β1-AR Blocker Research Articles

Baicalin reduces chronic stress-induced breast cancer metastasis via directly targeting β2-adrenergic receptor

Recent studies have shown that stress can substantially facilitate breast cancer metastasis, which can be reduced by nonselective β1/β2-adrenergic receptor (β1/β2-AR) blocker. However, several side effects were identified. Thus, it is extremely warranted to explore more effective and better-tolerated β2-AR blocker. Currently, we demonstrated that baicalin (BA), a major bioactive component of Scutellaria baicalensis Georgi, could significantly attenuate stress hormones especially epinephrine (Epi)-induced breast cancer cell migration and invasion in vitro. Mechanistically, we identified that β2-AR was a direct target of BA via the drug affinity responsive target stability (DARTS) combined with mass spectrum assay, and BA photoaffinity probe with pull-down assay, which was further confirmed by a couple of biophysical and biochemical assays. Furthermore, we demonstrated that BA could directly bind to the Phe-193 and Phe-289 of β2-AR, subsequently inhibit cyclic adenosine monophosphate-protein kinase A-focal adhesion kinase (cAMP-PKA-FAK) pathway, and thus impede epithelial-mesenchymal transition (EMT), thereby hindering the metastatic progression of the chronic stress coupled with syngeneic and xenograft in vivo orthotopic and tail vein mouse model. These findings firstly identify BA as a potential β2-AR inhibitor in the treatment of stress-induced breast cancer metastasis.

Phenylacetyl glutamine (PAGln) enhances cardiomyocyte death after myocardial infarction through β1 adrenergic receptor.

This study aims to explore the roles of phenylacetyl glutamine (PAGln) on myocardial infarction (MI) pathogenesis. Here, using targeted metabolomics analysis, it was found that the plasma metabolite PAGln was upregulated in coronary artery disease (CAD) patients and MI mice and could be an independent risk factor for CAD. In vivo and in vitro functional experiments revealed that PAGln pretreatment enhanced MI-induced myocardial injury and cardiac fibrosis, as evident by the increased infarct size, cardiomyocyte death, and the upregulated expression of cardiac fibrosis markers (Col1a1 and α-SMA). Combined with RNA-sequencing analysis and G protein-coupled receptor (GPCR) inhibitor, we found that the GPCR signaling activation is essential for PAGln-mediated effects on cardiomyocyte death. Furthermore, drug affinity responsive target stability and cellular thermal shift assay demonstrated that PAGln could interact with β1-adrenergic receptor (AR). Moreover, β1-AR blocker treatment indeed extended the cardiac remodeling after PAGln-enhanced MI. These results suggest that PAGln might be a potential therapeutic target for extending the cardiac remodeling window in MI patients that signals via β1-AR.

β3AR-Dependent Brain-Derived Neurotrophic Factor (BDNF) Generation Limits Chronic Postischemic Heart Failure.

Loss of brain-derived neurotrophic factor (BDNF)/TrkB (tropomyosin kinase receptor B) signaling accounts for brain and cardiac disorders. In neurons, β-adrenergic receptor stimulation enhances local BDNF expression. It is unclear if this occurs in a pathophysiological relevant manner in the heart, especially in the β-adrenergic receptor-desensitized postischemic myocardium. Nor is it fully understood whether and how TrkB agonists counter chronic postischemic left ventricle (LV) decompensation, a significant unmet clinical milestone. We conducted in vitro studies using neonatal rat and adult murine cardiomyocytes, SH-SY5Y neuronal cells, and umbilical vein endothelial cells. We assessed myocardial ischemia (MI) impact in wild type, β3AR knockout, or myocyte-selective BDNF knockout (myoBDNF KO) mice in vivo (via coronary ligation [MI]) or in isolated hearts with global ischemia-reperfusion (I/R). In wild type hearts, BDNF levels rose early after MI (<24 hours), plummeting at 4 weeks when LV dysfunction, adrenergic denervation, and impaired angiogenesis ensued. The TrkB agonist, LM22A-4, countered all these adverse effects. Compared with wild type, isolated myoBDNF KO hearts displayed worse infarct size/LV dysfunction after I/R injury and modest benefits from LM22A-4. In vitro, LM22A-4 promoted neurite outgrowth and neovascularization, boosting myocyte function, effects reproduced by 7,8-dihydroxyflavone, a chemically unrelated TrkB agonist. Superfusing myocytes with the β3AR-agonist, BRL-37344, increased myocyte BDNF content, while β3AR signaling underscored BDNF generation/protection in post-MI hearts. Accordingly, the β1AR blocker, metoprolol, via upregulated β3ARs, improved chronic post-MI LV dysfunction, enriching the myocardium with BDNF. Last, BRL-37344-imparted benefits were nearly abolished in isolated I/R injured myoBDNF KO hearts. BDNF loss underscores chronic postischemic heart failure. TrkB agonists can improve ischemic LV dysfunction via replenished myocardial BDNF content. Direct cardiac β3AR stimulation, or β-blockers (via upregulated β3AR), is another BDNF-based means to fend off chronic postischemic heart failure.

Neutrophil β1 adrenoceptor blockade blunts stroke-associated neuroinflammation.

Reperfusion therapy is the standard of care for ischaemic stroke; however, there is a need to identify new therapeutic targets able to ameliorate cerebral damage. Neutrophil β1 adrenoceptors (β1AR) have been linked to neutrophil migration during exacerbated inflammation. Given the central role of neutrophils in cerebral damage during stroke, we hypothesize that β1AR blockade will improve stroke outcomes. Rats were subjected to middle cerebral artery occlusion-reperfusion to evaluate the effect on stroke of the selective β1AR blocker metoprolol (12.5mg·kg-1 ) when injected i.v. 10 min before reperfusion. Magnetic resonance imaging and histopathology analysis showed that pre-reperfusion i.v. metoprolol reduced infarct size. This effect was accompanied by reduced cytotoxic oedema at 24 h and vasogenic oedema at 7days. Metoprolol-treated rats showed reduced brain neutrophil infiltration and those which infiltrated displayed a high proportion of anti-inflammatory phenotype (N2, YM1+ ). Additional inflammatory models demonstrated that metoprolol specifically blocked neutrophil migration via β1AR and excluded a significant effect on the glia compartment. Consistently, metoprolol did not protect the brain in neutrophil-depleted rats upon stroke. In patients suffering an ischaemic stroke, β1AR blockade by metoprolol reduced circulating neutrophil-platelet co-aggregates. Our findings describe that β1AR blockade ameliorates cerebral damage by targeting neutrophils, identifying a novel therapeutic target to improve outcomes in patients with stroke. This therapeutic strategy is in the earliest stages of the translational pathway and should be further explored.

AKT-mTOR signaling-mediated rescue of PRKAG2 R302Q mutant-induced familial hypertrophic cardiomyopathy by treatment with β-adrenergic receptor (β-AR) blocker metoprolol.

Protein kinase AMP-activated non-catalytic subunit gamma 2 gene (PRKAG2) cardiac syndrome, caused by mutations in PRKAG2, often shows myocardial hypertrophy and abnormal glycogen deposition in cardiomyocytes. However, it remains incurable due to a lack of a management guideline for treatment. We constructed a fluorescently labeled adenovirus carrying the wild-type or R302Q mutant of the PRKAG2 gene, infected neonatal rat cardiomyocytes (NRCMs) and H9C2 cell lines, and then analyzed changes in AMP-activated protein kinase (AMPK) activity, cell hypertrophy, glycogen storage, and cell proliferation when presence or absence of metoprolol or protein kinase A (PKA) inhibition H89, and then analyzed the changes in AKT-mTOR signal transduction activity. Overexpression of PRKAG2 R302Q in primary cardiomyocytes increased the activity of AMPK, induced cellular hypertrophy and glycogen storage, and promoted the phosphorylation levels of AKT-mTOR signaling pathway. Application of either β1-adrenergic receptor (β1-AR) blocker metoprolol or PKA inhibitor H89 to the cardiomyocytes rescued the hypertrophic cardiomyopathy (HCM)-like phenotypes induced by PRKAG2 R302Q, including suppression of both AKT-mTOR phosphorylation and AMPK activity. The current study not only determined the mechanism regulating HCM induced by PRKAG2 R302Q mutant, but also demonstrated a therapeutic strategy using β1-AR blocker to treat the patients with PRKAG2 cardiac syndrome.

Propranolol inhibits stemness of hemangioma through Jagged1.

BackgroundPropranolol is used clinically to treat infantile hemangioma (IH), although the exact mechanism that underlies its effectiveness is not fully understood. The Jagged1/Notch signaling pathway is downstream of the β2-adrenergic receptor (β2-AR). Propranolol is a non-selective β2-AR blocker that was shown to inhibit demethylation adrenaline-induced Jagged1 expression. A previous study has shown that propranolol dose-dependently inhibits the growth of IH. However, the effects of propranolol on stemness of IH are not known and are thus addressed in the current study.MethodsWe analyzed the expression of Jagged1 and Notch3 in IH specimens, using genetic tools to alter Notch signaling. The transduced IH cells were treated with different doses of propranolol, and the effects on IH cell proliferation, migration, and potential for tumor sphere formation were investigated. The effects of altered Notch signaling on tumor formation in vivo were also assessed.ResultsNotch3 and Jagged1 were significantly upregulated in IH. Augmented Notch signaling in IH cells increased cell proliferation, migration, the potential for tumor sphere formation and in vivo tumor formation. On the other hand, reduced Notch signaling in IH cells decreased cell proliferation, migration, the potential for tumor sphere formation and in vivo tumor formation.ConclusionsJagged1/Notch signaling regulated the stemness of IH, and propranolol inhibited it through suppression of Notch signaling.

Estrogen Attenuates Chronic Stress-Induced Cardiomyopathy by Adaptively Regulating Macrophage Polarizations via β2-Adrenergic Receptor Modulation.

Clinical demographics have demonstrated that postmenopausal women are predisposed to chronic stress-induced cardiomyopathy (CSC) and this has been associated with the decrease of estrogen. Meanwhile, recent studies have implicated unsolved myocardial proinflammatory responses, which are characterized by enormous CD86+ macrophage infiltrations as an underlying disease mechanism expediting the pathological remodeling of the heart during chronic stress. However, we had previously demonstrated that estrogen confers cardioprotection via the modulation of cardiomyocytes β2-adrenoceptors (β2AR)-Gs/Gi pathways during stress to lessen the incidence of stress-induced cardiovascular diseases in premenopausal women. Intriguingly, macrophages express β2AR profoundly as well; as such, we sought to elucidate the possibilities of estrogen modulating β2AR-Gs/Gi pathway to confer cardioprotection during stress via immunomodulation. To do this, ovariectomy (OVX) and sham operations (Sham) were performed on female Sprague-Dawley (SD) rats. Two weeks after OVX, the rats were injected with 40 μg/kg/day of estradiol (E2). Next, on day 36 after OVX, chronic stress was induced by a daily subcutaneous injection of 5 mg/kg/day of isoproterenol (ISO). The effect of E2 on relevant clinical cardiac function indexes (LVSP, LVEDP, + dp/dt and −dp/dt), myocardial architecture (cardiomyocyte diameter and fibrosis), β2AR alterations, and macrophage (CD86+ and CD206+) infiltrations were assessed. In vitro, peritoneal macrophages (PMΦ) were isolated from wild-type and β2AR-knockout female mice. The PMΦ were treated with ISO, E2, and β2AR blocker ICI 118,551 for 24 h, and flow cytometric evaluations were done to assess their phenotypic expression. E2 deficiency permitted the induction of CSC, which was characterized by cardiac dysfunctions, maladaptive myocardial hypertrophy, unresolved proinflammatory responses, and fibrosis. Nonetheless, E2 presence/supplementation during stress averted all the aforementioned adverse effects of chronic stress while preventing excessive depletion of β2AR. Also, we demonstrated that E2 facilitates timely resolution of myocardial proinflammation to permit reparative functions by enhancing the polarization of CD86+ to CD206+ macrophages. However, this adaptive immunomodulation is hampered when β2AR is inhibited. Taken together, the outcomes of this study show that E2 confers cardioprotection to prevent CSC via adaptive immunomodulation of macrophage phenotypes, and β2AR-mediated signaling is crucial for the polarizations of CD86+ to CD206+ macrophages.

Effects of a β2-adrenergic receptor blocker on experimental periodontitis in spontaneously hypertensive rats

AimsRecent studies have reported a relationship between periodontal disease and hypertension, and previous evidence suggests that the sympathetic nervous system plays an important role in the control of bone metabolism. This study sought to evaluate the effect of the beta-2 adrenergic receptor (β2-AR) blocker butoxamine on experimental periodontitis in a rat model. Materials and methodsWistar-Kyoto and spontaneously hypertensive rats (n = 6 per group) were orally administered butoxamine 1 mg/kg/day and experimental periodontitis was induced by applying an orthodontic ligature wire. The rats were sacrificed after 4 weeks and the residual alveolar bone was measured using micro-computed tomography (micro-CT) imaging analysis software for histological analysis. Key findingsMicro-CT imaging analysis showed a higher ratio of residual alveolar bone, BV/TV, and Tb.N in both Wistar-Kyoto and spontaneously hypertensive rats treated with butoxamine compared with the corresponding control rats. In histological analysis, compared with the Wistar-Kyoto and spontaneously hypertensive rat control groups, the corresponding butoxamine-treated groups showed a lower ratio of attachment level, lower values of osteoclast number and surface. Significanceβ2-AR blockers maintained the alveolar bone mass and attachment level by suppressing osteoclast activity. Thus, β2-AR blockers may be effective in preventing periodontitis.

Abstract 13100: Rescue Myocardial Aging Caused a Progressive Decline in Cardiac Function and β-Adrenergic Reserve With a Selective Beta3-Adrenergic Receptor Antagonist, but Not Beta1-Adrenergic Receptor Blocker in a Murine Model

Background: Cardiac aging (CA) leads to the progressive decline in cardiac function- increasing risk for heart failure (HF) and cardiac mortality. Both down-regulation of β 1 -adrenergic receptors (AR) and up-regulation of a newly-described inhibitory pathway mediated by β 3 -AR may play a crucial role in the development of CA. β-AR blockers are a mainstay in the therapy of several cardiovascular diseases, including HF. However the therapeutic effects of different β blockers in CA are unknown. We tested the hypothesis that up-regulation of cardiac β 3 -AR with enhanced negative inotropic actions, rather than the desensitization of β 1 -AR, is the critical determinant of the dysfunctional β-AR regulation that occurs in CA. Thus, β 3 -AR antagonists (β 3 ANT), but not β 1 Blockade, may reverse the progression of age-associated deficits in LV myocyte functional performance. Methods: LV myocyte basal and isoproterenol (ISO)-stimulated functional and [Ca 2+ ] i transient ([Ca 2+ ] iT ) responses were compared in 4 groups (7/group) of wild-type (WT) mice: 1) YWT , young (Y, 6 mo); 2) Aβ 3 ANT , aged (A, 26 mo) treated with β 3 ANT (L-748,337, 10 -7 M/kg/day, mini-pump) for 12 weeks (W);3) Aβ 1 Blockade , metoprolol XL (1.5 mg/kg/day, po), a selective β 1 blocker was given for 12 W and 4) AWT , aged sham control. Results: Compared with YWT, AWT myocytes had about 30% decreases in basal cell contraction (dL/dt max , 85.9 vs 122.3 μm/s) and relaxation (dR/dt max , 69.4 vs 99.2 μm/s) with reduced the peak [Ca 2+ ] iT (0.17 vs 0.21). ISO (10 -8 M)-stimulated increases in dL/dt max (33% vs 69%), dR/dt max (31% vs 62%) and [Ca 2+ ] iT (15% vs 34%) also significantly reduced. In Aβ 1 Blockade group, metoprolol therapy failed to improve basal dL/dt max (86.3 μm/s), dR/dt max (68.9 μm/s) and [Ca 2+ ] iT (0.18). ISO-stimulated increases in dL/dt max (34%), dR/dt max (35%) and [Ca 2+ ] iT (17%) remained attenuated. By contrary, Aβ 3 ANT myocytes showed normal basal cell contraction (131.0 μm/s), relaxation (101.9 μm/s) and [Ca 2+ ] iT (0.21) with preserved ISO-stimulated increases in dL/dt max (68%), dR/dt max (59%) and [Ca 2+ ] iT (32%). Conclusions: Chronic β 3 -AR antagonist, but not β 1 -AR blocker leads to the preservation of myocyte function, [Ca 2+ ] iT , and β-adrenergic responsiveness in a murine model of cardiac aging.

Overexpression of β-Adrenergic Receptors and the Suppressive Effect of β2-Adrenergic Receptor Blockade in Oral Squamous Cell Carcinoma

The purpose of this project was to investigate the expression of β-adrenergic receptors in oral squamous cell carcinoma (OSCC) and the tumor suppressive activity of β2-adrenergic receptor (β2-AR) blockade. Samples of 15 normal oral mucosal epithelial tissues, 60 surgically resected OSCC tissues, and 60 adjacent para-carcinoma tissues were collected. The expression of β1-adrenergic receptor and β2-AR was detected by real-time quantitative polymerase chain reaction and the Western blot test.SCC9 and Cal27cell lines and primary OSCC cells also were included and treated with ICI-118,551 (MedChemExpress, Monmouth Junction, NJ), a selective β2-AR blocker.In addition, the Cal27cell line was treated with propranolol (a nonselective β-adrenergic receptor blocker) to verify the suppressive effect of β2-AR blockade. For invivo assays, Cal27cells were subcutaneously injected in the tongue flank of nude mice. ICI-118,551 was orally administered to the mice in the treatment group daily. High-throughput sequencing was used to screen for changes in gene expression. Real-time quantitative polymerase chain reaction and the Western blot test both showed that β1-adrenergic receptor and β2-AR were overexpressed in OSCC tissues and cells. A relationship was found between β2-AR and a more advanced clinical stage, as well as preoperative lymphatic metastasis. After treatment with ICI-118,551 or propranolol, the capacities for proliferation, invasion, and metastasis of OSCC cells were significantly inhibited. Tumor size was significantly different between the ICI-118,551 and control groups. The survival time in the ICI-118,551 group also was prolonged significantly. Moreover, high-throughput sequencing identified 19 affected signaling pathways, including mitogen-activated protein kinase and PI3K-Akt.We confirmed a significant change to the expression of several genes closely related to the progression of cancer. This study showed that β2-AR is related to a more advanced clinical stage and preoperative lymphatic metastasis. Additionally, a β2-AR blocker has a significant suppressive effect in OSCC.

Suppression of tooth movement-induced sclerostin expression using β-adrenergic receptor blockers.

Regulation of bone metabolism by the sympathetic nervous system has recently been clarified. Tooth movement is increased by increased bone metabolic turnover due to sympathetic activation. This study aimed to compare the effects of the β-adrenergic receptor (β-AR) blockers atenolol (β1-AR blocker), butoxamine (β2-AR blocker) and propranolol (non-selective β-AR blocker) on tooth movement in spontaneously hypertensive rats (SHR) with sympathicotonia. Spontaneously hypertensive rats were divided into the following four groups: an SHR control group and groups treated with 0.1mg/kg atenolol, 1mg/kg butoxamine or 1mg/kg propranolol (n=6 rats/group). Atenolol, butoxamine or propranolol was administered daily to each treatment group, and orthodontic force was applied using a closed-coil spring. Finally, immunohistochemical analysis was performed for receptor activator of nuclear factor kappa-B ligand (RANKL) and sclerostin (SOST). Atenolol, butoxamine and propranolol inhibited tooth movement and increased maxillary alveolar bone volume. Histological analysis revealed that these β-AR blockers decreased osteoclast activity on the compression side. Furthermore, immunohistochemical analysis revealed that atenolol, butoxamine and propranolol decreased the number of RANKL- and SOST-positive osteocytes on the compression side. β-AR blockers decreased tooth movement and downregulated SOST in osteocytes, accompanied by increasing alveolar bone resorption.

Isoproterenol-induced heart failure in rat heart: Evaluation and cardioprotective strategy

It is well established that cardiac remodelling plays a pivotal role in the development of heart failure (HF), a leading cause of death worldwide. Meanwhile, a preclinical rat model of isoproterenol (Iso)-induced HF causes sympathetic hyperactivity, an important factor of cardiac remodelling, hemodynamic changes and cardiac fibrosis development. Several studies showed that metoprolol, a β1-adrenergic receptor (β1-AR) blocker prevents cardiac dysfunction following prolonged β-AR stimulation in different preclinical models. We evaluated the cardioprotective effects of metoprolol on cardiac remodelling and diastolic function in a severe Iso-induced HF model using echocardiography and cardiac pressure measurement. Rats received Iso injections (Iso; 5 mg/kg/day, N = 5), metoprolol (Meto; 24 mg/kg/day) + Iso 5 (Iso + Meto, N = 6), or vehicle ( N = 9) for 7 days. Left ventricular (LV) dimensions and function were followed-up by echocardiography before and after the treatment to assess LV hypertrophy. After echocardiography, LV end-diastolic pressure (LVEDP) was recorded to assess ventricular compliance and rat hearts were used for cardiac remodelling analysis. One week of Iso injections significantly increased LVEDP (+ 48%) and was associated with a major LV hypertrophy, characterised by a significant increase in LV mass (+ 33%) and a thickening of the LV walls observed by echocardiography. Simultaneous treatment with metoprolol prevented diastolic dysfunction and LV remodelling. Hypertrophy and fibrosis will be analysed in each group by histology (on-going experiments). Metoprolol afforded a cardioprotective effect on cardiac remodelling and diastolic dysfunction in Iso-induced HF model. Echocardiography and LVEDP measurements validate the reversibility of hypertrophic HF, and Iso-induced HF developed in rat could represent an effective and predictive preclinical model for evaluating antihypertrophic and antifibrotic agents.

β1-adrenergic receptors mediate plasma acyl-ghrelin elevation and depressive-like behavior induced by chronic psychosocial stress.

The ghrelin system is a key component of the mood and metabolic responses to chronic psychosocial stress. For example, circulating acyl-ghrelin rises in several rodent and human stress models, administered acyl-ghrelin induces antidepressant-like behavioral responses in mice, and mice with deleted ghrelin receptors (GHSRs) exhibit exaggerated depressive-like behaviors, changed eating behaviors, and altered metabolism in response to chronic stress. However, the mechanisms mediating stress-induced rises in ghrelin are unknown and ghrelin's antidepressant-like efficacy in the setting of chronic stress is incompletely characterized. Here, we used a pharmacological approach in combination with a 10-day chronic social defeat stress (CSDS) model in male mice to investigate whether the sympathoadrenal system is involved in the ghrelin response to stress. We also examined the antidepressant-like efficacy of administered ghrelin and the synthetic GHSR agonist GHRP-2 during and/or after CSDS. We found that administration of the β1-adrenergic receptor (β1AR) blocker atenolol during CSDS blunts the elevation of plasma acyl-ghrelin and exaggerates depressive-like behavior. Neither acute injection of acyl-ghrelin directly following CSDS nor its chronic administration during or after CSDS nor chronic delivery of GHRP-2 during and after CSDS improved stress-induced depressive-like behavior. Thus, β1ARs drive the acyl-ghrelin response to CSDS, but supplementing the natural increases in acyl-ghrelin with exogenous acyl-ghrelin or GHSR agonist does not further enhance the antidepressant-like actions of the endogenous ghrelin system in the setting of CSDS.

Beta3-Adrenergic Blockade Restores Erythropoietic Homeostasis in Anemia of Critical Illness

IntroductionDuring anemia of critical illness such as burn injury, erythropoietin (Epo) resistance is accompanied by impaired bone marrow (BM) erythropoiesis. The non-selective β1, β2 -adrenergic receptor (AR) blocker propranolol was effective in mitigating MafB expression in multi potential progenitors and rescuing their erythrocyte commitment in burn patients. However, peripheral HGB levels did not reflect the effect of propranolol perhaps due to defective maturation of late erythroblasts, not regulated by propranolol. Subsequently, we reported that early commitment and late maturation stages are independently orchestrated via discrete β2- and β3-AR mechanisms respectively in the BM of mice with a parallel increase in blood HGB levels. Nonetheless, it is not clear whether commitment of progenitors or maturation of erythroblasts is essential to restore erythropoietic homeostasis after burn injury. In adult mice, the spleen serves as an extra medullary erythropoietic organ under hypoxia and also functions as a primary organ of erythropoietic homeostasis (Bozzini CE et al. Am J Physiol. 1970; 219(3):724). We examined in mice, erythropoietic responses to burn injury in two organs, the spleen and BM to understand the reciprocal relationship, if any, in erythropoiesis between the two adrenergically innervated organs in response to propranolol, and selective β2- and β3- AR blockers. As variation in the size of RBCs is an indication of ineffective maturation, we also evaluated coefficient of variation of RBC size (RDW) in peripheral blood from mice. To validate our animal studies, we also measured RBC parameters in human burn patients.MethodsAnimal study: B6D2F1 mice were randomized into 2 groups, 15% TBSA (total burn surface area) scald burn and sham burn. Burn mice were given β-AR blockers or saline either via Alzet pumps eight hours after injury or by daily injections. All treatments were terminated 24 hours before harvest. Animals were euthanized to obtain spleen, femurs and blood on post burn days (PBD) as specified in results. Single cell suspensions from spleen and BM were characterized for early erythroblasts (CD71+Ter119neg), orthochromatic erythroblasts (Ter119+CD71+Syto16+), reticulocytes (Ter119+CD71+Syto16neg), erythrocytes (Ter119+), and non-erythroid cells (Ter119neg CD71neg) by flow cytometry, and expressed as 103/106 total splenocytes or total BM cells. RDW was measured using HemaTrue analyzer.Human study: Retrospective analysis was done on 19 adult burn patients enrolled in IRB approved study. All patients received standard burn care (SBC) during the study period of seven weeks. As part of another clinical trial, 9/19 patients had received propranolol along with SBC (SBC+PR). Cohorts matched for age, gender and %TBSA.ResultsAnimal study: Splenic erythropoiesis was increased after burn injury on PBD 3, 7, 14 and 21 as measured by erythrocyte and erythroblast production. Propranolol treatment for 14 days via Alzet pump alone or in combination with Epo was not sufficient to reduce splenic erythrocytes and erythroblasts that were elevated after burn injury. Between the selective blockers, only SR59320A (β3-AR) and not butoxamine (β2-AR) significantly reduced splenic erythropoiesis. This was accompanied by a decrease in splenomegaly and a reciprocal increase in BM erythropoiesis only with SR59230A treatment. RDW was significantly increased in blood after burn injury, while MCV remained within normal range. Interestingly, only SR59230A treatment and not propranolol or butoxamine reversed RDW to sham levels.Human study: Hemoglobin levels were higher at PBD 1-3 (due to hemo concentration) and decreased significantly in both groups through PBD 30-48. Mean RDW was normal at PBD 1-3 (Normal range =11.5% -14.5%) in all burn patients. Although RDW seemed to stabilize initially in SBC+PR treated patients at PBD 7-10, it increased to SBC levels by PBD 30-48. Mean corpuscular volume (MCV) remained within normal limits.ConclusionEffective maturation of late erythroblasts in BM is essential for the spleen to sense erythropoietic homeostasis, which is reestablished with β3-AR blockade after burn injury. Results emphasize only β3-AR antagonist restores RDW to sham levels and not β1/ β2-AR blocker. Increased RDW in burn patients corroborates with animal studies. Overall, this model system is suitable to study mechanisms of early and late erythropoiesis after burn injury. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Selective β2-adrenoreceptor signaling regulates osteoclastogenesis via modulating RANKL production and neuropeptides expression in osteocytic MLO-Y4 cells.

The β2-adrenergic receptor (β2-AR) signaling on bone cells is the major contributor in the effect of the sympathetic nervous system on bone turnover. However, it remains unclear whether receptor activator of nuclear factor κ-Β ligand(RANKL) modulation and neuropeptides expression in osteocytes are responsible for the mechanism. This study used β2-AR stimulation to investigate cell cycle and proliferation, the gene and protein expression of RANKL, and osteoprotegerin (OPG), as well as neuropeptides regulation in osteocytic MLO-Y4 cells. Clenbuterol (CLE; a β2-AR agonist) slightly promoted the growth of MLO-Y4 cells in a concentration-dependent effect but had no effect on the proliferation index. And the concentration of 10-8 M showed a significant increase in the S-phase fraction on day 3 in comparison with the control. Additionally, CLE-promoted osteoclast formation and bone resorption in osteocytic MLO-Y4 cell-RAW264.7 cell cocultures. RANKL expression level and the ratio of RANKL to OPG in MLO-Y4 cells were enhanced in CLE treatment but were rescued by blocking β2-AR signaling. However, neuropeptide Y and α-calcitonin gene-related peptide, two neurogenic markers, were inhibited in CLE treatment of MLO-Y4 cells, which was reversed by a β2-AR blocker. The results indicate that osteocytic β2-AR plays an important role in the regulation of RANKL/OPG and neuropeptides expression, and β2-AR signaling in osteocytes can be used as a new valuable target for osteoclast-related pathologic disease.

Blockade of Host β2-Adrenergic Receptor Enhances Graft-versus-Tumor Effect through Modulating APCs.

Allogeneic hematopoietic cell transplantation is a potential curative therapy for hematologic malignancies. Host APCs are pivotal to the desired graft-versus-tumor (GVT) effect. Recent studies have shown that β2-adrenergic receptor (β2AR) signaling can have an important impact on immune cell function, including dendritic cells (DCs). In this article, we demonstrate that pretreatment of host mice with a β2AR blocker significantly increases the GVT effect of donor CD8+ T cells by decreasing tumor burden without increasing graft-versus-host disease. β2AR-deficient host mice have significantly increased effector memory and central memory CD8+ T cells and improved reconstitution of T cells, including CD4+Foxp3+ regulatory T cells. Notably, β2AR deficiency induces increased CD11c+ DC development. Also, β2AR-deficient bone marrow-derived DCs induce higher CD8+ T cell proliferation and improved tumor killing in vitro. Metabolic profiling shows that β2AR deficiency renders DCs more immunogenic through upregulation of mTOR activity and reduction of STAT3 phosphorylation. Altogether, these findings demonstrate an important role for host β2AR signaling in suppressing T cell reconstitution and GVT activity.

Regional β-adrenergic regulation of action potential duration and arrhythmias in the pig right ventricle

The right ventricular outflow tract (RVOT) is the main origin of idiopathic ventricular tachycardia (VT). Although these arrhythmias appear to be cAMP-dependent, the actual mechanisms leading to VT remain uncertain. Here we studied β-adrenergic regulation of action potential duration (APD) in the healthy pig right ventricle (RV). To determine regional β-adrenergic regulation of APD and arrhythmias in the RVOT. The RV of 4 pig hearts were isolated and perfused via both the coronary arteries. Epicardial electrical activity was optically mapped (di-4-ANEPPS 20 μM) and APD measured at 80% repolarization (APD80) in the RVOT and posterior RV free wall region (RVFW). Regional mRNA and protein expression of β-adrenergic receptors was determined respectively by RT-PCR and Western-Blot. The expression of β1-adrenergic receptor (β1-AR) was lower in the RVOT at the mRNA level ( P < 0.05) but remained unchanged at the protein level compared to the RVFW while β2-adrenergic receptor (β2-AR) expression remained similar in both regions. At lower concentrations (0.6–10 nM), isoprenaline (ISO) led to a prolongation of APD80 in the RVOT and the RVFW. Higher ISO concentrations (500 nM–1 μM) led to a larger APD80 shortening in the RVOT than in the RVFW and triggered VT episodes that originated from the RVOT or the anterior RV. β2-AR blockade (ICI 118,551 100 nM) accelerated while β1-AR blocker Metoprolol (4 μM) slowed or abolished VT. Similar ISO concentrations triggered bursts of Ca2+ transients in RVOT myocytes that stopped upon ISO washout while these events remained rare in RVFW myocytes. β-adrenergic stimulation differentially regulates APD80 in the RVOT and RVFW. Although β1-AR expression was similar in the 2 regions, ISO preferentially shortened APD and triggered arrhythmia in the RVOT. These results suggest the presence of a high sensitivity of RVOT electrophysiology to β-adrenergic stimulation.

Estrogen replacement attenuates stress-induced pressor responses through vasorelaxation via β2-adrenoceptors in peripheral arteries of ovariectomized rats.

We examined whether chronic estrogen replacement has an inhibitory effect on stress-induced pressor responses via activation of β2-adrenoceptor (AR) in peripheral arteries of ovariectomized rats. Female Wistar rats aged 9 wk were ovariectomized. After 4 wk, pellets containing either 17β-estradiol (E2) or placebo (Pla) were subcutaneously implanted into the rats. After 4 wk of treatment, rats underwent cage-switch stress, and, in a separate experiment, a subset received an infusion of isoproterenol (ISO) with or without pretreatment with the β1-AR blocker atenolol or the β2-AR blocker butoxamine. In addition, the isolated mesenteric artery was used to assess the concentration-related relaxing responses to ISO and the β1- or β2-AR mRNA level. The cage-switch stress-induced pressor response was significantly attenuated in the E2-treated group compared with the Pla-treated group. Pretreatment with atenolol reduced blood pressure responses in both groups. However, butoxamine enhanced the pressor response only in the E2-treated group, resulting in no difference between the two groups. In addition, the intravenous ISO-induced depressor response was significantly enhanced in the E2-treated group compared with the Pla-treated group. Furthermore, the difference in the depressor response was abolished by pretreatment with butoxamine but not by atenolol. In the isolated mesenteric artery, butoxamine caused a rightward shift in ISO-induced concentration-related relaxation in the E2-treated group. The β2-AR mRNA level in the mesenteric artery was higher in the E2-treated group than in the Pla-treated group. These results suggest that estrogen replacement attenuated the stress-induced pressor response probably by suppressing vasoconstriction via activation of β2-ARs in peripheral arteries of ovariectomized rats. NEW & NOTEWORTHY In this study, we show, for the first time, that estrogen replacement has an inhibitory effect on the psychological stress-induced pressor response through vasorelaxation via β2-adrenoceptors, probably due to overexpression of β2-adrenoceptor mRNA, in peripheral arteries of ovariectomized rats.

The role of β-adrenergic receptors and p38MAPK signaling pathways in physiological processes of cardiosphere-derived cells.

The effects of β adrenergic receptors (β-ARs) and p38 mitogen-activated protein kinases (MAPK) pathways on cardiosphere-derived cells (CDCs) are largely unknown. This study aimed to investigate the roles of β-ARs and p38MAPK pathways on the proliferation, apoptosis, and differentiation capacity of CDCs. The CDCs were treated with β1-AR blocker (Met group), β2-AR antagonist (ICI group), and p38MAPK inhibitor (SB group), non-selective β-AR blocker (PRO group), and β-AR agonist (ISO group). The viability, apoptotic rate and differentiation status of CDCs were determined by MST-1 assay, flow cytometery, and Western blot, respectively. The CDCs viability significantly reduced in ICI group (all P < 0.05), and SB group had a significant high viability after 48 h treatment (P < 0.05). Compared with control group, all treated groups had a low apoptotic rate. After treatment for 72 h, ISO treatment elevated the expression of Nkx2.5, and could partially or fully attenuate the inhibitory effects of β-AR antagonists and/or p38MAPK inhibitor. A similar overall trend of protein expression levels among all groups could be observed between protein pairs of cTnT and β1-AR as well as c-Kit and β2-AR, respectively. These results suggested that β-ARs and p38MAPK signaling pathways play crucial roles in the proliferation and differentiation of CDCs. Our findings should be helpful for better understanding the molecular mechanism underlying the physiological processes of CDCs.

Ventricular action potential adaptation to regular exercise: role of β-adrenergic and KATP channel function.

Regular exercise training is known to affect the action potential duration (APD) and improve heart function, but involvement of β-adrenergic receptor (β-AR) subtypes and/or the ATP-sensitive K+ (KATP) channel is unknown. To address this, female and male Sprague-Dawley rats were randomly assigned to voluntary wheel-running or control groups; they were anesthetized after 6-8 wk of training, and myocytes were isolated. Exercise training significantly increased APD of apex and base myocytes at 1 Hz and decreased APD at 10 Hz. Ca2+ transient durations reflected the changes in APD, while Ca2+ transient amplitudes were unaffected by wheel running. The nonselective β-AR agonist isoproterenol shortened the myocyte APD, an effect reduced by wheel running. The isoproterenol-induced shortening of APD was largely reversed by the selective β1-AR blocker atenolol, but not the β2-AR blocker ICI 118,551, providing evidence that wheel running reduced the sensitivity of the β1-AR. At 10 Hz, the KATP channel inhibitor glibenclamide prolonged the myocyte APD more in exercise-trained than control rats, implicating a role for this channel in the exercise-induced APD shortening at 10 Hz. A novel finding of this work was the dual importance of altered β1-AR responsiveness and KATP channel function in the training-induced regulation of APD. Of physiological importance to the beating heart, the reduced response to adrenergic agonists would enhance cardiac contractility at resting rates, where sympathetic drive is low, by prolonging APD and Ca2+ influx; during exercise, an increase in KATP channel activity would shorten APD and, thus, protect the heart against Ca2+ overload or inadequate filling.NEW & NOTEWORTHY Our data demonstrated that regular exercise prolonged the action potential and Ca2+ transient durations in myocytes isolated from apex and base regions at 1-Hz and shortened both at 10-Hz stimulation. Novel findings were that wheel running shifted the β-adrenergic receptor agonist dose-response curve rightward compared with controls by reducing β1-adrenergic receptor responsiveness and that, at the high activation rate, myocytes from trained animals showed higher KATP channel function.