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Piloerection (goosebumps) requires concerted actions of the hair follicle, the arrector pili muscle (APM), and the sympathetic nerve, providing a model to study interactions across epithelium, mesenchyme, and nerves. Here, we show that APMs and sympathetic nerves form a dual-component niche to modulate hair follicle stem cell (HFSC) activity. Sympathetic nerves form synapse-like structures with HFSCs and regulate HFSCs through norepinephrine, whereas APMs maintain sympathetic innervation to HFSCs. Without norepinephrine signaling, HFSCs enter deep quiescence by down-regulating the cell cycle and metabolism while up-regulating quiescence regulators Foxp1 and Fgf18. During development, HFSC progeny secretes Sonic Hedgehog (SHH) to direct the formation of this APM-sympathetic nerve niche, which in turn controls hair follicle regeneration in adults. Our results reveal a reciprocal interdependence between a regenerative tissue and its niche at different stages and demonstrate sympathetic nerves can modulate stem cells through synapse-like connections and neurotransmitters to couple tissue production with demands., Competing Interests: Declaration of Interests A provisional patent application has been filed by the President and Fellows of Harvard College based on this work., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Reperfusion as a therapeutic intervention for acute myocardial infarction-induced cardiac injury itself induces further cardiomyocyte death. β-arrestin (βarr)-biased β-adrenergic receptor (βAR) activation promotes survival signaling responses in vitro; thus, we hypothesize that this pathway can mitigate cardiomyocyte death at the time of reperfusion to better preserve function. However, a lack of efficacious βarr-biased orthosteric small molecules has prevented investigation into whether this pathway relays protection against ischemic injury in vivo. We recently demonstrated that the pepducin ICL1-9, a small lipidated peptide fragment designed from the first intracellular loop of β2AR, allosterically engaged pro-survival signaling cascades in a βarr-dependent manner in vitro. Thus, in this study we tested whether ICL1-9 relays cardioprotection against ischemia/reperfusion (I/R)-induced injury in vivo. Methods: Wild-type (WT) C57BL/6, β2AR knockout (KO), βarr1KO and βarr2KO mice received intracardiac injections of either ICL1-9 or a scrambled control pepducin (Scr) at the time of ischemia (30 min) followed by reperfusion for either 24 h, to assess infarct size and cardiomyocyte death, or 4 weeks, to monitor the impact of ICL1-9 on long-term cardiac structure and function. Neonatal rat ventricular myocytes (NRVM) were used to assess the impact of ICL1-9 versus Scr pepducin on cardiomyocyte survival and mitochondrial superoxide formation in response to either serum deprivation or hypoxia/reoxygenation (H/R) in vitro and to investigate the associated mechanism(s). Results: Intramyocardial injection of ICL1-9 at the time of I/R reduced infarct size, cardiomyocyte death and improved cardiac function in a β2AR- and βarr-dependent manner, which led to improved contractile function early and less fibrotic remodeling over time. Mechanistically, ICL1-9 attenuated mitochondrial superoxide production and promoted cardiomyocyte survival in a RhoA/ROCK-dependent manner. RhoA activation could be detected in cardiomyocytes and whole heart up to 24 h post-treatment, demonstrating the stability of ICL1-9 effects on βarr-dependent β2AR signaling. Conclusion: Pepducin-based allosteric modulation of βarr-dependent β2AR signaling represents a novel therapeutic approach to reduce reperfusion-induced cardiac injury and relay long-term cardiac remodeling benefits., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Background and Aim: β 2 -Adrenoceptors (β 2 -ARs) are G protein-coupled receptors (GPCRs) expressed in the major insulin target tissues. The interplay between β 2 -AR and insulin pathways is involved in the maintenance of glucose homeostasis. The aim of this study was to explore the consequences of β 2 -ARs deletion on insulin sensitivity and insulin signaling cascade in metabolically active tissues., Methods and Results: We evaluated glucose homeostasis in skeletal muscle and liver of β 2 -AR-null mice (β 2 -AR -/- ) by performing in vivo (glucose tolerance test and insulin tolerance test) and ex vivo (glucose uptake and glycogen determination) experiments. β 2 -AR gene deletion is associated with hepatic insulin resistance and preserved skeletal muscle insulin sensitivity. Importantly, we demonstrate that hepatic β 2 -AR regulates insulin-induced AKT activation via Grb2-mediated SRC recruitment through a G i -independent mechanism., Conclusions: β-AR stimulation contributes to the development of early stages of insulin resistance progression in the liver. Our findings indicate that the cross-talk between β 2 -AR and insulin signaling represents a fundamental target towards the development of novel therapeutic approaches to treat type 2 diabetes and metabolic syndrome., (Copyright © 2017 The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.)

Aims: Compartmentalization of cAMP and PKA activity in cardiac muscle cells plays a key role in maintaining basal and enhanced contractility stimulated by sympathetic nerve activity. In cardiomyocytes, activation of adrenergic receptor increases cAMP production, which is countered by the hydrolytic activity of selective phosphodiesterases (PDEs). The intracellular regional dynamics of cAMP production and hydrolysis modulate downstream signals resulting in different biological responses. The interplay between beta receptors (βARs) signalling and phosphodiesterase 5 (PDE5) activity remains to be addressed., Methods and Results: Using combined strategies with pharmacological inhibitors and genetic deletion of PDEs and βAR isoforms, we revealed a specific pool of cAMP that is under dual regulation by PDE2 and, indirectly, PDE5 activity. Inhibition of PDE5 with sildenafil produces a cGMP-dependent activation of PDE2 that attenuates cAMP generation induced by βAR agonists, with concomitant modulation of stimulated contraction rate and calcium transients. PDE2 haploinsufficiency abolished the effects of sildenafil. The negative chronotropic effect of PDE5 inhibition through PDE2 activation was also observed in sinoatrial node tissue from adult mice. PDE5 inhibition selectively lowered contraction rate stimulated by β2AR, but not β1AR activation, supporting a compartmentalization of the cGMP-modulated pool of cAMP., Conclusion: These data identify a new effect of PDE5 inhibitors on the modulation of cardiomyocyte response to adrenergic stimulation via PDE5-PDE2-mediated cross-talk., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.)

The counter-regulatory effects of insulin and catecholamines on carbohydrate and lipid metabolism are well studied, whereas the details of insulin regulation of β adrenergic receptor (βAR) signaling pathway in heart remain unknown. Here, we characterize a novel signaling pathway of insulin receptor (IR) to G protein-coupled receptor kinase 2 (GRK2) in the heart. Insulin stimulates recruitment of GRK2 to β2AR, which induces β2AR phosphorylation at the GRK sites of serine 355/356 and subsequently β2AR internalization. Insulin thereby suppresses βAR-induced cAMP-PKA activities and contractile response in neonatal and adult mouse cardiomyocytes. Deletion of insulin receptor substrate 2 (IRS2) disrupts the complex of IR and GRK2, which attenuates insulin-mediated β2AR phosphorylation at the GRK sites and β2AR internalization, and the counter-regulation effects of insulin on βAR signaling. These data indicate the requirements of IRS2 and GRK2 for insulin to stimulate counter-regulation of βAR via β2AR phosphorylation and internalization in cardiomyocytes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Inhibition of TNFα protects the retina against diabetic-like changes in rodent models. The mechanism by which TNFα induces deleterious retinal changes is not known. Previously, we have shown that TNFα can inhibit normal insulin signal transduction, leading to increased apoptosis in both retinal endothelial cells (REC) and Müller cells. Additionally, β2-adrenergic receptor knockout mice (β2KO) have increased TNFα levels and decreased insulin receptor activity. In this study, we hypothesized that inhibition of TNFα in β2KO mice would increase normal insulin signaling, leading to improved retinal function., Methods: C57BL6 or β2KO mice were left untreated or treated with etanercept (0.3 mg/kg subcutaneously, 3× a week) for 2 months. Electroretinogram analyses were done before treatment was initiated and after two months of treatment with etanercept on all mice. Western blot or ELISA analyses were done on whole retinal lysates from all four groups of mice for TNFα, suppressor of cytokine signaling 3 (SOCS3), insulin receptor, and apoptotic proteins., Results: Etanercept significantly reduced TNFα levels in β2KO mice, leading to increased insulin receptor phosphorylation on tyrosine 1150/1151. SOCS3 levels were increased in β2KO mice, which were reduced after etanercept treatment. Pro-apoptotic proteins were reduced in etanercept-treated β2KO mice. Etanercept improved ERG amplitudes in β2KO mice., Conclusions: Inhibition of TNFα by etanercept protects the retina likely through reduced TNFα-mediated insulin resistance, leading to reduced apoptosis.

Objective: Sustained β-adrenergic stimulation is a hallmark of sympathetic hyperactivity in cardiovascular diseases. It is associated with oxidative stress and altered vasoconstrictor tone. This study investigated the β-adrenoceptor subtype and the signaling pathways implicated in the vascular effects of β-adrenoceptor overactivation., Methods and Results: Mice lacking the β1- or β2-adrenoceptor subtype (β1KO, β2KO) and wild-type (WT) were treated with isoproterenol (ISO, 15 μg.g(-1) x day(-1), 7 days). ISO significantly enhanced the maximal vasoconstrictor response (Emax) of the aorta to phenylephrine in WT (+34%) and β1KO mice (+35%) but not in β2KO mice. The nitric oxide synthase (NOS) inhibitor L-NAME abolished the differences in phenylephrine response between the groups, suggesting that ISO impaired basal NO availability in the aorta of WT and β1KO mice. Superoxide dismutase (SOD), pertussis toxin (PTx) or PD 98,059 (p-ERK 1/2 inhibitor) incubation reversed the hypercontractility of aortic rings from ISO-treated WT mice; aortic contraction of ISO-treated β2KO mice was not altered. Immunoblotting revealed increased aortic expression of Giα-3 protein (+50%) and phosphorylated ERK1/2 (+90%) and decreased eNOS dimer/monomer ratio in ISO-treated WT mice. ISO enhanced the fluorescence response to dihydroethidium (+100%) in aortas from WT mice, indicating oxidative stress that was normalized by SOD, PTx and L-NAME. The ISO effects were abolished in β2KO mice., Conclusions: The β2-adrenoceptor/Giα signaling pathway is implicated in the enhanced vasoconstrictor response and eNOS uncoupling-mediated oxidative stress due to ISO treatment. Thus, long-term β2-AR activation might results in endothelial dysfunction.

Xamoterol, a partial β(1)-adrenergic receptor agonist, has been reported to impair the retrieval of hippocampus-dependent spatial reference memory in rats. In contrast, xamoterol restores memory retrieval in gene-targeted mice lacking norepinephrine (NE) and in a transgenic mouse model of Down syndrome in which NE levels are reduced. Restoration of retrieval by xamoterol in these two models complements the observation that NE and β(1) signaling are required for hippocampus-dependent retrieval of contextual and spatial reference memory in wild-type mice and rats. Additional evidence indicates that cAMP-mediated PKA and Epac signaling are required for the retrieval of hippocampus-dependent memory. As a result, we hypothesized that xamoterol has effects in addition to the stimulation of β(1) receptors that, at higher doses, act to counter the effects of β(1) signaling. Here we report that xamoterol-induced disruption of memory retrieval depends on β(2)-adrenergic receptor signaling. Interestingly, the impairment of memory retrieval by xamoterol is blocked by pretreatment with pertussis toxin, an uncoupling agent for G(i/o) signaling, suggesting that β(2) signaling opposes β(1) signaling during memory retrieval at the level of G protein and cAMP signaling. Finally, similar to the time-dependent roles for NE, β(1), and cAMP signaling in hippocampus-dependent memory retrieval, xamoterol only impairs retrieval for several days after training, indicating that its effects are also limited by the age of the memory. We conclude that the disruption of memory retrieval by xamoterol is mediated by G(i/o)-coupled β(2) signaling, which opposes the G(s)-coupled β(1) signaling that is transiently required for hippocampus-dependent emotional memory retrieval.

Sustained β-adrenergic receptors (βAR) activation leads to cardiac hypertrophy and prevents left ventricular (LV) atrophy during LV unloading. The immediate signaling pathways downstream from βAR stimulation, however, have not been well investigated. The current study was to examine the early cardiac signaling mechanism(s) following βAR stimulation. In adult C57BL/6 mice, acute βAR stimulation induced significant increases in PI3K activity and activation of Akt and ERK1/2 in the heart, but not in lungs or livers. In contrast, the same treatment did not elicit these changes in β(1)/β(2)AR double knockout mice. We further showed the specificity of β(2)AR in this crosstalk as treatment with formoterol, a β(2)AR-selective agonist, but not dobutamine, a predominantly β(1)AR agonist, activated cardiac Akt and ERK1/2. Acute βAR stimulation also significantly increased the phosphorylation of mTOR (the mammalian target of rapamycin), P70S6K, ribosomal protein S6, GSK-3α/β (glycogen synthase kinase-3α/β), and FOXO1/3a (the forkhead box family of transcription factors 1 and 3a). Moreover, acute βAR stimulation time-dependently decreased the mRNA levels of the muscle-specific E3 ligases atrogin-1 and muscle ring finger protein-1 (MuRF1) in mouse heart. Our results indicate that acute βAR stimulation in vivo affects multiple cardiac signaling cascades, including the PI3K signaling pathway, ERK1/2, atrogin-1 and MuRF1. These data 1) provide convincing evidence for the crosstalk between βAR and PI3K signaling pathways; 2) confirm the β(2)AR specificity in this crosstalk in vivo; and 3) identify novel signaling factors involved in cardiac hypertrophy and LV unloading. Understanding of the intricate interplay between β(2)AR activation and these signaling cascades should provide critical clues to the pathogenesis of cardiac hypertrophy and enable identification of targets for early clinical interaction of cardiac lesions.

Background and Purpose: The role of beta-adrenoceptors in heart disease remains controversial. Although beta-blockers ameliorate the progression of heart disease, the mechanism remains undefined. We investigated the effect of beta-adrenoceptors on cardiac hypertrophic growth using beta(1)- and beta(2)-adrenoreceptor knockout and wild-type (WT) mice., Experimental Approach: Mice were subjected to aortic banding or sham surgery, and their cardiac function was determined by echocardiography and micromanometry., Key Results: At 4 and 12 weeks after aortic banding, the left ventricle:body mass ratio was increased by 80-87% in wild-type mice, but only by 15% in knockouts, relative to sham-operated groups. Despite the blunted hypertrophic growth, ventricular function in knockouts was maintained. WT mice responded to pressure overload with up-regulation of gene expression of inflammatory cytokines and fibrogenic growth factors, and with severe cardiac fibrosis. All these effects were absent in the knockout animals., Conclusion and Implications: Our findings of a markedly attenuated cardiac hypertrophy and fibrosis following pressure overload in this knockout model emphasize that beta-adrenoceptor signalling plays a central role in cardiac hypertrophy and maladaptation following pressure overload.

This study examines the role of the beta2-adrenergic receptor (beta2-AR) in cardioprotection. The beta2-AR couples to Gs and Gi proteins. Gs activates PKA, which phosphorylates the receptor and switches beta2-AR coupling from Gs to Gi. Prior to 20 min of global ischemia, mouse hearts were either perfused for 30 min without treatment (control), treated with 10 nmol/L of isoproterenol (ISO) for 5 min followed by 5 min washout, or preconditioned with 4 cycles of 5 min ischemia and 5 min reflow (PC). Recovery of left ventricular developed pressure (LVDP) and infarct size were measured. Intermittent ISO treatment improved post-ischemic recovery of LVDP (58.5+/-4.8% vs. 22.0+/-6.3% in control) and reduced infarct size (31.0+/-2.4% vs. 53.0+/-4.6% in control). The Gi inhibitor pertussis toxin blocked the ISO-induced improvement in postischemic LVDP and infarct size. To test the role of beta2-AR in PC, we studied mice lacking beta2-AR (beta2-AR-/-) and found that PC had no effect on postischemic LVDP or infarct size in beta2-AR-/-. To test whether PKA is required for the PC and ISO-induced protection, hearts were treated with the PKA inhibitors PKI and H-89. We found that PKI and H-89 blocked the PC- and ISO-induced improvement in postischemic LVDP and infarct size. These data show an important role for beta2-AR in cardioprotection and support the novel hypothesis that preconditioning involves switching of beta2-AR coupling from Gs to Gi.

The sympathetic nervous system modulates cardiac contractility and rate by activating beta-adrenergic receptors (beta AR) expressed on cardiac myocytes and specialized cells in the sinoatrial node and the conduction system. Recent clinical studies have suggested that beta-adrenergic receptors also play a role in cardiac remodeling that occurs in the pathogenesis of cardiomyopathy. Both beta(1) and beta(2) adrenergic receptors are expressed in human and murine hearts. We have examined the effect of beta AR activation on the spontaneous contraction rate of neonatal myocyte cultures from wild-type and beta receptor knockout (KO) mice (beta(1)AR-KO, beta(2)AR-KO and beta(1)beta(2)AR-KO mice). Stimulation of the beta(1)AR in beta(2)AR-KO myocytes produces the greatest increase in contraction rate through a signaling pathway that requires protein kinase A (PKA) activation. In contrast, stimulation of the beta(2)AR in beta(1)AR-KO myocytes results in a biphasic effect on contraction rate with an initial increase in rate that does not require PKA, followed by a decrease in rate that involves coupling to a pertussis toxin sensitive G protein. A small isoproterenol-induced decrease in contraction rate observed in beta(1)beta(2)AR-KO myocytes can be attributed to the beta(3)AR. These studies show that all three beta AR subtypes are expressed in neonatal cardiac myocytes, and the beta(1)AR and beta(2)AR couple to distinct signaling pathways.

The second messenger 3':5'-cyclic adenosine monophosphate (cAMP) inhibits the proliferation of human B lymphocytes. In lymphoid malignancies, cAMP levels or the number of beta 2-adrenergic receptors seem to be decreased. In order to explore this phenomenon further, the function of the beta 2-adrenergic receptor complex was examined in mononuclear leucocytes (MNLs) from patients with B-cell chronic lymphocytic leukaemia (CLL). Peripheral blood MNLs from 25 CLL patients (16 male, nine female: aged 62 +/- 9 years) and 10 healthy volunteers (seven male, three female; aged 47 +/- 19 years) were used. The binding characteristics of beta 2-adrenergic receptors (beta 2-AR) on MNLs were determined by radioligand binding assays with [125I]-cyanopindolol ([125I]-CYP). The number of high-affinity binding sites for [125I]-CYP was significantly lower in CLL patients (313 +/- 300 sites per cell; mean +/- SD) than in control subjects (1479 +/- 1268 sites per cell). Moreover, the density of beta 2-AR decreased with disease progression, from Binet stage A (371 +/- 236, n = 13) to B (236 +/- 136, n = 7) and C (141 +/- 59, n = 5) (P < 0.05; Kruskal-Wallis analysis). Functional analyses of the beta 2-AR complex were performed by measuring the cellular cAMP content of MNLs in response to different stimulators. The cAMP production of MNLs upon isoprenaline stimulation (ISO; 10 min, 10(-4) mol L-1) was slightly lower in CLL patients (12.5 +/- 7.04 pmol 10(-6) cells) than in control subjects (15.91 +/- 10.08 pmol 10(-6) cells), and decreased with CLL progression (stage A 14 +/- 7; stage B 13.66 +/- 3.91; stage C 3.07 +/- 0.79 pmol 10(-6) cells). In contrast, cAMP accumulation in response to cholera toxin (CHO; 10(-4) gml-1, 120 min) was not different in control subjects (70.07 +/- 31.30 pmol 10(-6) cells) and CLL patients (stage A 95.24 +/- 123.07, stage B 70.76 +/- 57.37, stage C 33.21 +/- 33.73 pmol 10(-6) cells). When stimulated with forskolin (100 mumol L-1, 15 min), control MNLs produced about ten-fold more cAMP than CLL MNLs (188.56 +/- 92.53 vs. 17.88 +/- 10.32 pmol 10(-6) cells); this response was not stage dependent. Taken together, the results show that the beta 2-AR transmembrane signalling is impaired in CLL patients. The correlation of some beta 2-AR signalling defects with disease progression suggests that they may contribute to the disease progression of CLL patients.