Sodium Channel Research Articles

Role of modified calcium montmorillonite and 5 A zeolite in microstructure and efflorescence formation of metakaolin-based geopolymer

To reduce the efflorescence extent of metakaolin (MK)-based geopolymer, this study introduced thermally activated modified calcium montmorillonite (MT) and 5 A zeolite (ZT) into MK-based geopolymer, and the optimal mixing ratio was acquired by response surface methodology. The effect of the two modified clay minerals on the microstructural and mechanical properties of the MT-ZT-MK geopolymer was explored by compressive strength testing, phase analysis (XRD and FTIR), pore structure analysis (MIP), and morphology analysis (SEM-EDS). To investigate the performance and mechanism of efflorescence inhibition of the ternary geopolymer, the cation concentration in the leaching solution was detected by inductively coupled plasma atomic emission spectrometry (ICP-OES), and the efflorescence area of the geopolymer was calculated using Image Pro Plus (IPP) software. The results of the experiment showed that the optimal mixing ratio, as determined by RSM, was 1.5 wt% of modified MT and 2.9 wt% of modified ZT, with the highest compressive strength at 65.1 MPa and the lowest degree of efflorescence after 28 days of curing. It can be concluded that the two clays gradually exhibit the effect of cation exchange and internal curing. The pore structure is optimized and (N, C)-A-S-H gels are formed with the extension of curing time. On the other hand, the lesser extent of efflorescence possibly is attributed to the restricted channels for Na+ in the matrix to undergo carbonation with CO2 in the air.

ELUCIDATING THE DIFFERENTIAL IMPACTS OF EQUIVALENT GATING-CHARGE MUTATIONS IN VOLTAGE-GATED SODIUM CHANNELS.

Voltage-gated sodium (Nav) channels are pivotal for cellular signaling and mutations in Nav channels can lead to excitability disorders in cardiac, muscular, and neural tissues. A major cluster of pathological mutations localizes in the voltage-sensing domains (VSDs), resulting in either gain-of-function (GoF), loss-of-function (LoF) effects, or both. However, the mechanism behind this functional divergence of mutations at equivalent positions remains elusive. Through hotspot analysis, we identified three gating charges (R1, R2, and R3) as major mutational hotspots in VSDs. The same amino-acid substitutions at equivalent gating-charge positions in VSDI and VSDII of the cardiac sodium channel Nav1.5 show differential gating-property impacts in electrophysiology measurements. We conducted 120 μs molecular dynamics (MD) simulations on wild-type and six mutants to elucidate the structural basis of their differential impacts. Our μs-scale MD simulations with applied external electric fields captured VSD state transitions and revealed the differential structural dynamics between equivalent R-to-Q mutants. Notably, we observed transient leaky conformations in some mutants during structural transitions, offering a detailed structural explanation for gating-pore currents. Our salt-bridge network analysis uncovered VSD-specific and state-dependent interactions among gating charges, countercharges, and lipids. This detailed analysis elucidated how mutations disrupt critical electrostatic interactions, thereby altering VSD permeability and modulating gating properties. By demonstrating the crucial importance of considering the specific structural context of each mutation, our study represents a significant leap forward in understanding structure-function relationships in Nav channels. Our work establishes a robust framework for future investigations into the molecular basis of ion channel-related disorders.

A venom peptide-induced NaV channel modulation mechanism involving the interplay between fixed channel charges and ionic gradients

Venoms are used by arthropods either to immobilise prey or as defence against predators. Our study focuses on the venom peptide, Ta3a, from the African ant species, Tetramorium africanum and its effects on voltage-gated sodium (NaV) channels, which are ion channels responsible for the generation of electrical signals in electrically excitable cells, such as neurons. Using the NaV1.7 isoform as our model NaV channel we show that Ta3a prolongs single channel active periods with increased open probability and induces non-inactivating whole-cell currents. Ta3a-affected NaV1.7 channels exhibit a leftward (hyperpolarising) shift in activation threshold, constitutive activity even in the absence of an activating voltage stimulus, and at cell membrane voltages where channels are normally silent. Current-voltage experiments show that Ta3a shifts the voltage at which NaV current changes direction (reversal potential) by altering the local ionic concentration of permeant ions (Na+) rather than changing the channel’s preference for ionic species. We propose a model where Ta3a maintains the positively charged voltage-sensing (S4) domains of the channel in the activated configuration where their electric field is exposed to the extracellular membrane surface to create an ionic bilayer comprising S4 domains and mobile anions (Cl−). This bilayer has a depolarising effect on the cell membrane, thus reducing the amount of externally applied voltage required for channel activation.

Modulating voltage-gated sodium channels to enhance differentiation and sensitize glioblastoma cells to chemotherapy

BackgroundGlioblastoma (GBM) stands as the most prevalent and aggressive form of adult gliomas. Despite the implementation of intensive therapeutic approaches involving surgery, radiation, and chemotherapy, Glioblastoma Stem Cells contribute to tumor recurrence and poor prognosis. The induction of Glioblastoma Stem Cells differentiation by manipulating the transcriptional machinery has emerged as a promising strategy for GBM treatment. Here, we explored an innovative approach by investigating the role of the depolarized resting membrane potential (RMP) observed in patient-derived GBM sphereforming cell (GSCs), which allows them to maintain a stemness profile when they reside in the G0 phase of the cell cycle.MethodsWe conducted molecular biology and electrophysiological experiments, both in vitro and in vivo, to examine the functional expression of the voltage-gated sodium channel (Nav) in GSCs, particularly focusing on its cell cycle-dependent functional expression. Nav activity was pharmacologically manipulated, and its effects on GSCs behavior were assessed by live imaging cell cycle analysis, self-renewal assays, and chemosensitivity assays. Mechanistic insights into the role of Nav in regulating GBM stemness were investigated through pathway analysis in vitro and through tumor proliferation assay in vivo.ResultsWe demonstrated that Nav is functionally expressed by GSCs mainly during the G0 phase of the cell cycle, suggesting its pivotal role in modulating the RMP. The pharmacological blockade of Nav made GBM cells more susceptible to temozolomide (TMZ), a standard drug for this type of tumor, by inducing cell cycle re-entry from G0 phase to G1/S transition. Additionally, inhibition of Nav substantially influenced the self-renewal and multipotency features of GSCs, concomitantly enhancing their degree of differentiation. Finally, our data suggested that Nav positively regulates GBM stemness by depolarizing the RMP and suppressing the ERK signaling pathway. Of note, in vivo proliferation assessment confirmed the increased susceptibility to TMZ following pharmacological blockade of Nav.ConclusionsThis insight positions Nav as a promising prognostic biomarker and therapeutic target for GBM patients, particularly in conjunction with temozolomide treatment.

An artificial intelligence accelerated virtual screening platform for drug discovery

Structure-based virtual screening is a key tool in early drug discovery, with growing interest in the screening of multi-billion chemical compound libraries. However, the success of virtual screening crucially depends on the accuracy of the binding pose and binding affinity predicted by computational docking. Here we develop a highly accurate structure-based virtual screen method, RosettaVS, for predicting docking poses and binding affinities. Our approach outperforms other state-of-the-art methods on a wide range of benchmarks, partially due to our ability to model receptor flexibility. We incorporate this into a new open-source artificial intelligence accelerated virtual screening platform for drug discovery. Using this platform, we screen multi-billion compound libraries against two unrelated targets, a ubiquitin ligase target KLHDC2 and the human voltage-gated sodium channel NaV1.7. For both targets, we discover hit compounds, including seven hits (14% hit rate) to KLHDC2 and four hits (44% hit rate) to NaV1.7, all with single digit micromolar binding affinities. Screening in both cases is completed in less than seven days. Finally, a high resolution X-ray crystallographic structure validates the predicted docking pose for the KLHDC2 ligand complex, demonstrating the effectiveness of our method in lead discovery.

Exploring the interplay between extracellular pH and Dronedarone's pharmacological effects on cardiac function

Dronedarone (DRN) is a clinically used drug to mitigate arrhythmias with multichannel block properties, including the sodium channel Nav1.5. Extracellular acidification is known to change the pharmacological properties of several antiarrhythmic drugs. Here, we explore how modification in extracellular pH (pHe) shapes the pharmacological profile of DRN upon Nav1.5 sodium current (INa) and in the ex vivo heart preparation. Embryonic human kidney cells (HEK293T/17) were used to transiently express the human isoform of Nav1.5 α-subunit. Patch-Clamp technique was employed to study INa. Neurotoxin-II (ATX-II) was used to induce the late sodium current (INaLate). Additionally, ex vivo Wistar male rat preparations in the Langendorff system were utilized to study electrocardiogram (ECG) waves. DRN preferentially binds to the closed state inactivation mode of Nav1.5 at pHe 7.0. The recovery from INa inactivation was delayed in the presence of DRN in both pHe 7.0 and 7.4, and the use-dependent properties were distinct at pHe 7.0 and 7.4. However, the potency of DRN upon the peak INa, the voltage dependence for activation, and the steady-state inactivation curves were not altered in both pHe tested. Also, the pHe did not change the ability of DRN to block INaLate. Lastly, DRN in a concentration and pH dependent manner modulated the QRS complex, QT and RR interval in clinically relevant concentration. Thus, the pharmacological properties of DRN upon Nav1.5 and ex vivo heart preparation partially depend on the pHe. The pHe changed the biological effect of DRN in the heart electrical function in relevant clinical concentration.

Molecular Detection of kdr and superkdr Mutation Sites and Analysis of the Binding Modes of Pyrethroid Insecticides with Voltage-Gated Sodium Channels in the Plant Bug Lygus pratensis (Hemiptera: Miridae).

This study identified genetic mutations linked to resistance to pyrethroid insecticides in the plant pest Lygus pratensis. The voltage-gated sodium channel (VGSC) gene was cloned, revealing two mutations (Met918Thr and Leu1014Phe) in laboratory strains and field populations from Inner Mongolia, resulting in variable pyrethroid resistance. A 3D model of LpVGSC was created using homology modeling, and pyrethroid binding patterns were analyzed via molecular docking. Molecular dynamics simulations confirmed structural stability changes and binding stability of pyrethroids to VGSC sites. Mutation frequencies of homozygous and heterozygous genotypes did not exceed 40 and 20%, respectively. Toxicity tests showed high resistance to λ-cyhalothrin (LC50:401.31 ng/cm2). The kdr (L1014F) and superkdr (M918T) mutations weakened interaction forces, reducing pyrethroid binding. M918T and L1014F mutations are predicted to reduce Type I pyrethroid affinity, suggesting Type II pyrethroids may be more effective against resistant strains. These findings aid in resistance management and insecticide design.

Comparative genomics uncovers the evolutionary dynamics of detoxification and insecticide target genes across 11 phlebotomine sand flies.

Sand flies infect more than one million people annually with Leishmania parasites and other bacterial and viral pathogens. Progress in understanding sand fly adaptations to xenobiotics has been hampered by the limited availability of genomic resources. To address this gap, we sequenced, assembled and annotated the transcriptomes of 11 phlebotomine sand fly species. Subsequently, we leveraged these genomic resources to generate novel evolutionary insights pertaining to their adaptations to xenobiotics, including those contributing to insecticide resistance. Specifically, we annotated over 2,700 sand fly detoxification genes and conducted large-scale phylogenetic comparisons to uncover the evolutionary dynamics of the five major detoxification gene families: Cytochrome P450s (CYPs), Glutathione-S-Transferases (GSTs), UDP-Glycosyltransferases (UGTs), Carboxyl/Cholinesterases (CCEs) and ATP-Binding Cassette (ABC) transporters. Using this comparative approach we show that sand flies have evolved diverse CYP and GST gene repertoires, with notable lineage-specific expansions in gene groups evolutionarily related to known xenobiotic metabolizers. Furthermore, we show that sand flies have conserved orthologs of a) CYP4G genes involved in cuticular hydrocarbon biosynthesis, b) ABCB genes involved in xenobiotic toxicity and c) two primary insecticide targets, acetylcholinesterase-1 (Ace1) and Voltage Gated Sodium Channel (VGSC). The biological insights and genomic resources produced in this study provide a foundation for generating and testing hypotheses regarding the molecular mechanisms underlying sand fly adaptations to xenobiotics.

Acid-sensing ion channel 3 is a new potential therapeutic target for the control of glioblastoma cancer stem cells growth

Glioblastoma (GBM) is the most common malignant primary brain cancer that, despite recent advances in the understanding of its pathogenesis, remains incurable. GBM contains a subpopulation of cells with stem cell-like properties called cancer stem cells (CSCs). Several studies have demonstrated that CSCs are resistant to conventional chemotherapy and radiation thus representing important targets for novel anti-cancer therapies. Proton sensing receptors expressed by CSCs could represent important factors involved in the adaptation of tumours to the extracellular environment. Accordingly, the expression of acid-sensing ion channels (ASICs), proton-gated sodium channels mainly expressed in the neurons of peripheral (PNS) and central nervous system (CNS), has been demonstrated in several tumours and linked to an increase in cell migration and proliferation. In this paper we report that the ASIC3 isoform, usually absent in the CNS and present in the PNS, is enriched in human GBM CSCs while poorly expressed in the healthy human brain. We propose here a novel therapeutic strategy based on the pharmacological activation of ASIC3, which induces a significant GBM CSCs damage while being non-toxic for neurons. This approach might offer a promising and appealing new translational pathway for the treatment of glioblastoma.

Role of protein domains in trafficking and localization of the voltage-gated sodium channel β2 subunit

The voltage-gated sodium (NaV) channel is critical for cardiomyocyte function since it is responsible for action potential initiation and its propagation throughout the cell. It consists of a protein complex made of a pore forming α subunit and associated β subunits, which regulate α subunit function and subcellular localization. We previously showed the implication of N-linked glycosylation and S-acylation of β2 in its polarized trafficking. Here, we present evidence of β2 dimerization. Moreover, we demonstrate the implication of the cytoplasmic tail, extracellular loop, and transmembrane domain on proper β2 folding and export to the cell surface of polarized Madin-Darby canine kidney cells. Substantial alteration, or lack of any of these domains, leads to accumulation of β2 in the endoplasmic reticulum, along with impaired complex N-glycosylation, which is needed for its efficient surface delivery. We also show that these alterations to β2 affected to certain extent NaV1.5 surface localization. Conversely, however, NaV1.5 had little or no influence on β2 trafficking, its localization to the surface, or homodimer formation. Altogether, our data link the architecture of the β2 domains to the establishment of its proper subcellular localization. These findings could provide valuable insights to gain a deeper comprehension of the elusive biology of β subunits in excitable cells, such as neurons and cardiomyocytes.

Voltage-gated sodium channel 1.6 as a novel target for amyotrophic lateral sclerosis treatment

Voltage-gated sodium channel 1.6 as a novel target for amyotrophic lateral sclerosis treatment

Possible mechanism of action potential propagation mediated by static electric field: A novel assumption of understanding nerve interaction and ephaptic coupling

The generation and propagation of physical signals in living biosystems are continuous issues. Traditional Hodgkin-Huxley model based on ionic current conduction could not explain the fast transmission of action potential in myelinated axons and factors influencing action potential velocity. We propose that the ion flow induced by Nav channel generates near field quasi-static electric field at extracellular space, termed as an ephaptic field which is able to excite nearby passive axons. Our simulation indicates that the static electric field produced by sodium ion channels in one node of Ranvier is improbable to stimulate the ion channels in the adjacent neighboring node. However, the ion channel ring in one node of Ranvier could induce the shift of membrane potential (0.01 mV) on the node at nearby axons (100 μm) in a bundle of axon synchronously, suggesting zig-zag propagation of action potential. Together with the superposition effect of ephaptic feedback field generated by the synchronized movement of adjacent parallel axons stimulate the adjacent node of the original axon, strengthen the action potential to travel in a zig-zag pattern. Our model also provides an explanation for the rapid velocity of action potential propagation reported in experimental studies.

Mad honey (wild honey) poisoning: clinical case series from Nepal.

Mad honey is commonly used for hypertension, and coronary artery disease, and as a sexual stimulant. Patients with mad honey poisoning present with dizziness, nausea, syncope, blurred vision, bradycardia, and hypotension with ECG findings of sinus bradycardia, complete AV block, and ST elevation. Here, the authors report five cases admitted to our tertiary care center following the consumption of mad honey. The amount of ingestion of honey varies from 1 to 2 teaspoons (~10-20ml). Most of the cases presented with chief complaints of nausea, dizziness, and vomiting, and all the cases had hypotension and bradycardia. Two cases were admitted to the ward and three of them were admitted to the ICU for further management. They were managed with intravenous fluid, injection atropine along with adjunctive vasopressor and oxygen whenever necessary. Mad honey contains grayanotoxin extracted from the nectar of Rhododendron species. This honey contains grayanotoxin, which binds to sodium channels in its open state causing hyperpolarization of the sodium channel predominantly causing gastrointestinal, neurological, and respiratory symptoms. Intravenous fluids and injection atropine are the mainstays of management in an ICU setup. Some also may require vasopressors. Mad honey poisoning is rare, and limited cases have been reported in Nepal. Physicians should consider mad honey poisoning in cases with ingestion history and clinical symptoms, as it may be a clinical diagnosis due to limited lab tests for grayanotoxin intoxication. Supportive management still forms the cornerstone for its management after diagnosis.

Abstract P351: Obesity in Early Life Leads to Endothelial and Erectile Dysfunction in Young Adult Rats.

Background: Erectile dysfunction (ED) is largely associated with obesity, however, the consequences of early life obesity for ED in young adulthood are unknown. We hypothesize that obesity induced by preweaning overfeeding impairs vascular and erectile function and affects blood pressure in young adult male rats (5-month-old). Methods: At postnatal day (PND) 1, Wistar rats were divided into a normal litter (NL, 5 female + 5 male pups/dam) or small litter (SL, 2 male + 1 female pup/dam) during lactation. After weaning, SL pups had increased body weight. Next, rats from both groups returned to normal size colonies (4-5 rats per cage) and were fed standard chow. At PND160, we evaluated body weight and fat pad deposition, mean arterial pressure (MAP) by tail-cuff, and pudendal artery (PA) and corpus cavernosum (CC) reactivity. Concentration-response curves to phenylephrine (PE) or acetylcholine (ACh) were performed in both preparations. In the CC, contraction and relaxation to electrical field stimulation (EFS) were obtained. Concentration-response curves were analyzed using non-linear regression analysis. RNA sequencing (RNAseq) analyses were performed in both PA and CC. Data were analyzed by Student’s t-test and presented as mean ± S.E.M. Statistical significance was set at p<0.05. Results: At PND160 no differences in body weight were observed in SL (594±14g) compared to NL (559±13g), however, there was an increase in fat deposition in SL (retroperitoneal fat: NL 7.47±0.69g vs SL 11.25±1.23g; perigonadal fat: NL 10.05±0.55g vs SL 13.24±1.16g). MAP was higher in SL (120.5±1.20 mmHg vs 114.9±0.58 NL). In the PA, ACh-induced relaxation was reduced in SL (Emax: 28±6%) vs NL (Emax: 68±5.5%), with no changes in the contraction to PE. In the CC, EFS-induced relaxation was significantly decreased in SL rats compared to NL rats (16 Hz: NL 3.06 ± 0.28% vs SL 2.39 ± 0.53%). RNAseq revealed that, in PA from SL rats, 289 and 224 genes were down and upregulated, respectively, while in the CC from SL rats, 115 genes were downregulated, and 110 genes were upregulated. Among them, hcn2 gene (which encodes HCN2, the hyperpolarization-activated cyclic nucleotide-gated potassium and sodium channel 2) was strongly downregulated in both PA and CC, and this may affect mechanotransduction. Conclusion: Our data shows that early-life obesity causes long-lasting vascular and cavernous damage that might be associated with endothelial and erectile dysfunction in young adulthood.

Bacterial production of ProTx-I, a gating modifier toxin that inhibits voltage-gated sodium (NaV) and TRPA1 channels

Bacterial production of ProTx-I, a gating modifier toxin that inhibits voltage-gated sodium (NaV) and TRPA1 channels

Abstract 37: Targeted Deletion of NKCC1 in Vascular Smooth Muscle Cells Lowers Blood Pressure in Normotensive and Hypertensive Mice

Background: Salt plays a major role in blood pressure homeostasis and the pathogenesis of arterial hypertension. While numerous mechanisms have been described by which Na + can influence arterial blood pressure, the role of Cl - in blood pressure regulation is still largely unexplored. The Na + -K + -2Cl - cotransporter NKCC1 is an important Cl - import pathway in vascular smooth muscle cells (VSMC). To investigate the role of NKCC1 in blood pressure regulation, we generated mice with an inducible VSMC-specific disruption of Slc12a2 . Methods: Mean arterial blood pressure and heart rate were measured by radiotelemetry in unrestrained mice lacking vascular NKCC1 (KO) and wildtype littermates (WT). Cardiac function was assessed by echocardiography. Plasma renin activity and aldosterone concentrations were quantified by radioimmunoassay. Protein expression of renal sodium transporters and channels was analyzed by Western blotting. Results: Mean arterial blood pressure was markedly decreased in Nkcc1 -deficient mice 6 weeks after induction of gene disruption (from 116±2 mm Hg to 103±4 mm Hg, n=18, p<0.001, 2-way ANOVA and Bonferroni post-test), while it did not change in WT control animals (n=17). Heart rate, stroke volume, fractional shortening, and ejection fraction remained unchanged compared to non-induced controls. We did not observe a compensatory increase in plasma renin activity or aldosterone levels, and the blood pressure response to ANG II receptor 1 antagonism by losartan treatment for 7 days was similar in both groups (ΔMAP KO -8.8±3.0 mm Hg, n=8, ΔMAP WT -12.4±1.9 mm Hg, n=7; p>0.05, 2-way ANOVA). In the kidney, the relative expression of total NCC (1.5- fold , n=7-8; p=<0.05, Student’s t-test) and pNCCThr53 (1.9- fold , n=7-8; p=<0.05, Student’s t-test) were modestly increased. The hypotensive effect of Nkcc1 disruption was maintained after induction of hypertension by either infusion of ANG II for 10 days (ΔMAP KO 43.8±6.5 mm Hg, n=6, ΔMAP WT 50.2±7.2 mm Hg, n=4; p>0.05, 2-way ANOVA) or a high salt diet for 10 days (ΔMAP KO 21.7±7.0 mm Hg, n=8, ΔMAP WT 34.0±7.3 mm Hg, n=5; p>0.05, 2-way ANOVA). Conclusions: Our findings indicate that NKCC1 in vascular smooth muscle contributes significantly to the maintenance of arterial blood pressure. Inhibition of NKCC1 in VSMC may provide additional blood pressure reduction in resistant hypertension.

δ-Conotoxin Structure Prediction and Analysis through Large-Scale Comparative and Deep Learning Modeling Approaches.

The δ-conotoxins, a class of peptides produced in the venom of cone snails, are of interest due to their ability to inhibit the inactivation of voltage-gated sodium channels causing paralysis and other neurological responses, but difficulties in their isolation and synthesis have made structural characterization challenging. Taking advantage of recent breakthroughs in computational algorithms for structure prediction that have made modeling especially useful when experimental data is sparse, this work uses both the deep-learning-based algorithm AlphaFold and comparative modeling method RosettaCM to model and analyze 18 previously uncharacterized δ-conotoxins derived from piscivorous, vermivorous, and molluscivorous cone snails. The models provide useful insights into the structural aspects of these peptides and suggest features likely to be significant in influencing their binding and different pharmacological activities against their targets, with implications for drug development. Additionally, the described protocol provides a roadmap for the modeling of similar disulfide-rich peptides by these complementary methods.

Fibroblast Growth Factor (FGF) 13

Fibroblast Growth Factor (FGF) 13, also referred to as FGF homologous factor (FHF) 2, is a member of the FGF11 subfamily that is characterized as having sequence similarities to classical FGF receptor (FGFR)-binding FGFs, but functionally do not bind FGFRs. In this primer mini-review, we summarize current knowledge regarding FGF13 expression, mutant analyses, and gene and protein structure. Similar to other FHFs, FGF13 has been considered a non-secreted protein that lacks an amino signal and is prominently expressed in developing and mature neurons of the central and peripheral nervous systems, as well as the heart. The expression of FGF13 is not limited to early embryonic stages and has been shown to persist in adult tissues. As well, FGF13 is known to localize subcellularly, both within the cytoplasm and the nucleus. FGF13 is extremely adaptable, as it interacts with MAPK scaffolding protein islet brain 2 (IB2), stabilizes microtubules, or binds to voltage-gated sodium channels. Fgf13 mutant mouse lines display various neurological pathologies. Through sequence mapping, FGF13 is considered a candidate causative gene that is mutated in multiple human X-linked neurological diseases.

Abstract P177: High salt modulates Antigen Presenting Cell TNF-alpha production in an Isolevuglandin-dependent manner in Hypertensive Humans

Salt intake is a predisposing factor to high blood pressure in people with salt sensitivity, but the mechanisms are not fully understood. Previously, we found that the epithelial sodium channel facilitates sodium entry into antigen-presenting cells (APCs), leading to the production of Isolevuglandins (IsoLGs) and hypertension via proinflammatory cytokines. TNF-alpha is a major proinflammatory cytokine that is produced in response to oxidative stress. We hypothesize that high salt intake-associated increased production of IsoLGs activates TNF-alpha-dependent mechanisms to contribute to salt-sensitive hypertension. To test this hypothesis, we conducted bulk RNA sequencing on isolated human monocytes with and without high salt. We also performed in vivo studies on humans living with hypertension using the rigorous Weinberger salt loading and depletion protocol at baseline (B), salt loading (SL), and salt depletion (SD) timepoints. We found that in vitro high salt treatment increased human monocyte expression of TNF-α, TNFRSF1A and TNFRSF1B (117.09 ± 23.71 vs 357.82 ± 59.62, p=0.001; 4560 ± 430.02 vs 6189 ± 461.11, p=0.0014 and 43824.73 ± 6462.55 vs 54585.64 ± 5383.58, p=0.0336 respectively, [n=11]) when compared to normal salt. In vivo studies on 16 hypertensive subjects reveal mean blood pressures as baseline (SBP: 137.7 ± 3.1, DBP: 85±2.2, MAP 104.7±2.1 [mmHg]), salt-loading (SBP: 141.3 ± 2.9, DBP: 86.1±2.3, MAP 105.6±2.2 [mmHg]), and salt-depletion (SBP: 138.7 ± 2.9, DBP: 87±2.4, MAP 104.7±2.1 [mmHg]). Using flow cytometry, we determined the buildup of Isolevuglandin adducts in APCs as follows: B: 57.6±6.4, SL: 59.8±4.6, SD: 46.0±7.1 (dendritic cells); B: 78.6±5.8, SL: 77.3±5.7, SD: 61.0±6.9 (CD14 + cells); B: 83.2±4.9, SL: 74.0±6.4, SD: 63.3±8.2 (CD14 + 16 + cells) and B: 63.3± 5.7, SL: 54.2±6.1, SD: 46.8±7.9 (CD16+ cells). In response to salt loading, patient plasma levels of TNF-α were B: 13.8±0.5 pg/mL, SL: 14.1±0.6 pg/mL, and SD: 15.3±1.2 pg/mL respectively. Finally, in vivo single-cell transcriptomic analysis in peripheral blood mononuclear cells revealed that in response to high salt treatment, TNF expression changes dynamically with blood pressure in salt-sensitive, but not salt-resistant patients. These preliminary findings suggest that TNF-α plays a role in salt-sensitive hypertension, possibly following IsoLG-driven reactive oxygen species generation. Further studies are needed to delineate specific dependent mechanisms by which this occurs.

Abstract 49: Activator protein 1 (AP-1) Complex in Antigen Presenting Cells contributes to Salt-Sensitive Blood Pressure in Humans

Salt-sensitivity of blood pressure (SSBP) is an independent risk factor for cardiovascular morbidity and mortality. The exact mechanism by which salt intake increases blood pressure is complex and not fully understood. We previously found that sodium entry into antigen-presenting cells (APCs) via the epithelial sodium channel (ENaC) activates proinflammatory cytokines to activate T cells and modulate salt-sensitive hypertension. The activator protein 1 (AP-1) transcriptional factors (FOS/JUN) have been implicated in activating the pro-inflammatory pathway, but its role in SSBP is unknown. We hypothesized that high salt activates the AP-1 signaling pathway and inflammation in APCs and contributes to SSBP. Our bulk RNA-sequencing data in human monocytes, demonstrated that high salt increases the expression of the AP-1 gene family, FOS (2,378.1 ± 480.7 vs 6,494.0 ± 945.5, p= 0.0009), and JUN (7,313.9 ± 984.9 vs11,370.0 ± 1,286.3, p= 0.0015) compared to normal salt-treated monocytes. In additional experiments, we enrolled patients with hypertension and phenotype them for SSBP using an established inpatient protocol of salt-loading/depletion and performed single-cell transcriptomic analyses on peripheral blood mononuclear cells (PBMCs). We observed expression of the e FOS (r= 0.5041, p= 0.0464) and JUN (r= 0.7083, p= 0.0021) genes changes in concert with blood pressure in salt-sensitive (SS) but not in salt-resistant (SR) patients. To confirm whether genes expression translates to protein expression, we cultured total splenocytes from salt-sensitive SV/129 mice for 24 hours in either normal (150 mM) or high salt media (190 mM) and performed flow cytometry. Compared to normal salt, high salt-induced a significant increase in the expression of FOS-JUN genes in monocytes of the spleen. We also adoptively transferred PBMCs from salt-sensitive (SS) and salt-resistant (SR) hypertensive individuals into immunodeficient NSG mice, treated with the high salt diet for three weeks and performed flow cytometry on immune cells of the kidney, aorta, and spleen. We found that immune cells from SS people demonstrated a higher propensity to infiltrate mouse tissues with increased expression of FOS-JUN genes than immune cells from SR people. These preliminary findings disclose the role of the AP-1 gene family in salt-sensitive hypertension and may provide a potential therapeutic target for the treatment and diagnosis of SSBP.