Changing the academic culture: valuing patents and commercialization toward tenure and career advancement.

There is national and international recognition of the importance of innovation, technology transfer, and entrepreneurship for sustained economic revival. With the decline of industrial research laboratories in the United States, research universities are being asked to play a central role in our knowledge-centered economy by the technology transfer of their discoveries, innovations, and inventions. In response to this challenge, innovation ecologies at and around universities are starting to change. However, the change has been slow and limited. The authors believe this can be attributed partially to a lack of change in incentives for the central stakeholder, the faculty member. The authors have taken the position that universities should expand their criteria to treat patents, licensing, and commercialization activity by faculty as an important consideration for merit, tenure, and career advancement, along with publishing, teaching, and service. This position is placed in a historical context with a look at the history of tenure in the United States, patents, and licensing at universities, the current status of university tenure and career advancement processes, and models for the future.

O-GlcNAcylation of AMPA receptor GluA2 is associated with a novel form of long-term depression at hippocampal synapses.

Serine phosphorylation of AMPA receptor (AMPAR) subunits GluA1 and GluA2 modulates AMPAR trafficking during long-term changes in strength of hippocampal excitatory transmission required for normal learning and memory. The post-translational addition and removal of O-linked ß-N-acetylglucosamine (O-GlcNAc) also occurs on serine residues. This, together with the high expression of the enzymes O-GlcNAc transferase (OGT) and ß-N-acetylglucosamindase (O-GlcNAcase), suggests a potential role for O-GlcNAcylation in modifying synaptic efficacy and cognition. Furthermore, because key synaptic proteins are O-GlcNAcylated, this modification may be as important to brain function as phosphorylation, yet its physiological significance remains unknown. We report that acutely increasing O-GlcNAcylation in Sprague Dawley rat hippocampal slices induces an NMDA receptor and protein kinase C-independent long-term depression (LTD) at hippocampal CA3-CA1 synapses (O-GcNAc LTD). This LTD requires AMPAR GluA2 subunits, which we demonstrate are O-GlcNAcylated. Increasing O-GlcNAcylation interferes with long-term potentiation, and in hippocampal behavioral assays, it prevents novel object recognition and placement without affecting contextual fear conditioning. Our findings provide evidence that O-GlcNAcylation dynamically modulates hippocampal synaptic function and learning and memory, and suggest that altered O-GlcNAc levels could underlie cognitive dysfunction in neurological diseases.

Stromal interaction molecule 1 is essential for normal cardiac homeostasis through modulation of ER and mitochondrial function.

The endoplasmic reticulum (ER) Ca(2+) sensor stromal interaction molecule 1 (STIM1) has been implicated as a key mediator of store-dependent and store-independent Ca(2+) entry pathways and maintenance of ER structure. STIM1 is present in embryonic, neonatal, and adult cardiomyocytes and has been strongly implicated in hypertrophic signaling; however, the physiological role of STIM1 in the adult heart remains unknown. We, therefore, developed a novel cardiomyocyte-restricted STIM1 knockout ((cr)STIM1-KO) mouse. In cardiomyocytes isolated from (cr)STIM1-KO mice, STIM1 expression was reduced by ∼92% with no change in the expression of related store-operated Ca(2+) entry proteins, STIM2, and Orai1. Immunoblot analyses revealed that (cr)STIM1-KO hearts exhibited increased ER stress from 12 wk, as indicated by increased levels of the transcription factor C/EBP homologous protein (CHOP), one of the terminal markers of ER stress. Transmission electron microscopy revealed ER dilatation, mitochondrial disorganization, and increased numbers of smaller mitochondria in (cr)STIM1-KO hearts, which was associated with increased mitochondrial fission. Using serial echocardiography and histological analyses, we observed a progressive decline in cardiac function in (cr)STIM1-KO mice, starting at 20 wk of age, which was associated with marked left ventricular dilatation by 36 wk. In addition, we observed the presence of an inflammatory infiltrate and evidence of cardiac fibrosis from 20 wk in (cr)STIM1-KO mice, which progressively worsened by 36 wk. These data demonstrate for the first time that STIM1 plays an essential role in normal cardiac function in the adult heart, which may be important for the regulation of ER and mitochondrial function.

Modification of STIM1 by O-linked N-acetylglucosamine (O-GlcNAc) attenuates store-operated calcium entry in neonatal cardiomyocytes.

Store-operated calcium entry (SOCE) is a major Ca(2+) signaling pathway responsible for regulating numerous transcriptional events. In cardiomyocytes SOCE has been shown to play an important role in regulating hypertrophic signaling pathways, including nuclear translocation of NFAT. Acute activation of pathways leading to O-GlcNAc synthesis have been shown to impair SOCE-mediated transcription and in diabetes, where O-GlcNAc levels are chronically elevated, cardiac hypertrophic signaling is also impaired. Therefore the goal of this study was to determine whether changes in cardiomyocyte O-GlcNAc levels impaired the function of STIM1, a widely recognized mediator of SOCE. We demonstrated that acute activation of SOCE in neonatal cardiomyocytes resulted in STIM1 puncta formation, which was inhibited in a dose-dependent manner by increasing O-GlcNAc synthesis with glucosamine or inhibiting O-GlcNAcase with thiamet-G. Glucosamine and thiamet-G also inhibited SOCE and were associated with increased O-GlcNAc modification of STIM1. These results suggest that activation of cardiomyocyte O-GlcNAcylation attenuates SOCE via STIM1 O-GlcNAcylation and that this may represent a new mechanism by which increased O-GlcNAc levels regulate Ca(2+)-mediated events in cardiomyocytes. Further, since SOCE is a fundamental mechanism underlying Ca(2+) signaling in most cells and tissues, it is possible that STIM1 represents a nexus linking protein O-GlcNAcylation with Ca(2+)-mediated transcription.

Glucose deprivation-induced increase in protein O-GlcNAcylation in cardiomyocytes is calcium-dependent.

The posttranslational modification of nuclear and cytosolic proteins by O-linked ß-N-acetylglucosamine (O-GlcNAc) has been shown to play an important role in cellular response to stress. Although increases in O-GlcNAc levels have typically been thought to be substrate-driven, studies in several transformed cell lines reported that glucose deprivation increased O-GlcNAc levels by a number of different mechanisms. A major goal of this study therefore was to determine whether in primary cells, such as neonatal cardiomyocytes, glucose deprivation increases O-GlcNAc levels and if so by what mechanism. Glucose deprivation significantly increased cardiomyocyte O-GlcNAc levels in a time-dependent manner and was associated with decreased O-GlcNAcase (OGA) but not O-GlcNAc transferase (OGT) protein. This response was unaffected by either the addition of pyruvate as an alternative energy source or by the p38 MAPK inhibitor SB203580. However, the response to glucose deprivation was blocked completely by glucosamine, but not by inhibition of OGA with 2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate. Interestingly, the CaMKII inhibitor KN93 also significantly reduced the response to glucose deprivation. Lowering extracellular Ca(2+) with EGTA or blocking store operated Ca(2+) entry with SKF96365 also attenuated the glucose deprivation-induced increase in O-GlcNAc. In C2C12 and HEK293 cells both glucose deprivation and heat shock increased O-GlcNAc levels, and CaMKII inhibitor KN93 attenuated the response to both stresses. These results suggest that increased intracellular calcium and subsequent activation of CaMKII play a key role in regulating the stress-induced increase in cellular O-GlcNAc levels.

STIM1/Orai1-mediated SOCE: current perspectives and potential roles in cardiac function and pathology.

Store-operated Ca²âº entry (SOCE) is critical for Ca²âº signaling in nonexcitable cells; however, its role in the regulation of cardiomyocyte Ca²âº homeostasis has only recently been investigated. The increased understanding of the role of stromal interaction molecule 1 (STIM1) in regulating SOCE combined with recent studies demonstrating the presence of STIM1 in cardiomyocytes provides support that this pathway co-exists in the heart with the more widely recognized Ca²âº handling pathways associated with excitation-contraction coupling. There is now substantial evidence that STIM1-mediated SOCE plays a key role in mediating cardiomyocyte hypertrophy, both in vitro and in vivo, and there is growing support for the contribution of SOCE to Ca²âº overload associated with ischemia/reperfusion injury. Here, we provide an overview of our current understanding of the molecular regulation of SOCE and discuss the evidence supporting the role of STIM1/Orai1-mediated SOCE in regulating cardiomyocyte function.

The role of protein O-linked beta-N-acetylglucosamine in mediating cardiac stress responses.

The modification of serine and threonine residues of nuclear and cytoplasmic proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc) has emerged as a highly dynamic post-translational modification that plays a critical role in regulating numerous biological processes. Much of our understanding of the mechanisms underlying the role of O-GlcNAc on cellular function has been in the context of its adverse effects in mediating a range of chronic disease processes, including diabetes, cancer and neurodegenerative diseases. However, at the cellular level it has been shown that O-GlcNAc levels are increased in response to stress; augmentation of this response improved cell survival while attenuation decreased cell viability. Thus, it has become apparent that strategies that augment O-GlcNAc levels are pro-survival, whereas those that reduce O-GlcNAc levels decrease cell survival. There is a long history demonstrating the effectiveness of acute glucose-insulin-potassium (GIK) treatment and to a lesser extent glutamine in protecting against a range of stresses, including myocardial ischemia. A common feature of these approaches for metabolic cardioprotection is that they both have the potential to stimulate O-GlcNAc synthesis. Consequently, here we examine the links between metabolic cardioprotection with the ischemic cardioprotection associated with acute increases in O-GlcNAc levels. Some of the protective mechanisms associated with activation of O-GlcNAcylation appear to be transcriptionally mediated; however, there is also strong evidence to suggest that transcriptionally independent mechanisms also play a critical role. In this context we discuss the potential link between O-GlcNAcylation and cardiomyocyte calcium homeostasis including the role of non-voltage gated, capacitative calcium entry as a potential mechanism contributing to this protection.

Increased O-linked beta-N-acetylglucosamine levels on proteins improves survival, reduces inflammation and organ damage 24 hours after trauma-hemorrhage in rats.

OBJECTIVE: To evaluate the effects of O-linked beta-N-acetylglucosamine (O-GlcNAc) levels on survival, inflammation, and organ damage 24 hrs after trauma-hemorrhage. We have previously shown that increasing protein O-GlcNAc levels by different mechanisms reduced inflammatory responses and improved organ function 2 hrs after trauma-hemorrhage. DESIGN: Prospective, randomized, controlled study. SETTING: Animal research laboratory. SUBJECTS: Male, adult Sprague-Dawley rats. INTERVENTIONS: Overnight fasted animals were subjected to either sham surgery or trauma-hemorrhage and during the resuscitation phase received glucosamine (270 mg/kg) to increase O-GlcNAc synthesis or O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenyl carbamate (PUGNAc, 7 mg/kg) to inhibit O-GlcNAc removal, or mannitol as control. MEASUREMENTS AND MAIN RESULTS: Survival was followed up for 24 hrs. Surviving rats were euthanized and inflammatory responses, and end organ injuries were assessed. Both glucosamine and PUGNAc increased 24-hr survival compared with controls (control: 53%, GN: 85%, PUGNAc: 86%, log-rank test, p < .05). PUGNAc attenuated the trauma-hemorrhage-induced increase in serum interleukin-6 (sham surgery: 8 +/- 6, control: 181 +/- 36, PUGNAc: 42 +/- 22 pg/mL, p < .05), alanine transaminase (sham surgery: 95 +/- 14, control: 297 +/- 56, PUGNAc: 126 +/- 21 IU, p < .05), aspartate transaminase (sham surgery: 536 +/- 110, control: 1661 +/- 215, PUGNAc: 897 +/- 155 IU, p < .05), and lactate dehydrogenase (sham surgery: 160 +/- 18, control: 1499 +/- 311, PUGNAc: 357 +/- 99 IU, p < .05); however, glucosamine had no effect on these serum parameters. Furthermore, PUGNAc but not glucosamine maintained O-GlcNAc levels in liver and lung and significantly attenuated the NF-kappaB DNA activation in the liver. In the liver and heart, increased inducible nitric oxide synthase expression was also attenuated in the PUGNAc-treated group. CONCLUSIONS: These results demonstrate that increasing O-GlcNAc with either glucosamine or PUGNAc improved 24-hr survival after trauma-hemorrhage. However, only PUGNAc treatment attenuated significantly the subsequent tissue injury and inflammatory responses, suggesting that inhibition of O-GlcNAc removal may represent a new therapeutic approach for the treatment of hypovolemic shock.

Hexosamine biosynthesis and protein O-glycosylation: the first line of defense against stress, ischemia, and trauma.

An early and rapid response to severe injury or trauma is the development of hyperglycemia, which has long been thought to be an essential survival response by providing fuel for vital organ systems and facilitating mobilization of interstitial fluid reserves by increasing osmolarity. However, glucose can also be metabolized via the hexosamine biosynthesis pathway (HBP), leading to the synthesis of uridine diphosphate N-acetyl-glucosamine(UDP-GlcNAc). UDP-GlcNAc is a substrate for the addition, via an O-linkage, of a single N-acetylglucosamine to serine or threonine residues of nuclear and cytoplasmic proteins (O-glycosylation, O-GlcNAc). There is increasing appreciation that protein O-glycosylation is a highly dynamic posttranslational modification that plays a key role in signal transduction pathways. Sustained increases in O-GlocNAc have been implicated in the development of diabetes and diabetic complications; however, recent studies have demonstrated that stress leads to a transient increase in O-GlcNAc levels that is associated with increased tolerance to stress. Indeed, activation of pathways leading to O-GlcNAc formation improves cell survival after I/R injury, whereas inhibition of O-GlcNAc formation decreases cell survival. In addition, in rodent models of trauma-hemorrhage, increasing O-GlcNAc levels during resuscitation improves cardiac function and organ perfusion and attenuates the inflammatory response. At the cellular level, increasing O-GlcNAc levels attenuates nuclear factor-kappaB activation. It is noteworthy that other metabolic-based treatments for severe injury such as glucose-insulin-potassium and glutamine also lead to increased HBP flux and O-GlcNAc levels. The goal of this review is to summarize our current understanding of the role of the HBP and O-GlcNAc on the regulation of cell function and survival and to present evidence to support the notion that activation of these pathways represents a novel treatment strategy for severe injury and trauma.

Role of protein O-linked N-acetyl-glucosamine in mediating cell function and survival in the cardiovascular system.

There is growing recognition that the O-linked attachment of N-acetyl-glucosamine (O-GlcNAc) on serine and threonine residues of nuclear and cytoplasmic proteins is a highly dynamic post-translational modification that plays a key role in signal transduction pathways. Numerous proteins have been identified as targets of O-GlcNAc modifications including kinases, phosphatases, transcription factors, metabolic enzymes, chaperons, and cytoskeletal proteins. Modulation of O-GlcNAc levels has been shown to modify DNA binding, enzyme activity, protein-protein interactions, the half-life of proteins, and subcellular localization. The level of O-GlcNAc is regulated in part by the metabolism of glucose via the hexosamine biosynthesis pathway (HBP), and the metabolic abnormalities associated with insulin resistance and diabetes, such as hyperglycemia, hyperlipidemia, and hyperinsulinemia, are all associated with increased flux through the HBP and elevated O-GlcNAc levels. Increased HBP flux and O-GlcNAc levels have been implicated in the impaired relaxation of isolated cardiomyocytes, blunted response to angiotensin II and phenylephrine, hyperglycemia-induced cardiomyocyte apoptosis, and endothelial and vascular cell dysfunction. In contrast to these adverse effects, recent studies have also shown that O-GlcNAc levels increase in response to acute stress and that this is associated with increased cell survival. Thus, while the relationship between O-GlcNAc levels and cellular function is complex and not well-understood, it is clear that these pathways play a critical role in the regulation of cell function and survival in the cardiovascular system and may be implicated in the adverse effects of metabolic disease on the heart.

Glucosamine administration improves survival rate after severe hemorrhagic shock combined with trauma in rats.

We have previously shown that glucosamine administration resulted in higher cardiac output and improved tissue perfusion after trauma-hemorrhage with resuscitation in rats, which was associated with the increased levels of protein O-linked-N-acetylglucosamine (O-GlcNAc). The purpose of the study was to evaluate the effect of glucosamine on the survival, without resuscitation, in rats. Adult male rats underwent midline laparotomy and 55% of total blood volume was withdrawn for 25 min under isoflurane anesthesia. At the end of the hemorrhage period, 2.5 mL of 150 mM glucosamine or equivalent osmolarity of mannitol solution was injected intravenously for 10 min. The survival time, mean blood pressure, heart rate, and central body temperature were monitored continuously; then, the O-GlcNAc levels in heart, brain, liver, and muscle were measured by means of Western blot analysis. Glucosamine administration significantly increased the survival rate in comparison with mannitol administration (percentage of survival after 2 h, 47% vs. 20%; P < 0.05). The mean arterial pressure was significantly higher in the glucosamine group for 18 min after treatment. The protein O-GlcNAc levels, assessed 30 min after glucosamine treatment, were significantly increased in the heart, brain, and liver. These data demonstrate that i.v. glucosamine administration improves the survival rate after trauma-hemorrhage without resuscitation; this effect may be related to the glucosamine-induced increase in protein O-glycosylation. Furthermore, the increase in mean arterial pressure may suggest a vasoactive and/or positive inotropic effect of glucosamine in hypovolemic shock.

The protective effects of PUGNAc on cardiac function after trauma-hemorrhage are mediated via increased protein O-GlcNAc levels.

We have previously shown that administration of glucosamine after trauma-hemorrhage (TH) improved cardiac output and organ perfusion, and this was associated with increased levels of O-linked N-acetylglucosamine (O-GlcNAc) on proteins in the heart and brain. An alternative means of increasing O-GlcNAc levels is by inhibition of O-linked N-acetylglucosaminidase, which catalyzes the removal of N-acetylglucosamine from proteins, with O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino-N-phenylcarbamate (PUGNAc). The goal of this study, therefore, was to determine whether PUGNAc administration after TH also improves recovery of organ perfusion and function. Fasted male rats were bled to and maintained at a mean arterial blood pressure of 40 mmHg for 90 min, followed by fluid resuscitation. Intravenous administration of PUGNAc (200 micromol/kg body weight) 30 min after the onset of resuscitation significantly improved cardiac output compared with the vehicle controls (12.3 +/- 1.3 mL/min per 100 g body weight vs. 25.5 +/- 2.0 mL/min per 100 g body weight; P < 0.05), decreased total peripheral resistance (6.6 +/- 0.8 mmHg/mL per minute per 100 g body weight vs. 3.7 +/- 0.3 mmHg/mL per minute per 100 g body weight; P < 0.05), and increased perfusion of critical organ systems, including the kidney and liver, determined at 2 h after the end of resuscitation. Treatment with PUGNAc also attenuated the TH-induced increase in plasma IL-6 levels (864 +/- 112 pg/mL vs. 392 +/- 188 pg/mL; P < 0.05) and TNF-alpha levels (216 +/- 21 pg/mL vs. 94 +/- 11 pg/mL; P < 0.05) and significantly increased O-GlcNAc levels in the heart, liver, and kidney. Thus, PUGNAc, like glucosamine, improves cardiac function and organ perfusion and reduced the level of circulating IL-6 and TNF-alpha after TH. The similar effects of glucosamine and PUGNAc support the notion that the protection associated with both interventions is mediated via increased protein O-GlcNAc levels.

Glucosamine administration during resuscitation improves organ function after trauma hemorrhage.

Stress-induced hyperglycemia is necessary for maximal rates of survival after severe hemorrhage; however, the responsible mechanisms are not clear. One consequence of hyperglycemia is an increase in hexosamine biosynthesis, which leads to increases in levels of O-linked attachment of N-acetyl-glucosamine (O-GlcNAc) on nuclear and cytoplasmic proteins. This modification has been shown to lead to improved survival of isolated cells after stress. In view of this, we hypothesized that glucosamine (GlcNH2), which more selectively increases the levels of O-GlcNAc administration after shock, will have salutary effects on organ function after trauma hemorrhage (TH). Fasted male rats that underwent midline laparotomy were bled to a mean arterial blood pressure of 40 mmHg for 90 min and then resuscitated with Ringer lactate (four times the shed blood volume). Administration of 2.5 mL of 150 mmol L GlcNH2 midway during resuscitation improved cardiac output 2-fold compared with controls that received 2.5 mL of 150 mmol L NaCl. GlcNH2 also improved perfusion of various organs systems, including kidney and brain, and attenuated the TH-induced increase in serum levels of IL-6 (902+/-224 vs. 585+/-103 pg mL) and TNF-alpha (540+/-81 vs. 345+/-110 pg mL) (values are mean+/-SD). GlcNH2 administration resulted in significant increase in protein-associated O-GlcNAc in the heart and brain after TH. Thus, GlcNH2 administered during resuscitation improves recovery from TH, as assessed by cardiac function, organ perfusion, and levels of circulating inflammatory cytokines. This protection correlates with enhanced levels of nucleocytoplasmic protein O-GlcNAcylation and suggests that increased O-GlcNAc could be the mechanism that links stress-induced hyperglycemia to improved outcomes.

Hexosamine pathway is responsible for inhibition by diabetes of phenylephrine-induced inotropy.

Hyperglycemia diminishes positive inotropic responses to agonists that activate phospholipase C (PLC) and generate inositol trisphosphate (1,4,5). The mechanisms underlying both the inotropic responses and hyperglycemia's effects on them remain undetermined, but data from isolated cardiomyocytes suggest the involvement of capacitative Ca(2+) entry (CCE), the influx of Ca(2+) through plasma membrane channels activated in response to depletion of endoplasmic or sarcoplasmic reticulum Ca(2+) stores. In neonatal rat cardiomyocytes, hyperglycemia decreased CCE induced by PLC-mediated agonists. The attenuation of CCE was also seen with glucosamine, and the inhibition by hyperglycemia was prevented by azaserine, thereby implicating hexosamine biosynthesis as the responsible metabolic pathway. In the current study, the importance of hexosamine metabolites to hyperglycemia's effects on inotropic responses was examined in isolated perfused rat hearts. The inhibition by hyperglycemia of phenylephrine-induced inotropy was reversed with azaserine and mimicked by glucosamine. An independent inhibitor of CCE, SKF96365, was also effective in blunting inotropy. These treatments did not inhibit inotropy induced by activation of adenylate cyclase through beta-adrenergic receptors. These data thus implicate CCE in responses to PLC-mediated agonists in the intact heart and point to the hexosamine pathway's negative effect on CCE as being central to the inhibition seen with hyperglycemia.

Hyperglycemia inhibits capacitative calcium entry and hypertrophy in neonatal cardiomyocytes.

Hyperglycemia alters cardiac function and often leads to diabetic cardiomyopathy as cardiomyocyte apoptosis causes a hypertrophied heart to deteriorate to dilation and failure. Paradoxically, many short-term animal models of hyperglycemia protect against ischemia-induced damage, including apoptosis, by limiting Ca(2+) overload. We have determined that, like nonexcitable cells, both neonatal and adult cardiomyocytes respond to depletion of sarcoplasmic/endoplasmic reticulum Ca(2+) stores with an influx of extracellular Ca(2+) through channels distinct from voltage-gated Ca(2+) channels, a process termed capacitative Ca(2+) entry (CCE). Here, we demonstrate that in neonatal rat cardiomyocytes, hyperglycemia decreased CCE induced by angiotensin II or the Ca(2+)ATPase inhibitor thapsigargin. Hyperglycemia also significantly blunted Ca(2+)-dependent hypertrophic responses by approximately 60%, as well as the Ca(2+)-sensitive nuclear translocation of a chimeric protein bearing the nuclear localization signal of a nuclear factor of activated T-cells transcription factor. The attenuation of CCE by hyperglycemia was prevented by azaserine, an inhibitor of hexosamine biosynthesis, and partially by inhibitors of oxidative stress. This complements previous work showing that increasing hexosamine metabolites in neonatal cardiomyocytes also inhibited CCE. The inhibition of CCE by hyperglycemia thus provides a likely explanation for the transition to diabetic cardiomyopathy as well as to the protection afforded to injury after ischemia/reperfusion in diabetic models.

Protein O-GlcNAcylation: A critical regulator of the cellular response to stress.

The post-translational modification of serine and threonine residues of nuclear and cytoplasmic proteins by the O-linked attachment of the monosaccharide ß-N-acetyl-glucosamine (O-GlcNAc) is a highly dynamic and ubiquitous protein modification that plays a critical role in regulating numerous biological processes. Much of our understanding of the mechanisms underlying the role of O-GlcNAc on cellular function has been in the context of chronic disease processes. However, there is increasing evidence that O-GlcNAc levels are increased in response to stress and that acute augmentation of this response is cytoprotective, at least in the short term. Conversely, a reduction in O-GlcNAc levels appears to be associated with decreased cell survival in response to an acute stress. Here we summarize our current understanding of protein O-GlcNAcylation on the cellular response to stress and in mediating cellular protective mechanisms focusing primarily on the cardiovascular system as an example. We consider the potential link between O-GlcNAcylation and cardiomyocyte calcium homeostasis and explore the parallels between O-GlcNAc signaling and redox signaling. We also discuss the apparent paradox between the reported adverse effects of increased O-GlcNAcylation with its recently reported role in mediating cell survival mechanisms.

Glucosamine improves cardiac function following trauma-hemorrhage by increased protein O-GlcNAcylation and attenuation of NF-{kappa}B signaling.

We have previously demonstrated that in a rat model of trauma-hemorrhage (T-H), glucosamine administration during resuscitation improved cardiac function, reduced circulating levels of inflammatory cytokines, and increased tissue levels of O-linked N-acetylglucosamine (O-GlcNAc) on proteins. The mechanism(s) by which glucosamine mediated its protective effect were not determined; therefore, the goal of this study was to test the hypothesis that glucosamine treatment attenuated the activation of the nuclear factor-kappaB (NF-kappaB) signaling pathway in the heart via an increase in protein O-GlcNAc levels. Fasted male rats were subjected to T-H by bleeding to a mean arterial blood pressure of 40 mmHg for 90 min followed by resuscitation. Glucosamine treatment during resuscitation significantly attenuated the T-H-induced increase in cardiac levels of TNF-alpha and IL-6 mRNA, IkappaB-alpha phosphorylation, NF-kappaB, NF-kappaB DNA binding activity, ICAM-1, and MPO activity. LPS (2 microg/ml) increased the levels of IkappaB-alpha phosphorylation, TNF-alpha, ICAM-1, and NF-kappaB in primary cultured cardiomyocytes, which was significantly attenuated by glucosamine treatment and overexpression of O-GlcNAc transferase; both interventions also significantly increased O-GlcNAc levels. In contrast, the transfection of neonatal rat ventricular myocytes with OGT small-interfering RNA decreased O-GlcNAc transferase and O-GlcNAc levels and enhanced the LPS-induced increase in IkappaB-alpha phosphorylation. Glucosamine treatment of macrophage cell line RAW 264.7 also increased O-GlcNAc levels and attenuated the LPS-induced activation of NF-kappaB. These results demonstrate that the modulation of O-GlcNAc levels alters the response of cardiomyocytes to the activation of the NF-kappaB pathway, which may contribute to the glucosamine-mediated improvement in cardiac function following hemorrhagic shock.

Overexpression of TRPC3 increases apoptosis but not necrosis in response to ischemia-reperfusion in adult mouse cardiomyocytes.

An increase in cytosolic Ca2+ via a capacitative calcium entry (CCE)-mediated pathway, attributed to members of the transient receptor potential (TRP) superfamily, TRPC1 and TRPC3, has been reported to play an important role in regulating cardiomyocyte hypertrophy. Increased cytosolic Ca2+ also plays a critical role in mediating cell death in response to ischemia-reperfusion (I/R). Therefore, we tested the hypothesis that overexpression of TRPC3 in cardiomyocytes will increase sensitivity to I/R injury. Adult cardiomyocytes isolated from wild-type (WT) mice and from mice overexpressing TRPC3 in the heart were subjected to 90 min of ischemia and 3 h of reperfusion. After I/R, viability was 51 +/- 1% in WT mice and 42 +/- 5% in transgenic mice (P < 0.05). Apoptosis assessed by annexin V was significantly increased in the TRPC3 group compared with WT (32 +/- 1% vs. 21 +/- 3%; P < 0.05); however, there was no significant difference in necrosis between groups. Treatment of TRPC3 cells with the CCE inhibitor SKF-96365 (0.5 microM) significantly improved cellular viability (54 +/- 4%) and decreased apoptosis (15 +/- 4%); in contrast, the L-type Ca2+ channel inhibitor verapamil (10 microM) had no effect. Calpain-mediated cleavage of alpha-fodrin was increased approximately threefold in the transgenic group following I/R compared with WT (P < 0.05); this was significantly attenuated by SKF-96365. The calpain inhibitor PD-150606 (25 microM) attenuated the increase in both alpha-fodrin cleavage and apoptosis in the TPRC3 group. Increased TRPC3 expression also increased sensitivity to Ca2+ overload stress, but it did not affect the response to TNF-alpha-induced apoptosis. These results suggest that CCE mediated via TRPC may play a role in cardiomyocyte apoptosis following I/R due, at least in part, to increased calpain activation.