Adrenergic Receptor Research Articles

Isoform-level expression of the constitutive androstane receptor (CAR or NR1I3) transcription factor better predicts the mRNA expression of the cytochrome P450s in human liver samples.

Many factors cause inter-person variability in the activity and expression of liver cytochrome P450 (CYP) drug-metabolizing enzymes, leading to variable drug exposure and treatment outcomes. Several liver-enriched transcription factors (TFs) are associated with CYP expression, with estrogen receptor alpha (ESR1) and constitutive androstane receptor (CAR or NR1I3) being the two top factors. ESR1 and NR1I3 undergo extensive alternative splicing that results in numerous splice isoforms, but how these splice isoforms associate with CYP expression is unknown. Here, we quantified 18 NR1I3 splice isoforms and the three most abundant ESR1 isoforms in 260 liver samples derived from African Americans (AA, n=125) and European Americans (EA, n=135). Our results showed variable splice isoform populations in the liver for both NR1I3 and ESR1. Multiple linear regression analyses revealed that, compared to gene-level NR1I3, isoform-level NR1I3 expression better predicted the mRNA expression of most CYPs and three UDP-glucuronosyltransferases (UGTs), while ESR1 isoforms improved predictive models for the UGTs and CYP2D6, but not for most CYPs. Also, different NR1I3 isoforms were associated with different CYPs, and the associations varied depending on sample ancestry. Surprisingly, non-canonical NR1I3 isoforms having retained introns (introns 2 or 6) were abundantly expressed and associated with the expression of most CYPs and UGTs, whereas the reference isoform (NR1I3-205) only associated with CYP2D6. Moreover, NR1I3 isoform diversity increased during the differentiation of induced pluripotent stem cells to hepatocytes, paralleling increasing CYP expression. These results suggest that isoform-level TF expression may help to explain variation in CYP or UGT expression between individuals. Significance Statement We quantified 18 NR1I3 splice isoforms and three ESR1 splice isoforms in 260 liver samples derived from AA and EA donors and found variable NR1I3 and ESR1 splice isoform expression in the liver. Multiple linear regression analysis showed that, compared to gene-level expression, isoform-level expression of NR1I3 and ESR1 better predicted the mRNA expression of some CYPs and UGTs, highlighting the importance of isoform-level analyses to enhance our understanding of gene transcriptional regulatory networks controlling the expression of drug-metabolizing enzymes.

Ligand-induced conformational changes in the β1-adrenergic receptor revealed by hydrogen-deuterium exchange mass spectrometry.

G Protein Coupled Receptors (GPCRs) constitute the largest family of signalling proteins responsible for translating extracellular stimuli into intracellular functions. They play crucial roles in numerous physiological processes and are major targets for drug discovery. Dysregulation of GPCRs is implicated in various diseases, making understanding their structural dynamics critical for therapeutic development. Here, we use Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) to explore the structural dynamics of the turkey β1-adrenergic receptor (tβ1AR) bound with nine different ligands, including agonists, partial agonists, and antagonists. We find that these ligands induce distinct dynamic patterns across the receptor, which can be grouped by compound modality. Notably, full agonist binding destabilises the intracellular loop 1 (ICL1), while antagonist binding stabilises it, highlighting ICL1's role in G protein recruitment. Our findings indicate that the conserved L72 residue in ICL1 is crucial for maintaining receptor structural integrity and stabilising the GDP-bound state. Overall, our results provide a platform for determining drug modality and highlight how HDX-MS can be used to dissect receptor ligand interaction properties and GPCR mechanism.

Targeting Sodium in Heart Failure.

A dominant event determining the course of heart failure (HF) includes the disruption of the delicate sodium (Na+) and water balance leading to (Na+) and water retention and edema formation. Although incomplete decongestion adversely affects outcomes, it is unknown whether interventions directly targeting (Na+), such as strict dietary (Na+) restriction, intravenous hypertonic saline, and diuretics, reverse this effect. As a result, it is imperative to implement (Na+)-targeting interventions in selected HF patients with established congestion on top of quadruple therapy with angiotensin receptor neprilysin inhibitor, β-adrenergic receptor blocker, mineralocorticoid receptor antagonist, and sodium glucose cotransporter 2 inhibitor, which dramatically improves outcomes. The limited effectiveness of (Na+)-targeting treatments may be partly due to the fact that the current metrics of HF severity have a limited capacity of foreseeing and averting episodes of congestion and guiding (Na+)-targeting treatments, which often leads to dysnatremias, adversely affecting outcomes. Recent evidence suggests that spot urinary sodium measurements may be used as a guide to monitor (Na+)-targeting interventions both in chronic and acute HF. Further, the classical (2)-compartment model of (Na+) storage has been displaced by the (3)-compartment model emphasizing the non-osmotic accumulation of (Na+), chiefly in the skin. 23(Na+) magnetic resonance imaging (MRI) enables the accurate and reliable quantification of tissue (Na+). Another promising approach enabling tissue (Na+) monitoring is based on wearable devices employing ion-selective electrodes for electrolyte detection, including (Na+) and (Cl-). Undoubtably, further studies using 23(Na+)-MRI technology and wearable sensors are required to learn more about the clinical significance of tissue (Na+) storage and (Na+)-related mechanisms of morbidity and mortality in HF.

Distinct role of GRK3 in platelet activation by desensitization of G protein-coupled receptors.

Many platelet agonists mediate their cellular effects through G protein-coupled receptors (GPCRs) to induce platelet activation, and GPCR kinases (GRKs) have been demonstrated to have crucial roles in most GPCR functions in other cell types. Here, we investigated the functional role of GRK3 and the molecular basis for the regulation of GPCR desensitization by GRK3 in platelets. We used mice lacking GRK3 as well as β-arrestin2 which has been shown to be important in GPCR function in platelets. Platelet aggregation and dense granule secretion induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in both GRK3 -/- and β-arrestin2 -/- platelets compared to wild-type (WT) platelets, while non-GPCR agonist collagen-induced platelet aggregation and secretion were not affected. We have previously shown that GRK6 is not involved in the regulation of Gq-coupled 5HT2A and Gz-coupled a2A adrenergic receptors. Interestingly, in contrast to GRK6, platelet aggregation induced by co-stimulation of serotonin and epinephrine which activate 5-HT2A and a2A adrenergic receptors, respectively, was significantly potentiated in GRK3 -/- platelets, suggesting that GRK3 was involved in general GPCR regulation. In addition, platelet aggregation in response to the second challenge of ADP was restored in GRK3 -/- platelets whereas re-stimulation of the agonist failed to induce aggregation in WT platelets, confirming that GRK3 contributes to general GPCR desensitization. Finally, 2-MeSADP- and AYPGKF-induced AKT and ERK phosphorylation were significantly potentiated in GRK3 -/- platelets. GRK3 plays a crucial role in the regulation of platelet activation through general GPCR desensitization in platelets.

Catecholamines Attenuate LPS-Induced Inflammation through β2 Adrenergic Receptor Activation- and PKA Phosphorylation-Mediated TLR4 Downregulation in Macrophages.

Inflammation is a tightly regulated process involving immune receptor recognition, immune cell migration, inflammatory mediator secretion, and pathogen elimination, all essential for combating infection and restoring damaged tissue. However, excessive inflammatory responses drive various human diseases. The autonomic nervous system (ANS) is known to regulate inflammatory responses; however, the detailed mechanisms underlying this regulation remain incompletely understood. Herein, we aimed to study the anti-inflammatory effects and mechanism of action of the ANS in RAW264.7 cells. Quantitative PCR and immunoblotting assays were used to assess lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNFα) expression. The anti-inflammatory effects of catecholamines (adrenaline, noradrenaline, and dopamine) and acetylcholine were examined in LPS-treated cells to identify the receptors involved. Catecholamines inhibited LPS-induced TNFα expression by activating the β2 adrenergic receptor (β2-AR). β2-AR activation in turn downregulated the expression of Toll-like receptor 4 (TLR4) by stimulating protein kinase A (PKA) phosphorylation, resulting in the suppression of TNFα levels. Collectively, our findings reveal a novel mechanism underlying the inhibitory effect of catecholamines on LPS-induced inflammatory responses, whereby β2-AR activation and PKA phosphorylation downregulate TLR4 expression in macrophages. These findings could provide valuable insights for the treatment of inflammatory diseases and anti-inflammatory drug development.

FDG Uptake in Brown Adipose Tissue Activated by a Long-Acting β2-Adrenergic Receptor Agonist Inhaler Prescribed for Bronchial Asthma.

We herein report a case of an elderly woman in whom brown adipose tissue (BAT) was visible on 18 F-FDG PET imaging performed in the summer. In this patient, none of the well-known factors that induce BAT activation in adults, including cold exposure, catecholamine-producing tumors, or β3-adrenergic receptor agonists administration, were present. Daily inhalation of a long-acting β2-adrenergic agonist for treatment of her bronchial asthma was considered the cause of her BAT visualization. This case suggests the need to consider β2-adrenergic receptor agonists as a cause of BAT visualization.

New Brønsted–Lowry acid Co(II) complexes: Synthesis, characterization, and investigation of electrochemical application for sensing of salbutamol

In recent years, the employment of transition metal complexes for working electrode surface modification for sensor fabrication has become an area of research direction. Based on this, in this study, three Co(II) complexes: Co(phen)Cl2 (CoC12H8N2Cl2) (C1), and Brønsted–Lowry acids Co(phen)(HTA)Cl (CoC16H13N2O6Cl)(C2), and Co(phen)(HTA)2 (CoC20H18N2O12)(C3) were synthesized and characterized using various techniques such as UV–Vis, FT-IR, ICP OES, TGA, DTA, pXRD as well as electrolytic conductivity, and acid-base property analysis. C and C3 were found polycrystalline whose size falls within the nano range of 50 and 51 nm, respectively. The synthesis was intended to get materials for voltammetrically modifying glassy carbon electrodes for the determination of salbutamol. Salbutamolis a short-acting β2adrenergic receptor agonist that is applied for opening the medium and large airways in the lungs and eventually treats asthma among other problems. The electrochemical application of the synthesized materials was studied by fabricating through electropolymerization of C1, C2, and C3 on glassy carbon electrodes. The poly(C1)/GCE, poly(C2)/GCE, and poly(C3)/GCE) revealed effective surface area is 0.174 cm2, 0.183 cm2, and 0.281 cm2, respectively, and electrochemical catalysis of the materials increased with the number of the tartrate. The Rct values are 5686, 242, 216, and 185 Ω cm2, while the Cdl values are 9.93 × 10−8, 5.35 × 10−6, 1.25 × 10−5, and 2.72 × 10−5, for bare GCE, poly(C1)/GCE, poly(C2)/GCE and poly(C3)/GCE, respectively. Similarly, the rate of charge transfer (k°) increased significantly on the modified electrode compared to the bare GCE. Salbutamol demonstrated a single well-shaped irreversible oxidative peak at the modified electrodes in the ascending order of poly(C1)/GCE, poly(C2)/GCE, and poly(C3)/GCE when compared to a bare GCE.

Diverse pathways in GPCR-mediated activation of Ca2+ mobilization in HEK293 cells

G protein-coupled receptors (GPCRs) transduce extracellular stimuli into intracellular signaling. Ca2+ is a well-known second messenger that can be induced by GPCR activation through the primary canonical pathways involving Gαq- and Gβγ-mediated activation of phospholipase C-β (PLCβ). While some Gs-coupled receptors are shown to trigger Ca2+ mobilization, underlying mechanisms remain elusive. Here we evaluated whether Gs-coupled receptors including the β2-adrenergic receptor (β2AR) and the prostaglandin EP2 and EP4 receptors (EP2R and EP4R) that are endogenously expressed in HEK293 cells utilize common pathways for mediating Ca2+ mobilization. For the β2AR, we found an essential role for Gq in agonist-promoted Ca2+ mobilization while genetic or pharmacological inhibition of Gs or Gi had minimal effect. β-agonist-promoted Ca2+ mobilization was effectively blocked by the Gq-selective inhibitor YM-254890 and was not observed in ΔGαq/11 or ΔPLCβ cells. Bioluminescence resonance energy transfer analysis also suggests agonist-dependent association of the β2AR with Gq. For the EP2R, which couples to Gs, agonist treatment induced Ca2+ mobilization in a pertussis toxin (PTX)-sensitive but YM-254890-insensitive manner. In contrast, EP4R, which couples to Gs and Gi, exhibited Ca2+ mobilization that was sensitive to both PTX and YM-254890. Interestingly, both EP2R and EP4R were largely unable to induce Ca2+ mobilization in ΔGαs or ΔPLCβ cells, supporting a strong dependency on Gs signaling in HEK293 cells. Taken together, we identify differences in the signaling pathways that are utilized to mediate Ca2+ mobilization in HEK293 cells where the β2AR primarily utilizes Gq, EP2R uses Gs and Gi, and EP4R utilizes Gs, Gi and Gq.

Regulation of β-Adrenergic Receptors in the Heart: A Review on Emerging Therapeutic Strategies for Heart Failure.

The prolonged overstimulation of β-adrenergic receptors (β-ARs), a member of the G protein-coupled receptor (GPCR) family, causes abnormalities in the density and functionality of the receptor and contributes to cardiac dysfunctions, leading to the development and progression of heart diseases, especially heart failure (HF). Despite recent advancements in HF therapy, mortality and morbidity rates continue to be high. Treatment with β-AR antagonists (β-blockers) has improved clinical outcomes and reduced overall hospitalization and mortality rates. However, several barriers in the management of HF remain, providing opportunities to develop new strategies that focus on the functions and signal transduction of β-ARs involved in the pathogenesis of HF. As β-AR can signal through multiple pathways influenced by different receptor subtypes, expression levels, and signaling components such as G proteins, G protein-coupled receptor kinases (GRKs), β-arrestins, and downstream effectors, it presents a complex mechanism that could be targeted in HF management. In this narrative review, we focus on the regulation of β-ARs at the receptor, G protein, and effector loci, as well as their signal transductions in the physiology and pathophysiology of the heart. The discovery of potential ligands for β-AR that activate cardioprotective pathways while limiting off-target signaling is promising for the treatment of HF. However, applying findings from preclinical animal models to human patients faces several challenges, including species differences, the genetic variability of β-ARs, and the complexity and heterogeneity of humans. In this review, we also summarize recent updates and future research on the regulation of β-ARs in the molecular basis of HF and highlight potential therapeutic strategies for HF.

Critical Management in a Resource-Limited Setting of Amitraz Overdose Case Report

Background: Amitraz poisoning, though rare, poses significant health risks, particularly in developing regions like Africa where its use as an insecticide and acaricide is widespread. Case Presentation: A 24-year-old female in Southwest Uganda was brought to the Emergency Department after intentional ingestion of Amitraz. Initial assessment revealed somnolence, bradycardia, hypotension, and respiratory distress. Despite decontamination, intravenous fluids, and atropine administration, the patient's condition deteriorated, necessitating intubation and transfer to an ICU. Discussion: Amitraz toxicity manifests primarily through central α2 adrenergic receptor agonism, resembling clonidine overdose, with symptoms including hypotension, bradycardia, altered mental status, and respiratory depression. Differentiating Amitraz poisoning from organophosphate toxicity is crucial due to overlapping clinical features but distinct management strategies. Supportive care, including airway management and cardiovascular support, is essential. Conclusion: This case highlights the critical need for awareness and preparedness for Amitraz poisoning in resource-limited settings. Effective supportive care can lead to successful outcomes despite severe initial presentations. Further education and preventive measures are recommended to mitigate risks, especially among vulnerable populations.

The β2-adrenergic biased agonist nebivolol inhibits the development of Th17 and the response of memory Th17 cells in an NF-κB-dependent manner.

Adrenergic receptors regulate metabolic, cardiovascular, and immunological functions in response to the sympathetic nervous system. The effect of β2-adrenergic receptor (AR) as a high expression receptor on different subpopulations of T cells is complex and varies depending on the type of ligand and context. While traditional β2-AR agonists generally suppress T cells, they potentially enhance IL-17A production by Th17 cells. The effects of pharmacological drugs that count as biased agonists of AR like nebivolol are not completely understood. We investigated the impact of nebivolol on human memory CD4+ T (Th1, Th2, Th17) cells and polarized naive Th17 cells, highlighting its potential for IL-17A suppression via a non-canonical β2-AR cell signaling pathway. The effects of nebivolol were tested on healthy human peripheral blood mononuclear cells, purified memory Th cells, and polarized naive Th17 cells activated with anti-CD3/anti-CD28/anti-CD2 ImmunoCult reagent. IFN-γ, IL-4, and IL-17A, which are primarily derived from Th1, Th2, and Th17 cells, respectively, were quantified by ELISA and flow cytometry. IL-10 was measured by ELISA. Gene expression of RORC, ADRB1, ADRB2, and ADRB3 was evaluated by qPCR. The ADRB2 gene was knocked out in memory Th cells using CRISPR/Cas9. Protein expression of phosphorylated serine133-CREB and phosphorylated NF-κB p65 was assessed by Western blot. Proliferation was assessed by fluorescent dye loading and flow cytometry. Nebivolol treatment decreased IL-17A and IFN-γ secretion by activated memory Th cells and elevated IL-4 levels. Nebivolol reduced the proportion of IL-17A+ Th cells and downregulated RORC expression. Unlike the β2-AR agonist terbutaline, nebivolol inhibited the shift of naive CD4+ T cells toward the Th17 phenotype. IL-10 and the proliferation index remained unchanged. Nebivolol-treated β2-knockout memory Th cells showed significant inhibition of β2-AR-mediated signaling, evidenced by the absence of IL-17A suppression compared to controls. Phosphorylation of the NF-κB p65 subunit was inhibited by nebivolol, but CREB phosphorylation was not changed, suggesting a selective transcriptional control. The findings demonstrate that nebivolol acts through a β2-AR-mediated signaling pathway, as a distinctive anti-inflammatory agent capable of selectively shifting Th17 cells and suppressing the phosphorylation of NF-κB. This highlights nebivolol's potential for therapeutic interventions in chronic autoimmune conditions with elevated IL-17A levels.

Leonurine Inhibits Hepatic Lipid Synthesis to Ameliorate NAFLD via the ADRA1a/AMPK/SCD1 Axis.

Leonurine is a natural product unique to the Lamiaceae plant Leonurus japonicus Houtt., and it has attracted attention due to its anti-oxidative stress, anti-apoptosis, anti-fibrosis, and metabolic regulation properties. Also, it plays an important role in the prevention and treatment of nonalcoholic fatty liver disease (NAFLD) through a variety of biological mechanisms, but its mechanism of action remains to be elucidated. Therefore, this study aims to preliminarily explore the mechanisms of action of leonurine in NAFLD. Mice were randomly divided into four groups: the normal control (NC) group, the Model (M) group, the leonurine treatment (LH) group, and the fenofibrate treatment (FB) group. The NAFLD model was induced by a high-fat high-sugar diet (HFHSD) for 12 weeks, and liver pathological changes and biochemical indices were observed after 12 weeks. Transcriptomic analysis results indicated that leonurine intervention reversed the high-fat high-sugar diet-induced changes in lipid metabolism-related genes such as stearoyl-CoA desaturase 1 (Scd1), Spermine Synthase (Sms), AP-1 Transcription Factor Subunit (Fos), Oxysterol Binding Protein Like 5 (Osbpl5), and FK506 binding protein 5 (Fkbp5) in liver tissues. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis results suggest that leonurine may exert its lipid-lowering effects through the AMP-activated protein kinase (AMPK) signaling pathway. Liver lipidomic analysis showed that leonurine could alter the abundance of lipid molecules related to fatty acyl (FAs) and glycerophospholipids (GPs) such as TxB3, carnitine C12-OH, carnitine C18:1-OH, and LPC (20:3/0:0). Molecular biology experiments and molecular docking techniques verified that leonurine might improve hepatic lipid metabolism through the alpha-1A adrenergic receptor (ADRA1a)/AMPK/SCD1 axis. In summary, the present study explored the mechanism by which leonurine ameliorated NAFLD by inhibiting hepatic lipid synthesis via the ADRA1a/AMPK/SCD1 axis.

The Mechanism Involved in the Inhibition of Resveratrol and Genistein on the Contractility of Isolated Rat Uterus Smooth Muscle

Purpose: This study aimed to compare the effects of the phytoestrogens resveratrol (RES) and genistein (GEN) on the contractility of isolated uterine smooth muscle from rats, focusing on both spontaneous and stimulated contractions, and to investigate the underlying mechanisms. Methods: Uterine strips were suspended vertically in perfusion chambers containing Kreb’s solution, various concentrations of RES and GEN were added to the ex vivo uterine strips, and contractions were measured before and after incubation with RES or GEN. Results: (1) Both RES and GEN inhibited K+-induced contractions in a dose-dependent manner; the β/β2-adrenoceptor antagonist propranolol (PRO), ICI118551, the ATP-dependent K+ channel blocker glibenclamide (HB-419) and the NO synthase inhibitor N-nitro-L-arginine (L-NNA) diminished the inhibitory effects of RES and GEN on K+-induced contractions. (2) RES and GEN also dose-dependently inhibited PGF2α-induced uterine contractions. (3) The inhibitory effects of RES and GEN were observed in spontaneous contractile activities as well; PRO, ICI118551, HB-419 and L-NNA attenuated the inhibitory effects of RES and GEN on the spontaneous contractions of isolated uterine muscle strips. (4) RES and GEN significantly decreased the cumulative concentration response of Ca2+ and shifted the Ca2+ cumulative concentration–response curves to the right in high-K+ Ca2+-free Kreb’s solution. (5) RES and GEN markedly reduced the first phasic contraction induced by oxytocin, acetylcholine, and prostaglandin F2α but did not alter the second phasic contraction caused by CaCl2 in Ca2+-free Kreb’s solution. Conclusions: RES and GEN can directly inhibit both spontaneous and activated contractions of isolated uterine smooth muscle. The mechanisms underlying the inhibitory effects of RES and GEN likely involve β adrenergic receptor activation, reduced Ca2+ influx and release, the activation of ATP-dependent K+ channels and increased NO production.

Left ventricular systolic function after inhalation of beta-2 agonists in healthy athletes

Inhaled beta-2 adrenoceptor agonists (iβ2A) are routinely used as bronchodilators in the treatment of asthma. However, their cardiac effects in athletes are scarcely examined. Thus, the aim of this study was to evaluate the effects of iβ2A on left ventricular (LV) systolic function (SF) by echocardiography in healthy, non-asthmatic female and male endurance athletes. A randomized, double-blinded, placebo-controlled, balanced, 4-way complete block cross-over study was conducted. Twenty-four healthy athletes (12f/12m: 22.9 ± 2.7/24.4 ± 4.6 years) randomly completed 4 study arms (placebo; salbutamol; formoterol; formoterol + salbutamol). After inhalation of the study medication, the participants performed a 10-min time trial (TT) on a bicycle ergometer. After each TT an echocardiography was performed to determine LVSF. Blood samples were collected pre, post, 3 h and 24 h post TT. In females, total serum concentrations for salbutamol and formoterol were higher. LV ejection fraction (LVEF) and LV global longitudinal strain (LVendoGLS) showed a treatment effect for the whole study group (p < 0.0001) and a sex effect on LVEF (p = 0.0085). In women, there was a significant treatment effect for all medication arms (at least p ≤ 0.01) both on LVEF and LVendoGLS. In men only formoterol and formoterol + salbutamol displayed a treatment effect on LVEF (p = 0.0427, p = 0.0330; respectively), whereas on LVendoGLS only formoterol + salbutamol was significant (p = 0.0473). The iβ2A significantly influenced LVSF after an acute bout of exercise in healthy endurance athletes. These effects were even more pronounced when combining both iβ2A that supports a dose-dependent effect on cardiac function. Moreover, female athletes had higher serum concentrations of β2 agonists and stronger effects on LVSF compared to male athletes. This is mainly explained by differences in body weight and related plasma volume and may indicate a potential risk when increasing dose above the tested concentrations. Trial registration: At the European Union Drug Regulating Authorities Clinical Trials (Eudra CT) with the number 201,500,559,819 (registered prospectively on 09/12/2015) and at the German register for clinical studies (DRKS number 00010574 registered retrospectively on 16/11/2021).

Safety and efficacy of up to 60h of iv istaroxime in pre-cardiogenic shock patients: Design of the SEISMiC trial.

Cardiogenic shock (CS) is linked to high morbidity and mortality rates, posing a challenge for clinicians. Interventions to improve tissue perfusion and blood pressure are crucial to prevent further deterioration. Unfortunately, current inotropes, which act through adrenergic receptor stimulation, are associated with malignant arrhythmias and poorer outcomes. Due to its unique mechanism of action, istaroxime should improve haemodynamics without adrenergic overactivation. The SEISMiC study is designed to examine the safety and efficacy (haemodynamic effect) of istaroxime administrated in pre-CS patients. The SEISMiC study is a multinational, multicentre, randomized, double-blind, placebo-controlled safety and efficacy study with two parts (A and B). The study enrols patients hospitalized for decompensated heart failure (pre-CS, not related to myocardial ischaemia) with persistent hypotension [systolic blood pressure (SBP) 70-100mmHg for at least 2h] and clinically confirmed congestion, NT-proBNP ≥1400pg/mL, and LVEF≤40%. Subjects must not have taken intravenous (iv) vasopressors, inotropes or digoxin in the past 6h. Eligible patients are randomized to receive IV infusion of istaroxime (different doses and regimens in Parts A and B) or placebo for up to 60h. Central haemodynamics, ECG Holter monitoring, cardiac ultrasound and biomarkers are recorded at predefined time points during the trial. The study's primary efficacy endpoint is the SBP area under the curve from baseline curve from baseline to 6 and 24h in the combined SEISMiC Parts A and B population. Key secondary efficacy endpoints include haemodynamic, laboratory and clinical measures in SEISMiC B alone in the combined SEISMiC A and B studies. The study results will contribute to our understanding of the role of istaroxime in pre-CS patients and potentially provide insight into the drug's haemodynamic effects and safety in this population.

7690 Acute hormonal signaling-induced 3D genome organization controls transcriptional dynamics during adipocyte thermogenesis

Abstract Disclosure: Y. Zhang: None. R. Zheng: None. T. Tsuji: None. Y. Xing: None. C. Wang: None. J. Darcy: None. K. Chen: None. Y. Tseng: None. Brown adipose tissue (BAT) maintains body temperature by dissipating energy as heat, presenting a promising target against obesity and its associated metabolic disorders. When exposed to cold, the β3-adrenergic receptor (β3-AR) in brown adipocytes is stimulated, initiating a sequential signaling cascade and activating a thermogenic gene program essential for heat generation. Despite remarkable progress has been made towards understanding the engagement of transcriptional and epigenetic regulators during thermogenesis in response to β3-AR stimulation, the precise impact of three-dimensional (3D) genome organization, an increasingly important modulator of gene expression, on the activation of thermogenic gene program remains elusive. In this study, we employed high-resolution Micro-C in murine brown adipocytes to interrogate the rapidity of 3D genome reorganization in response to β3-AR hormonal signaling and its impact on transcriptional dynamics during thermogenesis. Our findings unveiled substantial rewiring of chromatin loops within 4 hours of β3-AR stimulation. These dynamic changes in chromatin loops, particularly enhancer-promoter (E-P) interactions, were coupled with gene activation during thermogenesis. Furthermore, we uncovered the mechanisms by which β3-AR hormonal signaling mediates 3D chromatin reorganization to regulate the thermogenic gene program. We found that β3-AR hormonal signaling induced the phosphorylation of p18Hamlet, a subunit of the SRCAP complex, facilitating the deposition of the histone variant H2A.Z into regulatory DNA regions. This nucleosome incorporation of H2A.Z further mediated nucleosome rearrangement, favoring the formation of nucleosome-depleted regions (NDRs) and enhancing the accessibility of regulatory DNA regions. As a result, H2A.Z chromatin incorporation facilitated interactions between cis-regulatory regions and gene promoters, such as E-P interactions, thereby activating thermogenic gene expression in response to β3-AR stimulation. Loss of H2A.Z disrupted loop dynamics, impeding gene activation and BAT thermogenic capacity. The conserved role of H2A.Z in human thermogenesis was validated, and we identified adiposity-related SNPs in human homologous loop anchors that could potentially be targeted for treating obesity. Taken together, our results unveil a previously underappreciated aspect of acute hormonal signaling-induced chromatin dynamics in orchestrating the thermogenic program in brown adipocytes. These findings not only enhance our understanding of molecular regulation of thermogenesis but also shed light on the role of rapid and dynamic chromatin reconfiguration in regulating the cellular response to external stimuli. Our study bridges a critical gap between 3D genome organization and gene activation in the thermogenic program mediated by acute β3-AR hormonal signaling. Presentation: 6/2/2024

Design, synthesis and biological evaluation of arylpropylamine derivatives as potential multi-target antidepressants

In this study, a series of novel arylpropylamine derivatives were designed, synthesized and evaluated as potential multi-target antidepressants. Among them, compound (R)-13j displayed unique pharmacological features, exhibiting excellent inhibitory potency against serotonin and noradrenaline transporters (SERT/NET) and high affinity for 5-HT2A/2C receptor, and showing low affinity for histamine H1, adrenergic α1 receptors and hERG channels (to reduce QT interval prolongation). Molecular docking studies provided a rational binding model of (R)-13j in complex with SERT and 5-HT2A/2C receptor. In animal models, compound (R)-13j dose-dependently reduced the immobility time in the tail suspension test (TST) and the forced swimming test (FST) in mice, with higher efficacy when compared to duloxetine, and showed no stimulatory effect on the locomotor activity. Moreover, compound (R)-13j significantly shortened the immobility time in the ACTH-induced rat model of treatment-resistant depression (TRD). Furthermore, compound (R)-13j also exhibited a higher threshold for acute toxicity than duloxetine. In addition, compound (R)-13j possessed a favorable pharmacokinetic profile in mice. Taken together, compound (R)-13j may constitute a novel class of drugs for the treatment of depression.

8028 Targeted Therapy-Induced ER And HER2 Chromatin Binding: A Novel Driver Of Poor Response To Treatment In Triple Positive Breast Cancer

Abstract Disclosure: S. Tam: None. M.D. McCoy: None. S. Bahnassy: None. R.B. Riggins: None. Triple positive breast cancer (TPBC) is a unique variant of breast cancer characterized by co-expression of two oncogenic drivers, estrogen receptor alpha (ER) and human epidermal growth factor receptor 2 (HER2). Both drivers promote tumor progression and serve as targets for effective small molecule and monoclonal antibody (mAb) therapies. Previous studies showed that the co-expression of ER reduces the efficacy of anti-HER2 targeted therapy in TPBC, and vice versa. ER is a well-established nuclear receptor and transcription factor. HER2 mainly functions as a transmembrane receptor, activating cytosolic pro-tumorigenic signaling pathways. However, less-studied nuclear HER2 is involved in the transcriptional regulation of several genes, leading to increased BC cell growth and survival. In this study, we investigate the subcellular localization and transcriptional regulatory functions of ER and HER2 in the unique context of TPBC, using cell fractionation, immunofluorescence, chromatin immunoprecipitation (ChIP)-qPCR, and integrated analysis of ER ChIP-seq/RNA-seq data. We hypothesize that crosstalk between ER and HER2 in the nucleus may, in part, underlie poor response to targeted therapy. At baseline, ER has strong nuclear localization in TPBC cells, BT474 and MDA-MB-361, but is predominantly cytosolic in ER+ MCF7 cells. Activated, phospho-HER2 is also localized to the nucleus of TPBC and HER2+ SkBr3 cells at baseline. Treatment with the anti-HER2 mAb combination of trastuzumab and pertuzumab (TP) induces ER binding to specific ER binding sites (ERBSs) in the enhancers or promoters of PGR, TFF1, and FOXC1 in BT474, but this is not observed in MCF7 or SkBr3 cells. Unlike TP, which increases HER2 nuclear localization but not ERBS recruitment, fulvestrant (a selective ER degrader) induces HER2 binding at the same ERBSs in BT474, but not MCF7 or SkBr3 cells. Collectively, these results suggest that the inhibition of either ER or HER2 can promote chromatin binding of the other at ERBSs. Integration of ER ChIP-seq peaks and differentially expressed genes from RNA-seq of BT474 cells treated with E2 vs control following short-term estrogen deprivation shows the ER cistrome is enriched for genes associated with RNA binding, RNA processing, and ribosome biogenesis. Interestingly, nuclear HER2 is reported to increase BC cell growth by activating transcription of ribosomal RNA genes. By contrast, the ER cistrome in MCF7 cells is enriched for genes associated with cell cycle regulation. In summary, anti-HER2 therapy induces recruitment of ER to well-established ERBSs, while anti-ER treatment causes recruitment of HER2 to these same sites in TPBC. Ongoing and future work leverages HER2 cistrome data to further define nuclear HER2/ER functional interactions, at the level of transcriptional regulation, that may contribute to poor treatment outcomes in TPBC. Presentation: 6/2/2024

8028 Targeted Therapy-Induced ER and HER2 Chromatin Binding: a Novel Driver of Poor Response to Treatment in Triple Positive Breast Cancer

Abstract Disclosure: S. Tam: None. M.D. McCoy: None. S. Bahnassy: None. R.B. Riggins: None. Triple positive breast cancer (TPBC) is a unique variant of breast cancer characterized by co-expression of two oncogenic drivers, estrogen receptor alpha (ER) and human epidermal growth factor receptor 2 (HER2). Both drivers promote tumor progression and serve as targets for effective small molecule and monoclonal antibody (mAb) therapies. Previous studies showed that the co-expression of ER reduces the efficacy of anti-HER2 targeted therapy in TPBC, and vice versa. ER is a well-established nuclear receptor and transcription factor. HER2 mainly functions as a transmembrane receptor, activating cytosolic pro-tumorigenic signaling pathways. However, less-studied nuclear HER2 is involved in the transcriptional regulation of several genes, leading to increased BC cell growth and survival. In this study, we investigate the subcellular localization and transcriptional regulatory functions of ER and HER2 in the unique context of TPBC, using cell fractionation, immunofluorescence, chromatin immunoprecipitation (ChIP)-qPCR, and integrated analysis of ER ChIP-seq/RNA-seq data. We hypothesize that crosstalk between ER and HER2 in the nucleus may, in part, underlie poor response to targeted therapy. At baseline, ER has strong nuclear localization in TPBC cells, BT474 and MDA-MB-361, but is predominantly cytosolic in ER+ MCF7 cells. Activated, phospho-HER2 is also localized to the nucleus of TPBC and HER2+ SkBr3 cells at baseline. Treatment with the anti-HER2 mAb combination of trastuzumab and pertuzumab (TP) induces ER binding to specific ER binding sites (ERBSs) in the enhancers or promoters of PGR, TFF1, and FOXC1 in BT474, but this is not observed in MCF7 or SkBr3 cells. Unlike TP, which increases HER2 nuclear localization but not ERBS recruitment, fulvestrant (a selective ER degrader) induces HER2 binding at the same ERBSs in BT474, but not MCF7 or SkBr3 cells. Collectively, these results suggest that the inhibition of either ER or HER2 can promote chromatin binding of the other at ERBSs. Integration of ER ChIP-seq peaks and differentially expressed genes from RNA-seq of BT474 cells treated with E2 vs control following short-term estrogen deprivation shows the ER cistrome is enriched for genes associated with RNA binding, RNA processing, and ribosome biogenesis. Interestingly, nuclear HER2 is reported to increase BC cell growth by activating transcription of ribosomal RNA genes. By contrast, the ER cistrome in MCF7 cells is enriched for genes associated with cell cycle regulation. In summary, anti-HER2 therapy induces recruitment of ER to well-established ERBSs, while anti-ER treatment causes recruitment of HER2 to these same sites in TPBC. Ongoing and future work leverages HER2 cistrome data to further define nuclear HER2/ER functional interactions, at the level of transcriptional regulation, that may contribute to poor treatment outcomes in TPBC. Presentation: 6/2/2024

7951 The Response of HPT Axis &amp; BAT to Cold Exposure Is Delayed in Female Rats Compared to Males

Abstract Disclosure: A. Gutiérrez-Mata: None. L. Jaimes-Hoy: None. I. Sotelo-Rivera: None. F. Romero-Arteaga: None. R.M. Uribe: None. J.L. Charli: None. P. Joseph-Bravo: None. Cold exposure activates the sympathetic nervous system releasing NE in thermogenic brown adipose tissue (BAT) and in parallel, the Hypothalamus-Pituitary-Thyroid (HPT) axis, increasing synthesis and release of hypothalamic-TRH, pituitary-TSH, and thyroid hormones T4 and T3; T4 in concert with NE induce brown adipose tissue (BAT) thermogenesis through stimulation of deiodinase 2 (Dio2), and uncoupling protein 1 (Ucp1) expression. These changes are detected within the first hour in male rats [J Neuroendo 12:861, 2014] while in females, only in ovariectomized [J Neuroendocrinol 21:439, 2009]. Given the thermogenic effect of ovarian hormones, we hypothesized that females could respond to longer exposures. Female Wistar rats were exposed individually to 5°C for 1 to 5h and compared to male’s acute response (15 min to 4h); controls were moved individually to a nearby room at 22°C for similar times. Rats were euthanized by guillotine, rectal temperature was immediately measured, trunk blood collected, serum separated, and tissues kept at -70°C. Hormone concentrations were quantified in serum by RIA and ELISA, and gene expression in paraventricular nucleus (PVN), anterior pituitary (AP), and BAT by RT-PCR or qPCR. Body temperature of cold-exposed males was higher than isolated controls since the first 30min, Trh expression increased at 1h in the paraventricular nucleus (PVN), TSH release from 15 min up to 2 hours, T4 levels peaked until 4h, and T3 increased after 2h as in [Endocrinology 58:140, 1993]; Trhr1 in AP reached its lowest levels at 2h. In BAT, Dio2 expression peaked at 1h and subsequently decreased. Ucp1 increased from 30 min and remained elevated until 4h, Adrb3, and Ppargc1a expressions were highest at 4h. Cold exposure in females decreased body temperature after 4 hours when Trh PVN expression increased and that of TRH receptor 1 (Trhr1) in AP was lowest; TRH degrading enzyme (Trhde) expression peaked after 2h of cold and subsequently decreased at 4h. TSH in serum increased at 2h and then returned to control levels, T4 slightly increased at 3h, and T3 peaked at 5h. Progesterone serum concentration increased from 2h and peaked at 4h. No changes were found in estradiol serum concentration. In BAT, Dio2 expression increased from 2 to 4h, Ucp1 at 3 and 5h while that of β3 adrenergic receptor (Adrb3) decreased at 2h but rose reaching a peak at 4h. In contrast, expression of Pgc1α -the major transcriptional coactivator of Ucp1- rapidly increased (1h) decreasing afterward. The data suggest that when the ambient temperature decreases, the sympathetic system is activated including stimulation of progesterone, and supports a delayed response of female HPT axis to cold exposure, compared to male’s leading to the proposal that when female body temperature diminishes, the HPT axis and thyroid hormone-induced thermogenesis are activated.Funding: DGAPA IN217422, PAEP, CONAHCYT 790605. Presentation: 6/2/2024