RESUMEN
Glutamine is a critical amino acid that serves as an energy source, building block, and signaling molecule for the heart tissue and the immune system. However, the role of glutamine metabolism in regulating cardiac remodeling following myocardial infarction (MI) is unknown. In this study, we show in adult male mice that glutamine metabolism is altered both in the remote (contractile) area and in infiltrating macrophages in the infarct area after permanent left anterior descending artery occlusion. We found that metabolites related to glutamine metabolism were differentially altered in macrophages at days 1, 3, and 7 after MI using untargeted metabolomics. Glutamine metabolism in live cells was increased after MI relative to no MI controls. Gene expression in the remote area of the heart indicated a loss of glutamine metabolism. Glutamine administration improved left ventricle (LV) function at days 1, 3, and 7 after MI, which was associated with improved contractile and metabolic gene expression. Conversely, administration of BPTES, a pharmacological inhibitor of glutaminase-1, worsened LV function after MI. Neither glutamine nor BPTES administration impacted gene expression or bioenergetics of macrophages isolated from the infarct area. Our results indicate that glutamine metabolism plays a critical role in maintaining LV contractile function following MI and that glutamine administration improves LV function. Glutamine metabolism may also play a role in regulating macrophage function, but macrophages are not responsive to exogenous pharmacological manipulation of glutamine metabolism.NEW & NOTEWORTHY Glutamine metabolism is altered in both infarct macrophages and the remote left ventricle (LV) following myocardial infarction (MI). Supplemental glutamine improves LV function following MI while inhibiting glutamine metabolism with BPTES worsens LV function. Supplemental glutamine or BPTES does not impact macrophage immunometabolic phenotypes after MI.
Asunto(s)
Glutamina , Macrófagos , Ratones Endogámicos C57BL , Infarto del Miocardio , Función Ventricular Izquierda , Animales , Glutamina/metabolismo , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Macrófagos/metabolismo , Macrófagos/inmunología , Masculino , Función Ventricular Izquierda/efectos de los fármacos , Ratones , Remodelación Ventricular/efectos de los fármacos , Glutaminasa/metabolismo , Glutaminasa/antagonistas & inhibidores , Glutaminasa/genética , Miocardio/metabolismo , Miocardio/patología , Miocardio/inmunología , Inflamación/metabolismo , Inflamación/patología , Metabolismo Energético/efectos de los fármacosRESUMEN
Subclinical leaflet thrombosis (SLT) is a potentially serious complication of aortic valve replacement with a bioprosthetic valve in which blood clots form on the replacement valve. SLT is associated with increased risk of transient ischemic attacks and strokes and can progress to clinical leaflet thrombosis. SLT following aortic valve replacement also may be related to subsequent structural valve deterioration, which can impair the durability of the valve replacement. Because of the difficulty in clinical imaging of SLT, models are needed to determine the mechanisms of SLT and could eventually predict which patients will develop SLT. To this end, we develop methods to simulate leaflet thrombosis that combine fluid-structure interaction and a simplified thrombosis model that allows for deposition along the moving leaflets. Additionally, this model can be adapted to model deposition or absorption along other moving boundaries. We present convergence results and quantify the model's ability to realize changes in valve opening and pressures. These new approaches are an important advancement in our tools for modeling thrombosis because they incorporate both adhesion to the surface of the moving leaflets and feedback to the fluid-structure interaction.
Asunto(s)
Estenosis de la Válvula Aórtica , Prótesis Valvulares Cardíacas , Trombosis , Reemplazo de la Válvula Aórtica Transcatéter , Humanos , Válvula Aórtica/cirugía , Reemplazo de la Válvula Aórtica Transcatéter/efectos adversos , Reemplazo de la Válvula Aórtica Transcatéter/métodos , Trombosis/cirugía , Estenosis de la Válvula Aórtica/etiología , Prótesis Valvulares Cardíacas/efectos adversosRESUMEN
The bed nucleus of the stria terminalis (BNST) is a critical mediator of stress responses and anxiety-like behaviors. Neurons expressing protein kinase C delta (BNSTPKCδ) are an abundant but understudied subpopulation implicated in inhibiting feeding, but which have conflicting reports about their role in anxiety-like behaviors. We have previously shown that expression of PKCδ is dynamically regulated by stress and that BNSTPKCδ cells are recruited during bouts of active stress coping. Here, we first show that in vivo activation of this population is mildly aversive. This aversion was insensitive to prior restraint stress exposure. Further investigation revealed that unlike other BNST subpopulations, BNSTPKCδ cells do not exhibit increased cfos expression following restraint stress. Ex vivo current clamp recordings also indicate they are resistant to firing. To elucidate their afferent control, we next used rabies tracing with whole-brain imaging and channelrhodopsin-assisted circuit mapping, finding that BNSTPKCδ cells receive abundant input from affective, arousal, and sensory regions including the basolateral amygdala (BLA) paraventricular thalamus (PVT) and central amygdala PKCδ-expressing cells (CeAPKCδ). Given these findings, we used in vivo optogenetics and fiber photometry to further examine BNSTPKCδ cells in the context of stress and anxiety-like behavior. We found that BNSTPKCδ cell activity is associated with increased anxiety-like behavior in the elevated plus maze, increases following footshock, and unlike other BNST subpopulations, does not desensitize to repeated stress exposure. Taken together, we propose a model in which BNSTPKCδ cells may serve as threat detectors, integrating exteroceptive and interoceptive information to inform stress coping behaviors.
Asunto(s)
Núcleo Amigdalino Central , Núcleos Septales , Núcleos Septales/metabolismo , Ansiedad , Núcleo Amigdalino Central/metabolismo , Neuronas/fisiología , AfectoRESUMEN
Stress coping strategies represent critical responses to environmental challenges, and active coping has been linked to stress resilience in humans. Understanding the neuroadaptations that support these strategies may provide insights into adaptive and maladaptive stress responses. NMDA receptors (NMDARs) play key roles in neuroadaptation, and NMDARs have been specifically implicated in stress responsiveness. Constitutive knockout mice have been used to implicate the GluN2D NMDAR subunit in regulation of stress-sensitive and affective behavior, but the brain regions in which GluN2D expression changes drive these effects remain unknown. Here we report that following an acute restraint stressor, GluN2D subunit expression is specifically decreased in the bed nucleus of the stria terminalis (BNST), a key region involved in stress processing, in male but not female mice, with no differences found in the thalamus or ventral hippocampus in either sex. Rodents engage in active struggling events during restraint stress that may represent active coping strategies to stress. Thus, we assessed active coping bouts during acute and chronic restraint stress sessions in GluN2D knockout mice. During the first restraint session, GluN2D knockout mice exhibited a pronounced decrease in struggling bouts during restraint stress relative to wild-type littermates, consistent with a role of GluN2D in active coping responses to stress. Repeated, daily restraint sessions revealed a sex-specific role of GluN2D expression on certain aspects of active coping behaviors, with male GluN2D KO mice exhibiting a decrease in total coping bouts measured across five sessions. However, BNST-specific knockdown of GluN2D in male mice did not alter active coping bouts, suggesting either a multi-synaptic role of GluN2D and/or a developmental role of GluN2D in this behavior. Altogether, these data are consistent with a growing literature suggesting that exploration of GluN2D control of stress circuit actions may lead to a novel therapeutic target to consider for stress-related mood disorders.
Asunto(s)
Receptores de N-Metil-D-Aspartato , Núcleos Septales , Animales , Femenino , Masculino , Ratones , Adaptación Psicológica , Encéfalo/metabolismo , Hipocampo/metabolismo , Ratones Noqueados , Receptores de N-Metil-D-Aspartato/metabolismo , Restricción Física , Núcleos Septales/metabolismo , Estrés PsicológicoRESUMEN
Transcatheter aortic valve replacement (TAVR) first received FDA approval for high-risk surgical patients in 2011 and has been approved for low-risk surgical patients since 2019. It is now the most common type of aortic valve replacement, and its use continues to accelerate. Computer modeling and simulation (CM&S) is a tool to aid in TAVR device design, regulatory approval, and indication in patient-specific care. This study introduces a computational fluid-structure interaction (FSI) model of TAVR with Medtronic's CoreValve Evolut R device using the immersed finite element-difference (IFED) method. We perform dynamic simulations of crimping and deployment of the Evolut R, as well as device behavior across the cardiac cycle in a patient-specific aortic root anatomy reconstructed from computed tomography (CT) image data. These IFED simulations, which incorporate biomechanics models fit to experimental tensile test data, automatically capture the contact within the device and between the self-expanding stent and native anatomy. Further, we apply realistic driving and loading conditions based on clinical measurements of human ventricular and aortic pressures and flow rates to demonstrate that our Evolut R model supports a physiological diastolic pressure load and provides informative clinical performance predictions.
Asunto(s)
Estenosis de la Válvula Aórtica , Prótesis Valvulares Cardíacas , Reemplazo de la Válvula Aórtica Transcatéter , Humanos , Reemplazo de la Válvula Aórtica Transcatéter/efectos adversos , Análisis de Elementos Finitos , Válvula Aórtica/diagnóstico por imagen , Válvula Aórtica/cirugía , Diseño de Prótesis , Resultado del TratamientoRESUMEN
α2a-adrenergic receptor (α2a-AR) agonists are candidate substance use disorder therapeutics due to their ability to recruit noradrenergic autoreceptors to dampen stress system engagement. However, we recently found that postsynaptic α2a-ARs are required for stress-induced reinstatement of cocaine-conditioned behavior. Understanding the ensembles recruited by these postsynaptic receptors (heteroceptors) is necessary to understand noradrenergic circuit control. We utilized a variety of approaches in FosTRAP (Targeted Recombination in Active Populations) mice to define an ensemble of cells activated by the α2a-AR partial agonist guanfacine ("Guansembles") in the bed nucleus of the stria terminalis (BST/BNST), a region key to stress-induced reinstatement of drug seeking. We define BNST "Guansembles" and show they differ from restraint stress-activated cells. Guanfacine produced inhibition of cAMP-dependent signaling in Guansembles, while chronic restraint stress increased cAMP-dependent signaling. Guanfacine both excited and inhibited aspects of Guansemble neuronal activity. Further, while some stressors produced overall reductions in Guansemble activity, active coping events during restraint stress and exposure to unexpected shocks were both associated with Guansemble recruitment. Using viral tracing, we define a BNST Guansemble afferent network that includes regions involved in the interplay of stress and homeostatic functions. Finally, we show that activation of Guansembles produces alterations in behavior on the elevated plus maze consistent with task-specific anxiety-like behavior. Overall, we define a population of BNST neurons recruited by α2a-AR signaling that opposes the behavioral action of canonical autoreceptor α2a-AR populations and which are differentially recruited by distinct stressors. Moreover, we demonstrate stressor-specific physiological responses in a specific neuronal population.
Asunto(s)
Núcleos Septales , Trastornos Relacionados con Sustancias , Ratones , Animales , Guanfacina/farmacología , Norepinefrina/farmacología , Neuronas , Transducción de SeñalRESUMEN
Recent findings indicate that glutamate receptors are regulated at the epigenetic level through the posttranslational modification of histones and through DNA methylation. Furthermore, dysregulation of these marks in the context of neurological disease has been shown to influence glutamate receptor function. Over the past two decades, an appreciation for the essential role epigenetic mechanisms play in nervous system function has led to the development of many methods and tools to map, quantitate, and manipulate these chromatin marks. Here we describe two popular methods used to quantitate DNA methylation levels at the gene or nucleotide level. The first, cloning-based bisulfite sequencing involves modification of DNA samples using the chemical sodium bisulfite (BS) , which deaminates all unmethylated cytosines to form uracil. Subsequent PCR amplification converts the uracils to thymine, leaving any cytosines in the PCR product representative of methylation. Fragments are then cloned and sequenced to quantitate the percentage of methylation at each cytosine. The second technique, methyl-binding domain capture (MBDCap), involves shearing the genomic DNA into fragments via sonication. Samples are then incubated with magnetic beads conjugated to methyl-binding domain (MBD) peptides to bind and enrich fragments containing methylated CpGs. Quantitation of DNA methylation levels are then measured indirectly using qRT-PCR with primers specific to the region of interest. Because these methods do not require advanced technical knowledge and can be performed with common laboratory equipment, they are great options for interrogating DNA methylation patterns at the level of the gene, the regulatory region, or in the case of bisulfite sequencing, the nucleotide.
Asunto(s)
Metilación de ADN , Regulación de la Expresión Génica , Reacción en Cadena de la Polimerasa/métodos , Receptores de Glutamato/fisiología , Secuencias Reguladoras de Ácido Ribonucleico , Análisis de Secuencia de ADN/métodos , Sulfitos/química , Epigénesis Genética , Humanos , Receptores de Glutamato/genéticaRESUMEN
Epigenetic modifications, such as DNA cytosine methylation, contribute to the mechanisms underlying learning and memory by coordinating adaptive gene expression and neuronal plasticity. Transcription-dependent plasticity regulated by DNA methylation includes synaptic plasticity and homeostatic synaptic scaling. Memory-related plasticity also includes alterations in intrinsic membrane excitability mediated by changes in the abundance or activity of ion channels in the plasma membrane, which sets the threshold for action potential generation. We found that prolonged inhibition of DNA methyltransferase (DNMT) activity increased intrinsic membrane excitability of cultured cortical pyramidal neurons. Knockdown of the cytosine demethylase TET1 or inhibition of RNA polymerase blocked the increased membrane excitability caused by DNMT inhibition, suggesting that this effect was mediated by subsequent cytosine demethylation and de novo transcription. Prolonged DNMT inhibition blunted the medium component of the after-hyperpolarization potential, an effect that would increase neuronal excitability, and was associated with reduced expression of the genes encoding small-conductance Ca(2+)-activated K(+) (SK) channels. Furthermore, the specific SK channel blocker apamin increased neuronal excitability but was ineffective after DNMT inhibition. Our results suggested that DNMT inhibition enables transcriptional changes that culminate in decreased expression of SK channel-encoding genes and decreased activity of SK channels, thus providing a mechanism for the regulation of neuronal intrinsic membrane excitability by dynamic DNA cytosine methylation. This study has implications for human neurological and psychiatric diseases associated with dysregulated intrinsic excitability.
Asunto(s)
Metilación de ADN/fisiología , Epigénesis Genética/fisiología , Potenciales de la Membrana/fisiología , Células Piramidales/metabolismo , Animales , Apamina/farmacología , Línea Celular , Metilación de ADN/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Humanos , Potenciales de la Membrana/efectos de los fármacos , Células Piramidales/citología , Ratas , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismoRESUMEN
Epigenetic mechanisms such as DNA methylation are essential regulators of the function and information storage capacity of neurons. DNA methylation is highly dynamic in the developing and adult brain, and is actively regulated by neuronal activity and behavioural experiences. However, it is presently unclear how methylation status at individual genes is targeted for modification. Here, we report that extra-coding RNAs (ecRNAs) interact with DNA methyltransferases and regulate neuronal DNA methylation. Expression of ecRNA species is associated with gene promoter hypomethylation, is altered by neuronal activity, and is overrepresented at genes involved in neuronal function. Knockdown of the Fos ecRNA locus results in gene hypermethylation and mRNA silencing, and hippocampal expression of Fos ecRNA is required for long-term fear memory formation in rats. These results suggest that ecRNAs are fundamental regulators of DNA methylation patterns in neuronal systems, and reveal a promising avenue for therapeutic targeting in neuropsychiatric disease states.
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Región CA1 Hipocampal/metabolismo , Metilación de ADN , Epigénesis Genética , Neuronas/metabolismo , Proteínas Oncogénicas v-fos/genética , ARN Mensajero/genética , Animales , Región CA1 Hipocampal/citología , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Islas de CpG , Miedo/fisiología , Humanos , Inyecciones Intraventriculares , Masculino , Neuronas/citología , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Proteínas Oncogénicas v-fos/antagonistas & inhibidores , Proteínas Oncogénicas v-fos/metabolismo , Cultivo Primario de Células , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Técnicas EstereotáxicasRESUMEN
The Hedgehog (Hh) pathway was first defined by its role in segment polarity in the Drosophila melanogaster embryonic epidermis and has since been linked to many aspects of vertebrate development and disease. In humans, mutation of the Patched1 (PTCH1) gene, which encodes an inhibitor of Hh signaling, leads to tumors of the skin and pediatric brain. Despite the high level of conservation between the vertebrate and invertebrate Hh pathways, studies in Drosophila have yet to find direct evidence that ptc limits organ size. Here we report identification of Drosophila ptc in a screen for mutations that require a synergistic apoptotic block in order to drive overgrowth. Developing imaginal discs containing clones of ptc mutant cells immortalized by the concurrent loss of the Apaf-1-related killer (Ark) gene are overgrown due, in large part, to the overgrowth of wild type portions of these discs. This phenotype correlates with overexpression of the morphogen Dpp in ptc,Ark double-mutant cells, leading to elevated phosphorylation of the Dpp pathway effector Mad (p-Mad) in cells surrounding ptc,Ark mutant clones. p-Mad functions with the Hippo pathway oncoprotein Yorkie (Yki) to induce expression of the pro-growth/anti-apoptotic microRNA bantam. Accordingly, Yki activity is elevated among wild type cells surrounding ptc,Ark clones and alleles of bantam and yki dominantly suppress the enlarged-disc phenotype produced by loss of ptc. These data suggest that ptc can regulate Yki in a non-cell autonomous manner and reveal an intercellular link between the Hh and Hippo pathways that may contribute to growth-regulatory properties of the Hh pathway in development and disease.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/fisiología , Discos Imaginales/fisiología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Receptores de Superficie Celular/genética , Transactivadores/genética , Transactivadores/metabolismo , Animales , Proliferación Celular , Drosophila/genética , Drosophila/crecimiento & desarrollo , Drosophila/metabolismo , Femenino , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Discos Imaginales/crecimiento & desarrollo , Discos Imaginales/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , MicroARNs/genética , MicroARNs/metabolismo , Mutación , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Superficie Celular/metabolismo , Proteínas Señalizadoras YAPRESUMEN
RATIONALE: 17ß-Estradiol (E2) attenuates hypoxic pulmonary vasoconstriction and hypoxic pulmonary hypertension (HPH) through an unknown mechanism that may involve estrogen receptors (ER) or E2 conversion to catecholestradiols and methoxyestradiols with previously unrecognized effects on cardiopulmonary vascular remodeling. OBJECTIVES: To determine the mechanism by which E2 exerts protective effects in HPH. METHODS: Male rats were exposed to hypobaric hypoxia while treated with E2 (75 µg/kg/d) or vehicle. Subgroups were cotreated with pharmacologic ER-antagonist or with inhibitors of E2-metabolite conversion. Complementary studies were performed in rats cotreated with selective ERα- or ERß-antagonist. Hemodynamic and pulmonary artery (PA) and right ventricular (RV) remodeling parameters, including cell proliferation, cell cycle, and autophagy, were measured in vivo and in cultured primary rat PA endothelial cells. MEASUREMENTS AND MAIN RESULTS: E2 significantly attenuated HPH endpoints. Hypoxia increased ERß but not ERα lung vascular expression. Co-treatment with nonselective ER inhibitor or ERα-specific antagonist rendered hypoxic animals resistant to the beneficial effects of E2 on cardiopulmonary hemodynamics, whereas ERα- and ERß-specific antagonists opposed the remodeling effects of E2. In contrast, inhibition of E2-metabolite conversion did not abolish E2 protection. E2-treated hypoxic animals exhibited reduced ERK1/2 activation and increased expression of cell-cycle inhibitor p27(Kip1) in lungs and RV, with up-regulation of lung autophagy. E2-induced signaling was recapitulated in hypoxic but not normoxic endothelial cells, and was associated with decreased vascular endothelial growth factor secretion and cell proliferation. CONCLUSIONS: E2 attenuates hemodynamic and remodeling parameters in HPH in an ER-dependent manner, through direct antiproliferative mechanisms on vascular cells, which may provide novel nonhormonal therapeutic targets for HPH.