAc levels, albeit through as yet unknown circulating factors (43). A number of other cardioprotective interventions have also been associated with increased O-GlcNAc levels (44 -46), raising the intriguing possibility that activation of O-GlcNAcylation could represent a nodal point linking diverse cardioprotective strategies.
The mechanisms leading to increased O-GlcNAc levels in the heart in response to ischemia or ischemic preconditioning are not well understood. Wang et al. (47) demonstrated a causal link between the unfolded protein response, activation of Xbp1s (X-box-binding protein 1, spliced), and increased O-GlcNAc synthesis. They found that Xbp1s transcriptionally activated GFAT1 under a range of different stress conditions, including ischemia/reperfusion. Moreover, not only was Xbp1s required for increased HBP flux and O-GlcNAcylation following ischemia/reperfusion, but increased expression of Xbp1s alone was sufficient for HBP-dependent cardioprotection. It should be noted, however, that other mechanisms known to be activated following ischemia/reperfusion potentially contribute altered O-GlcNAc levels. For example, it has been shown that stress-induced increases in cardiomyocyte O-GlcNAc levels were mediated at least in part by Ca 2ϩ -dependent activation of Ca 2ϩ /calmodulin-dependent protein kinase II (CaMKII) (48).
The role of O-GlcNAc within different cellular organelles in cardiomyocytes is also beginning to attract attention. Of particular relevance to the impact of O-GlcNAcylation in the heart is the role of O-GlcNAc modification of mitochondrial proteins. For example, respiratory chain complex proteins, such as subunit NDUFA9 of complex I, subunits core 1 and core 2 of complex III, and the mitochondrial DNA-encoded subunit I of complex IV (cytochrome c oxidase 1), as well as DRP1 (dynamin-related protein 1), a regulator of mitochondrial fission, are O-GlcNAcylated in cardiomyocytes (49,50). Increased O-GlcNAcylation of these proteins has been linked to the adverse effects of hyperglycemia on mitochondrial function (49,50). Hearts from rats with a low running capacity have higher O-GlcNAcylation of complexes I and IV and the voltagedependent anion channel (VDAC) compared with those from rats with a high running capacity (51). An earlier study by Jones et al. (34) also demonstrated that VDAC was an O-GlcNAc target in the heart. Further support for a functional role of mitochondrial O-GlcNAcylation was provided by Palaniappan et al. (52), who reported that VDAC2 was O-GlcNAcylated in both mouse embryonic fibroblasts and Jurkat cells. Importantly, they also found that mitochondrial dysfunction and cell death induced by global increases in O-GlcNAc were dependent on the presence of VDAC2. Whether this is also true in cardiomyocytes has yet to be determined. Although evidence suggests that changes in O-GlcNAcylation influence mitochondrial function, how matrix proteins are O-GlcNAcylated remains a mystery. Despite reports that the 103-kDa isoform of OGT resides within mitochondria (53), it is unclear how the precursors for O-GlcNAc synthesis enter the mitochondria. In addition, a mitochondrial OGA, which would be required for an active mitochondrial O-GlcNAc cycle, has yet to be identified.
Overall, these findings demonstrate that changes in O-GlcNAc levels exert apparently divergent effects on the heart. Although the mechanisms remain poorly understood, current evidence suggests that this is likely due to a complex combination of the duration of exposure, the presence or absence of an additional cellular insult, the nature of the specific stress, and the hormonal milieu surrounding the cardiomyocyte.

O-GlcNAcylation and Vascular Function
Much of the early work examining the role of O-GlcNAcylation in vascular function was in the context of identifying specific mechanisms underlying the adverse effects of hyperglycemia. An important example of this is the work by Brownlee and colleagues (54), who reported that hyperglycemia increased O-GlcNAcylation of endothelial NOS (eNOS) in bovine aortic endothelial cells, which was associated with a decrease in phosphorylation at Ser-1177, the site responsible for activation of the enzyme. They also found that eNOS activity was reduced in aortas from diabetic rats, with no change in eNOS protein levels. An inverse correlation between levels of eNOS O-GlcNAcylation and phosphorylation has also been reported in human coronary artery endothelial cells (55). Furthermore, insulin-stimulated phosphorylation of eNOS was significantly attenuated by both hyperglycemia and direct activation of the HBP by glucosamine (55). Interestingly, Musicki et al. (56) demonstrated that increased O-GlcNAcylation of Ser-1177 in eNOS attenuated the shear stress-induced increase in penile blood flow during diabetes. More recently, Beleznai and Bagi (57) reported that increased O-GlcNAcylation contributed to impaired NO-mediated arteriolar dilation following hyperglycemia, supporting the concept that O-GlcNAcylation may be a significant contributing factor to microvascular disease in diabetic patients. Vascular calcification is another complication contributing to the increased morbidity and mortality of patients with diabetes. A recent study showed that calcifica-tion was enhanced in response to increased O-GlcNAcylation and that this was mediated through O-GlcNAc-induced activation of Akt (58).
Using intact rat aorta, Lima et al. (59) reported that augmenting O-GlcNAc levels by inhibiting OGA enhanced reactivity to vasoconstrictors, such as phenylephrine and serotonin, was associated with decreased phosphorylation of both eNOS and Akt. Endothelin-1-induced vasoconstriction was found to be O-GlcNAc-dependent, and the RhoA/Rho kinase pathway was a potential downstream mediator of O-GlcNAc signaling in the vasculature (60). Similarly, Kim et al. (61) reported that glucosamine augmented vessel contraction in endothelium-denuded aortic rings, which was prevented by inhibition of OGT. Overall, these studies suggest that increased vascular O-GlcNAc levels may contribute to vascular dysfunction that occurs with hypertension.
Similar to studies described in the heart, several reports suggest that increased O-GlcNAcylation also functions in a vasoprotective role. Following endoluminal injury of the carotid artery, O-GlcNAc levels decreased rapidly and remained depressed for at least 24 h (62). Treatment with glucosamine or PUGNAc increased O-GlcNAc levels in injured arteries, which was associated with lower acute inflammatory responses compared with untreated groups. In addition, prolonged treatment with glucosamine attenuated neointima formation (62). Subsequent studies in vascular smooth muscle cells demonstrated that increased O-GlcNAcylation attenuated TNF-␣-induced expression of inflammatory mediators, which was associated with increased O-GlcNAcylation of NF-B p65 and inhibition of NF-B signaling (63). Increasing O-GlcNAc levels also attenuated TNF-␣-mediated hypercontractility and endothelial dysfunction in isolated aortic rings, which were accompanied by a decrease in inducible NOS expression and lower protein nitrosylation (64).
Taken together, these data suggest that O-GlcNAc plays a pivotal but complex role in the vasculature. As with studies in cardiomyocytes and non-cardiac cells, the presence and activation of O-GlcNAc in the vasculature regulate both cytoprotection and inflammation. O-GlcNAc cycling also regulates vascu- lar-specific processes, such as calcification, vasoconstriction, and vasodilation.

Role of O-GlcNAc in Hypertrophic Signaling
It is now well established that activation of the Ca 2ϩ -sensitive phosphatase calcineurin, which dephosphorylates nuclear factor of activated T-cells (NFAT), leading to NFAT nuclear translocation, is a critical step in the initial transcriptional response to pathological hypertrophic stimuli (65). Marchase and colleagues (66,67) reported that agonist-induced nuclear translocation of NFAT in cardiomyocytes was inhibited by hyperglycemia. The effects of hyperglycemia were attenuated by inhibition of GFAT and mimicked by activation of the HBP with glucosamine, supporting the notion that O-GlcNAcylation might influence hypertrophic signaling. This was confirmed in subsequent studies in which both glucosamine and PUGNAc attenuated the angiotensin II-induced increase in intracellular Ca 2ϩ and increased O-GlcNAc levels (68). Inhibition of the HBP also restored the sensitivity of diabetic cardiomyocytes to both angiotensin II and phenylephrine (17). Collectively, these observations raise the possibility that the adverse response of the diabetic heart to stress could be a consequence of O-GlcNAc-mediated alterations in the normal hypertrophic signaling pathways.
Interestingly, Facundo et al. (69) reported that O-GlcNAc signaling was required for NFAT-mediated regulation of cardiomyocyte hypertrophy. They demonstrated in neonatal cardiomyocytes that activation of hypertrophic signaling by phenylephrine was associated with an increase in O-GlcNAc levels. Moreover, when this response was blunted either by inhibition of the HBP or by increased expression of OGA, NFAT activation and nuclear translocation were attenuated, and hypertrophic responses were blunted (69). These observations are consistent with a number of reports demonstrating that pathological hypertrophy is associated with increased overall O-GlcNAcylation (70,71). Conversely, however, a genetic deletion of OGT in cardiomyocytes induced postnatal pathological cardiac hypertrophy and cardiac dysfunction in the absence of any stress or additional stimuli (72).
The apparent paradox of increased O-GlcNAc levels being required for hypertrophic signaling while also inhibiting the same pathway is similar to the reports of increasing O-GlcNAc levels having both adverse and beneficial effects on cell survival. One possible explanation for these observations is that under normal conditions, a transient increase in O-GlcNAcylation in response to a hypertrophic stimulus plays a key role in activation of NFAT, but under conditions of chronically elevated O-GlcNAc levels or impaired O-GlcNAc cycling as seen in diabetes, this acute transient response is impaired.
In contrast to pathological hypertrophy, physiological cardiac hypertrophy occurs in response to exercise training. The first studies to examine the effects of exercise on cardiac O-GlcNAc levels found that following an intense swimming training protocol in rats, O-GlcNAc levels were decreased in both non-diabetic and type 1 diabetic hearts (19,51). In contrast, Cox and Marsh (20) recently reported that moderateintensity treadmill training actually increased cardiac O-GlcNAcylation in type 2 diabetic mouse hearts. The same group also found that a single bout of intense exercise altered the nucleocytosolic distributions of O-GlcNAc in the non-diabetic heart in the absence of any overall change in cardiac O-GlcNAcylation (73). Moreover, exercise decreased O-GlcNAcylation of OGT and reduced the association of OGT with REST (repressor element 1-silencing transcription factor), a corepressor of hypertrophic gene transcription (73).
Collectively, these data suggest that acute perturbations of O-GlcNAc likely modulate both pathological and physiological hypertrophic remodeling. Moreover, it appears that impaired O-GlcNAc cycling resulting in either a chronic excess or lack of O-GlcNAc negatively affects the normal hypertrophic response and accelerates the progression to heart failure.

O-GlcNAc and Ca 2؉ Signaling
Regulation of cellular Ca 2ϩ homeostasis is critical for all cells. This is particularly true in excitable cells, such as cardiomyocytes, where large rapid excursions in intracellular Ca 2ϩ concentration are required for normal contractile function. Thus, in addition to contractile function, Ca 2ϩ also plays a central role in regulating cardiomyocyte metabolism, protein folding, and Ca 2ϩ -dependent signaling pathways. Early studies by Davidoff and colleagues (74 -77) demonstrated that a brief duration of diabetes resulted in impaired contractile function of isolated cardiomyocytes, characterized primarily by slower relaxation and decreased sarcoplasmic/endoplasmic reticulum Ca 2ϩ -ATPase (SERCA) activity. These observations could be mimicked by exposure of normal cardiomyocytes to hyperglycemia; the reduction in SERCA activity occurred in the absence of changes in SERCA protein levels or phospholamban phosphorylation (74 -77). Interestingly, they also found that glucosamine had the same effects as high glucose on cardiomyocyte relaxation (76), suggesting for the first time a potential link between SERCA activity and the HBP. More recently, phospholamban has been shown to be O-GlcNAcylated (78), which attenuated its phosphorylation. Prolonged periods of diabetes resulted in a decrease in cardiac SERCA protein levels, which was associated with O-GlcNAcylation of Sp1 (15). Overexpression of OGA restored SERCA levels and normalized contractile function both in isolated cardiomyocytes exposed to high glucose (14) and in diabetic hearts (15,22). Of particular note, Erickson et al. (21) recently reported that diabetic hearts exhibit increased O-GlcNAcylation of CaMKII, resulting in activation of this Ca 2ϩ -dependent kinase. These observations have potentially wide-ranging implications, given the key role of CaMKII in the regulation of cardiac physiology and pathophysiology (80).
As mentioned above, the Ca 2ϩ -activated phosphatase calcineurin plays a central role in activating pathological hypertrophic signaling through activation of NFAT activation and subsequent nuclear translocation. In non-excitable cells, agonist-mediated Ca 2ϩ signaling, including NFAT activation, is known to be mediated through a store-operated calcium entry (SOCE) pathway (81). Although SOCE has been largely ignored in the heart, a number of studies have recently shown that STIM1, a central component of SOCE, not only is present in cardiomyocytes, but also plays a role in regulating pathological hypertrophy (82)(83)(84). Interestingly, Zhu-Mauldin et al. (85) showed that STIM1 is a target for O-GlcNAcylation and that increased O-GlcNAc levels impaired both STIM1 function and SOCE. These observations provide a potential mechanism by which increased O-GlcNAcylation blunts hypertrophic signaling.
Thus, there is increasing evidence that O-GlcNAcylation plays an important role in regulating the function of several proteins involved in regulating cardiomyocyte Ca 2ϩ homeostasis, Ca 2ϩ signaling, and sarcoplasmic reticulum function. This represents an underexplored mechanism for modulating cardiomyocyte Ca 2ϩ levels and potentially a new intersection between nutrient and Ca 2ϩ signaling.

O-GlcNAc and Epigenetics
Although protein O-GlcNAcylation is now firmly established as an essential mediator of intracellular signal transduction, there is increasing evidence to support the notion that OGT is a vital component of chromatin-protein complexes and that O-GlcNAc regulates multiple epigenetic processes. A role for O-GlcNAc in epigenetic regulation is not new, as several key papers over 10 years ago showed that O-GlcNAc, OGT, and OGA all play important roles in gene transcription machinery (86 -89). However, subsequent studies have demonstrated an even more substantial and complex role for O-GlcNAc and OGT in epigenetic regulation with the identification of OGT as a member of the polycomb group complex in Drosophila (90) and of histones H2A, H2B, and H4 as O-GlcNAc targets (91). Analyses by Love and colleagues (92) using anti-O-GlcNAc ChIP-on-chip whole-genome tiling arrays detected over 800 promoters where O-GlcNAc cycling occurs. They also found that these were primarily genes associated with phosphatidylinositol 1,4,5-trisphosphate signaling, hexosamine biosynthesis, and lipid/carbohydrate metabolism (92). O-GlcNAcylation of histone residues has been shown to have a direct effect on ubiquitination, phosphorylation, methylation, and acetylation of amino acids that are key regulators of transcriptional activation and repression (91,93,94). For example, the TET (ten-eleven translocation methylcytosine dioxygenase) proteins are 5methylcytosine oxidases that demethylate DNA. Interactions between OGT and TET proteins and O-GlcNAcylation of histone modifiers, such as MLL5, EZH2, and mSin3A, help to finetune epigenetic processes (95)(96)(97)(98).
There is substantial evidence demonstrating that epigenetic processes regulate cardiac cell function and are altered in response to various stimuli (99); however, a role for O-GlcNAcylation in regulating cardiac epigenetics had not been explored until recently. Marsh and co-workers (20,73,79) reported for the first time that mSin3A and REST are O-GlcNAcylated in mouse heart; moreover, not only are HDAC1, HDAC2, HDAC4, and HDAC5 O-GlcNAcylated, but also OGT interacts with all of these proteins. Although there was no impact of a high fat/high sugar diet on cardiac O-GlcNAc levels (79), acute exercise decreased the OGT/REST association in non-diabetic mouse hearts (73). In diabetic hearts, Cox and Marsh (20) observed an increase in mSin3A O-GlcNAcylation irrespective of training status, as well as an increased association between OGT/REST in the sedentary diabetic heart that was attenuated with exercise training.
The above results demonstrate for the first time a role for O-GlcNAc and OGT in epigenetic regulation in cardiomyocytes, providing novel insights into our understanding of cardiac remodeling. Identifying the O-GlcNAc-dependent epigenetic processes in the heart and determining how the downstream effects differ from non-cardiac cells will provide a greater understanding of the regulation of gene transcription in the heart. Importantly, characterizing the extent to which O-GlcNAc-mediated epigenetic processes are plastic, reversible, and heritable will enable us to determine whether the cardiac effects of O-GlcNAc can be transferred to subsequent generations.

Conclusions
Initial investigations into the role of O-GlcNAc in the heart focused primarily on the observation that hyperglycemia in diabetes was associated with a chronic sustained elevation of protein O-GlcNAcylation and was therefore detrimental. Conversely, subsequent work emerged to demonstrate that acute up-regulation of O-GlcNAcylation is cardioprotective and thus beneficial. Recent work has also shown that O-GlcNAc is an integral component of processes that control gene transcription and cellular metabolism, function, and growth, and that O-GlcNAc cycling is critical for normal function of the cardiovascular system. These exciting findings indicate a complex role of O-GlcNAc in the normal regulation of the cardiovascular system (Fig. 2); consequently, alterations in O-GlcNAc levels cannot be simply interpreted as either good or bad.
A number of studies have explored therapeutic approaches designed either to decrease O-GlcNAc levels in diabetes or to acutely increase them in the setting of ischemia/reperfusion. It is clear, however, that there are complex multilayered interactions between O-GlcNAcylation and cardiomyocyte function, which preclude a systemic or even cardiomyocyte-targeted approach. As a result, a more thorough and nuanced understanding is required of the role of O-GlcNAc cycling in the regulation of normal cardiovascular function and the development of cardiovascular disease before effective therapeutic approaches can be developed.
Acknowledgments-We appreciate the contributions of current and past members of our laboratories as well as numerous collaborators who have played critical roles in helping advance our understanding of the role of O-GlcNAcylation in regulating the cardiovascular system. We also thank Drs. Adam Wende and Martin Young for stimulating discussions and careful review of the manuscript prior to submission.