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GABAergic transmission is influenced by post-translational modifications, like phosphorylation, impacting channel conductance, allosteric modulator sensitivity, and membrane trafficking. O-GlcNAcylation is a post-translational modification involving the O-linked attachment of ß-N-acetylglucosamine on serine/threonine residues. Previously we reported an acute increase in O-GlcNAcylation elicits a long-term depression of evoked GABAAR inhibitory postsynaptic currents (eIPSCs) onto hippocampal principal cells. Importantly, O-GlcNAcylation and phosphorylation can co-occur or compete for the same residue; whether they interact in modulating GABAergic IPSCs is unknown. We tested this by recording IPSCs from hippocampal principal cells and pharmacologically increased O-GlcNAcylation, before or after increasing serine phosphorylation using the adenylate cyclase activator, forskolin. Although forskolin had no significant effect on baseline eIPSC amplitude, we found that a prior increase in O-GlcNAcylation unmasks a forskolin-dependent increase in eIPSC amplitude, reversing the O-GlcNAc-induced eIPSC depression. Inhibition of adenylate cyclase or protein kinase A did not prevent the potentiating effect of forskolin, indicating serine phosphorylation is not the mechanism. Surprisingly, increasing O-GlcNAcylation also unmasked a potentiating effect of the neurosteroids 5α-pregnane-3α,21-diol-20-one (THDOC) and progesterone on eIPSC amplitude in about half of the recorded cells, mimicking forskolin. Our findings show that under conditions of heightened O-GlcNAcylation, the neurosteroid site on synaptic GABAARs is possibly accessible to agonists, permitting strengthening of synaptic inhibition.
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Colforsina , Hipocampo , Receptores de GABA-A , Sinapses , Colforsina/farmacologia , Animais , Hipocampo/metabolismo , Hipocampo/efeitos dos fármacos , Receptores de GABA-A/metabolismo , Sinapses/metabolismo , Sinapses/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Ratos , Neuroesteroides/metabolismo , Neuroesteroides/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Masculino , Transmissão Sináptica/efeitos dos fármacos , Ratos Sprague-DawleyRESUMO
GABAergic transmission is influenced by post-translational modifications, like phosphorylation, impacting channel conductance, allosteric modulator sensitivity, and membrane trafficking. O-GlcNAcylation is a post-translational modification involving the O-linked attachment of ß-N-acetylglucosamine on serine/threonine residues. Previously we reported an acute increase in O-GlcNAcylation elicits a long-term depression of evoked GABAAR inhibitory post synaptic currents (eIPSCs) onto hippocampal principal cells. Importantly, O-GlcNAcylation and phosphorylation can co-occur or compete for the same residue; whether they interact in modulating GABAergic IPSCs is unknown. We tested this by recording IPSCs from hippocampal principal cells and pharmacologically increased O-GlcNAcylation, before or after increasing serine phosphorylation using the adenylate cyclase activator, forskolin. Although forskolin had no significant effect on baseline eIPSC amplitude, we found that a prior increase in O-GlcNAcylation unmasks a forskolin-dependent increase in eIPSC amplitude, reversing the O-GlcNAc-induced eIPSC depression. Inhibition of adenylate cyclase or protein kinase A did not prevent the potentiating effect of forskolin, indicating serine phosphorylation is not the mechanism. Surprisingly, increasing O-GlcNAcylation also unmasked a potentiating effect of the neurosteroids 5α-pregnane-3α,21-diol-20-one (THDOC) and progesterone on eIPSC amplitude, mimicking forskolin. Our findings show under conditions of heightened O-GlcNAcylation, the neurosteroid site on synaptic GABAARs is accessible to agonists, permitting strengthening of synaptic inhibition.
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Protein glycosylation, which involves the attachment of carbohydrates to proteins, is one of the most abundant protein co-translational and post-translational modifications. Advances in technology have substantially increased our knowledge of the biosynthetic pathways involved in protein glycosylation, as well as how changes in glycosylation can affect cell function. In addition, our understanding of the role of protein glycosylation in disease processes is growing, particularly in the context of immune system function, infectious diseases, neurodegeneration and cancer. Several decades ago, cell surface glycoproteins were found to have an important role in regulating ion transport across the cardiac sarcolemma. However, with very few exceptions, our understanding of how changes in protein glycosylation influence cardiovascular (patho)physiology remains remarkably limited. Therefore, in this Review, we aim to provide an overview of N-linked and O-linked protein glycosylation, including intracellular O-linked N-acetylglucosamine protein modification. We discuss our current understanding of how all forms of protein glycosylation contribute to normal cardiovascular function and their roles in cardiovascular disease. Finally, we highlight potential gaps in our knowledge about the effects of protein glycosylation on the heart and vascular system, highlighting areas for future research.
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Doenças Cardiovasculares , Processamento de Proteína Pós-Traducional , Humanos , Glicosilação , Doenças Cardiovasculares/metabolismo , Animais , Sistema Cardiovascular/metabolismo , Sistema Cardiovascular/fisiopatologiaRESUMO
GABAergic transmission is influenced by post-translational modifications, like phosphorylation, impacting channel conductance, allosteric modulator sensitivity, and membrane trafficking. O-GlcNAcylation is a post-translational modification involving the O-linked attachment of ß-N-acetylglucosamine on serine/threonine residues. Previously we reported an acute increase in O-GlcNAcylation elicits a long-term depression of evoked GABAAR inhibitory post synaptic currents (eIPSCs) onto hippocampal principal cells. Importantly O-GlcNAcylation and phosphorylation can co-occur or compete for the same residue; whether they interact in modulating GABAergic IPSCs is unknown. We tested this by recording IPSCs from hippocampal principal cells and pharmacologically increased O-GlcNAcylation, before or after increasing serine phosphorylation using the adenylate cyclase activator, forskolin. Although forskolin had no significant effect on baseline eIPSC amplitude, we found that a prior increase in O-GlcNAcylation unmasks a forskolin-dependent increase in eIPSC amplitude, reversing the O-GlcNAc-induced eIPSC depression. Inhibition of adenylate cyclase or protein kinase A did not prevent the potentiating effect of forskolin, indicating serine phosphorylation is not the mechanism. Surprisingly, increasing O-GlcNAcylation also unmasked a potentiating effect of the neurosteroids 5α-pregnane-3α,21-diol-20-one (THDOC) and progesterone on eIPSC amplitude, mimicking forskolin. Our findings show under conditions of heightened O-GlcNAcylation, the neurosteroid site on synaptic GABAARs is accessible to agonists, permitting strengthening of synaptic inhibition.
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Mitochondria morphology and function, and their quality control by mitophagy, are essential for heart function. We investigated whether these are influenced by time of the day (TOD), sex, and fed or fasting status, using transmission electron microscopy (EM), mitochondrial electron transport chain (ETC) activity, and mito-QC reporter mice. We observed peak mitochondrial number at ZT8 in the fed state, which was dependent on the intrinsic cardiac circadian clock, as hearts from cardiomyocyte-specific BMAL1 knockout (CBK) mice exhibit different TOD responses. In contrast to mitochondrial number, mitochondrial ETC activities do not fluctuate across TOD, but decrease immediately and significantly in response to fasting. Concurrent with the loss of ETC activities, ETC proteins were decreased with fasting, simultaneous with significant increases of mitophagy, mitochondrial antioxidant protein SOD2, and the fission protein DRP1. Fasting-induced mitophagy was lost in CBK mice, indicating a direct role of BMAL1 in regulating mitophagy. This is the first of its kind report to demonstrate the interactions between sex, fasting, and TOD on cardiac mitochondrial structure, function and mitophagy. These studies provide a foundation for future investigations of mitochondrial functional perturbation in aging and heart diseases.
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Fatores de Transcrição ARNTL , Miócitos Cardíacos , Camundongos , Animais , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Miócitos Cardíacos/metabolismo , Mitocôndrias/metabolismo , Camundongos Knockout , Jejum , Dinâmica Mitocondrial/fisiologiaRESUMO
Background Lifestyle and metabolic diseases influence the severity and pathogenesis of cardiovascular disease through numerous mechanisms, including regulation via posttranslational modifications. A specific posttranslational modification, the addition of O-linked ß-N acetylglucosamine (O-GlcNAcylation), has been implicated in molecular mechanisms of both physiological and pathologic adaptations. The current study aimed to test the hypothesis that in cardiomyocytes, sustained protein O-GlcNAcylation contributes to cardiac adaptations, and its progression to pathophysiology. Methods and Results Using a naturally occurring dominant-negative O-GlcNAcase (dnOGA) inducible cardiomyocyte-specific overexpression transgenic mouse model, we induced dnOGA in 8- to 10-week-old mouse hearts. We examined the effects of 2-week and 24-week dnOGA overexpression, which progressed to a 1.8-fold increase in protein O-GlcNAcylation. Two-week increases in protein O-GlcNAc levels did not alter heart weight or function; however, 24-week increases in protein O-GlcNAcylation led to cardiac hypertrophy, mitochondrial dysfunction, fibrosis, and diastolic dysfunction. Interestingly, systolic function was maintained in 24-week dnOGA overexpression, despite several changes in gene expression associated with cardiovascular disease. Specifically, mRNA-sequencing analysis revealed several gene signatures, including reduction of mitochondrial oxidative phosphorylation, fatty acid, and glucose metabolism pathways, and antioxidant response pathways after 24-week dnOGA overexpression. Conclusions This study indicates that moderate increases in cardiomyocyte protein O-GlcNAcylation leads to a differential response with an initial reduction of metabolic pathways (2-week), which leads to cardiac remodeling (24-week). Moreover, the mouse model showed evidence of diastolic dysfunction consistent with a heart failure with preserved ejection fraction. These findings provide insight into the adaptive versus maladaptive responses to increased O-GlcNAcylation in heart.
Assuntos
Doenças Cardiovasculares , Miócitos Cardíacos , Camundongos , Animais , Miócitos Cardíacos/metabolismo , Acetilglucosamina/metabolismo , Doenças Cardiovasculares/metabolismo , Glicosilação , Cardiomegalia/genética , Cardiomegalia/metabolismo , Processamento de Proteína Pós-Traducional , Mitocôndrias/metabolismo , Modelos Animais de Doenças , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismoRESUMO
The modification of serine and threonine amino acids of proteins by O-linked N-acetylglucosamine (O-GlcNAc) regulates the activity, stability, function, and subcellular localization of proteins. Dysregulation of O-GlcNAc homeostasis is well established as a hallmark of various cardiac diseases, including cardiac hypertrophy, heart failure, complications associated with diabetes, and responses to acute injuries such as oxidative stress and ischemia-reperfusion. Given the limited availability of site-specific O-GlcNAc antibodies, studies of changes in O-GlcNAcylation in the heart frequently use pan-O-GlcNAc antibodies for semiquantitative evaluation of overall O-GlcNAc levels. However, there is a high degree of variability in many published cardiac O-GlcNAc blots. For example, many blots often have regions that lack O-GlcNAc positive staining of proteins either below 50 or above 100 kDa. In some O-GlcNAc blots, only a few protein bands are detected, while in others, intense bands around 75 kDa dominate the gel due to nonspecific IgM band staining, making it difficult to visualize less intense bands. Therefore, the goal of this study was to develop a modifiable protocol that optimizes O-GlcNAc positive banding of proteins in cardiac tissue extracts. We showed that O-GlcNAc blots using CTD110.6 antibody of proteins ranging from <30 to â¼450 kDa could be obtained while also limiting nonspecific staining. We also show that some myofilament proteins are recognized by the CTD110.6 antibody. Therefore, by protocol optimization using the widely available CTD110.6 antibody, we found that it is possible to obtain pan-O-GlcNAc blots of cardiac tissue, which minimizes common limitations associated with this technique.NEW & NOTEWORTHY The post-translational modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc) is recognized as mediating cardiac pathophysiology. However, there is considerable variability in the quality of O-GlcNAc immunoblots used to evaluate changes in cardiac O-GlcNAc levels. Here we show that with relatively minor changes to a commonly used protocol it is possible to minimize the intensity of nonspecific bands while also reproducibly generating O-GlcNAc immunoblots covering a range of molecular weights from <30 to â¼450 kDa.
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Acetilglucosamina , Proteínas , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Proteínas/metabolismo , Coração , Anticorpos , Immunoblotting , Processamento de Proteína Pós-Traducional , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismoRESUMO
In this issue of Cell Reports Methods, Sunden et al. develop an enzymatic assay to measure UDP-GlcNAc levels from cells and tissue.1 By reporting on the level of the substrate itself, this approach can potentially enhance the fields' understanding of UDP-GlcNAc concentration under a variety of conditions.
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Ensaios Enzimáticos , Difosfato de UridinaRESUMO
Enhancing protein O-GlcNAcylation by pharmacological inhibition of the enzyme O-GlcNAcase (OGA), which removes the O-GlcNAc modification from proteins, has been explored in mouse models of amyloid-beta and tau pathology. However, the O-GlcNAcylation-dependent link between gene expression and neurological behavior remains to be explored. Using chronic administration of Thiamet G (TG, an OGA inhibitor) in vivo, we used a protocol designed to relate behavior with the transcriptome and selected biochemical parameters from the cortex of individual animals. TG-treated mice showed improved working memory as measured using a Y-maze test. RNA sequencing analysis revealed 151 top differentially expressed genes with a Log2fold change >0.33 and adjusted p-value <0.05. Top TG-dependent upregulated genes were related to learning, cognition and behavior, while top downregulated genes were related to IL-17 signaling, inflammatory response and chemotaxis. Additional pathway analysis uncovered 3 pathways, involving gene expression including 14 cytochrome c oxidase subunits/regulatory components, chaperones or assembly factors, and 5 mTOR (mechanistic target of rapamycin) signaling factors. Multivariate Kendall correlation analyses of behavioral tests and the top TG-dependent differentially expressed genes revealed 91 statistically significant correlations in saline-treated mice and 70 statistically significant correlations in TG-treated mice. These analyses provide a network regulation landscape that is important in relating the transcriptome to behavior and the potential impact of the O-GlcNAC pathway.
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Processamento de Proteína Pós-Traducional , Transdução de Sinais , Camundongos , Animais , Modelos Animais de Doenças , Sirolimo , Expressão GênicaRESUMO
Autophagy is important for protein and organelle quality control. Growing evidence demonstrates that autophagy is tightly controlled by transcriptional mechanisms, including repression by zinc finger containing KRAB and SCAN domains 3 (ZKSCAN3). We hypothesize that cardiomyocyte-specific ZKSCAN3 knockout (Z3K) disrupts autophagy activation and repression balance and exacerbates cardiac pressure-overload-induced remodeling following transverse aortic constriction (TAC). Indeed, Z3K mice had an enhanced mortality compared to control (Con) mice following TAC. Z3K-TAC mice that survived exhibited a lower body weight compared to Z3K-Sham. Although both Con and Z3K mice exhibited cardiac hypertrophy after TAC, Z3K mice exhibited TAC-induced increase of left ventricular posterior wall thickness at end diastole (LVPWd). Conversely, Con-TAC mice exhibited decreases in PWT%, fractional shortening (FS%), and ejection fraction (EF%). Autophagy genes (Tfeb, Lc3b, and Ctsd) were decreased by the loss of ZKSCAN3. TAC suppressed Zkscan3, Tfeb, Lc3b, and Ctsd in Con mice, but not in Z3K. The Myh6/Myh7 ratio, which is related to cardiac remodeling, was decreased by the loss of ZKSCAN3. Although Ppargc1a mRNA and citrate synthase activities were decreased by TAC in both genotypes, mitochondrial electron transport chain activity did not change. Bi-variant analyses show that while in Con-Sham, the levels of autophagy and cardiac remodeling mRNAs form a strong correlation network, such was disrupted in Con-TAC, Z3K-Sham, and Z3K-TAC. Ppargc1a also forms different links in Con-sham, Con-TAC, Z3K-Sham, and Z3K-TAC. We conclude that ZKSCAN3 in cardiomyocytes reprograms autophagy and cardiac remodeling gene transcription, and their relationships with mitochondrial activities in response to TAC-induced pressure overload.
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Estenose da Valva Aórtica , Miócitos Cardíacos , Camundongos , Animais , Miócitos Cardíacos/metabolismo , Remodelação Ventricular , Cardiomegalia/metabolismo , Ventrículos do Coração/metabolismo , Proteínas , Camundongos Knockout , Camundongos Endogâmicos C57BL , Fatores de Transcrição/genéticaRESUMO
Tight spatiotemporal regulation of intracellular Ca2+ plays a critical role in regulating diverse cellular functions including cell survival, metabolism, and transcription. As a result, eukaryotic cells have developed a wide variety of mechanisms for controlling Ca2+ influx and efflux across the plasma membrane as well as Ca2+ release and uptake from intracellular stores. The STIM and Orai protein families comprising of STIM1, STIM2, Orai1, Orai2, and Orai3, are evolutionarily highly conserved proteins that are core components of all mammalian Ca2+ signaling systems. STIM1 and Orai1 are considered key players in the regulation of Store Operated Calcium Entry (SOCE), where release of Ca2+ from intracellular stores such as the Endoplasmic/Sarcoplasmic reticulum (ER/SR) triggers Ca2+ influx across the plasma membrane. SOCE, which has been widely characterized in non-excitable cells, plays a central role in Ca2+-dependent transcriptional regulation. In addition to their role in Ca2+ signaling, STIM1 and Orai1 have been shown to contribute to the regulation of metabolism and mitochondrial function. STIM and Orai proteins are also subject to redox modifications, which influence their activities. Considering their ubiquitous expression, there has been increasing interest in the roles of STIM and Orai proteins in excitable cells such as neurons and myocytes. While controversy remains as to the importance of SOCE in excitable cells, STIM1 and Orai1 are essential for cellular homeostasis and their disruption is linked to various diseases associated with aging such as cardiovascular disease and neurodegeneration. The recent identification of splice variants for most STIM and Orai isoforms while complicating our understanding of their function, may also provide insight into some of the current contradictions on their roles. Therefore, the goal of this review is to describe our current understanding of the molecular regulation of STIM and Orai proteins and their roles in normal physiology and diseases of aging, with a particular focus on heart disease and neurodegeneration.
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The accumulation of neurotoxic proteins characteristic of age-related neurodegenerative pathologies such as Alzheimer's and Parkinson's diseases is associated with the perturbation of metabolism, bioenergetics, and mitochondrial quality control. One approach to exploit these interactions therapeutically is to target the pathways that regulate metabolism. In this respect, the nutrient-sensing hexosamine biosynthesis pathway is of particular interest since it introduces a protein post-translational modification known as O-GlcNAcylation, which modifies different proteins in control versus neurodegenerative disease postmortem brains. A potent inhibitor of the O-GlcNAcase enzyme that removes the modification from proteins, Thiamet G (TG), has been proposed to have potential benefits in Alzheimer's disease. We tested whether key factors in the O-GlcNAcylation are correlated with mitochondrial electron transport and proteins related to the autophagy/lysosomal pathways in the cortex of male and female mice with and without exposure to TG (10 mg/kg i.p.). Mitochondrial complex activities were measured in the protein homogenates, and a panel of metabolic, autophagy/lysosomal proteins and O-GlcNAcylation enzymes were assessed by either enzyme activity assay or by western blot analysis. We found that the networks associated with O-GlcNAcylation enzymes and activities with mitochondrial parameters, autophagy-related proteins as well as neurodegenerative disease-related proteins exhibited sex and TG dependent differences. Taken together, these studies provide a framework of interconnectivity for multiple O-GlcNAc-dependent pathways in mouse brain of relevance to aging and sex/age-dependent neurodegenerative pathogenesis and response to potential therapies.
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Doença de Alzheimer , Doenças Neurodegenerativas , Doença de Alzheimer/metabolismo , Animais , Autofagia , Metabolismo Energético , Feminino , Masculino , Camundongos , Processamento de Proteína Pós-TraducionalRESUMO
Stromal interaction molecule 1 (STIM1) is a major regulator of store-operated calcium entry in non-excitable cells. Recent studies have suggested that STIM1 plays a role in pathological hypertrophy; however, the physiological role of STIM1 in the heart is not well understood. We have shown that mice with a cardiomyocyte deletion of STIM1 (cr STIM1-/- ) develop ER stress, mitochondrial, and metabolic abnormalities, and dilated cardiomyopathy. However, the specific signaling pathways and kinases regulated by STIM1 are largely unknown. Therefore, we used a discovery-based kinomics approach to identify kinases differentially regulated by STIM1. Twelve-week male control and cr STIM1-/- mice were injected with saline or phenylephrine (PE, 15 mg/kg, s.c, 15 min), and hearts obtained for analysis of the Serine/threonine kinome. Primary analysis was performed using BioNavigator 6.0 (PamGene), using scoring from the Kinexus PhosphoNET database and GeneGo network modeling, and confirmed using standard immunoblotting. Kinomics revealed significantly lower PKG and protein kinase C (PKC) signaling in the hearts of the cr STIM1-/- in comparison to control hearts, confirmed by immunoblotting for the calcium-dependent PKC isoform PKCα and its downstream target MARCKS. Similar reductions in cr STIM1-/- hearts were found for the kinases: MEK1/2, AMPK, and PDPK1, and in the activity of the Ca2+ -dependent phosphatase, calcineurin. Electrocardiogram analysis also revealed that cr STIM1-/- mice have significantly lower HR and prolonged QT interval. In conclusion, we have shown several calcium-dependent kinases and phosphatases are regulated by STIM1 in the adult mouse heart. This has important implications in understanding how STIM1 contributes to the regulation of cardiac physiology and pathophysiology.
Assuntos
Miócitos Cardíacos/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Potenciais de Ação , Animais , Calcineurina/metabolismo , Sinalização do Cálcio , Células Cultivadas , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Estresse do Retículo Endoplasmático , Frequência Cardíaca , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase C/metabolismo , Molécula 1 de Interação Estromal/genéticaRESUMO
AIMS: The glucose-driven enzymatic modification of myocardial proteins by the sugar moiety, ß-N-acetylglucosamine (O-GlcNAc), is increased in pre-clinical models of diabetes, implicating protein O-GlcNAc modification in diabetes-induced heart failure. Our aim was to specifically examine cardiac manipulation of the two regulatory enzymes of this process on the cardiac phenotype, in the presence and absence of diabetes, utilising cardiac-targeted recombinant-adeno-associated viral-vector-6 (rAAV6)-mediated gene delivery. METHODS AND RESULTS: In human myocardium, total protein O-GlcNAc modification was elevated in diabetic relative to non-diabetic patients, and correlated with left ventricular (LV) dysfunction. The impact of rAAV6-delivered O-GlcNAc transferase (rAAV6-OGT, facilitating protein O-GlcNAcylation), O-GlcNAcase (rAAV6-OGA, facilitating de-O-GlcNAcylation), and empty vector (null) were determined in non-diabetic and diabetic mice. In non-diabetic mice, rAAV6-OGT was sufficient to impair LV diastolic function and induce maladaptive cardiac remodelling, including cardiac fibrosis and increased Myh-7 and Nppa pro-hypertrophic gene expression, recapitulating characteristics of diabetic cardiomyopathy. In contrast, rAAV6-OGA (but not rAAV6-OGT) rescued LV diastolic function and adverse cardiac remodelling in diabetic mice. Molecular insights implicated impaired cardiac PI3K(p110α)-Akt signalling as a potential contributing mechanism to the detrimental consequences of rAAV6-OGT in vivo. In contrast, rAAV6-OGA preserved PI3K(p110α)-Akt signalling in diabetic mouse myocardium in vivo and prevented high glucose-induced impairments in mitochondrial respiration in human cardiomyocytes in vitro. CONCLUSION: Maladaptive protein O-GlcNAc modification is evident in human diabetic myocardium, and is a critical regulator of the diabetic heart phenotype. Selective targeting of cardiac protein O-GlcNAcylation to restore physiological O-GlcNAc balance may represent a novel therapeutic approach for diabetes-induced heart failure.
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Antígenos de Neoplasias/metabolismo , Cardiomiopatias Diabéticas/enzimologia , Histona Acetiltransferases/metabolismo , Hialuronoglucosaminidase/metabolismo , Miócitos Cardíacos/enzimologia , N-Acetilglucosaminiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Disfunção Ventricular Esquerda/enzimologia , Função Ventricular Esquerda , Remodelação Ventricular , Idoso , Animais , Antígenos de Neoplasias/genética , Linhagem Celular , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Cardiomiopatias Diabéticas/genética , Cardiomiopatias Diabéticas/patologia , Cardiomiopatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Feminino , Fibrose , Regulação da Expressão Gênica , Glicosilação , Histona Acetiltransferases/genética , Humanos , Hialuronoglucosaminidase/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Miócitos Cardíacos/patologia , N-Acetilglucosaminiltransferases/genética , Fenótipo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/fisiopatologiaRESUMO
The modification of proteins by O-linked ß-N-acetylglucosamine (O-GlcNAc) is associated with the regulation of numerous cellular processes. Despite the importance of O-GlcNAc in mediating cellular function our understanding of the mechanisms that regulate O-GlcNAc levels is limited. One factor known to regulate protein O-GlcNAc levels is nutrient availability; however, the fact that nutrient deficient states such as ischemia increase O-GlcNAc levels suggests that other factors also contribute to regulating O-GlcNAc levels. We have previously reported that in unstressed cardiomyocytes exogenous NAD+ resulted in a time and dose dependent decrease in O-GlcNAc levels. Therefore, we postulated that NAD+ and cellular O-GlcNAc levels may be coordinately regulated. Using glucose deprivation as a model system in an immortalized human ventricular cell line, we examined the influence of extracellular NAD+ on cellular O-GlcNAc levels and ER stress in the presence and absence of glucose. We found that NAD+ completely blocked the increase in O-GlcNAc induced by glucose deprivation and suppressed the activation of ER stress. The NAD+ metabolite cyclic ADP-ribose (cADPR) had similar effects on O-GlcNAc and ER stress suggesting a common underlying mechanism. cADPR is a ryanodine receptor (RyR) agonist and like caffeine, which also activates the RyR, both mimicked the effects of NAD+. SERCA inhibition, which also reduces ER/SR Ca2+ levels had similar effects to both NAD+ and cADPR on O-GlcNAc and ER stress responses to glucose deprivation. The observation that NAD+, cADPR, and caffeine all attenuated the increase in O-GlcNAc and ER stress in response to glucose deprivation, suggests a potential common mechanism, linked to ER/SR Ca2+ levels, underlying their activation. Moreover, we showed that TRPM2, a plasma membrane cation channel was necessary for the cellular responses to glucose deprivation. Collectively, these findings support a novel Ca2+-dependent mechanism underlying glucose deprivation induced increase in O-GlcNAc and ER stress.
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Essentially all biological processes fluctuate over the course of the day, manifesting as time-of-day-dependent variations with regards to the way in which organ systems respond to normal behaviors. For example, basic, translational, and epidemiologic studies indicate that temporal partitioning of metabolic processes governs the fate of dietary nutrients, in a manner in which concentrating caloric intake towards the end of the day is detrimental to both cardiometabolic and cardiovascular parameters. Despite appreciation that branched chain amino acids impact risk for obesity, diabetes mellitus, and heart failure, it is currently unknown whether the time-of-day at which dietary BCAAs are consumed influence cardiometabolic/cardiovascular outcomes. Here, we report that feeding mice a BCAA-enriched meal at the end of the active period (i.e., last 4 h of the dark phase) rapidly increases cardiac protein synthesis and mass, as well as cardiomyocyte size; consumption of the same meal at the beginning of the active period (i.e., first 4 h of the dark phase) is without effect. This was associated with a greater BCAA-induced activation of mTOR signaling in the heart at the end of the active period; pharmacological inhibition of mTOR (through rapamycin) blocked BCAA-induced augmentation of cardiac mass and cardiomyocyte size. Moreover, genetic disruption of the cardiomyocyte circadian clock abolished time-of-day-dependent fluctuations in BCAA-responsiveness. Finally, we report that repetitive consumption of BCAA-enriched meals at the end of the active period accelerated adverse cardiac remodeling and contractile dysfunction in mice subjected to transverse aortic constriction. Thus, our data demonstrate that the timing of BCAA consumption has significant implications for cardiac health and disease.
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Aminoácidos de Cadeia Ramificada/metabolismo , Metabolismo Energético , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Vigília , Fatores de Transcrição ARNTL/deficiência , Animais , Biomarcadores , Relógios Circadianos , Suscetibilidade a Doenças , Ingestão de Alimentos , Camundongos , Camundongos Knockout , Biossíntese de Proteínas , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Remodelação Ventricular/genéticaRESUMO
Transmission electron microscopy (TEM) has long been an important technique, capable of high degree resolution and visualization of subcellular structures and organization. Over the last 20 years, TEM has gained popularity in the cardiovascular field to visualize changes at the nanometer scale in cardiac ultrastructure during cardiovascular development, aging, and a broad range of pathologies. Recently, the cardiovascular TEM enabled the studying of several signaling processes impacting mitochondrial function, such as mitochondrial fission/fusion, autophagy, mitophagy, lysosomal degradation, and lipophagy. The goals of this review are to provide an overview of the current usage of TEM to study cardiac ultrastructural changes; to understand how TEM aided the visualization of mitochondria, autophagy, and mitophagy under normal and cardiovascular disease conditions; and to discuss the overall advantages and disadvantages of TEM and potential future capabilities and advancements in the field.
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O-linked conjugation of ß-N-acetyl-glucosamine (O-GlcNAc) to serine and threonine residues is a post-translational modification process that senses nutrient availability and cellular stress and regulates diverse biological processes that are involved in neurodegenerative diseases and provide potential targets for therapeutics development. However, very little is known of the networks involved in the brain that are responsive to changes in the O-GlcNAc proteome. Pharmacological increase of protein O-GlcNAcylation by Thiamet G (TG) has been shown to decrease tau phosphorylation and neurotoxicity, and proposed as a therapy in Alzheimer's disease (AD). However, acute TG exposure impairs learning and memory, and protein O-GlcNAcylation is increased in the aging rat brain and in Parkinson's disease (PD) brains. To define the cortical O-GlcNAc proteome that responds to TG, we injected young adult mice with either saline or TG and performed mass spectrometry analysis for detection of O-GlcNAcylated peptides. This approach identified 506 unique peptides corresponding to 278 proteins that are O-GlcNAcylated. Of the 506 unique peptides, 85 peptides are elevated by > 1.5 fold in O-GlcNAcylation levels in response to TG. Using pathway analyses, we found TG-dependent enrichment of O-GlcNAcylated synaptic proteins, trafficking, Notch/Wnt signaling, HDAC signaling, and circadian clock proteins. Significant changes in the O-GlcNAcylation of DNAJC6/AUXI, and PICALM, proteins that are risk factors for PD and/or AD respectively, were detected. We compared our study with two key prior O-GlcNAc proteome studies using mouse cerebral tissue and human AD brains. Among those identified to be increased by TG, 15 are also identified to be increased in human AD brains compared to control, including those involved in cytoskeleton, autophagy, chromatin organization and mitochondrial dysfunction. These studies provide insights regarding neurodegenerative diseases therapeutic targets.
RESUMO
In the mid-1980s, the identification of serine and threonine residues on nuclear and cytoplasmic proteins modified by a N-acetylglucosamine moiety (O-GlcNAc) via an O-linkage overturned the widely held assumption that glycosylation only occurred in the endoplasmic reticulum, Golgi apparatus, and secretory pathways. In contrast to traditional glycosylation, the O-GlcNAc modification does not lead to complex, branched glycan structures and is rapidly cycled on and off proteins by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. Since its discovery, O-GlcNAcylation has been shown to contribute to numerous cellular functions, including signaling, protein localization and stability, transcription, chromatin remodeling, mitochondrial function, and cell survival. Dysregulation in O-GlcNAc cycling has been implicated in the progression of a wide range of diseases, such as diabetes, diabetic complications, cancer, cardiovascular, and neurodegenerative diseases. This review will outline our current understanding of the processes involved in regulating O-GlcNAc turnover, the role of O-GlcNAcylation in regulating cellular physiology, and how dysregulation in O-GlcNAc cycling contributes to pathophysiological processes.
Assuntos
Acetilglucosamina/genética , Fenômenos Fisiológicos Celulares/genética , N-Acetilglucosaminiltransferases/genética , Processamento de Proteína Pós-Traducional/genética , Acetilglucosamina/metabolismo , Animais , Glicosilação , Humanos , N-Acetilglucosaminiltransferases/metabolismoRESUMO
The post-translational modification of serine and threonine residues of proteins by O-linked N-acetylglucosamine (O-GlcNAc) regulates diverse cellular processes in the cardiovascular system. UDP-GlcNAc is a substrate for O-GlcNAc transferase, which catalyzes the attachment of O-GlcNAc to proteins. O-GlcNAcase catalyzes the removal of O-GlcNAc from proteins. UDP-GlcNAc is the end product of the hexosamine biosynthesis pathway, which is regulated primarily by glucose-6-phosphate-Glutamine:fructose-6-phosphate amidotransferase (GFAT). GFAT catalyzes the formation of glucosamine-6-phosphate from fructose-6-phosphate and glutamine. Whereas O-GlcNAc is essential for cell viability, sustained increases in O-GlcNAc levels have been implicated in the etiology of many chronic diseases and is associated with glucose toxicity and diabetic complications in various organs including the cardiovascular system. This review provides an overview of the regulation of protein O-GlcNAcylation followed by a discussion of potential mechanisms by which dysregulation in O-GlcNAc cycling contributes to the adverse effects of diabetes on the cardiovascular system.