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1.  Race, Common Genetic Variation, and Therapeutic Response Disparities in Heart Failure 
JACC. Heart failure  2014;2(6):561-572.
Because of its relatively recent evolution, Homo sapiens exhibits relatively little within-species genomic diversity. However, because of genome size, a proportionally small amount of variation creates ample opportunity for both rare mutations that may be disease-causative as well as more common genetic variation that may be important in disease modification or pharmacogenetics. Primarily because of the East African origin of modern humans, individuals of African ancestry (AA) exhibit greater degrees of genetic diversity than more recently established populations, such as those of European ancestry (EA) or Asian ancestry. These population effects extend to differences in the frequency of common gene variants that may be important in heart failure natural history or therapy. For cell-signaling mechanisms important in heart failure, we review and present new data on genetic variation between AA and EA populations. The data indicate that 1) neurohormonal signaling mechanisms frequently (16 of the 19 investigated polymorphisms) exhibit racial differences in the allele frequencies of variants comprising key constituents, 2) some of these differences in allele frequency may differentially affect the natural history of heart failure in AA vs. EA individuals, and 3) in many cases these differences likely play a role in observed racial differences in drug or device response.
doi:10.1016/j.jchf.2014.06.010
PMCID: PMC4302116  PMID: 25443111
Genetic polymorphisms; pharmacogenetics; racial ancestry; heart failure
2.  Prevention of Atrial Fibrillation by Bucindolol Is Dependent on the Beta1389 Arg/Gly Adrenergic Receptor Polymorphism 
JACC. Heart failure  2013;1(4):338-344.
Objectives
This study assessed the impact of bucindolol, a beta-blocker/sympatholytic agent, on the development of atrial fibrillation (AF) in advanced chronic heart failure with reduced left ventricular ejection fraction (HFREF) patients enrolled in the BEST (Beta-Blocker Evaluation of Survival Trial).
Background
β-Blockers have modest efficacy for AF prevention in HFREF patients. Bucindolol’s effects on HF and ventricular arrhythmic endpoints are genetically modulated by β1- and α2c-adrenergic receptor (AR) polymorphisms that can be used to subdivide HFREF populations into those with bucindolol effectiveness levels that are enhanced, unchanged, or lost.
Methods
BEST enrolled 2,708 New York Heart Association (NYHA) class III to IV HFREF patients. A substudy in which 1,040 patients’ DNA was genotyped for the β1-AR position 389 Arg/Gly and the α2c322–325 wild type (Wt)/deletion (Del) polymorphisms, and new-onset AF was assessed from adverse event case report forms or electrocardiograms at baseline and at 3 and 12 months.
Results
In the entire cohort, bucindolol reduced the rate of new-onset AF compared to placebo by 41% (hazard ratio [HR]: 0.59 [95% confidence interval (CI): 0.44 to 0.79], p = 0.0004). In the 493 β1389 arginine homozygotes (Arg/Arg) in the DNA substudy, bucindolol reduced new-onset AF by 74% (HR: 0.26 [95% CI: 0.12 to 0.57]), with no effect in β1389 Gly carriers (HR: 1.01 [95% CI: 0.56 to 1.84], interaction test = 0.008). When β1389 Gly carriers were subdivided by α2c Wt homozygotes (n = 413, HR: 0.94 [95% CI: 0.48 to 1.82], p = 0.84) or Del variant carriers (n = 134, HR: 1.33 [95% CI: 0.32 to 5.64], p = 0.70), there was a positive interaction test (p = 0.016) when analyzed with β1389 Arg homozygotes.
Conclusions
Bucindolol prevented new-onset AF; β1 and α2c polymorphisms predicted therapeutic response; and the 47% of patients who were β1389 Arg homozygotes had an enhanced effect size of 74%. (Beta-Blocker Evaluation in Survival Trial [BEST]; NCT00000560)
doi:10.1016/j.jchf.2013.04.002
PMCID: PMC3804380  PMID: 24159564
arrhythmia; beta adrenergic receptors; genetics; heart failure; norepinephrine
3.  Agonist-Promoted Homologous Desensitization of Human Airway Smooth Muscle Bitter Taste Receptors 
Bitter taste receptors (TAS2Rs) were shown to be expressed in human airway smooth muscle (ASM). They couple to specialized [Ca2+]i release, leading to membrane hyperpolarization, the relaxation of ASM, and marked bronchodilation. TAS2Rs are G-protein–coupled receptors, known to undergo rapid agonist-promoted desensitization that can limit therapeutic efficacy. Because TAS2Rs represent a new drug target for treating obstructive lung disease, we investigated their capacity for rapid desensitization, and assessed their potential mechanisms. The pretreatment of human ASM cells with the prototypic TAS2R agonist quinine resulted in a 31% ± 5.1% desensitization of the [Ca2+]i response from a subsequent exposure to quinine. No significant change in the endothelin-stimulated [Ca2+]i response was attributed to the short-term use of quinine, indicating a homologous form of desensitization. The TAS2R agonist saccharin also evoked desensitization, and cross-compound desensitization with quinine was evident. Desensitization of the [Ca2+]i response was attenuated by a dynamin inhibitor, suggesting that receptor internalization (a G-protein coupled receptor kinase [GRK]-mediated, β-arrestin–mediated process) plays an integral role in the desensitization of TAS2R. Desensitization was insensitive to antagonists of the second messenger kinases protein kinase A and protein kinase C. Using intact airways, short-term, agonist-promoted TAS2R desensitization of the relaxation response was also observed. Thus these receptors, which represent a potential novel target for direct bronchodilators, undergo a modest degree of agonist-promoted desensitization that may affect clinical efficacy. Collectively, the results of these mechanistic studies, along with the multiple serines and threonines in intracellular loop 3 and the cytoplasmic tail of TAS2Rs, suggest a GRK-mediated mode of desensitization.
doi:10.1165/rcmb.2011-0061OC
PMCID: PMC3361362  PMID: 21642585
airway smooth muscle relaxation; taste receptors; tachyphylaxis; phosphorylation; G-protein–coupled receptor kinases
4.  Genome Sequences of Rhinovirus A Isolates from Wisconsin Pediatric Respiratory Studies 
Genome Announcements  2014;2(2):e00200-14.
Full-length or nearly full-length RNA genome sequences for 98 rhinovirus (RV) A isolates (from the Enterovirus genus of the Picornaviridae family), representing 43 different genotypes, were resolved as part of ongoing studies to define RV genetic diversity and its potential link to respiratory disease.
doi:10.1128/genomeA.00200-14
PMCID: PMC3968333  PMID: 24675855
5.  Genome Sequences of Rhinovirus B Isolates from Wisconsin Pediatric Respiratory Studies 
Genome Announcements  2014;2(2):e00202-14.
Nearly full-length RNA genome sequences for 39 rhinovirus B isolates (RV-B), representing 13 different genotypes, were resolved as part of ongoing studies at the University of Wisconsin that attempt to link rhinovirus (RV) diversity and respiratory disease in infants.
doi:10.1128/genomeA.00202-14
PMCID: PMC3968335  PMID: 24675857
6.  Genome Sequences of Rhinovirus C Isolates from Wisconsin Pediatric Respiratory Studies 
Genome Announcements  2014;2(2):e00203-14.
Human rhinovirus (RV) isolates from the RV-C species are recently discovered infectious agents that are closely linked to asthma and wheezing etiologies in infants. Clinical study samples collected at the University of Wisconsin–Madison describe 41 nearly complete genome sequences representing 21 RV-C genotypes.
doi:10.1128/genomeA.00203-14
PMCID: PMC3968336  PMID: 24675858
7.  Sequencing and Analyses of All Known Human Rhinovirus Genomes Reveal Structure and Evolution 
Science (New York, N.Y.)  2009;324(5923):55-59.
Infection by human rhinovirus (HRV) is a major cause of upper and lower respiratory tract disease worldwide and displays considerable phenotypic variation. We examined diversity by completing the genome sequences for all known serotypes (n = 99). Superimposition of capsid crystal structure and optimal-energy RNA configurations established alignments and phylogeny. These revealed conserved motifs; clade-specific diversity, including a potential newly identified species (HRV-D); mutations in field isolates; and recombination. In analogy with poliovirus, a hypervariable 5′ untranslated region tract may affect virulence. A configuration consistent with nonscanning internal ribosome entry was found in all HRVs and may account for rapid translation. The data density from complete sequences of the reference HRVs provided high resolution for this degree of modeling and serves as a platform for full genome-based epidemiologic studies and antiviral or vaccine development.
doi:10.1126/science.1165557
PMCID: PMC3923423  PMID: 19213880
8.  Analysis of Complete Genome Sequences of Human Rhinovirus 
Human Rhinovirus (HRV) infection is the cause of about one-half of asthma and COPD exacerbations. With >100 serotypes in the HRV reference set an effort was undertaken to sequence their complete genomes so as to understand diversity, structural variation, and evolution of the virus. Analysis revealed conserved motifs, hypervariable regions, a potential fourth HRV species, within-serotype variation in field isolates, a non-scanning internal ribosome entry site, and evidence for HRV recombination. Techniques have now been developed using next generation sequencing to generate complete genomes from patient isolates with high throughput, deep coverage, and low costs. Thus relationships can now be sought between obstructive lung phenotypes and variation in HRV genomes in infected patients, and, potential novel therapeutic strategies developed based on HRV sequence.
doi:10.1016/j.jaci.2010.04.010
PMCID: PMC2893015  PMID: 20471068
Asthma; COPD; inflammation; virus
9.  Pharmacogenomics of β1-adrenergic Receptor Polymorphisms in Heart Failure 
Heart failure clinics  2010;6(1):27-33.
Synopsis
The major pathologic β-adrenergic (βAR) subtype in heart failure is the β1AR. Our laboratory has thus pursued genetic variation of the β1AR gene at the molecular, cellular, physiologic and clinical levels as the potential basis for interindividual variability in the response to β-blocker treatment in heart failure. This chapter will review these findings, with an emphasis on mechanism of action and future directions.
doi:10.1016/j.hfc.2009.08.011
PMCID: PMC2825120  PMID: 19945058
adrenergic; adenylyl cyclase; myocardium; β-blocker; transgenic
10.  Full-genome sequence and analysis of a novel human Rhinovirus strain within a divergent HRV-A clade 
Archives of virology  2009;155(1):83-87.
Genome sequences of human Rhinoviruses (HRV) have primarily been from stocks collected in the 1960’s, with genomes and phylogeny of modern HRVs remaining undefined. Here, two modern isolates (hrv-A101 and hrv-A101-v1) collected ~8 years apart were sequenced in their entirety. Incorporation into our full-genome HRV alignment with subsequent phylogenetic network inference indicated that these represent a unique HRV-A, localized within an early diverging clade. They appear to have resulted from recombination of the hrv-65 and hrv-78 lineages. These results support our contention that there are unrecognized distinct HRV-A strains, and that recombination is evident in currently circulating strains.
doi:10.1007/s00705-009-0549-8
PMCID: PMC2910715  PMID: 19936613
11.  Striated muscle tropomyosin isoforms differentially regulate cardiac performance and myofilament calcium sensitivity 
Tropomyosin (TM) plays a central role in calcium mediated striated muscle contraction. There are three muscle TM isoforms: α-TM, β-TM, and γ-TM. α-TM is the predominant cardiac and skeletal muscle isoform. β-TM is expressed in skeletal and embryonic cardiac muscle. γ-TM is expressed in slow-twitch musculature, but is not found in the heart. Our previous work established that muscle TM isoforms confer different physiological properties to the cardiac sarcomere. To determine whether one of these isoforms is dominant in dictating its functional properties, we generated single and double transgenic mice expressing β-TM and/or γ-TM in the heart, in addition to the endogenously expressed α-TM. Results show significant TM protein expression in the βγ-DTG hearts: α-TM: 36%, β-TM: 32%, and γ-TM: 32%. These βγ-DTG mice do not develop pathological abnormalities; however, they exhibit a hyper contractile phenotype with decreased myofilament calcium sensitivity, similar to γ-TM transgenic hearts. Biophysical studies indicate that γ-TM is more rigid than either α-TM or β-TM. This is the first report showing that with approximately equivalent levels of expression within the same tissue, there is a functional dominance of γ-TM over α-TM or β-TM in regulating physiological performance of the striated muscle sarcomere. In addition to the effect expression of γ-TM has on Ca2+ activation of the cardiac myofilaments, our data demonstrates an effect on cooperative activation of the thin filament by strongly bound rigor cross-bridges. This is significant in relation to current ideas on the control mechanism of the steep relation between Ca2+ and tension.
doi:10.1007/s10974-010-9228-3
PMCID: PMC3805252  PMID: 20803058
Tropomyosin isoforms; Thin filament regulation; Calcium sensitivity; Muscle contraction; Transgenic mice
12.  A polymorphism in the thyroid hormone receptor gene is associated with bronchodilator response in asthmatics 
The pharmacogenomics journal  2012;13(2):130-136.
A pro-asthmatic culture milieu and β2-agonist (isoproterenol) were previously shown to regulate the expression of select transcription factors (TFs) within human airway epithelial and smooth muscle cells. This study tests 1116 single nucleotide polymorphisms (SNPs) across 98 of these TF genes for association with bronchodilator response (BDR) in asthma patients. Genotyping was conducted using the Illumina HumanHap550v3 Beadchip in 403 non-Hispanic White asthmatic children and their parents. SNPs were evaluated for association with BDR using family and population-based analyses. Forty-two SNPs providing p values < 0.1 in both analyses were then genotyped in three adult asthma trials. One SNP 5’ of the thyroid hormone receptor beta gene was associated with BDR in the childhood population and two adult populations (p value = 0.0012). This investigation identified a novel locus for inter-individual variability in BDR and represents a translation of a cellular drug-response study to potential personalization of clinical asthma management.
doi:10.1038/tpj.2011.56
PMCID: PMC3349771  PMID: 22212731
Bronchodilator response; transcription factor; association; thyroid hormone receptor β; asthma; pharmacogenetics
13.  G PROTEIN RECEPTOR KINASE 4 (GRK4) POLYMORPHISMS: BETA-BLOCKER PHARMACOGENETICS AND TREATMENT RELATED OUTCOMES IN HYPERTENSION 
Hypertension  2012;60(4):957-964.
G protein-coupled receptor kinases (GRKs) are important regulatory proteins for many G protein-coupled receptors, but little is known about GRK4 pharmacogenetics. We hypothesized three nonsynonymous GRK4 SNPs, R65L (rs2960306), A142V (rs1024323) and A486V (rs1801058) would be associated with blood pressure response to atenolol, but not hydrochlorothiazide, and would be associated with long term cardiovascular outcomes (all cause, death, nonfatal myocardial infarction, nonfatal stroke) in participants treated with an atenolol-based versus verapamil-SR-based antihypertensive strategy. GRK4 SNPs were genotyped in 768 hypertensive participants from the Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) trial. In Caucasians and African Americans, increasing copies of the variant 65L-142V haplotype were associated with significantly reduced atenolol-induced diastolic blood pressure lowering (−9.1 ± 6.8 vs −6.8 ± 7.1 vs −5.3 ± 6.4 mmHg in participants with 0, 1 and 2 copies of 65L-142V respectively; p=0.0088). 1460 participants with hypertension and coronary artery disease from the INternational VErapamil SR / Trandolapril STudy (INVEST) were genotyped and variant alleles of all three GRK4 SNPs were associated with increased risk for adverse cardiovascular outcomes in an additive fashion, with 486V homozygotes reaching statistical significance (Odds ratio 2.29 [1.48–3.55], p=0.0002). These effects on adverse cardiovascular outcomes were independent of antihypertensive treatment. These results suggest the presence of GRK4 variant alleles may be important determinants of blood pressure response to atenolol and risk for adverse cardiovascular events. The associations with GRK4 variant alleles were stronger in patients who were also ADRB1 389R-homozygotes, suggesting a potential interaction between these two genes.
doi:10.1161/HYPERTENSIONAHA.112.198721
PMCID: PMC3462355  PMID: 22949529
hypertension; GRK4; atenolol; beta-blocker; outcomes; ADRB1; pharmacogenetics
14.  A Polymorphism of GRK5 Alters Agonist-Promoted Desensitization of β2-Adrenergic Receptors 
Pharmacogenetics and genomics  2008;18(8):729-732.
β-agonist treatment of asthma displays substantial interindividual variation, which has prompted polymorphism discovery and characterization of β2-adrenergic (β2AR) signaling genes. β2AR function undergoes desensitization during persistent agonist exposure due to receptor phosphorylation by G-protein coupled receptor kinases (GRKs). GRK5 was found to be highly expressed in airway smooth muscle, the tissue target for β-agonists. The coding region is polymorphic at codon 41, where Gln can be substituted by Leu (minor allele), but almost exclusively in those of African descent. In transfected cells, GRK5-Leu41 evoked a greater degree of agonist-promoted desensitization of adenylyl cyclase compared to GRK5-Gln41. Consistent with this functional effect, agonist-promoted β2AR phosphorylation was greater in cells expressing GRK5-Leu41, as was the rate of agonist-promoted receptor internalization. In studies with mutated β2AR lacking PKA-phosphorylation sites, this phenotype was confirmed as being GRK-specific. So, GRK5-Leu41 represents a gain-of-function polymorphism that evokes enhanced loss-of-function of β2AR during persistent agonist exposure, and thus may contribute to β-agonist variability in asthma treatment of African-Americans.
doi:10.1097/FPC.0b013e32830967e9
PMCID: PMC2699179  PMID: 18622265
Polymorphism; tachyphylaxis; β-agonist; kinases; desensitization; asthma
15.  Allele-Specific Binding of Airway Nuclear Extracts to Polymorphic β2-Adrenergic Receptor 5′ Sequence 
Like other intronless G protein–coupled receptor genes, the β2-adrenergic receptor (β2AR) has minimal genetic space for population variability, and has attained such via multiple coding and noncoding polymorphisms. Yet most clinical studies use the two nonsynonymous polymorphisms of the coding region for association analysis despite low levels of linkage disequilibrium with some promoter and 5′UTR polymorphisms. To assess the potential for allele-specific transcription factor binding to β2AR 5′-flanking sequence, 3′-biotin–labeled oligonucleotide duplexes were synthesized. Each was centered on variable sites representing major or minor alleles found in the human population with frequencies of 5% or greater (20 polymorphic sites). Electrophoretic mobility shift assays were performed using human airway smooth muscle or airway epithelial cell nuclear extracts. Many of these polymorphisms resulted in an alteration in binding, and both major allele and minor allele dominance were observed. For example, in airway smooth muscle nuclear extracts, 10 polymorphisms decreased and 2 increased binding, whereas 5 showed no differences. Concordance between airway smooth muscle and epithelial cell nuclear extract binding to polymorphic alleles was found in only ∼ 50% of cases. There was no tendency for the rare variants to be more likely to have altered nuclear extract binding compared to the more common variants. Taken together, these results provide potential mechanisms by which β2AR 5′-flanking polymorphisms affect obstructive lung phenotypes.
doi:10.1165/rcmb.2006-0394OC
PMCID: PMC1899344  PMID: 17255556
asthma; β; -agonist; polymorphism
16.  Pleiotropic β-Agonist–Promoted Receptor Conformations and Signals Independent of Intrinsic Activity 
β-Agonists used for treatment of obstructive lung disease have a variety of different structures but are typically classified by their intrinsic activities for stimulation of cAMP, and predictions are made concerning other downstream signals based on such a classification. We generated modified β2-adrenergic receptors with insertions of energy donor and acceptor moieties to monitor agonist-promoted conformational changes of the receptor using intramolecular bioluminescence resonance energy transfer in live cells. These studies suggested unique conformations stabilized by various agonists that were not based on their classic intrinsic activities. To address the cellular consequences of these differences, Gs-coupling, Gi-coupling (p44/p42 activation), G protein–coupled receptor kinase–mediated receptor phosphorylation, internalization, and down-regulation were assessed in response to isoproterenol, albuterol, terbutaline, metaproterenol, salmeterol, formoterol, and fenoterol. In virtually every case, agonists did not maintain the classic rank order, indicating that distinct signaling is evoked by β-agonists of different structures, which is unrelated to intrinsic activity. The extensive pleiotropy of agonist responses shown here suggests that classification of agonists by cAMP-based intrinsic activity is inadequate as it pertains to other intracellular events and that it may be possible to engineer a β-agonist that stabilizes conformations that evoke an ideal portfolio of signals for therapeutic purposes.
doi:10.1165/rcmb.2006-0257OC
PMCID: PMC1899310  PMID: 16980553
β-agonist; β-adrenergic receptor; adenylyl cyclase; G-protein; asthma
17.  Crosstalk between Gi and Gq/Gs pathways in airway smooth muscle regulates bronchial contractility and relaxation  
Journal of Clinical Investigation  2007;117(5):1391-1398.
Receptor-mediated airway smooth muscle (ASM) contraction via Gαq, and relaxation via Gαs, underlie the bronchospastic features of asthma and its treatment. Asthma models show increased ASM Gαi expression, considered the basis for the proasthmatic phenotypes of enhanced bronchial hyperreactivity to contraction mediated by M3-muscarinic receptors and diminished relaxation mediated by β2-adrenergic receptors (β2ARs). A causal effect between Gi expression and phenotype has not been established, nor have mechanisms whereby Gi modulates Gq/Gs signaling. To delineate isolated effects of altered Gi, transgenic mice were generated overexpressing Gαi2 or a Gαi2 peptide inhibitor in ASM. Unexpectedly, Gαi2 overexpression decreased contractility to methacholine, while Gαi2 inhibition enhanced contraction. These opposite phenotypes resulted from different crosstalk loci within the Gq signaling network: decreased phospholipase C and increased PKCα, respectively. Gαi2 overexpression decreased β2AR-mediated airway relaxation, while Gαi2 inhibition increased this response, consistent with physiologically relevant coupling of this receptor to both Gs and Gi. IL-13 transgenic mice (a model of asthma), which developed increased ASM Gαi, displayed marked increases in airway hyperresponsiveness when Gαi function was inhibited. Increased Gαi in asthma is therefore a double-edged sword: a compensatory event mitigating against bronchial hyperreactivity, but a mechanism that evokes β-agonist resistance. By selective intervention within these multipronged signaling modules, advantageous Gs/Gq activities could provide new asthma therapies.
doi:10.1172/JCI30489
PMCID: PMC1838924  PMID: 17415415
18.  Molecular Mechanisms of β2-Adrenergic Receptor Function and Regulation 
It is now clear that the β2-adrenergic receptor continuously oscillates between various conformations in the basal state, and that agonists act to stabilize one or more conformations. It is conceivable that synthetic agonists might be engineered to preferentially confine the receptor to certain conformations deemed clinically important while having a less stabilizing effect on unwanted conformations. In addition, studies of genetically engineered mice have revealed previously unrecognized cross-talk between the β2-receptor and phospholipase C, such that removal of the primary dilating pathway results in downregulation of constrictive pathways and overactivity of the dilating pathway increases the contractile response. These results indicate a dynamic interaction between β2-receptor activity and Gq-coupled receptors that constrict the airway. Potentially, then, during chronic β-agonist therapy, expression of phospholipase C is increased, the functions of Gq-coupled constrictive receptors are enhanced, and there may be an increased tendency for clinical decompensation due to asthma and chronic obstructive pulmonary disease triggers. Antagonists to these receptors might be able to act synergistically with chronic β-agonists to block the effect of phospholipase C. Alternatively, perhaps novel phospholipase C antagonists would provide the most efficacious approach to blocking the physiologic sequelae of cross-talk between the β2-receptor and phospholipase C.
doi:10.1513/pats.200504-027SR
PMCID: PMC2713324  PMID: 16267351
asthma; β-agonist; chronic obstructive pulmonary disease; G protein–coupled receptor; phospholipase C
19.  Airway smooth muscle prostaglandin-EP1 receptors directly modulate β2–adrenergic receptors within a unique heterodimeric complex 
Journal of Clinical Investigation  2006;116(5):1400-1409.
Multiple and paradoxical effects of airway smooth muscle (ASM) 7-transmembrane–spanning receptors activated during asthma, or by treatment with bronchodilators such as β2–adrenergic receptor (β2AR) agonists, indicate extensive receptor crosstalk. We examined the signaling of the prostanoid-EP1 receptor, since its endogenous agonist prostaglandin E2 is abundant in the airway, but its functional implications are poorly defined. Activation of EP1 failed to elicit ASM contraction in mouse trachea via this Gαq-coupled receptor. However, EP1 activation markedly reduced the bronchodilatory function of β2AR agonist, but not forskolin, indicating an early pathway interaction. Activation of EP1 reduced β2AR-stimulated cAMP in ASM but did not promote or augment β2AR phosphorylation or alter β2AR trafficking. Bioluminescence resonant energy transfer showed EP1 and β2AR formed heterodimers, which were further modified by EP1 agonist. In cell membrane [35S]GTPγS binding studies, the presence of the EP1 component of the dimer uncoupled β2AR from Gαs, an effect accentuated by EP1 agonist activation. Thus alone, EP1 does not appear to have a significant direct effect on airway tone but acts as a modulator of the β2AR, altering Gαs coupling via steric interactions imposed by the EP1:β2AR heterodimeric signaling complex and ultimately affecting β2AR-mediated bronchial relaxation. This mechanism may contribute to β-agonist resistance found in asthma.
doi:10.1172/JCI25840
PMCID: PMC1451203  PMID: 16670773
20.  Cardiac 7-transmembrane-spanning domain receptor portfolios: diversify, diversify, diversify 
Journal of Clinical Investigation  2006;116(4):875-877.
Enhanced signaling in myocytes by the G protein Gq has been implicated in cardiac hypertrophy and the transition to heart failure. α1-Adrenergic receptors (α1-ARs) are members of the 7-transmembrane-spanning domain (7-TM) receptor family and signal via interaction with Gq in the heart. The specific effects of a loss of α1-AR signaling in the heart are explored by O’Connell et al. in this issue of the JCI (see the related article beginning on page 1005). Paradoxically, gene ablation of the α1A and α1B subtypes in mice results in a maladaptive form of reactive cardiac hypertrophy from pressure overload, with a predisposition to heart failure. Thus signaling to the α1-AR (compared with signaling from other receptors such as angiotensin receptors, which also couple to Gq) appears to be specifically required for a normal hypertrophic response. This represents another example of how receptors that share common G proteins have diversified, developing unique signaling programs. These findings may have particular clinical relevance because of the widespread use of α1-AR antagonists in the treatment of hypertension and symptomatic prostate enlargement.
doi:10.1172/JCI28234
PMCID: PMC1421366  PMID: 16585959
21.  Genetic Variation, β-Blockers, and Perioperative Myocardial Infarction 
Anesthesiology  2011;115(6):1316-1327.
Perioperative myocardial infarction is a common and potentially fatal complication after noncardiac surgery, particular among patients with cardiovascular risk factors. β-blockers have been considered a mainstay in prevention and treatment of perioperative myocardial infarction, yet recent evidence suggests that β-blockers may have an unfavorable risk profile in this setting and the use has become controversial. What seems conspicuously absent from the current discussion is the appreciation of how much interindividual genetic variation influences the clinical response to β-blocker therapy. Genetic variation in the adrenergic signaling pathway is common, and has a major impact on adrenergic receptor function and β-blocker efficacy in other cardiovascular diseases such as heart failure and hypertension. Genetic variation in the cytochrome P450 2D6 enzyme, which is responsible for the metabolism of most β-blockers, is also important and can lead to poor metabolizing of β-blockers (potential toxicity) or their ultra-rapid degradation (decreased efficacy). Here, we review the molecular, cellular and physiologic consequences of polymorphisms in the adrenergic signaling pathway and CYP2D6 gene, and show that these are likely relevant factors influencing efficacy, safety and toxicity of β-blocker therapy in prevention and treatment of perioperative myocardial infarction.
doi:10.1097/ALN.0b013e3182315eb2
PMCID: PMC3226841  PMID: 21918425
22.  Antithetic regulation by β-adrenergic receptors of Gq receptor signaling via phospholipase C underlies the airway β-agonist paradox 
Journal of Clinical Investigation  2003;112(4):619-626.
β-adrenergic receptors (βARs) relax airway smooth muscle and bronchodilate, but chronic β-agonist treatment in asthma causes increased sensitivity to airway constriction (hyperreactivity) and is associated with exacerbations. This paradox was explored using mice with ablated βAR genes (βAR–/–) and transgenic mice overexpressing airway smooth muscle β2AR (β2AR-OE) representing two extremes: absence and persistent activity of airway βAR. Unexpectedly, βAR–/– mice, lacking these bronchodilating receptors, had markedly decreased bronchoconstrictive responses to methacholine and other Gq-coupled receptor agonists. In contrast, β2AR-OE mice had enhanced constrictive responses. Contraction to permeabilization with β-escin was unaltered by gene ablation or overexpression. Inositol phosphate accumulation by Gq-coupled M3-muscarinic, thromboxane-A2, and 5-HT2 receptors was desensitized in airway smooth muscle cells from βAR–/– mice and sensitized in cells from β2AR-OE mice. Thus, βAR antithetically regulates constrictive signals, affecting bronchomotor tone/reactivity by additional means other than direct dilatation. Studies of signaling elements in these pathways revealed the nodal point of this cross talk as phospholipase C-β1, whose expression was altered by βAR in a direction and magnitude consistent with the physiologic and cellular responses. These results establish a mechanism of the β-agonist paradox and identify a potential asthma modifier gene (phospholipase C-β1), which may also be a therapeutic target in asthma when chronic β-agonists are required.
doi:10.1172/JCI200318193
PMCID: PMC171392  PMID: 12925702
23.  Combinatorial Pharmacogenetic Interactions of Bucindolol and β1, α2C Adrenergic Receptor Polymorphisms 
PLoS ONE  2012;7(10):e44324.
Background
Pharmacogenetics involves complex interactions of gene products affecting pharmacodynamics and pharmacokinetics, but there is little information on the interaction of multiple genetic modifiers of drug response. Bucindolol is a β-blocker/sympatholytic agent whose efficacy is modulated by polymorphisms in the primary target (β1 adrenergic receptor [AR] Arg389 Gly on cardiac myocytes) and a secondary target modifier (α2C AR Ins [wild-type (Wt)] 322–325 deletion [Del] on cardiac adrenergic neurons). The major allele homozygotes and minor allele carriers of each polymorphism are respectively associated with efficacy enhancement and loss, creating the possibility for genotype combination interactions that can be measured by clinical trial methodology.
Methodology
In a 1,040 patient substudy of a bucindolol vs. placebo heart failure clinical trial, we tested the hypothesis that combinations of β1389 and α2C322–325 polymorphisms are additive for both efficacy enhancement and loss. Additionally, norepinephrine (NE) affinity for β1389 AR variants was measured in human explanted left ventricles.
Principal Findings
The combination of β1389 Arg+α2C322–325 Wt major allele homozygotes (47% of the trial population) was non-additive for efficacy enhancement across six clinical endpoints, with an average efficacy increase of 1.70-fold vs. 2.32-fold in β1389 Arg homozygotes+α2C322–325 Del minor allele carriers. In contrast, the minor allele carrier combination (13% subset) exhibited additive efficacy loss. These disparate effects are likely due to the higher proportion (42% vs. 8.7%, P = 0.009) of high-affinity NE binding sites in β1389 Arg vs. Gly ARs, which converts α2CDel minor allele-associated NE lowering from a therapeutic liability to a benefit.
Conclusions
On combination, the two sets of AR polymorphisms 1) influenced bucindolol efficacy seemingly unpredictably but consistent with their pharmacologic interactions, and 2) identified subpopulations with enhanced (β1389 Arg homozygotes), intermediate (β1389 Gly carriers+α2C322–325 Wt homozygotes), and no (β1389 Gly carriers+α2C322–325 Del carriers) efficacy.
doi:10.1371/journal.pone.0044324
PMCID: PMC3468617  PMID: 23071495
25.  Regulatory Haplotypes in ARG1 Are Associated with Altered Bronchodilator Response 
Rationale: β2-agonists, the most common treatment for asthma, have a wide interindividual variability in response, which is partially attributed to genetic factors. We previously identified single nucleotide polymorphisms in the arginase 1 (ARG1) gene, which are associated with β2-agonist bronchodilator response (BDR).
Objectives: To identify cis-acting haplotypes in the ARG1 locus that are associated with BDR in patients with asthma and regulate gene expression in vitro.
Methods: We resequenced ARG1 in 96 individuals and identified three common, 5′ haplotypes (denoted 1, 2, and 3). A haplotype-based association analysis of BDR was performed in three independent, adult asthma drug trial populations. Next, each haplotype was cloned into vectors containing a luciferase reporter gene and transfected into human airway epithelial cells (BEAS-2B) to ascertain its effect on gene expression.
Measurements and Main Results: BDR varied by haplotype in each of the three populations with asthma. Individuals with haplotype 1 were more likely to have higher BDR, compared to those with haplotypes 2 and 3, which is supported by odds ratios of 1.25 (95% confidence interval, 1.03–1.71) and 2.18 (95% confidence interval, 1.34–2.52), respectively. Luciferase expression was 50% greater in cells transfected with haplotype 1 compared to haplotypes 2 and 3.
Conclusions: The identified ARG1 haplotypes seem to alter BDR and differentially regulate gene expression with a concordance of decreased BDR and reporter activity from haplotypes 2 and 3. These findings may facilitate pharmacogenetic tests to predict individuals who may benefit from other therapeutic agents in addition to β2-agonists for optimal asthma management.
Clinical trial registered with www.clinicaltrials.gov (NCT00156819, NCT00046644, and NCT00073840).
doi:10.1164/rccm.201005-0758OC
PMCID: PMC3056223  PMID: 20851928
pharmacogenetics; asthma; β2-agonist

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