bluuebunny/crossref_metadata_2025 huggingface dataset:
The Therapeutic Potential of mGlu3 Antagonism in Correcting Hippocampal Long‐term Depression Impairments in a Mouse Model of MECP2 Duplication Syndrome
Authors: Stansley Branden , Gogliotti Rocco , Senter Rebecca , Fisher Nicole , Emmitte Kyle , Lindsley Craig , Conn P. Jeffrey , Niswender Colleen
Published: November 2023

Precise levels of the transcriptional regulator Methyl CpG Binding Protein 2 (MeCP2) are known to be a key determinant for proper neurological function. Mutations in the MECP2 gene results in a disease known as Rett syndrome; in contrast, genetic duplication of MECP2 leads to a disorder known as MECP2 Duplication syndrome. The latter genetic neurodevelopmental disorder has a wide symptomology including stereotypy, seizures, cognitive deficits, and autism. In rodent models of MECP2 Duplication Syndrome in which MeCP2 is overexpressed, increases in glutamatergic synapse formation in hippocampal neurons occur, as well as impairments in hippocampal synaptic plasticity, a neurological phenomenon thought to be involved in memory and cognition. In accordance with this notion, MeCP2 overexpression causes memory and cognitive deficits in mice. Interestingly, the metabotropic glutamate receptor 3 (mGlu3) has been shown to be involved in regulating synaptic plasticity and cognitive behaviors in mice, and mGlu3 expression appears to correlate with MeCP2 expression levels in the brain. We hypothesized that that an upregulation of mGlu3 leads to an impairment in synaptic plasticity and cognitive deficits in MeCP2 overexpressing mice. To investigate the mechanisms by which MeCP2 overexpression induces neurophysiological impairments, a MeCP2‐Tg1 mouse model was used. Our results indicate that mGlu3 is upregulated the hippocampus of MeCP2‐Tg1 mice, correlating with an increase in MeCP2 protein expression. Electrophysiological experiments found that paired‐pulse facilitation was enhanced and input‐output intensity was reduced at the SC‐CA1 hippocampal synapse in the MeCP2‐Tg1 mice compared to wild‐type controls. Furthermore, (S)‐3,5‐Dihydroxyphenylglycine (DHPG) induced‐long term depression (LTD) was impaired in MeCP2‐Tg1 mice. Interestingly, an mGlu3 negative allosteric modulator, VU0650786, was capable of normalizing DHPG‐induced LTD to the level of that observed in wild‐type controls. These results suggest that overexpression of mGlu3 may contribute to synaptic plasticity impairments within the hippocampus of MeCP2‐Tg1 mice. Future experiments are aimed at elucidating behavioral correlates of these neurophysiological abnormalities and their underlying mechanisms.

Support or Funding Information

T32NS007491 (BJS), rettsyndrome.org postdoctoral fellowship (RGG), Weatherstone predoctoral fellowship (BKS), T32 GM07628 (NF), R01 MH099269 (KAE), U54 MH084659 (CWL), R01 NS031373 (PJC), R21 MH102548 (CMN), Autism Speaks Treatment Award (CMN).
Kir4.1 (KCNJ10) Inhibition with a Newly Developed Antagonist Induces Diuresis in Rats
Authors: Kharade Sujay V. , Kurata Haruto , Hopkins Corey R. , Denton Jerod S.
Published: November 2023

Inward rectifier potassium (Kir) channels play fundamental roles in diverse physiological processes, and in some cases, represent novel drug targets for human diseases. Outside of the nervous system, Kir4.1 (encoded by KCNJ10) is prominently expressed in the basolateral membrane of the thick ascending limb of Henle, distal convoluted tubule, and collecting duct of nephron. Kir4.1 can form homotetrameric channels or heterotetrameric channels with Kir5.1, although the most common channel subtype in the kidney is the heteromeric Kir4.1/5.1 form. Genetic ablation of Kir4.1 in mice or humans causes severe salt and water wasting resembling clinical phenotypes associated with diuretic use, suggesting that Kir4.1 might represent a novel diuretic target. Therefore, the aim of this study was to develop a selective small‐molecule inhibitor of Kir4.1 with which to evaluate its therapeutic potential. From a high‐throughput screen of ~ 75,000 compounds from the Vanderbilt Institute of Chemical Biology library, we identified an inhibitor, termed VU992, which inhibits homotetrameric Kir4.1 with an IC50 of 760 nM and heterotetrameric Kir4.1/5.1 with an IC50 of approximately 25 μM (33‐fold selectivity). In addition, VU992 is at least 65‐fold selective for Kir4.1 over other renal Kir channels Kir1.1 and Kir7.1. SAR exploration revealed VU992 bears fundamental parts to inhibit efflux through Kir4.1 channel. Finally, oral administration of VU992 dose‐dependently increases urine output in volume‐loaded, freely behaving rats. This study shows proof‐of‐concept that homotetrameric Kir4.1 might represent a molecular target for a novel class of diuretic for the treatment of hypertension. In addition, its basolateral localization might offer advantages over other diuretic targets that are located on the luminal membrane of the nephron.

Support or Funding Information

NIH 5R21 NS073097‐01S1
Mitochondrial Topoisomerase Dysfunction promotes Mitochondrial DNA (mtDNA) damage leading to Myocardial Mitochondrial Dysfunction
Authors: Edwards John , Mitry Maria , Soler Amanda , Laurent Dimtri
Published: November 2023

Topoisomerases are highly conserved proteins that resolve the topological difficulties of DNA replication. In post mitotic cells, the mitochondrial genome continues to replicate about once a month and resolving the topology of mtDNA replication would be almost insurmountable in the absence of the three unique mitochondrial topoisomerases. We have previously reported that diabetes or alcoholism induces mitochondrial topoisomerase dysfunction to promote mtDNA degradation as an antecedent event to cell death. It remains unclear how chronic hyperglycemia causes this shift in the mitochondrial topoisomerase function from the physiologic to the pathological. Work by others has suggested that nuclear topoisomerases are sensitive to the oxidant stress molecules. To that end, we hypothesize that low levels of ROS alter mitochondrial topoisomerase function to accelerate DNA strand breakage. DNA relaxation of supercoiled DNA is a hallmark measurement of topoisomerase function. Supercoiled DNA incubated with isolated mitochondria generated the same isopane pattern of relaxation as compared to purified topoisomerase1 (Top1). Isolated mitochondria from cell lines in which Top1 or Topo‐2beta were knockout retained DNA relaxation function suggesting the presence of the three unique functional mitochondrial topoisomerases. In the absence of mitochondrial extracts; 125–1000 μM H2O2 did not alter DNA relaxation compared to vehicle controls. Myocardial mitochondrial extracts alone increased DNA relaxation compared to controls and this was significantly elevated (p<.05) in the presence of increasing concentrations of H2O2. Similar findings were obtained using a DNA cleavage assay; alone 60 μM H2O2 did not cause DNA cleavage but exacerbated DNA cleavage in the presence of mitochondrial extracts. Using the iron chelators, EDTA or deferoxamine, we determined that this is increase was not due to a Fenton effect. Using intact isolated intact mitochondria; incubation with H2O2 significantly increased mitochondrial superoxide levels indicating that H2O2 ready enters the mitochondria and is able to elevate intramitochondrial concentrations of superoxide. Previously we have reported that chronic hyperglycemia transiently elevated intramitochondrial superoxide levels in the H9c2 cell line. Collectively these results indicate that ROS alters mitochondrial topoisomerase function to increase DNA strand breakage. Separate from a direct impact of oxidative stress, our studies find that alteration of mitochondrial topoisomerase reaction mechanism accelerates and propagates an increase in mtDNA damage. These findings indicate that the functional state of the mitochondrial topoisomerases have important consequences for the myocardium and participate in the pathophysiology leading to the development of cardiomyopathy.

Support or Funding Information

Supported in part by NIH RO3HD065551
KvBeta2 Alters Kv1 Surface Expression and Modulates Electrophysiologic Properties in Ventricular Cardiomyocytes
Authors: Kilfoil Peter J , Raucci Frank J , Tipparaju Srinivas , Bhatnagar Aruni
Published: November 2023

Introduction

The voltage‐gated potassium (Kv) channels regulate the resting membrane potential and the repolarization phase of the cardiac action potential. Members of the Kv1 and Kv4 family associate with ancillary subunits, such as Kvβ 1–3, that modify channel kinetics, gating and trafficking. Although Kvβ1 has been shown to regulate Ito and IKslow currents in the heart, the role of Kvβ2 in regulating myocardial potassium conductances is unknown. In heterologous expression systems, Kvβ2 increases surface expression of Kv1 channels, shifts the activation potential of Kv1 currents to more polarized voltages, and increases the inactivation of inactivating Kv1 currents, but its role in regulating native cardiac channels remains unknown. Hence, to identify the role of Kvβ2 in the heart, we examined the distribution and activity of Kv1 and Kv4 channels in cardiac myocytes from wild‐type (WT) and Kvβ2‐null mice (KO).

Methods

Western blotting and confocal microscopy were used to determine the abundance and localization of Kvβ2, Kv1.4 and Kv1.5 proteins in heart and isolated cardiomyocytes. Action potentials and whole‐cell Kv currents were recorded from freshly isolated mouse cardiac myocytes using standard patch clamp methodologies.

Results

Immunoprecipitation of Kvβ2 from the heart resulted in co‐immunoprecipitation of Kv1.5 and Kv1.4. In comparison with WT cardiac myocytes, the surface expression of Kv1.5 was reduced in KO myocytes. Ventricular myocytes from KO mice had significantly lower peak Kv current density (p<0.01) and component Kv current densities as determined by triexponential curve fitting suggesting reduced IKur and Ito (p<0.01, n=34 cells, N=10 mice), consistent with a decrease in Kv1.4 and Kv1.5 channel density and a shift in the voltage‐dependence of the activation of the current. The duration of action potentials (APD 20, APD90) recorded from myocytes isolated from the right ventricle was significantly prolonged in KO mice (p<0.01, n=32 cells, N=11 mice) when compared with WT cardiac myocytes. Simulation of action potentials in CellML using experimental voltage clamp data resulted in similarly prolonged APs, as did partial pharmacological blockade of Kv1 channels with 4‐aminopyridine.

Conclusion

Mouse cardiac myocytes express Kvβ2, which interacts primarily with Kv1.4 and Kv1.5 channels. Association with Kvβ2 regulates Kv1 expression and function in the sarcolemma, and thereby, the repolarization phase of the action potential.
Immature Endothelial Cells Initiate Endothelin‐Mediated Constriction of Newborn Arteries
Authors: Chang Fumin , Flavahan Sheila , Flavahan Nicholas A
Published: November 2023

The early postnatal period is associated with remarkable changes in the morphology and function of endothelial cells. Newborn arterial endothelial cells have a highly unusual phenotype, which includes minimal NO activity, heightened Rho/ROCK signaling, prominent actin stress fibers and poorly developed cellular junctions. During the immediate postnatal period, arterial endothelial cells then acquire characteristics normally associated with mature protective cells including exuberant NO‐mediated dilation, complex endothelial cellular junctions and reorganization of stress fibers to a cortical actin network. Indeed, the atypical phenotype of immature newborn endothelial cells resembles dysfunctional endothelial cells that contribute to the development of vascular disease. The present experiments were performed to determine whether newborn endothelial cells might express and release endothelin‐1 (ET‐1) and initiate endothelium‐dependent constriction, hallmark features of pathological endothelial dysfunction. Carotid arteries were isolated from newborn (postnatal day 1, P1), P7 and P21 mice and mounted in pressure myographs at a constant transmural pressure of 20 mmHg (mean blood pressure for a P1 mouse). Exogenous ET‐1 caused similar concentration‐dependent constrictions of P1, P7 and P21 arteries. Endothelial stimulation with A23187 or thrombin caused powerful constriction in P1 arteries, no significant change in arterial diameter in P7 arteries, and powerful dilatation in P21 arteries. In P1 arteries, constriction to thrombin or A23187 was inhibited by endothelial denudation, by ET‐1 receptor antagonists (BQ123 or BQ123 plus BQ788), or by inhibition of endothelin converting enzyme (ECE) (phosphoramidon or SM19712). ET‐1 receptor antagonism did not significantly affect responses to thrombin or A23187 in more mature P7 and P21 arteries. Endothelial expression of ET‐1 peptides, assessed by immunofluorescent analysis, was significantly increased in P1 compared to P21 arteries. Endothelial stimulation with thrombin rapidly increased the endothelium‐dependent release of ET‐1 from P1 but not P21 arteries. Therefore, newborn endothelial cells express high levels of ET‐1 peptides, rapidly release ET‐1 in response to endothelial stimulation and initiate ET‐1 mediated endothelium‐dependent constriction. This activity is lost as the endothelium matures in the immediate postnatal period. The unusual phenotype of newborn endothelium including heightened activity of ET‐1 is reminiscent of pathological endothelial dysfunction, which may reflect reemergence of the newborn phenotype.

Support or Funding Information

NIH R01 HD078639
Vasopressin Gene Expression is Differently Regulated at the PVN Level of Salt‐loaded Wistar and SHR
Authors: Ribeiro Natalia , Jurdi el Chaar Laiali , Antunes Vagner Roberto
Published: November 2023

It is known that osmotic challenges leads to an increase in blood pressure that is associate with a simpathoexcitatory component; moreover, the paraventricular nucleus of hypothalamus (PVN) plays an important role in this response. Previous studies performed in our lab have shown that salt‐induced sympathoexcitation and hypertension involves arginine vasopressin (AVP) receptors activation at the PVN level. Thus, here we sought to evaluate if the high salt‐induced hypertension could be due a differential AVP mRNA expression into the PVN of SHR and Wistar. Institutional approved ethical committee protocol N° 111‐ Male SHR and Wistar (300 g). Were offered NaCl 2% in replacement of the drinking water, and kept in metabolic cages for 1, 4 or 7 days to evaluate hydroelectrolytic parameters. At the end, the animals were decapitated and the PVN was microdissected for RNA extraction and qPCR procedures. The results have shown a higher increase in the blood pressure (194±8 mmHg, n=3) in SHR exposed to 7 days of high salt intake when compared to 7 days salt‐loaded Wistars (131±4 mmHg, n=4). An upregulation of AVP gene expression in the PVN was observed only in SHR rats subjected to salt overload for 7 days (2,7±0,53 a.u, n=7) compared to the their normohydrated control group (0,86 ± 0,3, n=6) and Wistar animals exposure to the same period to salt overload (1,6±0,2 a.u, n=7). In conclusion, we can suggest that AVP mRNA expression in the PVN of salt‐loaded animals is time‐dependent and differently modulated in Wistar and SHR, which could be a causality relationship with higher blood pressure levels observed in salt‐loaded SHR.
The Exon Junction Complex proteins are non‐AMP kinase dependent sensors of energetic stress and regulate contractility and sarcomere structure in cardiac myocytes
Authors: Boateng Samuel , Pierrat Olivier , Paudyal Anju , koroleva Olga
Published: November 2023

The heart is known to undergo significant alterations in gene expression during stress and sometimes this adaptation can lead to heart failure. The mechanism by which cells select specific mRNAs for translation is not fully understood, but the Exon Junction Complex (EJC) plays a major role in this process in eukaryotes. The EJC selects the mRNA isoforms to be exported to the cytoplasm and determines whether they are then translated into protein. In this study we hypothesised that the EJC is involved in the regulation of gene expression during the stress response in cardiac myocytes. Using cultured rat neonatal myocytes and subcellular fractionation we examined the cellular distribution of two major components of the EJC in response to metabolic stress. In cardiac myocytes we found that there was a significant relocalisation of eIF4A3 and mago from the nucleus to cytoplasm following 18 hours of hypoxia. To examine the effect of metabolic stress on the EJC in more detail we treated cardiac myocytes with 50mM NaN3 for 4 hours to mimic the metabolic stress induced by hypoxia. Following NaN3 treatment there was a significant relocalisation of eIF4A3 and mago to the cytoplasm and this was associated with the formation of cytoplasmic stress granules. However, immunocytochemical staining with the stress granule marker PABP1 showed that cytoplasmic mago did not colocalise with stress granules. To examine whether the effects of metabolic stress on the EJC proteins was dependent on the metabolic sensor AMP kinase (AMPK), we treated myocytes with 1μM dorsomorphin in combination with NaN3. Dorsomorphin augmented the translocation of mago and eIF4A3 from the nucleus to the cytoplasm. This indicates that the EJC proteins respond to metabolic stress independently of AMPK activity. Moreover, the inhibition of AMPK was associated with increased metabolic cellular stress resulting in the augmented subcellular translocation of EJC proteins. Constructs of eIF4A3 tagged with green fluorescence protein on the N terminus resulted in a predominantly cytosolic protein distribution that did not relocalise in response to NaN3 treatment. This suggests that the N‐terminus of eIF4A3 is necessary for subcellular localisation. Finally, to determine the function of eIF4A3 in cardiac myocytes we knocked down the protein with siRNA. Protein knockdown of eIF4A3 resulted in cessation of cell contractility 96 hours post treatment. There was also a significant reduction in the number of intact sarcomeres suggesting that the contractile filaments were degrading in response to reduced eIF4A3. Taken together, these data suggest that eIF4A3 and other EJC members play an important role in the myocyte stress response, cell contractility and morphology.

Support or Funding Information

This work was supported by the British Heart Foundation project grant PG/10/64/28520 and PG/10/97/28666
Reperfusion Therapy with Rapamycin Prevents Myocardial Injury and restores Plasma Levels of Stress‐Sensitive microRNA‐191 in Conscious Diabetic Rabbits
Authors: Samidurai Arun , Ockaili Ramzi , Kukreja Rakesh C , Das Anindita
Published: November 2023

Background

Diabetes is a major risk factor for myocardial infarction (MI). Persistent activation of the mammalian target of rapamycin (mTOR) leads to diabetic complications and plays a critical role in myocardial reperfusion injury following ischemia. mTOR inhibition with rapamycin at reperfusion preserves cardiac function with reduction of myocardial infarction in type 2 diabetic mice. To demonstrate the clinical potential of these novel observations, we examined the effect of rapamycin in a conscious diabetic rabbit model of MI. In addition we examined whether rapamycin influences the plasma levels of stress‐sensitive microRNA‐191.

Methods and Results

To induce diabetes, alloxan monohydrate (125 mg/kg, i.v.) was administered in New Zealand white rabbits (n=10; average 3kg, 4 month old). Blood glucose levels increased to 345±66 mg/dL (n=10) following 2 weeks of alloxan administration. Subsequently, balloon occluders were implanted and 7 days later, these rabbits were subjected to a 30‐min ischemia and 3 days of reperfusion. Rapamycin (0.25 mg/kg, i.v.) or DMSO (vehicle) was infused 5 min before reperfusion. Plasma was collected at baseline, 1, 2, 4 and 24 hours during the procedure. The results show that rapamycin significantly reduced myocardial infarction in diabetic rabbit following I/R (Figure 1A) as assessed by triphenyltetrazolium chloride staining. Rapamycin also reduced cardiac troponin I level in blood at 1 hr and 2 hr of reperfusion as compared to vehicle alone (Figure 1B). Moreover the plasma levels of microRNA‐191 were decreased at 1 and 2 hr after I/R which were significantly elevated with rapamycin treatment.

Conclusion

The current translational study suggests that rapamycin could be a promising drug in attenuation of reperfusion injury in diabetic patients following acute MI. Also, micro‐RNA‐191 may have a role in attenuation of I/R injury following rapamycin treatment.

Support or Funding Information

NIH R37 HL051045, RO1 HL079424, RO1 HL093685, RO1 HL118808 (RCK)

A.D. Williams’ Fund of VCU UL1RR031990 (AD)

VCU National Center for Advancing Translational Sciences (UL1TR000058) and CCTR Endowment Fund (AD)
HIF‐1α Stabilization Inhibits Basal and Maximal Mitochondrial Respiration in Fetal Cardiac and Skeletal Myocytes
Authors: Saharan Sunil , Espinoza Herbert M , Brown Laura D , Jonker Sonnet S
Published: November 2023

Introduction

Hypoxia is the most common stress experienced by fetuses, and chronic hypoxia affects cardiac function as well as growth of fetal striated muscle. Cardiac muscle is active in utero, while skeletal muscle is relatively inactive, thus the metabolic effects of hypoxia may be differently mediated in these cells. Hypoxia signaling is mediated through both HIF‐1α and depletion of high‐energy phosphates, but the relative regulatory roles of these two signals on metabolism in fetal striated muscle is unknown.

Hypothesis

Chronic HIF‐1α stabilization and reduced mitochondrial proton gradient suppress oxidative respiration and promote glycolysis in fetal sheep cardiac and skeletal myocytes.

Methods

Near‐term fetal sheep cardiomyocytes and biceps femoris myoblasts were cultured (n=4) and myoblasts were differentiated to myotubes. Cells were treated for 48h with cobalt (II) chloride (to stabilize HIF‐1α) or carbonyl cyanide 4‐trifluoromethoxy phenylhydrazone (FCCP; to diminish the mitochondrial proton gradient). Doses were selected to achieve 80% cell survival; compounds and detached cells were washed out prior to metabolic assessment. The Seahorse XFe96 metabolic analyzer measured oxygen consumption rate (respiration) and H+ production rate (glycolysis), which were normalized to protein content. Data were analyzed within cell type by one‐way analysis of variance followed by Tukey's multiple comparisons test, and between cell type by Student's t‐test. Noted differences were significant at p<0.05.

Results

Basal ATP‐producing respiration (excluding proton leak) was 2.5 fold higher in cardiomyocytes than skeletal myotubes, and maximal respiration was 5.4 fold higher, while glycolysis was not different between cardiac and skeletal myocytes. In skeletal myotubes, basal ATP‐producing respiration was reduced 24% by FCCP and 77% by cobalt. In cardiomyocytes, ATP‐producing respiration was reduced 39% by cobalt. No significant changes occurred in glycolytic capacity following chronic cobalt or FCCP treatment.

Conclusion

Metabolic changes following treatment were similar between cardiac and skeletal muscle, although the metabolic capacity of fetal cardiomyocytes was many‐fold higher than skeletal myotubes. HIF‐1α signaling, as occurs in chronic hypoxia, significantly reduces the mitochondrial oxidative respiratory capacity of fetal striated muscle. This change may affect cardiac and skeletal muscle function and growth during and following birth.

Support or Funding Information

This research was supported by NICHD under awards numbered R01HD071068 and P01HD034430.
Hypoxic inhibition of mitochondrial oxygen consumption depends on HIF‐1α
Authors: Mairbaurl Heimo , Baloglu Emel , Zhang Ying
Published: November 2023

The AMP‐activated protein kinase (AMPK) couples cellular energy demand and ATP production by increasing PGC‐1α‐dependent expression of mitochondrial proteins. Hypoxia decreases mitochondrial activity. Here we tested the hypothesis that stimulation of AMPK, which indicates increased metabolic demand, blunts the impaired mitochondrial function in hypoxia to prevent functional impairment of cardiomyocytes. H‐10 cardiomyocytes were treated with AICAR to stimulate AMPK in normoxia and hypoxia (1.5 % O2).

AICAR, hypoxia, and their combination increased phosphorylated AMPK indicating its stimulation. AICAR induced a 3x increase in mitochondrial oxygen consumption (JO2), whereas hypoxia decreased JO2 (−20%) and prevented the AICAR‐induced increase. Hypoxia did not prevent the AICAR‐induced increase in pyruvate dehydrogenase (PDH) activity despite increased PDK mRNA expression. The HIF‐dependent genes GAPD (1.5x) and BNIP3 (4x) were increased in hypoxia, also in the presence of AICAR. Silencing HIF‐1α prevented the hypoxia‐induced inhibition of mitochondrial respiration in control and AICAR‐treated cells and significantly reduces upregulation of HIF‐dependent genes.

These results indicate that hypoxia prevents the AMP‐ and AMPK‐dependent stimulation of mitochondrial metabolism by HIF‐1α dependent mechanisms thus disproving our hypothesis. The inhibition by hypoxia seems to be caused by decreasing the expression of enzymes of the electron transfer chain by blocking signaling downstream of AMPK rather than by decreasing PDH‐mediated substrate flux into mitochondria. Together this indicates that hypoxia decreases cardiomyocyte oxidative metabolism despite increased metabolic demand.

Support or Funding Information

DFG Ma‐1503/29‐1
Pregnancy activates HIF‐1a and PPARγ in left ventricle of rats
Authors: Godoy‐Lugo Jose Arquimides , Hernandez‐Palomares Magally Luisa Elena , Rodriguez‐Martinez Daniel , Rosas‐Rodriguez Jesus Alfredo , Ortiz Rudy M , Soñanez‐Organis Jose Guadalupe
Published: November 2023

Pregnancy‐induced physiologic cardiac hypertrophy with important changes in the energetic metabolism to support its contractile demands and maintain viability. HIF‐1α and PPARγ activates glycolytic and glycerol‐lipid biosynthesis genes in response to pathologic hypertrophy, respectively. Therefore, these changes increase the rate of glycolysis and glucose‐to‐lipid ratio via glycerol‐3‐phosphate (G3P) pathway. However, the contribution of HIF‐1α and PPARγ to the pregnancy‐induced hypertrophy and its reversible process (postpartum) to heart metabolism are not defined. We evaluated HIF‐1α and PPARγ transcriptional activity, and quantified their target genes, key enzymes, and metabolic intermediates of glycolysis and lipid metabolism in the left ventricle of rats before, during, and after pregnancy. HIF‐1α and PPARγ activity increased 1.2‐ and 1.6‐fold, respectively, during pregnancy, and decreased to basal levels during postpartum. Expressions of mRNA for glycolytic (HK2, PFKM and GAPDH) and glycerol‐lipid biosynthesis (GPAT and GPD1) genes increased 1.6‐ to 14‐fold during pregnancy and returned to basal levels postpartum. While the increase in GPD1 expression translated to an increase in its activity, such was not the case for GAPDH suggestive of differential, post‐translational regulation of these proteins. Glycolytic (glucose, lactate and DHAP) and lipid biosynthesis (G3P and FFA) intermediates increased with pregnancy and were maintained postpartum. Results demonstrate that pregnancy induces the expression of glycolytic and glycerol‐lipid biosynthesis genes that translates to a shift in cardiac metabolism, which is sustained, at least acutely, postpartum and is associated with the activation of HIF‐1α and PPARγ.

Support or Funding Information

University of California Institute for Mexico and the United States and Mexico's National Council for Science and Technology (UC MEXUS‐CONACYT) and The Integral Program for Institutional Strengthening (PIFI).
Long‐term Sugar‐sweetened Beverage Intake elicits Early Metabolic Perturbations in Rat Hearts: a Bitter‐sweet Taste?
Authors: Driescher Natasha , Benade Janina , Joseph Danzil , Marnewick Jeanine , Bester Dirk , Essop M. Faadiel
Published: November 2023

Increased consumption of sugar‐sweetened beverages (SSB) is linked to the development of obesity, diabetes and heart diseases. However, the underlying mechanisms driving this process remain unclear. For this study we hypothesized that regular SSB intake elicits metabolic dysregulation and activates non‐oxidative glucose pathways (NOGPs), i.e. polyol pathway, hexosamine biosynthetic pathway (HBP), advanced glycation end‐products, and PKC; contributing to the onset of cardio‐metabolic complications. To further investigate this, we began by establishing and characterizing a rodent model of long‐term SSB consumption. Here male Wistar rats (~200 gr) were daily gavaged with 3–5.1 mL of a local SSB for a 3‐month period vs. two matched controls (water gavaging; caloric equivalent). Dietary behaviour and phenotypic changes were monitored on a weekly basis while we also measured metabolite levels, performed oral glucose tolerance tests (OGTT), determined the degree of oxidative stress and assessed NOGP activation in heart and liver tissues. These data demonstrate that SSB consumption did not cause significant changes in weight gain and food consumption, while fasting blood glucose levels remained unchanged. However, heart: body weight was higher vs. controls (p<0.05), while the OGTT analysis established that the area under the curve was significantly lower for the SSB group vs. controls. Myocardial cholesterol and triglyceride levels were not significantly altered. No differences were found between groups for oxidative stress markers (GSH/GSSG; malondialdehyde) evaluated in heart and liver tissues. Of note, SSB intake triggered the activation of the HBP and PKC pathway in heart tissues vs. controls (p<0.05). Our study successfully established a novel in vivo rat model of SSB consumption. These findings reveal that long‐term SSB intake results in dysregulation of potentially detrimental metabolic circuits (HBP, PKC pathway) in the heart that may place the organism at risk despite an apparently “healthy” phenotype.

Support or Funding Information

Research funded by the National Research Foundation and Stellenbosch University (South Africa).
Assessing the Relationship of Vitamin D with Iron Status in Children with Inflammatory Bowel Disease
Authors: Syed Sana , Smith Ellen M , Tangpricha Vin , Chesdachai Supavit , Kumar Archana , Prince Jarod , Ziegler Thomas R , Suchdev Parminder S , Kugathasan Subra
Published: November 2023

Inflammatory bowel disease (IBD) is a chronic intestinal condition with severalnutrition‐related comorbidities including anemia and vitamin D deficiency. Vitamin D has recently been linked to iron metabolism through down‐regulatory effects on hepcidin and hepcidin‐stimulatorypro‐inflammatory cytokines. It has been proposed that vitamin D may increase bioavailable iron for erythropoiesis and hemoglobin synthesis. We aimed to investigate the association of inflammation, anemia and iron biomarkers with vitamin D levels in pediatric IBD. We obtained cross‐sectional data from N=69 IBD patients aged 5 to <19y, in which iron biomarkers [reticulocyte hemoglobincontent (CHr), ferritin, soluble transferrin receptor (sTfR)], plasma25‐hydroxyvitamin D (25(OH)D), inflammatory biomarkers [C‐reactive protein(CRP), α1 acid glycoprotein (AGP), hepcidin] and hemoglobin (Hb) were collected. Multivariable logistic and linear models were used to examine the associations of vitamin D with inflammation, anemia and iron status. The prevalence of iron deficiency using ferritin < 15 μg/L or sTfR > 8.3mg/L was 65%, anemia was 36%, vitamin D deficiency was 38%, vitamin D insufficiency was 76% and 49% of subjects were inflamed (elevated CRP/AGP). 25(OH)D was positively associated with log(AGP), controlling for age, sex, race, Medicaid, BAZ, disease duration, RBP, and ferritin, (β(SE)=0.02(0.01), P=0.01), and inversely associated with log(hepcidin) (β(SE)=−0.02(0.01), P=0.05), controlling for the same covariates. When categorized by insufficiency/sufficiency, 25(OH)D < 30 ng/mL was associated with a 0.90 g/dL reduction in Hb concentration, compared to 25(OH)D ≥ 30 ng/mL, controlling for age, sex, race, medicaid, BAZ, disease duration, RBP, and ferritin (β(SE)=−0.90 (0.45), P=0.05). This association was most pronounced among those without elevations in inflammatory markers (β(SE)=−1.46(0.53), P=0.01). The association between vitamin D insufficiency and anemia was not significant overall, but when stratified by race, vitamin D insufficient African Americans had 20 times higher odds of anemia compared to vitamin D insufficient non‐African Americans (OR=20.18, 95% CI: 1.80, 226.5). In conclusion, Vitamin D insufficiency was associated with reduced hemoglobin concentrations in this population of children with IBD. Vitamin D may improve iron status by decreasing hepcidin, but the effect of inflammation status remains unclear. In non‐inflamed children with IBD, 25(OH) D and hemoglobin concentrations should be monitored. Among AAs with IBD, vitamin D status was related to anemia and merits further investigation.
Effects of Whole Egg Consumption on Vitamin E Absorption from Co‐Consumed, Mixed‐Vegetable Salad
Authors: Kim Jung Eun , Ferruzzi Mario G , Campbell Wayne W
Published: November 2023

The daily usual intake of vitamin E (VIT E) in nearly 90% of the US population is lower than the estimated average requirement. In addition to promoting increased VIT E intake, dietary approaches to increase the absorption of VIT E would help people meet their body's need. Vitamin E is co‐absorbed with dietary lipid, but very limited data exist on the impact of endogenous food sources of lipid on VIT E absorption. Co‐consuming whole egg with VIT E‐containing foods may increase VIT E absorption via the lipid contained in egg yolk. The objective of this study was to assess the effect of adding cooked whole egg to a raw mixed‐vegetable salad on VIT E absorption. Healthy young males (n=16) consumed the same salad (all served with 3g canola oil) with no egg (control, CON); with 75 g (1½ eggs) of scrambled whole egg (low egg, LE); and with 150 g (3 eggs) of scrambled whole egg (high egg, HE) (randomized, crossover design). The CON, LE, and HE trials contained 4.1 mg, 5.2 mg (1.1 mg from eggs), and 6.4 mg (2.3 mg from eggs) of VIT E (α‐tocopherol (α‐T) and γ‐tocopherol (γ‐T) combined), respectively, and included 3 g, 10.5 g (7.5 g from eggs), and 18 g (15 g from eggs) of total dietary lipids, respectively. Blood was collected hourly during 10 hours and triacylglycerol rich lipoprotein fractions (TRL) were isolated. α‐tocopherol and γ‐T concentrations in TRL were analyzed and composite areas under the curve (AUC) were calculated. α‐tocopherol AUC0–10h in TRL was higher for HE vs. LE and CON (981 ± 151a vs. 311 ± 151b vs. 117 ± 151b nmol • L−1 • 10h, p < 0.05) and γ‐T AUC0–10h in TRL was also higher for HE vs. CON (402 ± 50a vs. 226 ± 50ab vs. 72 ± 50b nmol • L−1 • 10h, p < 0.05). These findings support that consuming cooked whole eggs is an effective way to increase the absorption of α‐T and γ‐T from co‐consumed meals which naturally contain vitamin E such as a raw mixed‐vegetable salad. Thus, co‐consuming cooked whole eggs with a vegetable salad improves the nutritional value of the vegetables, which are under‐consumed by the majority of the US population.

Support or Funding Information

American Egg Board‐Egg Nutrition Center; NIH UL1TR001108; Purdue Ingestive Behavior Research Center
Impact of Microvesicles Pro‐Inflammatory Properties on Sepsis‐Induced Endothelium Dysfunction
Authors: Doursout Marie‐Francoise , Liang Yangyan , Wang Yao‐Wei W. , Devakottai Sundar , Payne JaNae A. , Wade Charles E. , Matijevic Nena
Published: November 2023

This is the first inter‐disciplinary proposal to assess the impact of microvesicles (MVs)‐ pro‐inflammatory properties on sepsis‐induced endothelial dysfunction. Sepsis is a clinical syndrome characterized by a systemic inflammatory response to infection, leading to endothelial dysfunction with increased endothelial permeability, increased levels of nitric oxide (NO), reduced NO availability, enhanced reactive oxygen species (ROS)‐induced oxidative stress and disseminated intravascular coagulation with microvascular thrombosis.

The overall goal of this research proposal was to assess phenotypic and pathophysiological implications of pro‐inflammatory MVs as strong biomarkers of sepsis‐induced vascular dysfunction over a 6 hour time course. We hypothesize that the pro‐inflammatory effects of isolated and characterized MVs from septic rats are tissue and time course‐specific. Ten rats were instrumented under isoflurane anesthesia to implant Tygon catheters into the femoral artery and vein to record mean arterial blood pressure (MAP), heart rate and for drug administration, respectively. Following recovery from surgery, rats were injected with lipopolysaccharide (LPS) at 20 mg/kg IV. Simultaneously to hemodynamic measurements, blood samples were collected from control (n=5) and experimental animals (n=5) into 0.129 mol/L sodium citrate to isolate and characterize leucocytes MVs and platelet MVs. Along with decreases in MAP, preliminary data show that LPS administration induced increases in MV counts (PMVs and LMVs) 4 hrs following LPS administration. LMVs increased from 39/μl to 68/μl and PMVs increased from 145/μl to 631/μl. We are currently exploring endothelial MVs in rat plasma subjected to LPS. This on‐going project is important because it will answer a critical question not explored until now: How the vascular system is affected in sepsis and consequently what will be the best treatment to improve endothelial dysfunction and clinical outcomes.
Beneficial cardiovascular effects of O‐GlcNAc increase in early phase of septic shock
Authors: Ferron Marine , Cadiet Julien , Prat Valentine , Erraud Angélique , Aillerie Virginie , Grabherr Amandine , Mevel Mathieu , Alraj Mohammed , Chatham John , Rozec Bertrand , Gauthier Chantal , Lauzier Benjamin
Published: November 2023

Background

O‐GlcNAcylation, a post‐translational modification, is the end product of the hexosamine biosynthetic pathway (HBP). Recent studies showed that increased concentration of N‐acetylglucosamine has beneficial effects in acute pathologies, such as hemorrhagic shock.

Objectives

We postulated that the increase in O‐GlcNAc at the early phase of septic shock, a systemic inflammation associated with a cardiovascular dysfunction, could improve the cardiovascular function.

Methods

Endotoxemic shock was induced in rats (n=6–9) by iv injection of lipopolysaccharide (LPS, 5 mg/kg) or saline (CTRL). One hour later, fluid resuscitation (FR, 15 ml/kg of colloid, iv) was associated or not with HBP substrate (glucosamine, GlcN, 180 mg/kg, iv) or an O‐GlcNAcase inhibitor (NButGT, 10 mg/kg, iv). Two hours later, echocardiography and arterial blood pressure measures were performed. Then, heart and blood were collected to evaluate biological parameters, inflammation and autophagy.

Results

LPS rats presented a hypotension corrected by FR and O‐GlcNAc increase (mean arterial pressure: CTRL 87±1, LPS 73±5*, LPS‐FR 93±6#, LPS‐FR‐NButGT 83±5, LPS‐FR‐GlcN 92±4mmHg, *: p<0.01 vs CTRL, #: p<0.01 vs LPS). LPS rats also presented a decrease of ejection fraction by 21% and diastolic function (E/A ratio) by 29%. These parameters were normalised by NButGT and tended to be improved by GlcN. In LPS rats, plasmatic troponin T and lactate were significantly increased by 31 and 1.7‐fold respectively, they were normalised under NButGT and GlcN treatments. Interestingly, increase in O‐GlcNAc was not associated with modification of autophagic pathway (Beclin‐1, LC3, Lamp‐2) or inflammation (TNFα, IL‐6).

Conclusions

Our results suggest that the increase in total O‐GlcNAc at the early phase of septic shock could be a suitable therapeutic strategy. However, it remains to be determined which cellular pathways are modulated by the O‐GlcNAc increase.
Scaffold‐dependent stimulation of CaV channels via C‐terminal phosphorylation promotes vasoconstriction in the diabetic vasculature
Authors: Nystoriak Matthew , Nieves‐Cintrón Madeline , Patriarchi Tommaso , Marotti Stefano , Grandi Eleonora , Dos Santos Fernandes Julia , Forbush Katherine , Hofmann Franz , Ward Sean , Scott John , Hell Johannes , Navedo Manuel
Published: November 2023

Dysregulated influx of calcium in arterial myocytes via L‐type CaV1.2 channels (LTCCs) has recently been linked to vascular dysfunction during diabetic hyperglycemia. Functional upregulation of LTCCs under hyperglycemic conditions involves cAMP‐dependent protein kinase (PKA) activity, yet the mechanistic details of how these pathological phenomena promote vasoconstriction is unclear. Here, we report that a subpopulation of CaV1.2 channels is located within nanometer proximity of PKA catalytic subunits at the sarcolemma of native murine and human arterial myocytes. This organization requires selective scaffolding of PKA RIIα by A‐kinase anchoring protein 150 (AKAP150; murine ortholog of human AKAP79). Potentiated LTCC activity in response to elevated D‐glucose was associated with enhanced phosphorylation of CaV1.2 at the putative PKA target site serine 1928 (S1928) in murine and human arterial myocytes, as well as in arteries from mice on a high‐fat diet (HFD) and diabetic human subjects. This modification was absent in myocytes from genetically engineered mice lacking AKAP150 or expressing mutant AKAP150 unable to target PKA. Supporting a functional role for S1928 phosphorylation, hyperglycemia‐ and HFD‐induced LTCC potentiation and vasoconstriction were nearly abolished in myocytes and arteries from mice expressing mutated CaV1.2 in which S1928 was replaced by alanine. These results demonstrate a requisite functional role for CaV1.2 S1928 phosphorylation in stimulation of vascular LTCC activity and vasoconstriction by targeted subsarcolemmal PKA signaling in hyperglycemic conditions and diabetes.

Support or Funding Information

This work was supported by grants from the National Institute of Health R01‐HL098200 and R01‐HL121059 and American Heart Association 14GRNT18730054 (to MFN), American Heart Association 13POST12730001 and the Lawrence J. and Florence A. DeGeorge Charitable Trust (to MAN), American Heart Association 14POST18380011 (to SM), American Heart Association Scientist Development Grant 15SDG24910015 (to EG), National Institute of Health R01‐DK54441 and R01‐DK105542 (to JDS), and National Institutes of Health R01‐MH097887 and R01‐NS078792 (to JWH).
Activation of Toll‐like Receptor 4 (TLR4) by High Mobility Group Box 1 (HMGB1) Leads to Oxidative Stress and Inflammation in Human Aortic Endothelial Cells
Authors: Szasz Theodora , Ogbi Safia , Webb R. Clinton
Published: November 2023

Endothelial dysfunction is a central mechanism in the vascular pathology associated with hypertension, diabetes and atherosclerosis. Key mechanisms mediating endothelial dysfunction, such as reduced NO bioavailability and increased reactive oxygen species (ROS) production are intimately linked to vascular inflammation. Recent evidence supports activation of the innate immune system by endogenous damage‐associated molecular patterns (DAMPs) as a causative factor of the chronic low‐grade sterile inflammation associated with these conditions. High mobility group box 1 (HMGB1) is a chromatin protein and a DAMP which activates Toll‐like receptor 4 (TLR4), leading to increased NFκB signaling, ROS and cytokine production in immune cells. We hypothesized that TLR4 activation by HMGB1 in human aortic endothelial cells (HAEC) leads to activation of oxidative stress and inflammatory pathways and contributes to endothelial dysfunction. Recombinant human HMGB1, time and concentration‐dependently, induced activation of TLR4 in cultured HAEC as evidenced by expression of its downstream partner MyD88. HMGB1 increased ERK phosphorylation and nuclear translocation of NFκB, as well as increased expression of cyclooxygenase (COX‐1 and COX‐2). NO production was decreased in the presence of HMGB1, however no changes were observed in eNOS phosphorylation. HMGB1 also induced an increase in ROS production, increase in the expression of Nox4 subunit of NADPH oxidase and upregulation of the mitochondrial isoform of superoxide dismutase (MnSOD). These effects closely mimicked those induced by the TLR4 agonist lipopolysaccharide (LPS) and were inhibited or reversed by pretreatment of HAEC with the TLR4 antagonist CLI‐095. Our results suggest that HMGB1 may contribute to the endothelial dysfunction associated with diabetes and cardiovascular disease via activation of inflammatory and oxidative stress pathways.

Support or Funding Information

NIDDK
Assessing the Migration and Morphology of Individual Neurons within Orderly Transcription Factor Domains in the Developing Zebrafish Hindbrain
Authors: Monen Joost , Fetcho Joseph
Published: November 2023

Neural networks within the spinal cord and hindbrain control much of the output of the nervous system. The discovery of a transcription factor code that gives rise to the cell types in spinal cord and hindbrain has revealed simple core patterns of organization that are shared by vertebrates. In zebrafish, transcription factor domains give rise to orderly stripes of neurons that extend longitudinally in hindbrain and exhibit a topographic, age related map of recruitment and input resistance. Neurons within these stripes are actively recruited in free swimming of five‐day‐old fish, suggesting that these neurons are wired to perform behaviors prior to the formation of discrete motor nuclei. To assess whether and to what extent neurons that makeup these orderly domains migrate and shift morphologically, we examine both gross morphology in fish with fluorescently labeled transcription factor types (alx [chx10], and dbx1b) and neurotransmitter phenotypes (vglut, glyt2), as well as individually labeled cells over several time points in vivo via multi‐photon microscopy over multiple time points from 3 days post fertilization up to 27 days post fertilization. We show that both the neurotransmitter and transcription factor stripes present in the early developing hindbrain undergo significant migration within the first 2 weeks of development leaving the stripe patterns of neurotransmitter phenotypes virtually undetectable and within transcription factors still apparent, but much less defined. Furthermore, single cells expressing dbx1b tracked over time demonstrate soma migration and while there is no correlation between a cells initial dorsoventral position in a stripe (a crude age indicator) and its final location, there is a correlation between the dorsoventral position of its arbors and the dorsoventral migration of its soma, suggesting that the dendritic and axonal connections established may dictate where a neuron ultimately settles.

Support or Funding Information

J.M. was supported in part by a Ruth L. Kirschstein National Research Service Award from the National Institute for Neurological Disorders and Stroke. This work was supported by an NIH grant to J.F.