A Cardiovascular Disease-Linked Gut Microbial Metabolite Acts via Adrenergic Receptors.

Using untargeted metabolomics (n = 1,162 subjects), the plasma metabolite (m/z = 265.1188) phenylacetylglutamine (PAGln) was discovered and then shown in an independent cohort (n = 4,000 subjects) to be associated with cardiovascular disease (CVD) and incident major adverse cardiovascular events (myocardial infarction, stroke, or death). A gut microbiota-derived metabolite, PAGln, was shown to enhance platelet activation-related phenotypes and thrombosis potential in whole blood, isolated platelets, and animal models of arterial injury. Functional and genetic engineering studies with human commensals, coupled with microbial colonization of germ-free mice, showed the microbial porA gene facilitates dietary phenylalanine conversion into phenylacetic acid, with subsequent host generation of PAGln and phenylacetylglycine (PAGly) fostering platelet responsiveness and thrombosis potential. Both gain- and loss-of-function studies employing genetic and pharmacological tools reveal PAGln mediates cellular events through G-protein coupled receptors, including α2A, α2B, and ß2-adrenergic receptors. PAGln thus represents a new CVD-promoting gut microbiota-dependent metabolite that signals via adrenergic receptors.

Sympathetic neuro-adipose connections mediate leptin-driven lipolysis.

Leptin is a hormone produced by the adipose tissue that acts in the brain, stimulating white fat breakdown. We find that the lipolytic effect of leptin is mediated through the action of sympathetic nerve fibers that innervate the adipose tissue. Using intravital two-photon microscopy, we observe that sympathetic nerve fibers establish neuro-adipose junctions, directly "enveloping" adipocytes. Local optogenetic stimulation of sympathetic inputs induces a local lipolytic response and depletion of white adipose mass. Conversely, genetic ablation of sympathetic inputs onto fat pads blocks leptin-stimulated phosphorylation of hormone-sensitive lipase and consequent lipolysis, as do knockouts of dopamine ß-hydroxylase, an enzyme required for catecholamine synthesis. Thus, neuro-adipose junctions are necessary and sufficient for the induction of lipolysis in white adipose tissue and are an efferent effector of leptin action. Direct activation of sympathetic inputs to adipose tissues may represent an alternative approach to induce fat loss, circumventing central leptin resistance. PAPERCLIP.

GDF15 promotes weight loss by enhancing energy expenditure in muscle.

Caloric restriction that promotes weight loss is an effective strategy for treating non-alcoholic fatty liver disease and improving insulin sensitivity in people with type 2 diabetes1. Despite its effectiveness, in most individuals, weight loss is usually not maintained partly due to physiological adaptations that suppress energy expenditure, a process known as adaptive thermogenesis, the mechanistic underpinnings of which are unclear2,3. Treatment of rodents fed a high-fat diet with recombinant growth differentiating factor 15 (GDF15) reduces obesity and improves glycaemic control through glial-cell-derived neurotrophic factor family receptor α-like (GFRAL)-dependent suppression of food intake4-7. Here we find that, in addition to suppressing appetite, GDF15 counteracts compensatory reductions in energy expenditure, eliciting greater weight loss and reductions in non-alcoholic fatty liver disease (NAFLD) compared to caloric restriction alone. This effect of GDF15 to maintain energy expenditure during calorie restriction requires a GFRAL-ß-adrenergic-dependent signalling axis that increases fatty acid oxidation and calcium futile cycling in the skeletal muscle of mice. These data indicate that therapeutic targeting of the GDF15-GFRAL pathway may be useful for maintaining energy expenditure in skeletal muscle during caloric restriction.

Mechanism of adrenergic CaV1.2 stimulation revealed by proximity proteomics.

Increased cardiac contractility during the fight-or-flight response is caused by ß-adrenergic augmentation of CaV1.2 voltage-gated calcium channels1-4. However, this augmentation persists in transgenic murine hearts expressing mutant CaV1.2 α1C and ß subunits that can no longer be phosphorylated by protein kinase A-an essential downstream mediator of ß-adrenergic signalling-suggesting that non-channel factors are also required. Here we identify the mechanism by which ß-adrenergic agonists stimulate voltage-gated calcium channels. We express α1C or ß2B subunits conjugated to ascorbate peroxidase5 in mouse hearts, and use multiplexed quantitative proteomics6,7 to track hundreds of proteins in the proximity of CaV1.2. We observe that the calcium-channel inhibitor Rad8,9, a monomeric G protein, is enriched in the CaV1.2 microenvironment but is depleted during ß-adrenergic stimulation. Phosphorylation by protein kinase A of specific serine residues on Rad decreases its affinity for ß subunits and relieves constitutive inhibition of CaV1.2, observed as an increase in channel open probability. Expression of Rad or its homologue Rem in HEK293T cells also imparts stimulation of CaV1.3 and CaV2.2 by protein kinase A, revealing an evolutionarily conserved mechanism that confers adrenergic modulation upon voltage-gated calcium channels.

ß-Adrenergic Receptors and Adipose Tissue Metabolism: Evolution of an Old Story.

The role of ß-adrenergic receptors (ßARs) in adipose tissue to promote lipolysis and the release of fatty acids and nonshivering thermogenesis in brown fat has been studied for so many decades that one would think there is nothing left to discover. With the rediscovery of brown fat in humans and renewed interest in UCP1 and uncoupled mitochondrial respiration, it seems that a review of adipose tissue as an organ, pivotal observations, and the investigators who made them would be instructive to understanding where the field stands now. The discovery of the ß3-adrenergic receptor was important for accurately defining the pharmacology of the adipocyte, while the clinical targeting of this receptor for obesity and metabolic disease has had its highs and lows. Many questions still remain about how ßARs regulate adipocyte metabolism and the signaling molecules through which they do it.

Astrocyte ß-Adrenergic Receptor Activity Regulates NMDA Receptor Signaling of Medial Prefrontal Cortex Pyramidal Neurons.

Glutamate spillover from the synapse is tightly regulated by astrocytes, limiting the activation of extrasynaptically located NMDA receptors (NMDAR). The processes of astrocytes are dynamic and can modulate synaptic physiology. Though norepinephrine (NE) and ß-adrenergic receptor (ß-AR) activity can modify astrocyte volume, this has yet to be confirmed outside of sensory cortical areas, nor has the effect of noradrenergic signaling on glutamate spillover and neuronal NMDAR activity been explored. We monitored changes to astrocyte process volume in response to noradrenergic agonists in the medial prefrontal cortex of male and female mice. Both NE and the ß-AR agonist isoproterenol (ISO) increased process volume by ∼20%, significantly higher than changes seen when astrocytes had G-protein signaling blocked by GDPßS. We measured the effect of ß-AR signaling on evoked NMDAR currents. While ISO did not affect single stimulus excitatory currents of Layer 5 pyramidal neurons, ISO reduced NMDAR currents evoked by 10 stimuli at 50 Hz, which elicits glutamate spillover, by 18%. After isolating extrasynaptic NMDARs by blocking synaptic NMDARs with the activity-dependent NMDAR blocker MK-801, ISO similarly reduced extrasynaptic NMDAR currents in response to 10 stimuli by 18%. Finally, blocking ß-AR signaling in the astrocyte network by loading them with GDPßS reversed the ISO effect on 10 stimuli-evoked NMDAR currents. These results demonstrate that astrocyte ß-AR activity reduces extrasynaptic NMDAR recruitment, suggesting that glutamate spillover is reduced.

Hypoxia Sensing of ß-Adrenergic Receptor Is Regulated by Endosomal PI3Kγ.

BACKGROUND: Impaired beta-adrenergic receptor (ß1 and ß2AR) function following hypoxia underlies ischemic heart failure/stroke. Activation of PI3Kγ (phosphoinositide 3-kinase γ) by beta-adrenergic receptor leads to feedback regulation of the receptor by hindering beta-adrenergic receptor dephosphorylation through inhibition of PP2A (protein phosphatase 2A). However, little is known about PI3Kγ feedback mechanism in regulating hypoxia-mediated ß1 and ß2AR dysfunction and cardiac remodeling. METHODS: Human embryonic kidney 293 cells or mouse adult cardiomyocytes and C57BL/6 (WT) or PI3Kγ knockout (KO) mice were subjected to hypoxia. Cardiac plasma membranes and endosomes were isolated and evaluated for ß1 and ß2AR density and function, PI3Kγ activity and ß1 and ß2AR-associated PP2A activity. Metabolic labeling was performed to assess ß1 and ß2AR phosphorylation and epinephrine/norepinephrine levels measured post-hypoxia. RESULTS: Hypoxia increased ß1 and ß2AR phosphorylation, reduced cAMP, and led to endosomal accumulation of phosphorylated ß2ARs in human embryonic kidney 293 cells and WT cardiomyocytes. Acute hypoxia in WT mice resulted in cardiac remodeling and loss of adenylyl cyclase activity associated with increased ß1 and ß2AR phosphorylation. This was agonist-independent as plasma and cardiac epinephrine and norepinephrine levels were unaltered. Unexpectedly, PI3Kγ activity was selectively increased in the endosomes of human embryonic kidney 293 cells and WT hearts post-hypoxia. Endosomal ß1- and ß2AR-associated PP2A activity was inhibited upon hypoxia in human embryonic kidney 293 cells and WT hearts showing regulation of beta-adrenergic receptors by PI3Kγ. This was accompanied with phosphorylation of endogenous inhibitor of protein phosphatase 2A whose phosphorylation by PI3Kγ inhibits PP2A. Increased ß1 and ß2AR-associated PP2A activity, decreased beta-adrenergic receptor phosphorylation, and normalized cardiac function was observed in PI3Kγ KO mice despite hypoxia. Compared to WT, PI3Kγ KO mice had preserved cardiac response to challenge with ß1AR-selective agonist dobutamine post-hypoxia. CONCLUSIONS: Agonist-independent activation of PI3Kγ underlies hypoxia sensing as its ablation leads to reduction in ß1- and ß2AR phosphorylation and amelioration of cardiac dysfunction.

ß3AR-Dependent Brain-Derived Neurotrophic Factor (BDNF) Generation Limits Chronic Postischemic Heart Failure.

BACKGROUND: Loss of brain-derived neurotrophic factor (BDNF)/TrkB (tropomyosin kinase receptor B) signaling accounts for brain and cardiac disorders. In neurons, ß-adrenergic receptor stimulation enhances local BDNF expression. It is unclear if this occurs in a pathophysiological relevant manner in the heart, especially in the ß-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. METHODS: 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, ß3AR 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). RESULTS: 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 ß3AR-agonist, BRL-37344, increased myocyte BDNF content, while ß3AR signaling underscored BDNF generation/protection in post-MI hearts. Accordingly, the ß1AR blocker, metoprolol, via upregulated ß3ARs, 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. CONCLUSIONS: BDNF loss underscores chronic postischemic heart failure. TrkB agonists can improve ischemic LV dysfunction via replenished myocardial BDNF content. Direct cardiac ß3AR stimulation, or ß-blockers (via upregulated ß3AR), is another BDNF-based means to fend off chronic postischemic heart failure.

Functional LTCC-ß2AR Complex Needs Caveolin-3 and Is Disrupted in Heart Failure.

BACKGROUND: Beta-2 adrenergic receptors (ß2ARs) but not beta-2 adrenergic receptors (ß1ARs) form a functional complex with L-type Ca2+ channels (LTCCs) on the cardiomyocyte membrane. However, how microdomain localization in the plasma membrane affects the function of these complexes is unknown. We aim to study the coupling between LTCC and ß adrenergic receptors in different cardiomyocyte microdomains, the distinct involvement of PKA and CAMKII (Ca2+/calmodulin-dependent protein kinase II) and explore how this functional complex is disrupted in heart failure. METHODS: Global signaling between LTCCs and ß adrenergic receptors was assessed with whole-cell current recordings and western blot analysis. Super-resolution scanning patch-clamp was used to explore the local coupling between single LTCCs and ß1AR or ß2AR in different membrane microdomains in control and failing cardiomyocytes. RESULTS: LTCC open probability (Po) showed an increase from 0.054±0.003 to 0.092±0.008 when ß2AR was locally stimulated in the proximity of the channel (<350 nm) in the transverse tubule microdomain. In failing cardiomyocytes, from both rodents and humans, this transverse tubule coupling between LTCC and ß2AR was lost. Interestingly, local stimulation of ß1AR did not elicit any change in the Po of LTCCs, indicating a lack of proximal functional interaction between the two, but we confirmed a general activation of LTCC via ß1AR. By using blockers of PKA and CaMKII and a Caveolin-3-knockout mouse model, we conclude that the ß2AR-LTCC regulation requires the presence of caveolin-3 and the activation of the CaMKII pathway. By contrast, at a cellular "global" level PKA plays a major role downstream ß1AR and results in an increase in LTCC current. CONCLUSIONS: Regulation of the LTCC activity by proximity coupling mechanisms occurs only via ß2AR, but not ß1AR. This may explain how ß2ARs tune the response of LTCCs to adrenergic stimulation in healthy conditions. This coupling is lost in heart failure; restoring it could improve the adrenergic response of failing cardiomyocytes.

Nitric Oxide Modulates Ca2+ Leak and Arrhythmias via S-Nitrosylation of CaMKII.

BACKGROUND: Nitric oxide (NO) has been identified as a signaling molecule generated during ß-adrenergic receptor stimulation in the heart. Furthermore, a role for NO in triggering spontaneous Ca2+ release via S-nitrosylation of CaMKIIδ (Ca2+/calmodulin kinase II delta) is emerging. NO donors are routinely used clinically for their cardioprotective effects on the heart, but it is unknown how NO donors modulate the proarrhythmic CaMKII to alter cardiac arrhythmia incidence. We test the role of S-nitrosylation of CaMKIIδ at the Cysteine-273 inhibitory site and cysteine-290 activating site in cardiac Ca2+ handling and arrhythmogenesis before and during ß-adrenergic receptor stimulation. METHODS: We measured Ca2+-handling in isolated cardiomyocytes from C57BL/6J wild-type (WT) mice and mice lacking CaMKIIδ expression (CaMKIIδ-KO) or with deletion of the S-nitrosylation site on CaMKIIδ at cysteine-273 or cysteine-290 (CaMKIIδ-C273S and -C290A knock-in mice). Cardiomyocytes were exposed to NO donors, S-nitrosoglutathione (GSNO; 150 µM), sodium nitroprusside (200 µM), and ß-adrenergic agonist isoproterenol (100 nmol/L). RESULTS: Both WT and CaMKIIδ-KO cardiomyocytes responded to isoproterenol with a full inotropic and lusitropic Ca2+ transient response as well as increased Ca2+ spark frequency. However, the increase in Ca2+ spark frequency was significantly attenuated in CaMKIIδ-KO cardiomyocytes. The protection from isoproterenol-induced Ca2+ sparks and waves was mimicked by GSNO pretreatment in WT cardiomyocytes but lost in CaMKIIδ-C273S cardiomyocytes. When GSNO was applied after isoproterenol, this protection was not observed in WT or CaMKIIδ-C273S but was apparent in CaMKIIδ-C290A. In Langendorff-perfused isolated hearts, GSNO pretreatment limited isoproterenol-induced arrhythmias in WT but not CaMKIIδ-C273S hearts, while GSNO exposure after isoproterenol sustained or exacerbated arrhythmic events. CONCLUSIONS: We conclude that prior S-nitrosylation of CaMKIIδ at cysteine-273 can limit subsequent ß-adrenergic receptor-induced arrhythmias, but that S-nitrosylation at cysteine-290 might worsen or sustain ß-adrenergic receptor-induced arrhythmias. This has important implications for the administration of NO donors in the clinical setting.

Meningococcus Hijacks a ß2-adrenoceptor/ß-Arrestin pathway to cross brain microvasculature endothelium.

Following pilus-mediated adhesion to human brain endothelial cells, meningococcus (N. meningitidis), the bacterium causing cerebrospinal meningitis, initiates signaling cascades, which eventually result in the opening of intercellular junctions, allowing meningeal colonization. The signaling receptor activated by the pathogen remained unknown. We report that N. meningitidis specifically stimulates a biased ß2-adrenoceptor/ß-arrestin signaling pathway in endothelial cells, which ultimately traps ß-arrestin-interacting partners, such as the Src tyrosine kinase and junctional proteins, under bacterial colonies. Cytoskeletal reorganization mediated by ß-arrestin-activated Src stabilizes bacterial adhesion to endothelial cells, whereas ß-arrestin-dependent delocalization of junctional proteins results in anatomical gaps used by bacteria to penetrate into tissues. Activation of ß-adrenoceptor endocytosis with specific agonists prevents signaling events downstream of N. meningitidis adhesion and inhibits bacterial crossing of the endothelial barrier. The identification of the mechanism used for hijacking host cell signaling machineries opens perspectives for treatment and prevention of meningococcal infection.

Environmental and genetic activation of a brain-adipocyte BDNF/leptin axis causes cancer remission and inhibition.

Cancer is influenced by its microenvironment, yet broader, environmental effects also play a role but remain poorly defined. We report here that mice living in an enriched housing environment show reduced tumor growth and increased remission. We found this effect in melanoma and colon cancer models, and that it was not caused by physical activity alone. Serum from animals held in an enriched environment (EE) inhibited cancer proliferation in vitro and was markedly lower in leptin. Hypothalamic brain-derived neurotrophic factor (BDNF) was selectively upregulated by EE, and its genetic overexpression reduced tumor burden, whereas BDNF knockdown blocked the effect of EE. Mechanistically, we show that hypothalamic BDNF downregulated leptin production in adipocytes via sympathoneural beta-adrenergic signaling. These results suggest that genetic or environmental activation of this BDNF/leptin axis may have therapeutic significance for cancer.

Thermal stress induces glycolytic beige fat formation via a myogenic state.

Environmental cues profoundly affect cellular plasticity in multicellular organisms. For instance, exercise promotes a glycolytic-to-oxidative fibre-type switch in skeletal muscle, and cold acclimation induces beige adipocyte biogenesis in adipose tissue. However, the molecular mechanisms by which physiological or pathological cues evoke developmental plasticity remain incompletely understood. Here we report a type of beige adipocyte that has a critical role in chronic cold adaptation in the absence of ß-adrenergic receptor signalling. This beige fat is distinct from conventional beige fat with respect to developmental origin and regulation, and displays enhanced glucose oxidation. We therefore refer to it as glycolytic beige fat. Mechanistically, we identify GA-binding protein α as a regulator of glycolytic beige adipocyte differentiation through a myogenic intermediate. Our study reveals a non-canonical adaptive mechanism by which thermal stress induces progenitor cell plasticity and recruits a distinct form of thermogenic cell that is required for energy homeostasis and survival.

Loss of adipose TET proteins enhances ß-adrenergic responses and protects against obesity by epigenetic regulation of ß3-AR expression.

ß-adrenergic receptor (ß-AR) signaling plays predominant roles in modulating energy expenditure by triggering lipolysis and thermogenesis in adipose tissue, thereby conferring obesity resistance. Obesity is associated with diminished ß3-adrenergic receptor (ß3-AR) expression and decreased ß-adrenergic responses, but the molecular mechanism coupling nutrient overload to catecholamine resistance remains poorly defined. Ten-eleven translocation (TET) proteins are dioxygenases that alter the methylation status of DNA by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine and further oxidized derivatives. Here, we show that TET proteins are pivotal epigenetic suppressors of ß3-AR expression in adipocytes, thereby attenuating the responsiveness to ß-adrenergic stimulation. Deletion of all three Tet genes in adipocytes led to increased ß3-AR expression and thereby enhanced the downstream ß-adrenergic responses, including lipolysis, thermogenic gene induction, oxidative metabolism, and fat browning in vitro and in vivo. In mouse adipose tissues, Tet expression was elevated after mice ate a high-fat diet. Mice with adipose-specific ablation of all TET proteins maintained higher levels of ß3-AR in both white and brown adipose tissues and remained sensitive to ß-AR stimuli under high-fat diet challenge, leading to augmented energy expenditure and decreased fat accumulation. Consequently, they exhibited improved cold tolerance and were substantially protected from diet-induced obesity, inflammation, and metabolic complications, including insulin resistance and hyperlipidemia. Mechanistically, TET proteins directly repressed ß3-AR transcription, mainly in an enzymatic activity-independent manner, and involved the recruitment of histone deacetylases to increase deacetylation of its promoter. Thus, the TET-histone deacetylase-ß3-AR axis could be targeted to treat obesity and related metabolic diseases.

An immune-sympathetic neuron communication axis guides adipose tissue browning in cancer-associated cachexia.

Cancer-associated cachexia (CAC) is a hypermetabolic syndrome characterized by unintended weight loss due to the atrophy of adipose tissue and skeletal muscle. A phenotypic switch from white to beige adipocytes, a phenomenon called browning, accelerates CAC by increasing the dissipation of energy as heat. Addressing the mechanisms of white adipose tissue (WAT) browning in CAC, we now show that cachexigenic tumors activate type 2 immunity in cachectic WAT, generating a neuroprotective environment that increases peripheral sympathetic activity. Increased sympathetic activation, in turn, results in increased neuronal catecholamine synthesis and secretion, ß-adrenergic activation of adipocytes, and induction of WAT browning. Two genetic mouse models validated this progression of events. 1) Interleukin-4 receptor deficiency impeded the alternative activation of macrophages, reduced sympathetic activity, and restrained WAT browning, and 2) reduced catecholamine synthesis in peripheral dopamine ß-hydroxylase (DBH)-deficient mice prevented cancer-induced WAT browning and adipose atrophy. Targeting the intraadipose macrophage-sympathetic neuron cross-talk represents a promising therapeutic approach to ameliorate cachexia in cancer patients.

ß-Adrenergic Signaling Promotes Morphological Maturation of Astrocytes in Female Mice.

Astrocytes play essential roles in the developing nervous system, including supporting synapse function. These astrocyte support functions emerge coincident with brain maturation and may be tailored in a region-specific manner. For example, gray matter astrocytes have elaborate synapse-associated processes and are morphologically and molecularly distinct from white matter astrocytes. This raises the question of whether there are unique environmental cues that promote gray matter astrocyte identity and synaptogenic function. We previously identified adrenergic receptors as preferentially enriched in developing gray versus white matter astrocytes, suggesting that noradrenergic signaling could be a cue that promotes the functional maturation of gray matter astrocytes. We first characterized noradrenergic projections during postnatal brain development in mouse and human, finding that process density was higher in the gray matter and increased concurrently with astrocyte maturation. RNA sequencing revealed that astrocytes in both species expressed α- and ß-adrenergic receptors. We found that stimulation of ß-adrenergic receptors increased primary branching of rodent astrocytes in vitro Conversely, astrocyte-conditional knockout of the ß1-adrenergic receptor reduced the size of gray matter astrocytes and led to dysregulated sensorimotor integration in female mice. These studies suggest that adrenergic signaling to developing astrocytes impacts their morphology and has implications for adult behavior, particularly in female animals. More broadly, they demonstrate a mechanism through which environmental cues impact astrocyte development. Given the key roles of norepinephrine in brain states, such as arousal, stress, and learning, these findings could prompt further inquiry into how developmental stressors impact astrocyte development and adult brain function.SIGNIFICANCE STATEMENT This study demonstrates a role for noradrenergic signaling in the development of gray matter astrocytes. We provide new evidence that the ß1-adrenergic receptor is robustly expressed by both mouse and human astrocytes, and that conditional KO of the ß1-adrenergic receptor from female mouse astrocytes impairs gray matter astrocyte maturation. Moreover, female conditional KO mice exhibit behavioral deficits in two paradigms that test sensorimotor function. Given the emerging interest in moving beyond RNA sequencing to probe specific pathways that underlie astrocyte heterogeneity, this study provides a foundation for future investigation into the effect of noradrenergic signaling on astrocyte functions in conditions where noradrenergic signaling is altered, such as stress, arousal, and learning.

Mechanism and application of ß-adrenoceptor blockers in soft tissue wound healing.

Soft tissue damage stimulates sympathetic nerves to release large amounts of catecholamine hormones which bind to ß-adrenergic receptors (ß-ARs) on the cell membrane surface. It activates the downstream effector molecules and impairs soft tissue wound healing. ß-blockers specifically inhibit ß-ARs activation in acute/chronic skin lesions and ulcerative hemangiomas. They also accelerate soft tissue wound healing by shortening the duration of inflammation, speeding keratinocyte migration and reepithelialization, promoting wound contraction and angiogenesis, and inhibiting bacterial virulence effects. In addition, ß-blockers shorten wound healing periods in patients with severe thermal damage by reducing the hypermetabolic response. While ß-blockers promote/inhibit corneal epithelial cell regeneration and restores limbal stem/progenitor cells function, it could well accelerate/delay corneal wound healing. Given these meaningful effects, a growing number of studies are focused on examining the efficacy and safety of ß-blockers in soft tissue wound repair, including acute and chronic wounds, severe thermal damage, ulcerated infantile hemangioma, corneal wounds, and other soft tissue disorders. However, an intensive investigation on their acting mechanisms is imperatively needed. The purpose of this article is to summerize the roles of ß-blockers in soft tissue wound healing and explore their clinical applications.

ß-Adrenergic signaling drives structural and functional maturation of mouse cardiomyocytes.

Cardiac maturation represents the last phase of heart development and is characterized by morphofunctional alterations that optimize the heart for efficient pumping. Its understanding provides important insights into cardiac regeneration therapies. Recent evidence implies that adrenergic signals are involved in the regulation of cardiac maturation, but the mechanistic underpinnings involved in this process are poorly understood. Herein, we explored the role of ß-adrenergic receptor (ß-AR) activation in determining structural and functional components of cardiomyocyte maturation. Temporal characterization of tyrosine hydroxylase and norepinephrine levels in the mouse heart revealed that sympathetic innervation develops during the first 3 wk of life, concurrent with the rise in ß-AR expression. To assess the impact of adrenergic inhibition on maturation, we treated mice with propranolol, isolated cardiomyocytes, and evaluated morphofunctional parameters. Propranolol treatment reduced heart weight, cardiomyocyte size, and cellular shortening, while it increased the pool of mononucleated myocytes, resulting in impaired maturation. No changes in t-tubules were observed in cells from propranolol mice. To establish a causal link between ß-AR signaling and cardiomyocyte maturation, mice were subjected to sympathectomy, followed or not by restoration with isoproterenol treatment. Cardiomyocytes from sympathectomyzed mice recapitulated the salient immaturity features of propranolol-treated mice, with the additional loss of t-tubules. Isoproterenol rescued the maturation deficits induced by sympathectomy, except for the t-tubule alterations. Our study identifies the ß-AR stimuli as a maturation promoting signal and implies that this pathway can be modulated to improve cardiac regeneration therapies.NEW & NOTEWORTHY Maturation involves a series of morphofunctional alterations vital to heart development. Its regulatory mechanisms are only now being unveiled. Evidence implies that adrenergic signaling regulates cardiac maturation, but the mechanisms are poorly understood. To address this point, we blocked ß-ARs or performed sympathectomy followed by rescue experiments with isoproterenol in neonatal mice. Our study identifies the ß-AR stimuli as a maturation signal for cardiomyocytes and highlights the importance of this pathway in cardiac regeneration therapies.

Stimulating ß-adrenergic receptors promotes synaptic potentiation by switching CaMKII movement from LTD to LTP mode.

Learning, memory, and cognition are thought to require synaptic plasticity, specifically including hippocampal long-term potentiation and depression (LTP and LTD). LTP versus LTD is induced by high-frequency stimulation versus low-frequency, but stimulating ß-adrenergic receptors (ßARs) enables LTP induction also by low-frequency stimulation (1 Hz) or theta frequencies (∼5 Hz) that do not cause plasticity by themselves. In contrast to high-frequency stimulation-LTP, such ßAR-LTP requires Ca2+-flux through L-type voltage-gated Ca2+-channels, not N-methyl-D-aspartate-type glutamate receptors. Surprisingly, we found that ßAR-LTP still required a nonionotropic scaffolding function of the N-methyl-D-aspartate-type glutamate receptor: the stimulus-induced binding of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) to its GluN2B subunit that mediates CaMKII movement to excitatory synapses. In hippocampal neurons, ß-adrenergic stimulation with isoproterenol (Iso) transformed LTD-type CaMKII movement to LTP-type movement, resulting in CaMKII movement to excitatory instead of inhibitory synapses. Additionally, Iso enabled induction of a major cell-biological feature of LTP in response to LTD stimuli: increased surface expression of GluA1 fused with super-ecliptic pHluorein. Like for ßAR-LTP in hippocampal slices, the Iso effects on CaMKII movement and surface expression of GluA1 fused with super-ecliptic pHluorein involved L-type Ca2+-channels and specifically required ß2-ARs. Taken together, these results indicate that Iso transforms LTD stimuli to LTP signals by switching CaMKII movement and GluN2B binding to LTP mode.

The mechanism of 25-hydroxycholesterol-mediated suppression of atrial ß1-adrenergic responses.

25-Hydroxycholesterol (25HC) is a biologically active oxysterol, whose production greatly increases during inflammation by macrophages and dendritic cells. The inflammatory reactions are frequently accompanied by changes in heart regulation, such as blunting of the cardiac ß-adrenergic receptor (AR) signaling. Here, the mechanism of 25HC-dependent modulation of responses to ß-AR activation was studied in the atria of mice. 25HC at the submicromolar levels decreased the ß-AR-mediated positive inotropic effect and enhancement of the Ca2+ transient amplitude, without changing NO production. Positive inotropic responses to ß1-AR (but not ß2-AR) activation were markedly attenuated by 25HC. The depressant action of 25HC on the ß1-AR-mediated responses was prevented by selective ß3-AR antagonists as well as inhibitors of Gi protein, Gßγ, G protein-coupled receptor kinase 2/3, or ß-arrestin. Simultaneously, blockers of protein kinase D and C as well as a phosphodiesterase inhibitor did not preclude the negative action of 25HC on the inotropic response to ß-AR activation. Thus, 25HC can suppress the ß1-AR-dependent effects via engaging ß3-AR, Gi protein, Gßγ, G protein-coupled receptor kinase, and ß-arrestin. This 25HC-dependent mechanism can contribute to the inflammatory-related alterations in the atrial ß-adrenergic signaling.