βAR Stimulation Research Articles

Crosstalk between β2- and α2-Adrenergic Receptors in the Regulation of B16F10 Melanoma Cell Proliferation.

Adrenergic receptors (AR) belong to the G protein-coupled receptor superfamily and regulate migration and proliferation in various cell types. The objective of this study was to evaluate whether β-AR stimulation affects the antiproliferative action of α2-AR agonists on B16F10 cells and, if so, to determine the relative contribution of β-AR subtypes. Using pharmacological approaches, evaluation of Ki-67 expression by flow cytometry and luciferase-based cAMP assay, we found that treatment with isoproterenol, a β-AR agonist, increased cAMP levels in B16F10 melanoma cells without affecting cell proliferation. Propranolol inhibited the cAMP response to isoproterenol. In addition, stimulation of α2-ARs with agonists such as clonidine, a well-known antihypertensive drug, decreased cancer cell proliferation. This effect on cell proliferation was suppressed by treatment with isoproterenol. In turn, the suppressive effects of isoproterenol were abolished by the treatment with either ICI 118,551, a β2-AR antagonist, or propranolol, suggesting that isoproterenol effects are mainly mediated by the β2-AR stimulation. We conclude that the crosstalk between the β2-AR and α2-AR signaling pathways regulates the proliferative activity of B16F10 cells and may therefore represent a therapeutic target for melanoma therapy.

Altered Cav1.2 trafficking contributes to β-adrenergic receptor hypo-responsivity in the aging myocardium

Progressive, age-associated CaV1.2 hypo-responsivity to β-adrenergic receptor (βAR)-stimulation occurs in both human and animal hearts, reducing inotropic reserve and diminishing their capacity to manage episodes of acute stress. Disruption of this crucial regulatory mechanism lowers the threshold for heart failure, yet the causal links between aging and βAR hypo-responsivity remain unclear. We have previously reported that a pre-synthesized pool of sub-sarcolemmal CaV1.2-containing vesicles/endosomes resides in cardiomyocytes and can be mobilized to the sarcolemma in response to βAR stimulation to tune EC-coupling.

Evaluation of β-adrenergic ligands for development of pharmacological heart failure and transparency models in zebrafish

Cardiovascular toxicity represents one of the most common reasons for clinical trial failure. Consequently, early identification of novel cardioprotective strategies could prevent the later-stage drug-induced cardiac side effects. The use of zebrafish (Danio rerio) in preclinical studies has greatly increased. High-throughput and low-cost of assays make zebrafish model ideal for initial drug discovery. A common strategy to induce heart failure is a chronic β-adrenergic (βAR) stimulation. Herein, we set out to test a panel of βAR agonists to develop a pharmacological heart failure model in zebrafish. We assessed βAR agonists with respect to the elicited mortality, changes in heart rate, and morphological alterations in zebrafish larvae according to Fish Embryo Acute Toxicity Test. Among the tested βAR agonists, epinephrine elicited the most potent onset of heart stimulation (EC50 = 0.05 mM), which corresponds with its physiological role as catecholamine. However, when used at ten-fold higher dose (0.5 mM), the same compound caused severe heart rate inhibition (−28.70 beats/min), which can be attributed to its cardiotoxicity. Further studies revealed that isoetharine abolished body pigmentation at the sublethal dose of 7.50 mM. Additionally, as a proof of concept that zebrafish can mimic human cardiac physiology, we tested βAR antagonists (propranolol, carvedilol, metoprolol, and labetalol) and verified that they inhibited fish heart rate in a similar fashion as in humans. In conclusion, we proposed two novel pharmacological models in zebrafish; i.e., epinephrine-dependent heart failure and isoetharine-dependent transparent zebrafish. We provided strong evidence that the zebrafish model constitutes a valuable tool for cardiovascular research.

Abstract 12089: Dual Actions of β 2 AR-Agonism Confer Protection Against Heart Failure and Renal Dysfunction via Inotropic and Lusitropic Effects and Normalized Cholesterol Homeostasis in a Mouse Model of Alport Syndrome

Introduction: Col4a3-/- Alport mice present a model of heart failure with preserved ejection fraction (HFpEF) secondary to chronic kidney disease(CKD) wherein etiological relationships have been established between hypertension, pulmonary edema, inflammation, cardiac hypertrophy and fibrosis, diastolic dysfunction and underlying abnormalities of elevated low-density lipoprotein receptor (LDLR) expression, excess LDL-cholesterol(LDL-C) accumulation, and mitochondrial dysfunction in renal tubules. Hypothesis: We tested the hypothesis that selective β2-Adrenoceptor (β2AR) modulation with salbutamol, a short-acting β2AR agonist, could alleviate symptoms of CKD and simultaneously augment cardiac function. Methods: Alport mice were injected i.p.with salbutamol or DMSO vehicle as a single bolus of 200μg/dose in short-term studies or daily with 100 μg/dose for 2 wks long-term. Cardiac and renal functions, cAMP levels, in vivo renal tubular LDL-C uptake and renal histology were evaluated post-injection. N=3-8 for all experiments. Results: Short-term, salbutamol improved renal function in parallel with decreased LDLR levels and reduced uptake of LDL-C into renal tubules. Long-term, cardiac diastolic function assessed by isovolumetric relaxation time, filling pressures, and myocardial performance index, and systolic function reflected by ejection fraction, stroke volume and cardiac output improved significantly in parallel with increased cardiac cAMP (p<.05). Mechanistically, in the kidney, salbutamol activated IDOL and hence lysosomal ubiquitination and degradation of LDLR via a novel β2AR-mediated, cAMP-independent pathway involving the Rac1/Cdc42 β1PixGEF. β1Pix reversibly sequesters IDOL into a complex with LDLR, thereby blocking the degradation pathway. β2AR stimulation dissipates the complex reactivating IDOL-mediated LDLR degradation thereby re-establishing LDL-C homeostasis and renal function. Using flow cytometry in 293T cells, ectopic expression of bPix stabilized membrane LDLR, sensitive to IDOL- but not PCSK-mediated degradation. Conclusions: β2AR agonism represents a potential treatment strategy to alleviate progression of CKD and HF associated with HFpEF phenogroup 3.

Immune cell β2-adrenergic receptors contribute to the development of heart failure.

β-Adrenergic receptors (βARs) regulate normal and pathophysiological heart function through their impact on contractility. βARs are also regulators of immune function where they play a unique role depending on the disease condition and immune cell type. Emerging evidence suggests an important role for the β2AR subtype in regulating remodeling in the pathological heart; however, the importance of these responses has never been examined. In heart failure, catecholamines are elevated, leading to chronic βAR activation and contributing to the detrimental effects in the heart. We hypothesized that immune cell β2AR plays a critical role in the development of heart failure in response to chronic catecholamine elevations through their regulation of immune cell infiltration. To test this, chimeric mice were generated by performing bone marrow transplant (BMT) experiments using wild-type (WT) or β2AR knockout (KO) donors. WT and β2ARKO BMT mice were chronically administered the βAR agonist isoproterenol. Immune cell recruitment to the heart was examined by histology and flow cytometry. Numerous changes in immune cell recruitment were observed with isoproterenol administration in WT BMT mice including proinflammatory myeloid populations and lymphocytes with macrophages made up the majority of immune cells in the heart and which were absent in β2ARKO BMT animal. β2ARKO BMT mice had decreased cardiomyocyte death, hypertrophy, and interstitial fibrosis following isoproterenol treatment, culminating in improved function. These findings demonstrate an important role for immune cell β2AR expression in the heart's response to chronically elevated catecholamines.NEW & NOTEWORTHY Immune cell β2-adrenergic receptors (β2ARs) are important for proinflammatory macrophage infiltration to the heart in a chronic isoproterenol administration model of heart failure. Mice lacking immune cell β2AR have decreased immune cell infiltration to their heart, primarily proinflammatory macrophage populations. This decrease culminated to decreased cardiac injury with lessened cardiomyocyte death, decreased interstitial fibrosis and hypertrophy, and improved function demonstrating that β2AR regulation of immune responses plays an important role in the heart's response to persistent βAR stimulation.

Isothermal microcalorimetry measures UCP1-mediated thermogenesis in mature brite adipocytes

The activation of thermogenesis in adipose tissue has emerged as an important target for the development of novel anti-obesity therapies. Using multi-well isothermal microcalorimetry, we have demonstrated that mature murine brown and brite adipocytes produce quantifiable heat upon β3-AR stimulation, independently of any anaerobic mechanisms. Additionally, in brite adipocytes lacking UCP1 protein, β3-AR stimulation still induces heat production, albeit to a much lower extent than in their wildtype counterparts, suggesting that UCP1 is an essential component of adrenergic induced thermogenesis in murine brite adipocytes exvivo. Similarly, we could observe an increase in heat production in human-derived adipocytes (hMADS) upon β-AR stimulation. Collectively, these results establish the use of isothermal microcalorimetry as a sensitive and accurate technique for measuring thermogenic responses in intact mature brite adipocytes from murine and human origin.

PDE1 Inhibition Modulates Cav1.2 Channel to Stimulate Cardiomyocyte Contraction.

[Figure: see text].

Abstract MP257: Dual Actions Of β 2 Ar-agonism Confer Protection Against Heart Failure And Renal Dysfunction Via Inotropic And Lusitropic Effects And Normalized Cholesterol Homeostasis In A Mouse Model Of Alport Syndrome

Background: Col4a3 -/- Alport mice present a model of heart failure with preserved ejection fraction (HFpEF) secondary to chronic kidney disease (CKD) wherein etiological relationships have been established between hypertension, pulmonary edema, inflammation, cardiac hypertrophy and fibrosis, diastolic dysfunction and underlying abnormalities of elevated low-density lipoprotein receptor (LDLR) expression, excess LDL-cholesterol (LDL-C) accumulation, and mitochondrial dysfunction in renal tubules. HFpEF is characteristically unresponsive to pharmacological intervention. Here, we tested the hypothesis that selective β 2 -Adrenoceptor (β 2 AR) modulation with salbutamol, a short-acting β 2 AR agonist, could alleviate symptoms of CKD and simultaneously augment cardiac function. Secondarily, we investigated the mechanism of actions of such β 2 AR-mediated therapeutics on cardiac and renal functions. Methods: Alport mice were injected intraperitoneally with salbutamol or DMSO vehicle as a single bolus of 200μg/dose in short-term studies or daily with 100 μg/dose for 2 weeks long-term. Cardiac and renal functions, cAMP levels, in vivo renal tubular LDL-C uptake and renal histology were evaluated post-injection. In vitro mechanistic studies were performed in HK-2, Alport dog smooth muscle and tubular epithelial cells differentiated from Alport patient-derived iPSCs. Protein-protein interactions were studied using co-immunoprecipitation experiments and LDL-C uptake was measured by live-cell imaging. Results: Short-term, salbutamol improved renal function in parallel with decreased LDLR levels and reduced uptake of LDL-C into renal tubules. Long-term, cardiac diastolic function assessed by isovolumetric relaxation time (IVRT), filling pressures (E/E’), and myocardial performance index, and systolic function reflected by ejection fraction, stroke volume and cardiac output improved significantly in parallel with increased cardiac cAMP. Mechanistically, in the kidney, salbutamol activated IDOL and hence lysosomal ubiquitination and degradation of LDLR via a novel β 2 AR-mediated, cAMP-independent pathway involving the Rac1/Cdc42 β 1 PixGEF. β 1 Pix reversibly sequesters IDOL into a complex with LDLR, thereby blocking the degradation pathway. β 2 AR stimulation dissipates the complex reactivating IDOL-mediated LDLR degradation thereby re-establishing LDL-C homeostasis and renal function. Using flow cytometry in HEK293T cells, ectopic expression of bPix stabilized membrane LDLR, sensitive to IDOL- but not PCSK-mediated degradation. Conclusions: β 2 AR agonism represents a potential treatment strategy to alleviate progression of CKD and heart failure associated with HFpEF phenogroup 3.

Nrf2 induces Ucp1 expression in adipocytes in response to β3-AR stimulation and enhances oxygen consumption in high-fat diet-fed obese mice

Coldinduced norepinephrine activates β3-adrenergic receptors (β3-AR) to stimulate the kinase cascade and cAMP-response element-binding protein, leading to the induction of thermogenic gene expression including uncoupling protein 1 (Ucp1). Here, we showed that stimulation of the β3-AR by its agonists isoproterenol and CL316,243 in adipocytes increased the expression of Ucp1 and Heme Oxygenase 1 (Hmox1), the principal Nrf2 target gene, suggesting the functional interaction of Nrf2 with β3-AR signaling. The activation of Nrf2 by tert-butylhydroquinone and reactive oxygen species (ROS) production by glucose oxidase induced both Ucp1 and Hmox1 expression. The increased expression of Ucp1 and Hmox1 was significantly reduced in the presence of a Nrf2 chemical inhibitor or in Nrf2-deleted (knockout) adipocytes. Furthermore, Nrf2 directly activated the Ucp1 promoter, and this required DNA regions located at −3.7 and −2.0 kb of the transcription start site. The CL316,243-induced Ucp1 expression in adipocytes and oxygen consumption in obese mice were partly compromised in the absence of Nrf2 expression. These data provide additional insight into the role of Nrf2 in β3-AR-mediated Ucp1 expression and energy expenditure, further highlighting the utility of Nrf2-mediated thermogenic stimulation as a therapeutic approach to diet-induced obesity.

β-Adrenergic Stimulation Synchronizes a Broad Spectrum of Action Potential Firing Rates of Cardiac Pacemaker Cells toward a Higher Population Average.

The heartbeat is initiated by pacemaker cells residing in the sinoatrial node (SAN). SAN cells generate spontaneous action potentials (APs), i.e., normal automaticity. The sympathetic nervous system increases the heart rate commensurate with the cardiac output demand via stimulation of SAN β-adrenergic receptors (βAR). While SAN cells reportedly represent a highly heterogeneous cell population, the current dogma is that, in response to βAR stimulation, all cells increase their spontaneous AP firing rate in a similar fashion. The aim of the present study was to investigate the cell-to-cell variability in the responses of a large population of SAN cells. We measured the βAR responses among 166 single SAN cells isolated from 33 guinea pig hearts. In contrast to the current dogma, the SAN cell responses to βAR stimulation substantially varied. In each cell, changes in the AP cycle length were highly correlated (R2 = 0.97) with the AP cycle length before βAR stimulation. While, as expected, on average, the cells increased their pacemaker rate, greater responses were observed in cells with slower basal rates, and vice versa: cells with higher basal rates showed smaller responses, no responses, or even decreased their rate. Thus, βAR stimulation synchronized the operation of the SAN cell population toward a higher average rate, rather than uniformly shifting the rate in each cell, creating a new paradigm of βAR-driven fight-or-flight responses among individual pacemaker cells.

Deletion of adipose triglyceride lipase abolishes blood flow increase after β3-adrenergic stimulation in visceral adipose tissue of mice.

Dynamic changes in adipose tissue blood flow (ATBF) with nutritional status play a role in the regulation of metabolic and endocrine functions. Activation of the sympathetic nervous system via β-adrenergic receptors (β-AR) contributes to the control of postprandial enhancement of ATBF. Herein, we sought to identify the role of each β-AR subtype in the regulation of ATBF in mice. We monitored the changes in visceral epididymal ATBF (VAT BF), induced by local infusion of dobutamine, salbutamol, and CL316,243 (a selective β1-, β2-, and β3-AR agonist, respectively) into VAT of lean CD-1 mice and global adipose triglyceride lipase (ATGL) knockout (KO) mice, using laser Doppler flowmetry. Administration of CL316,243, known to promote lipolysis in adipocytes, significantly increased VAT BF of CD-1 mice to a greater extent compared to that of the vehicle, whereas administration of dobutamine or salbutamol did not produce significant differences in VAT BF. The increase in VAT BF induced by β3-AR stimulation disappeared in ATGL KO mice as opposed to their wild-type (WT) littermates, implying a role of ATGL-mediated lipolysis in the regulation of VAT BF. Different vascular reactivities occurred despite no significant differences in vessel density and adiposity between the groups. Additionally, the expression levels of the angiogenesis-related genes were significantly higher in VAT of ATGL KO mice than in that of WT, implicating an association of ATBF responsiveness with angiogenic activity in VAT. Our findings suggest a potential role of β3-AR signaling in the regulation of VAT BF via ATGL-mediated lipolysis in mice.

Ivy leaves dry extract EA 575® mediates biased β2-adrenergic receptor signaling

Backgroundβ2-adrenergic receptor (β2-AR) stimulation activates the G protein/cAMP pathway, which is opposed by the GRK2/β-arrestin 2 pathway. The latter is undesirable in the treatment of respiratory diseases. Hypothesis/PurposeEA 575® is capable of mediating a biased β2-adrenergic signaling pathway. MethodsThe impact of the ivy leaves dry extract EA 575® on β2-adrenergic signaling was tested in a dynamic mass redistribution assay in HEK wild-type and in HEK β-arrestin knock-out cells. cAMP formation and recruitment of β-arrestin 2 were investigated using GloSensor™ and PathHunter® assays, respectively. NFκB transcriptional activity was determined in both HEK wild-type as well as HEK β-arrestin knock-out cells. ResultsEA 575® inhibits the recruitment of β-arrestin 2 and thereby enhances G protein/cAMP signaling under β2-stimulating conditions, as evidenced by a corresponding increase in cAMP formation. While β2-AR-mediated inhibition of NFκB transcriptional activity is β-arrestin-dependent, EA 575® leads to significant inhibition of NFκB transcriptional activity in β-arrestin knock-out cells and thus via a β-arrestin-independent signaling pathway. ConclusionEA 575® is the first active phytopharmaceutical ingredient for which biased β2-adrenergic activation has been described. This shift towards G protein/cAMP signaling provides the molecular basis for the clinically proven efficacy of EA 575® in the treatment of lower respiratory tract diseases. In this light, EA 575® could potentially reduce β-arrestin-mediated adverse effects in new combinatorial therapeutic approaches.

Response of the periodontal tissues to β-adrenergic stimulation

AimsStimulation of β-adrenergic receptors (βAR) in osteoblasts by isoproterenol (ISO) was shown to induce Vascular Endothelial Growth Factor (VEGF) and angiogenesis in long bones. We thus aimed to determine the vascular response of mandibular tissues to βAR stimulation regarding blood vessel formation. Main methodsSix-week-old wild-type C57BL6 female mice received daily intraperitoneal injections of ISO or phosphate buffered saline (PBS) for 1 month. Hemimandibles and tibias were collected for immunolocalization of endomucin, tyrosine hydroxylase (TH), neuropeptide Y (NPY) and norepinephrine transporter (NET). Moreover, Vegfa, Il-1 β, Il-6, Adrb2 and Rankl mRNA expression was assessed in mandibles and tibias 2 h after PBS or ISO treatment. Key findingsDespite similar sympathetic innervation and Adrb2 expression between mandibular tissues and tibias, with TH and NPY+ nerve fibers distributed around blood vessels, ISO treatment did not increase endomucin+ vessel area or the total number of endomucin+ vessels in any of the regions investigated (alveolar bone, periodontal ligament, and dental pulp). Consistent with these results, the expression of Vegfα, Il-6, Il-1β, and Rankl in the mandibular molar region did not change following ISO administration. We detected high expression of NET by immunofluorescence in mandible alveolar osteoblasts, osteocytes, and periodontal ligament fibroblasts, in addition to significantly higher Net expression by qPCR compared to the tibia from the same animals. SignificanceThese findings indicate a differential response to βAR agonists between mandibular and tibial tissues, since the angiogenic potential of sympathetic outflow observed in long bones is absent in periodontal tissues.

Repurposing beta-3 adrenergic receptor agonists for Alzheimer\u2019s disease: beneficial effects in a mouse model

BackgroundOld age, the most important risk factor for Alzheimer’s disease (AD), is associated with thermoregulatory deficits. Brown adipose tissue (BAT) is the main thermogenic driver in mammals and its stimulation, through β3 adrenergic receptor (β3AR) agonists or cold acclimation, counteracts metabolic deficits in rodents and humans. Studies in animal models show that AD neuropathology leads to thermoregulatory deficits, and cold-induced tau hyperphosphorylation is prevented by BAT stimulation through cold acclimation. Since metabolic disorders and AD share strong pathogenic links, we hypothesized that BAT stimulation through a β3AR agonist could exert benefits in AD as well.MethodsCL-316,243, a specific β3AR agonist, was administered to the triple transgenic mouse model of AD (3xTg-AD) and non-transgenic controls from 15 to 16 months of age at a dose of 1 mg/kg/day i.p.ResultsHere, we show that β3AR agonist administration decreased body weight and improved peripheral glucose metabolism and BAT thermogenesis in both non-transgenic and 3xTg-AD mice. One-month treatment with a β3AR agonist increased recognition index by 19% in 16-month-old 3xTg-AD mice compared to pre-treatment (14-month-old). Locomotion, anxiety, and tau pathology were not modified. Finally, insoluble Aβ42/Aβ40 ratio was decreased by 27% in the hippocampus of CL-316,243-injected 3xTg-AD mice.ConclusionsOverall, our results indicate that β3AR stimulation reverses memory deficits and shifts downward the insoluble Aβ42/Aβ40 ratio in 16-month-old 3xTg-AD mice. As β3AR agonists are being clinically developed for metabolic disorders, repurposing them in AD could be a valuable therapeutic strategy.

Golgi‐resident β1‐adrenergic Receptor Signaling to Cardiac Hypertrophy in Cardiac Myocytes in Vitro and in Failing Hearts in Vivo

Pathological cardiac hypertrophy contributes to a higher risk of developing cardiac dysfunction, such as heart failure, one of the major causes of morbidity or mortality in the western world. Chronic stimulation of Gs-coupled β1-adrenergic receptor (β1-AR) signaling by catecholamines during long-term pathological stress induces cardiac hypertrophy and ultimately heart failure. However, the mechanism of action remains incompletely understood. GPCRs at different intracellular compartments are rapidly emerging as novel mediators of distinct local signaling outputs. We have demonstrated activation of Golgi-β1-ARs generates a local pool of cAMP which, in turn, stimulates Golgi-localized phosphatidylinositol 4-phosphate (PI4P) hydrolysis via EPAC dependent PLCε activation, leading to cardiac hypertrophy in a cellular model. This novel prohypertrophic, phosphoinositide hydrolysis pathway is not accessed by plasma membrane β1-AR stimulation. Remarkably, specific inhibition of activation of these preexisting Golgi-resident β1-ARs prevents hypertrophy induced by a physiological neurotransmitter norepinephrine (NE) in cardiac myocytes in vitro. These data defined significant and unique roles of Golgi-β1-ARs in regulating downstream hypertrophic signaling in a biologically relevant cellular model. To determine if Golgi-β1-ARs play an important role in a whole animal model of pathological cardiac hypertrophy, we developed a Golgi targeted construct, eNos-mApple-Nb80 to deliver the β1-AR inhibitor, Nb80, specifically to the Golgi apparatus without interrupting surface β1-ARs. To deliver our target genes into animal heart tissue, I adopted recombinant adeno-associated virus 9 (rAAV9) gene delivery system. We confirmed selective localization to the Golgi apparatus of cardiac myocytes and equal expression of eNos-mApple-Nb80 and eNos-mApple (negative control without Nb80) in mouse hearts. Additionally, activation of the prohypertrophic Golgi PI4P hydrolysis pathway by dobutamine was inhibited in cardiac myocytes isolated from animals expressing eNos-mApple-Nb80. To evaluate the effect of inhibition of Golgi-localized β1-ARs in a mouse model of pressure overload-induced cardiac hypertrophy, we will perform transaortic constriction (TAC) on eNos-mApple-Nb80 expressing mice as well as eNos-mApple and GFP controls. After 6-8 weeks animals will be assessed by echocardiography to measure ejection fraction, followed by sacrifice of the mice and evaluation of heart size, heart weight to tibia length, myocyte area, and interstitial fibrosis. Heart tissues will also be analyzed by western blotting and mRNA analysis for activation of key signal transduction pathways and expression of hypertrophic genes. This will help us elucidate the role of Golgi β1-ARs in cardiac hypertrophy, informing new strategies for the development of β-blocker therapies for heart failure.

CAMP Imaging at Ryanodine Receptors Reveals β2-Adrenoceptor Driven Arrhythmias.

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β 3 Adrenergic Receptor Stimulation Promotes Reperfusion in Ischemic Limbs in a Murine Diabetic Model.

Aims/Hypothesis: Peripheral arterial disease (PAD) is a major burden, resulting in limb claudication, repeated surgical interventions and amputation. There is an unmet need for improved medical management of PAD that improves quality of life, maintains activities of daily life and reduces complications. Nitric oxide (NO)/redox balance is a key regulator of angiogenesis. We have previously shown beneficial effects of a β 3 adrenergic receptor (β 3AR) agonist on NO/redox balance. We hypothesized that β 3AR stimulation would have therapeutic potential in PAD by promoting limb angiogenesis. Methods: The effect of the β 3AR agonist CL 316,243 (1–1,000 nmol/L in vitro, 1 mg/kg/day s. c) was tested in established angiogenesis assays with human endothelial cells and patient-derived endothelial colony forming cells. Post-ischemia reperfusion was determined in streptozotocin and/or high fat diet-induced diabetic and non-diabetic mice in vivo using the hind limb ischemia model. Results: CL 316,243 caused accelerated recovery from hind limb ischemia in non-diabetic and type 1 and 2 diabetic mice. Increased eNOS activity and decreased superoxide generation were detected in hind limb ischemia calf muscle from CL 316, 243 treated mice vs. controls. The protective effect of CL 316,243 in diabetic mice was associated with >50% decreases in eNOS glutathionylation and nitrotyrosine levels. The β 3AR agonist directly promoted angiogenesis in endothelial cells in vitro. These pro-angiogenic effects were β 3AR and NOS-dependent. Conclusion/Interpretation: β 3AR stimulation increased angiogenesis in diabetic ischemic limbs, with demonstrable improvements in NO/redox balance and angiogenesis elicited by a selective agonist. The orally available β 3AR agonist, Mirabegron, used for overactive bladder syndrome, makes translation to a clinical trial by repurposing of a β 3AR agonist to target PAD immediately feasible.

Decreased Chlamydia muridarum genital infection and improved immune response in a β2-Adrenergic Receptor Knockout Mouse Stress Model

Chlamydia genital infection caused by Chlamydia trachomatis shows a high prevalence in low socioeconomic populations; however, whether stress is associated with the high prevalence of infection is not known. During chronic stress conditions, the interaction of beta2-adrenergic receptor (β2-AR) with norepinephrine (NE) is known to suppress the immune system in humans and animal models. However, the mechanism of suppression that leads to chlamydia infection is not well known. This study aims to measure the susceptibility of mice and cytokine production of a β2-AR knockout (KO) during Chlamydia muridarum genital infection. Stressed and non-stressed C57BL/6J wildtype (WT) and β2-AR gene KO mice will be infected with C. muridarum intravaginally post 21-day stressing period. Swabbing of mice at the 3-day interval for 42 days will be performed to determine C. muridarum counts and isolation. Moreover, splenic CD4+ T cells, bone marrow-derived dendritic cells at day 7 post-infection will be performed for immune response analysis by ELISA. We hypothesize that the deficiency of β2-AR leads to significantly less C. muridarum shedding from the genital tract of stressed KO and leads to an increase and decrease in the production of protective and suppressive cytokines, respectively. We anticipate results obtained will align with our central hypothesis that stressed WT mice have increased C. muridarum shedding of genital tracts and increased production of protective cytokines in stressed β2-AR KO, suggesting the importance of stress in β2-AR stimulation.

Pka and CaMKII Signaling Synergistically Promotes Atrial Arrhythmia in Populations of Human Atrial Tissues

The β-adrenergic receptor (βAR)/cAMP/PKA and multifunctional Ca2+-calmodulin-dependent protein kinase II (CaMKII) signaling pathways are key mediators of cardiac excitation-contraction coupling (ECC) and share multiple downstream targets. Hyperactivation of both signaling pathways contributes to the initiation and maintenance of atrial fibrillation (AF), the world's most common arrhythmia. Using novel populations of computational models coupling atrial electrophysiology and Ca2+ handling with detailed descriptions of βAR-PKA and CaMKII pathways, we have previously shown that PKA and CaMKII effects on atrial ECC proteins synergistically promote a vicious cycle of Ca2+and membrane potential instabilities. Since both kinases are known to affect tissue-level modifiers of atrial electrophysiology, here we sought to test whether PKA and CaMKII signaling also synergize to mediate arrhythmias in tissue, and to determine the mechanisms underlying the increased vulnerability. Populations of 1D strand models revealed increased vulnerability to unidirectional block with hyperactive CaMKII, mediated by Na+ channel loss of function and cell-to-cell uncoupling, which was exacerbated by activation of βARs. Simulations of 2D heterogeneous tissues demonstrated synergistic enhancement of arrhythmia vulnerability by βAR stimulation and CaMKII hyperactivation through dysregulating conduction and modifying source-sink mismatch, as well as promoting tissue repolarization dispersion and invoking spontaneous Ca2+-overload-mediated action potentials. CaMKII inhibition substantially reduced the vulnerability. Collectively, our simulations reveal synergy in PKA and CaMKII effects on tissue-level outcomes, and depict a novel paradigm for Ca2+-CaMKII-dependent involvement in both enhanced triggered activity and conduction disturbances in AF. These findings suggest that interrupting the vicious cycle of ever-increasing CaMKII and Ca2+ (e.g., via CaMKII inhibition) may be a valuable pharmacotherapy approach to counteract both AF triggers and functional substrate.

GRKs and Epac1 Interaction in Cardiac Remodeling and Heart Failure.

β-adrenergic receptors (β-ARs) play a major role in the physiological regulation of cardiac function through signaling routes tightly controlled by G protein-coupled receptor kinases (GRKs). Although the acute stimulation of β-ARs and the subsequent production of cyclic AMP (cAMP) have beneficial effects on cardiac function, chronic stimulation of β-ARs as observed under sympathetic overdrive promotes the development of pathological cardiac remodeling and heart failure (HF), a leading cause of mortality worldwide. This is accompanied by an alteration in cAMP compartmentalization and the activation of the exchange protein directly activated by cAMP 1 (Epac1) signaling. Among downstream signals of β-ARs, compelling evidence indicates that GRK2, GRK5, and Epac1 represent attractive therapeutic targets for cardiac disease. Here, we summarize the pathophysiological roles of GRK2, GRK5, and Epac1 in the heart. We focus on their signalosome and describe how under pathological settings, these proteins can cross-talk and are part of scaffolded nodal signaling systems that contribute to a decreased cardiac function and HF development.