Adrenergic Receptor Research Articles

Formoterol Reduces the Pro-Inflammatory Phenotype by Enhancing the Activity of Glutaminase in Monocyte-Derived Macrophages in the CVB3-Induced Viral Myocarditis.

Viral myocarditis (VMC) plays a significant role in heart failure, and there is currently a shortage of available targeted treatments. Macrophage phenotype and function are closely associated with the beta-2 adrenergic receptor (β2-AR). This research employed a BALB/c mouse model of VMC generated using Coxsackievirus B3 (CVB3), and the β2-AR agonist formoterol was administered as treatment. A bioinformatic analysis was conducted to identify the β2-AR in CCR2+MHCIIhigh monocyte-derived macrophages (MoMFs). Echocardiography and histopathological assessments were utilized to evaluate cardiac function and inflammation. The enzymatic activity of glutaminase (GLS) was quantified. Flow cytometry was employed to characterize the phenotype and function of the macrophages. Our study revealed that formoterol treatment effectively mitigated cardiac inflammation and fibrosis, improved cardiac function, and prolonged survival compared to the VMC group. Formoterol reduced the infiltration of CCR2+MHCIIhigh MoMFs in the heart, inhibited M1 phenotypic expression and activity, and reduced the percentage of Ly6Chigh monocytes in circulation. Additionally, formoterol stimulated M2 phenotypic expression and activity and increased the percentage of Ly6Clow monocytes in circulation. Additionally, the combination of NICB3344, a C-C motif chemokine receptor 2 inhibitor, with formoterol did not exhibit synergistic effects on reducing cardiac pathological scores or enhancing cardiac function. In vitro studies involving the use of lipopolysaccharide (LPS)-induced bone marrow-derived macrophages, revealed the ability of formoterol to suppress the M1 phenotype and functions induced by LPS while promoting the M2 phenotype and functions. Nevertheless, the observed effects were negated by the introduction of the GLS inhibitor BPTES. Formoterol potentially serves as a significant metabolic regulator in the differentiation process of cardiac MoMFs, influencing this process by controlling GLS activity. Targeting β2-AR exhibits potential as an effective approach for managing VMC. It is essential to acknowledge that these findings were derived under specific experimental conditions, with the current conclusions predominantly based on animal models. Future research is necessary to further investigate the feasibility of formoterol in clinical practice.

Perioperative pain management in dogs and cats: Attitudes and practices among Thai veterinarians

ObjectiveTo assess attitudes of Thai veterinarians towards perioperative pain management in dogs and cats, and explore associations between demographic characteristics and use of analgesics and pain assessment. Study designPaper-based survey distributed in person during various small animal practitioner conferences in 2022. MethodsThe questionnaire encompassed six sections: demographic information, use of analgesic techniques, postoperative pain evaluation, pain indicators, general opinions and confidence in managing postoperative pain in dogs and cats. Chi-square test, logistic regression model and Mann–Whitney U or Kruskal–Wallis tests were used for data analysis; p < 0.05 was considered significant. ResultsA total of 390 completed questionnaires were collected, with one discarded owing to potential erroneous responses. The most widely used analgesics were alpha-2 adrenergic receptor agonists (84%), postoperative non-steroidal anti-inflammatory drugs (NSAIDs; 83.5%) and preoperative opioids (74.3%). Carprofen (87.3%) and tolfenamic acid (80.9%) were the most frequently administered NSAIDs, while tramadol (86%) and morphine (71%) were the most widely used opioids. Preoperative and postoperative opioid administration were less common among males than females, with odds ratios (ORs) of 0.53 and 0.56, respectively (both p = 0.009). Veterinarians who graduated after 2014 were less likely to prescribe preoperative NSAIDs than those who graduated before 2009 (OR = 0.25; p < 0.001). Epidural anesthesia, nerve blocks and pre- and postincisional blocks were more frequently used by referral center veterinarians than those working in non-referral clinics (ORs = 30.5, 14.7, 4.29 and 8.85, respectively; all p < 0.001) and by veterinarians holding advanced degrees beyond the Doctor of Veterinary Medicine (DVM) compared with those with a DVM degree (OR = 6.25, 4.83, 2.25 and 5.75, respectively; all p < 0.001). Conclusions and clinical relevanceThis study reveals variations in perioperative pain management practices in dogs and cats among veterinarians, influenced by sex, graduation year, education and workplace.

Antihypertensive Activity Profile of Bangkirai Leaves (Shorea laevis Ridl.) Ethanol Extract on Angiotensin II (4ZUD) and β-Adrenergic (6PS5) Receptors

Hypertensive is a cardiovascular system disorder characterized by systolic pressure ≥ 140mmHg and diastolic ≥ 90mmHg. The increase in blod pressure is due to the mechanism of angiotensin II and β-adrenergic receptors, so in developing medicine for hypertension, compounds are needed to inhibit the activation of angiotensin II to angiotensin II, as well as β-adrenergic. Kalimantan, Indonesia has a biodiversity that has the potential as medicine such as Bangkirai (Shorea laevis Ridl.). Based on previous research on secondary metabolites, various compounds have been identified that have potential as new drug candidates, for example as antihypertensive. This study aims to determine the pharmacological profile and mechanism of angiotensin I inhibition at the 4ZUD and β-adrenergic receptors inhibition at the 6PS5 receptor, from ethanol extract of bangkirai leaves by carrying out molecular docking which begins with several stages including preparation and optimization of the structure of the test compound and structure preparation of 3D receptors 4ZUD and 6PS5. To find out the correctness of the method, validation was carried out on the Olmesartan ligand compound for 4ZUD and the propranolol ligand for 6PS5. Based on research carried out on both receptors, results were obtained for the test compounds in the form of MolDockScore, the pharmacological profile of absorption, distribution, metabolism, excretion, and toxicity. MolDockScore results show that the compound Colchicine, N-desacetyl-N-[4-hydroxy-3,5-dimethoxycinnamoyl] has the lowest values, namely -146.503 at the 4ZUD receptor dan -129.718 at the 6PS5 receptor, which is close to the natural ligand value compared to other metabolite compounds. Apart from that, it also provides good results based on the pharmacological profile of the 4ZUD and 6PS5 receptors including HIA (95.73%), Caco2 (35.14nm/second), and PPB (87.67%). Negative test results are also shown in the mutagenic toxicity profile (Ames Test Method), non-mutagenic, and carcinogenic tests which include genotoxic and nongenotoxic.

Allosteric Inhibition of the β2-Adrenergic Receptor: Design and Synthesis of Cmpd-15 Analogs.

We designed and synthesized 27 new amide and dipeptide derivatives containing a substituted phenylalanine as negative allosteric modulators (NAMs) for the beta-2 adrenergic receptor (β2AR). These analogs aimed to improve the activity of our lead compound, Cmpd-15, by introducing variations in three key regions: the meta-bromobenzyl methylbenzamide (S1), para-formamidophenylalanine (S2), and 1-cyclohexyl-1-phenylacetyl (S3) groups. The synthesis involved the Pd-catalyzed β-C(sp3)-H arylation of N-acetylglycine with 1-iodo-4-substituent-benzenes as the key step. GloSensor cAMP accumulation assay revealed that six analogs (A1, C5, C6, C13, C15 and C17) surpass Cmpd-15 in β2AR allosteric function. This highlights the crucial role of the S1 region (meta-bromobenzyl methylbenzamide) in β2AR allostery while suggesting potential replaceability of the S2 region (para-formamidophenylalanine). These findings serve as a valuable springboard for further optimizing Cmpd-15, potentially leading to smaller, more active, and more stable β2AR-targeting NAMs.

The extremely low-frequency electromagnetic field (50 Hz) can establish a new “set-point” for the activity of the locus coeruleus–noradrenergic (LC-NA) system in rat

Exposure of organisms to extremely low-frequency electromagnetic field (ELF-EMF; 50 Hz) has been increasing in recent decades, which is connected with dynamic technological development. ELF-EMF is considered a stress factor and its effects on organisms are still being investigated. We aimed to determine its impact on the locus coeruleus–noradrenergic (LC-NA) system enabling adaptation to stressful conditions. For this purpose, we exposed rats to 50 Hz ELF-EMF of 1 and 7 mT, 1 h/day for 7 days. The procedure was repeated three times to examine the organism's adaptive capabilities. Subsequently, the concentration of adrenaline, noradrenaline and its metabolite MHPG as well as the expression of the β2-adrenergic receptor was assessed. After the end of each exposure, part of the animals were subjected to a behavioural test to assess the influence of repeated ELF-EMF exposure on stress response to subsequent stress factors. Our research proved that mechanisms underlying the effects of ELF-EMF on stress response include the LC-NA system. ELF-EMF of 1 mT induced adaptive changes in the NA-LC system. However, exposure to 7 mT caused increased activity of the stress system which resulted in sensitization to subsequent, heterotypic (different from the one previously acting) stress factor. As ELF-EMF of 7 mT caused a profound decrease in β2-AR level would strongly inhibit the potential for neuroplastic processes in the hippocampus. Moreover, rats exposed to ELF-EMF of 7 mT showed moderately increased anxiety-related behaviour. Disturbances in NA-LC transmission may underlie the development of some neurodegenerative and psychiatric diseases which indicates the possible involvement of ELF-EMF in the pathogenesis of these disorders.

Preclinical in vitro and in vivo evaluation of [11C]ORM-13070 as PET ligand for alpha-2C adrenergic receptor occupancy using PET imaging in non-human primates.

This paper describes the preclinical validation of the radioligand [11C]ORM-13070 and its tritiated analog for addressing selectivity and occupancy of the selective alpha-2C adrenergic receptor (α2CR) antagonist BAY 292 in the cynomolgus brain. BAY 292 is a novel drug candidate being developed for the treatment of obstructive sleep apnea (OSA) via binding to central α2CR. In vitro autoradiography studies with sections from non-diseased post-mortem human caudate revealed an excellent specific binding window (>80%) using [3H]ORM-13070. BAY 292 bound to the same binding site as [3H]ORM-13070 and generated a good specific binding signal, with greater selectivity for α2CR. In non-human primates in vivo, [11C]ORM-13070 demonstrated a reversible behavior, with uptake at baseline highest in striatum (putamen, caudate, ventral striatum, and pallidum) and low in the cerebellar cortex, consistent with the known distribution of the α2CR. A dose dependent increase in receptor occupancy after BAY 292 administration was observed, confirming BBB penetration and target engagement. The estimated EC50 for BAY 292 is 33.39 ± 11.91 ng/mL. This study aimed to demonstrate the suitability of [11C]ORM-13070 as a PET-radioligand for the study of α2CR in the non-human primate brain, and to pave the way for future clinical PET tracer studies with BAY 292.

Impact of noradrenergic inhibition on neuroinflammation and pathophysiology in mouse models of Alzheimer's disease.

Norepinephrine (NE) modulates cognitive function, arousal, attention, and responses to novelty and stress, and also regulates neuroinflammation. We previously demonstrated behavioral and immunomodulatory effects of beta-adrenergic pharmacology in mouse models of Alzheimer's disease (AD). The current studies were designed to block noradrenergic signaling in 5XFAD mice through 1 ) chemogenetic inhibition of the locus coeruleus (LC), 2) pharmacologic blocking of β-adrenergic receptors, and 3) conditional deletion of β1- or β2-adrenergic receptors (adrb1 or adrb2) in microglia. First, brain-wide AD pathology was mapped in 3D by imaging immunolabeled, cleared 5XFAD brains to assess the overlap between Aβ pathology, reactive microglia, and the loss of tyrosine hydroxylase (TH) expression in the catecholaminergic system. To examine the effects of inhibiting the LC NE system in the 5XFAD model, inhibitory (Gi) DREADD receptors were expressed specifically in LC NE neurons. LC NE neurons were chronically inhibited through the subcutaneous pump administration of the DREADD agonist clozapine-N-oxide (CNO). Plasma and brains were collected for assessment of neuroinflammation and pathology. A separate cohort of 5XFAD mice was chronically dosed with the beta-adrenergic antagonist propranolol or vehicle and evaluated for behavior, as well as post-mortem neuroinflammation and pathology. Finally, we used 5XFAD mice with conditional deletion of either adrb1 or adrb2 in microglia to assess neuroinflammation and pathology mediated by β-adrenergic signaling. Using iDISCO, light sheet fluorescence microscopy, and novel analyses, we detected widespread microgliosis and amyloid pathology, along with modest TH downregulation in fibers across multiple brain regions, in contrast to the spatially limited TH downregulation observed in neurons. Both chemogenetic inhibition of LC adrenergic signaling and pharmacological inhibition of beta-adrenergic receptors potentiated neuroinflammation without altering amyloid beta pathology. Conditional deletion of adrb1 in microglia did not affect neuroinflammation. Conditional deletion of adrb2 in microglia attenuated inflammation and pathology in females but had no effect in males. Overall, these data support previous observations demonstrating the immunomodulatory effects of beta-adrenergic signaling in the pathophysiology of brain disorders and suggest that adrenergic receptors on cell types other than microglia, such as astrocytes, may predominantly mediate the disease-modifying effects of β-adrenergic agonists in the brain.

Mapping of β3-Adrenergic Receptor in Living Cells with a Ligand-Guided Fluorescent Probe.

Tracking the dynamic distribution of native proteins in living cells or tissues in real time is essential for understanding the functional mechanisms involved in their physiological or pathological processes. The β3-adrenergic receptor (β3-AR) has important biological functions. It is expected to be a diagnostic indicator for aging, yet current probes for β-ARs are unable to dynamically monitor β3-AR in real time, which impedes the progress of β3-AR research. Here, we developed a ligand-directed anchoring probe, β3-ARP, that precisely covalently anchors with native β3-AR in living cells, for the diagnosis of senescence. The ligand-directed anchoring probe selectively recognizes and traces β3-AR and can achieve dynamic observation and monitoring of β3-AR in living cells, including the interaction between lipid droplets and mitochondria. Moreover, we were able to directly probe the distribution of β3-AR in various organs of aging mice in situ and track the location of native β3-AR in different types of primary neuronal cells using β3-ARP and two-photon imaging, which revealed the aberrant accumulation of lipid droplets and the distribution of β3-AR in neurological diseases. This research has resulted in a new covalently anchoring fluorescent probe, β3-ARP, which could serve as a powerful tool to explore the link between β3-AR and age-related diseases.

Hypoxia-inducible factor-1α-deficient adipose-tissue macrophages produce the heat to mediate lipolysis of white adipose tissue through uncoupling protein-1

BackgroundUncoupling protein 1 (UCP1) is a proton uncoupler located across the mitochondrial membrane generally involved in thermogenesis of brown adipose tissues. Although UCP1 is known to be strongly expressed in brown adipocytes, recent evidence suggest that white adipocytes can also express UCP1 under certain circumstances such as cold- or β-adrenergic receptor-stimulation, allowing them to acquire brown adipocyte-like features thereby becoming 'beige’ adipocytes.ResultsIn this study, we report that UCP1 can be expressed in adipose-tissue macrophages (ATM) lacking functional hypoxia-inducible factor-1 (HIF-1) and this does not require cold- nor β-adrenergic receptor activation. By using myeloid-specific Hif-1α knockout (KO) mice, we observed that these mice were protected from diet-induced obesity and exhibited an improved thermogenic tolerance upon cold challenge. ATM isolated from white adipose tissues (WAT) of these mice fed with high fat diet exhibited significantly higher M2-polarization, decreased glycolysis, increased mitochondrial functions and acetyl-CoA levels, along with increased expression of Ucp1, peroxisome proliferator activated receptor-gamma co-activator-1a, and others involved in histone acetylation. Consistent with the increased Ucp1 gene expression, these ATM produced a significant amount of heat mediating lipolysis of co-cultured adipocytes liberating free fatty acid. Treating ATM with acetate, a substrate for acetyl-CoA synthesis was able to boost the heat production in wild-type or Hif-1α-deficient but not UCP1-deficient macrophages, indicating that UCP1 was necessary for the heat production in macrophages. Lastly, we observed a significant inverse correlation between the number of UCP1-expressing ATM in WAT and the body mass index of human individuals.ConclusionsUCP1-expressing ATM produce the heat to mediate lipolysis of adipocytes, indicating that this can be a novel strategy to treat and prevent diet-induced obesity.

Nephro- and Cardiotoxic Effects of Etoricoxib: Insights into Arachidonic Acid Metabolism and Beta-Adrenergic Receptor Expression in Experimental Mice.

Background: Etoricoxib is a widely used anti-inflammatory drug, but its safety profile concerning cardiovascular and renal health remains inadequately explored. This study aimed to assess the nephro- and cardiotoxic effects of etoricoxib in a murine model, with a focus on its impact on arachidonic acid-metabolizing enzymes and beta-adrenergic receptors associated with drug-induced toxicity. Methods: Thirty-five BALB/C mice were randomly assigned to five groups: control, low-dose etoricoxib, high-dose etoricoxib, low-dose celecoxib, and high-dose celecoxib (a well-known nephro- and cardiotoxic NSAID). The treatments were administered for 28 days, after which hearts and kidneys were excised for physical and histopathological analysis, and the expression of arachidonic acid-metabolizing enzymes (cytochrome P450s, lipoxygenases, cyclooxygenases) and beta-1 adrenergic receptor (adrb1) and angiotensin-converting enzyme (ace2) genes were quantified using quantitative reverse transcription PCR (qRT-PCR). Results: Etoricoxib administration resulted in dose-dependent nephro- and cardiotoxic effects. Renal histology revealed glomerular atrophy or hypertrophy and significant damage to the proximal and distal convoluted tubules, including epithelial flattening, cytoplasmic vacuolation, and luminal widening. Cardiac analysis showed disorganized muscle fibers and hyaline degeneration. These changes were associated with altered gene expression: the downregulation of cox2, cyp1a1, and cyp2c29 in the kidneys and the upregulation of cyp4a12, cox2, and adrb1, along with the downregulation of cyp2c29 and ace2 in the heart. Conclusions: Etoricoxib induces nephro- and cardiotoxicity, marked by alterations in arachidonic acid metabolism and beta-adrenergic signaling pathways. The drug affects the expression of arachidonic acid-metabolizing enzymes and adrb1 in the heart while downregulating cox2 and other related enzymes in the kidneys. These findings underscore the need for caution when prescribing etoricoxib, particularly in patients with pre-existing renal or cardiac conditions.

Innovative Approach of High-Throughput Screening in the Drug Discovery Quest for Chronic Bronchitis Treatment

Chronic bronchitis (CB) is a challenging condition that worsens over time. Patients continue to experience an abundance of symptoms despite their best attempts to manage them. The etiology of CB is goblet cell overproduction and mucus hypersecretion, which aggravates airflow obstruction by luminal blockage of small airways, epithelial remodeling and modification of airway surface tension that predisposes to collapse. Larger randomized controlled trials are required to validate the pilot data that are currently accessible and assess the treatment’s durability as therapies are still in the early stages of clinical development. To overcome the prevailing increase in CB with the rise in air pollution globally, this study proposes a de novo drug discovery candidate for the effective treatment of CB. With the aid of cutting-edge technology of high-throughput screening, a phytochemical, apigenin, has been administered to target the Beta-2 adrenergic receptor protein and utilized as an exemplary ligand to target the discovery of a suitable drug candidate for Beta-2 adrenergic receptor protein. The drug likeness scores, no evident toxicity and binding affinity –9.092 kcal/mol the “CHEMBL294878” proves to be a potential drug candidate discovered for CB. A therapeutic drug specifically meant to treat CB can be developed with the help of the computational data provided by the research presented in this study.

Beta-Adrenergic Activation of the Inward Rectifier K+ Current Is Mediated by the CaMKII Pathway in Canine Ventricular Cardiomyocytes.

Several ion currents in the mammalian ventricular myocardium are substantially regulated by the sympathetic nervous system via β-adrenergic receptor activation, including the slow delayed rectifier K+ current and the L-type calcium current. This study investigated the downstream mechanisms of β-adrenergic receptor stimulation by isoproterenol (ISO) on the inward rectifier (IK1) and the rapid delayed rectifier (IKr) K+ currents using action potential voltage clamp (APVC) and conventional voltage clamp techniques in isolated canine left ventricular cardiomyocytes. IK1 and IKr were dissected by 50 µM BaCl2 and 1 µM E-4031, respectively. Acute application of 10 nM ISO significantly increased IK1 under the plateau phase of the action potential (0-+20 mV) using APVC, and similar results were obtained with conventional voltage clamp. However, β-adrenergic receptor stimulation did not affect the peak current density flowing during terminal repolarization or the overall IK1 integral. The ISO-induced enhancement of IK1 was blocked by the calcium/calmodulin kinase II (CaMKII) inhibitor KN-93 (1 µM) but not by the protein kinase A inhibitor H-89 (3 µM). Neither KN-93 nor H-89 affected the IK1 density under baseline conditions (in the absence of ISO). In contrast, parameters of the IKr current were not affected by β-adrenergic receptor stimulation with ISO. These findings suggest that sympathetic activation enhances IK1 in canine left ventricular cells through the CaMKII pathway, while IKr remains unaffected under the experimental conditions used.

MTORC1 and 2 adrenergic regulation and function in brown adipose tissue.

Brown adipose tissue (BAT) thermogenesis results from the uncoupling of mitochondrial inner membrane proton gradient mediated by the uncoupling protein 1 (UCP-1), which is activated by lipolysis-derived fatty acids. Norepinephrine (NE) secreted by sympathetic innervation not only activates BAT lipolysis and UCP-1, but uniquely in brown adipocytes, promotes "futile" metabolic cycles and enhances BAT thermogenic capacity by increasing UCP-1 content, mitochondrial biogenesis and brown adipocyte hyperplasia. NE exerts these actions by triggering signaling in the canonical G protein coupled b adrenergic receptors, cAMP and protein kinase A (PKA) pathway which, in brown adipocyte, is under a complex and intricated crosstalk with important growth-promoting signaling pathways such as those of mechanistic target of rapamycin (mTOR) complexes 1 (mTORC1) and 2 (mTORC2). This article reviews evidence suggesting that mTOR complexes are modulated by and participate in the thermogenic, metabolic, and growth-promoting effects elicited by NE in BAT and discusses current gaps and future directions in this field of research.

Synergistic interaction between clonidine and ACPA on the modulation of anxiety-like behaviors in non-acute restraint stress and acute restraint stress conditions

The present research examined the possible role of α-2 adrenergic receptor drugs (clonidine, selective α-2 adrenergic receptor agonist, and yohimbine, competitive α-2 adrenoreceptor antagonist,) on the effect of arachidonylcyclopropylamide (ACPA), a cannabinoid CB1 receptor agonist, in non-acute restraint stress (NARS) and acute restraint stress (ARS) mice. The animals were unilaterally implanted with a cannula in the left lateral ventricle. ARS was carried out by movement restraint at a period of 4 h. An elevated plus-maze (EPM) apparatus was used to evaluate anxiety-like behaviors. The results indicated that induction of ARS for 4 h induced anxiogenic-like behavior due to the reduction of %OAT (the percentage of time spent in the open arms) in male mice. Additionally, ARS caused neuronal degeneration in the prefrontal cortex. On the other hand, alone intracerebroventricularly (i.c.v.) infusions of ACPA (0.5 µg/mouse) and clonidine (0.5 µg/mouse) increased %OAT, indicating an anxiolytic-like response in the NARS and ARS mice. In contrast, alone i.c.v. infusions of yohimbine (0.5 µg/mouse) decreased %OAT and %OAE (the percentage of entries to the open arms), proposing an anxiogenic-like effect in the NARS and ARS mice. When the subthreshold dose of ACPA and different doses of clonidine were co-injected, ACPA potentiated the anxiolytic-like behavior produced by clonidine in the ARS mice. On the other hand, when the ineffective dosage of ACPA and different dosages of yohimbine were co-infused, ACPA reversed the anxiogenic-like effect induced by yohimbine in the NARS and ARS mice. Moreover, the results revealed a synergistic effect between ACPA and clonidine upon induction of anxiolytic-like behaviors. It can be concluded that the interaction between clonidine and ACPA modulates the anxiety-like behaviors induced by stress in male mice.

Transactivation of the EGF receptor as a novel desensitization mechanism for G protein-coupled receptors, illustrated by dopamine D2-like and β2 adrenergic receptors

Transactivation of epidermal growth factor receptors (EGFR) provides intricate control over multiple regulatory cellular processes that merge the diversity of G protein-coupled receptors (GPCRs) with the robust signaling capacities of receptor tyrosine kinases. Contrary to the typical assertions, our findings demonstrate that EGFR transactivation contributes to the desensitization of GPCRs. Repeated agonist stimulation of certain GPCRs enhanced EGFR transactivation, triggering a series of cellular events associated with GPCR desensitization. This effect was observed in receptors undergoing desensitization (D3R, K149C-D2R, β2AR) but not in those resistant to desensitization (D2R, C147K-D3R, D4R, β2AR mutants lacking GRK2 or GRK6 phosphorylation sites). The EGFR inhibitor AG1478 prevented both desensitization and the associated cellular events. Similarly, these cellular events were also observed when cells were treated with EGF, but only in GPCRs that undergo desensitization. These findings suggest that EGFR transactivation diversifies pathways involved in ERK activation through the EGFR signaling system and also mediates GPCR desensitization. Alongside the widely accepted steric hindrance model, these findings offer new insights into understanding the mechanisms of GPCR desensitization, which occurs through complex cellular processes.

Effects of the restrictive cardiomyopathy-associated cardiac troponin I K178E mutation on cAMP signalling at the myofilament

Abstract Background Catecholaminergic stimulation of cardiac β-adrenergic receptors (β-ARs) and subsequent cAMP signalling control cardiac inotropy and lusitropy. cAMP signalling is known to be compartmentalised to optimise sympathetic control by producing heterogeneous cAMP signals and protein kinase A (PKA) activation at distinct subcellular sites. This signalling may be disrupted in diseases such as restrictive cardiomyopathy (RCM) characterised by diastolic dysfunction, which is associated with certain mutations including the K178E missense mutation in cardiac troponin I (TPNI). The impaired ventricular relaxation associated with this mutation is thought to arise from myofilament calcium hypersensitivity, which can be modulated by PKA-dependent TPNI phosphorylation. However, a detailed understanding of the pathological effects mediated by the K178E-TPNI mutation is lacking. Purpose This study aims to determine whether the K178E-TPNI mutation alters local cAMP/PKA signalling at TPNI, potentially affecting myofilament calcium sensitivity and cardiac myocyte relaxation. Methods Real-time imaging of cAMP was carried out using the Fluorescence Resonance Energy Transfer (FRET)-based sensor CUTie (cAMP Universal Tag for imaging experiments) [1] fused to wild type (WT)- or K178E-TPNI. The sensors were expressed by transfection in neonatal rat ventricular myocytes (NRVMs) and FRET-changes were recorded at the myofilament on application of the β-AR stimulant isoproterenol (ISO). We further assessed the involvement of cAMP hydrolysing phosphodiesterases (PDEs) in the regulation of cAMP levels selectively at TPNI. Results The peak FRET-change and FRET-change after 5mins on ISO application were significantly higher in NRVMs expressing the mutant compared to the WT sensor, as was the rate of cAMP accumulation. A strong trend showed that the rise in FRET following PDE4 inhibition was larger in NRVMs expressing the WT compared to the mutant sensor, suggesting the involvement of this isoform in degrading cAMP locally at TPNI and reduced PDE4 activity in the presence of the K178E mutation. Co-immunoprecipitation analysis in HEK293 cells demonstrated interaction between WT-TPNI and a PDE4D isoform, and a weaker interaction of this isoform with the K178E-TPNI mutant. Conclusions This study shows that the K178E-TPNI mutation attenuates the TPNI/PDE4D interaction and alters the magnitude and kinetics of the local cAMP response at the myofilament. This mechanistic insight may aid the development of therapeutics targeting the TPNI/PDE4D interaction to treat RCM-associated diastolic dysfunction.The TPNI-CUTie SensorFRET Response Trace

Temporal dynamics of cardiac remodelling in pressure overload: the key role of visceral adipose tissue

Abstract Background Myocardial fibrosis is a prominent pathological feature of cardiac remodelling in pressure overload. The underlying mechanisms of cardiac remodelling are complex including mechanical, inflammatory and neurohormonal factors. We have recently uncovered a pathological crosstalk between visceral adipose tissue (VAT) and heart in biological aging characterized by profibrotic proteins in both the VAT secretome and the plasma contributing to myocardial interstitial fibrosis. We hypothesize that a model of pressure overload induced by transverse aortic constriction (TAC) is responsible for VAT remodelling and a VAT profibrotic secretome, which in turn worsens cardiac fibrosis and alters cardiac function. Purpose To explore the effect of pressure overload on VAT and identify a crosstalk between heart and VAT. Methods We divided wild-type male mice (5-month-old, n=10/group) into two groups: a visceral Lipectomy group and a Sham group two weeks before a moderate TAC (26G; i.e. gradient between left ventricle and aorta of 30mmHg) or a sham surgery. We sacrificed the mice at 1- and 8-week after TAC. We performed in vivo metabolic tests, echocardiography, and invasive hemodynamics, followed by ex vivo tissue analysis. We then analysed the time-course of the cardiac transcriptomic signature in TAC vs control and the impact of visceral lipectomy in both groups. Results Our results show that TAC induced not only cardiac remodelling affecting the structure and function of the heart, but also VAT fibrosis and inflammation together with activation of adrenergic receptors (Adrb3). VAT became a major source of profibrotic (TGFb and osteopontin) and proinflammatory (TNFa, IL6 and PAI1) factors as early as 1 week after TAC. Interestingly, lipectomy prevented cardiac remodelling and dysfunction. After 1 week of TAC, cardiac transcriptomic profile showed a similar upregulation of profibrotic and prohypertrophic genes in TAC groups in presence or absence of lipectomy. After 8 weeks of TAC, lipectomy prevented the upregulation of those genes and transcriptomic profile of TAC+lipectomy was similar to sham group (Figure). PCR analysis confirmed the lower expression of profibrotic (TGFb, Osteopontin), prohypertrophic (Nppa) and contractility genes (Adr1b) in TAC+Lipectomy vs TAC group after 8 weeks of TAC. Conclusion Our results suggest that VAT is detrimental to cardiac structure and function by releasing a profibrotic and a proinflammatory secretome that aggravates the cardiac remodelling induced by TAC. Conversely, lipectomy prevents the early development of TAC-induced cardiac fibrosis.

Treatment of Post-COVID-19 POTS by inhibiting FcRn: a phase 2 randomized, placebo controlled, double-blind, proof of concept study with efgartigimod

Abstract Background While the underlying pathophysiology is unknown, post–COVID-19 postural orthostatic tachycardia syndrome (POTS) may be related to immune dysfunction and autoimmunity precipitated by SARS-CoV-2 infection. Various autoantibodies, including those targeting G-protein coupled receptors (GPCRs), have been observed in patients with POTS. Binding of these autoantibodies to adrenergic receptors, which are prototypical GPCRs, has been hypothesized to modulate receptor activity, increase sympathetic tone, and cause tachycardia. Moreover, IgG anti-GPCR autoantibodies, acting as partial agonists, are thought to decrease the effect of peripheral vasomotor control, leading to an increased sympathetic response to upright posture that can result in postural tachycardia in the absence of hypotension. Purpose We hypothesize that a reduction in autoantibody levels by efgartigimod, a FcRn-antagonist, will ameliorate the underlying immune-mediated pathogenesis and lead to clinical improvements in patients who developed POTS following COVID-19 infection. We propose a phase II multicenter, randomized, placebo-controlled, double-blind, proof-of-mechanism study initiated to evaluate the safety and efficacy of efgartigimod in adults with post–COVID-19 POTS. Methods Adult patients diagnosed with new-onset of POTS following SARS-CoV-2 infection are eligible for participation in the study. Prior COVID-19 must be confirmed by documentation of historical PCR test, and POTS diagnosis must meet consensus criteria. Study subjects must have moderate to severe autonomic symptoms (COMPASS-31 score ≥ 35 points at screening). Approximately 42 patients will be randomly allocated (2:1) to receive weekly intravenous efgartigimod or placebo for 24 weeks. At the end of the treatment period, participants who complete the study may roll over into an open-label extension (OLE). The co-primary endpoints are change from baseline to week 24 in the COMPASS-31 and Malmo POTS Symptom Score as well as safety and tolerability outcomes. Key secondary endpoints include assessments of disease activity, fatigue, cognitive function, walking capacity and quantitative autonomic testing. Additional exploratory endpoints include intraepidermal small fiber densities (optional), sudomotor innervation (optional) and peripheral blood biomarkers to define histopathological and immune determinants associated with treatment response. Conclusions This phase II study of efgartigimod will evaluate the effect of FcRn inhibition on disease pathology and the potential for therapeutic benefit in patients with post–COVID-19 POTS.

Single-cell transcriptomic analysis reveals dynamic activation of cellular signaling pathways regulating beige adipogenesis.

PDGFRA+ cells have been identified as adipocyte stem cells (ASCs) that differentiate into beige adipocytes in white adipose tissue (WAT) following thermogenic stimuli. To elucidate the molecular heterogeneity of ASCs, we conducted single-cell transcriptomic profiling of PDGFRA+ cells isolated from the inguinal WAT (iWAT) of mice treated with the beta3 adrenergic receptor agonist CL316243. Single-cell RNA-seq revealed nine major clusters, which were categorized into four groups: resting, proliferating, differentiating, and adipogenic factor-expressing cells (AFECs). Trajectory analysis revealed sequential activation of molecular pathways, including the Hedgehog and Notch signaling pathways, during beige adipogenesis. AFECs expressed Dpp4 and did not differentiate into adipocytes in culture or after transplantation. Furthermore, genetic lineage tracing studies indicated that DPP4+ cells did not differentiate into adipocytes in iWAT during CL316243-induced beige adipogenesis. However, high-fat diet feeding led to the recruitment of adipocytes from DPP4+ cells in iWAT. Overall, this study improved our understanding of the dynamic molecular basis of beige adipogenesis and the potential contribution of DPP4+ adipocyte lineages to the pathological expansion of WAT during diet-induced obesity.

Sympathetic innervation of interscapular brown adipose tissue is not a predominant mediator of OT-elicited reductions of body weight gain and adiposity in male diet-induced obese rats.

Recent studies indicate that central administration of oxytocin (OT) reduces body weight (BW) in high fat diet-induced obese (DIO) rodents by reducing energy intake and increasing energy expenditure (EE). Previous studies in our lab have shown that administration of OT into the fourth ventricle (4V; hindbrain) elicits weight loss and stimulates interscapular brown adipose tissue temperature (TIBAT) in DIO rats. We hypothesized that OT-elicited stimulation of sympathetic nervous system (SNS) activation of IBAT contributes to its ability to activate BAT and reduce BW in DIO rats. To test this, we determined the effect of disrupting SNS activation of IBAT on OT-elicited stimulation of TIBAT and reduction of BW in DIO rats. We first confirmed that bilateral surgical SNS denervation to IBAT was successful based on having achieved ≥ 60% reduction in IBAT norepinephrine (NE) content from DIO rats. NE content was selectively reduced in IBAT by 94.7 ± 2.7, 96.8 ± 1.8 and 85.9 ± 6.1% (P<0.05) at 1, 6 and 7-weeks post-denervation, respectively, and was unchanged in liver or inguinal white adipose tissue. We then measured the impact of bilateral surgical SNS denervation to IBAT on the ability of acute 4V OT (1, 5 μg) to stimulate TIBAT in DIO rats. We found that the high dose of 4V OT (5 μg) stimulated TIBAT similarly between sham and denervated rats (P=NS) and that the effects of 4V OT to stimulate TIBAT did not require beta-3 adrenergic receptor signaling. We subsequently measured the effect of bilateral surgical denervation of IBAT on the effect of chronic 4V OT (16 nmol/day) or vehicle infusion to reduce BW, adiposity, and energy intake in DIO rats. Chronic 4V OT reduced BW gain by -7.2 ± 9.6 g and -14.1 ± 8.8 g in sham and denervated rats (P<0.05 vs vehicle treatment), respectively, and this effect was similar between groups (P=NS). These effects were associated with reductions in adiposity and energy intake (P<0.05). Collectively, these findings support the hypothesis that sympathetic innervation of IBAT is not required for central OT to increase BAT thermogenesis and reduce BW gain and adiposity in male DIO rats.