Stephen B. Liggett

Use of regularly scheduled albuterol treatment in asthma: genotype-stratified, randomised, placebo-controlled cross-over trial.

BACKGROUND The issue of whether regular use of an inhaled beta2-adrenergic agonist worsens airflow and clinical outcomes in asthma is controversial. Retrospective studies have suggested that adverse effects occur in patients with a genetic polymorphism that results in homozygosity for arginine (Arg/Arg), rather than glycine (Gly/Gly), at aminoacid residue 16 of the beta2-adrenergic receptor. However, the existence of any genotype-dependent difference has not been tested in a prospective clinical trial. METHODS Patients with mild asthma, not using a controller medication, were enrolled in pairs matched for forced expiratory volume in 1 s (FEV1) according to whether they had the Arg/Arg (n=37; four of 41 matches withdrew before randomisation) or Gly/Gly (n=41) genotype. Regularly scheduled treatment with albuterol or placebo was given in a masked, cross-over design, for 16-week periods. During the study, as-needed albuterol use was discontinued and ipratropium bromide was used as needed. Morning peak expiratory flow rate (PEFR) was the primary outcome variable. The primary comparisons were between treatment period for each genotype; the secondary outcome was a treatment by genotype effect. Analyses were by intention to treat. FINDINGS During the run-in period, when albuterol use was kept to a minimum, patients with the Arg/Arg genotype had an increase in morning PEFR of 23 L/min (p=0.0162); the change in patients with the Gly/Gly genotype was not significant (2 L/min; p=0.8399). During randomised treatment, patients with the Gly/Gly genotype had an increase in morning PEFR during treatment with regularly scheduled albuterol compared with placebo (14 L/min [95% CI 3 to 25]; p=0.0175). By contrast, patients with the Arg/Arg genotype had lower morning PEFR during treatment with albuterol than during the placebo period, when albuterol use was limited (-10 L/min [-19 to -2]; p=0.0209). The genotype-attributable treatment difference was therefore -24 L/min (-37 to -12; p=0.0003). There were similar genotype-specific effects in FEV1, symptoms, and use of supplementary reliever medication. INTERPRETATION Genotype at the 16th aminoacid residue of the beta2-adrenergic receptor affects the long-term response to albuterol use. Bronchodilator treatments avoiding albuterol may be appropriate for patients with the Arg/Arg genotype.

A Gain-of-function Polymorphism in a G-protein Coupling Domain of the Human β1-Adrenergic Receptor

The β1-adrenergic receptor (β1AR) is a key cell surface signaling protein expressed in the heart and other organs that mediates the actions of catecholamines of the sympathetic nervous system. A polymorphism in the intracellular cytoplasmic tail near the seventh transmembrane-spanning segment of the human β1AR has been identified in a cohort of normal individuals. At amino acid position 389, Gly or Arg can be found (allele frequencies 0.26 and 0.74, respectively), the former previously considered as the human wild-type β1AR. Using site-directed mutagenesis to mimic the two variants, CHW-1102 cells were permanently transfected to express the Gly-389 and Arg-389 receptors. In functional studies with matched expression, the Arg-389 receptors had slightly higher basal levels of adenylyl cyclase activities (10.7 ± 1.2 versus 6.1 ± 0.4 pmol/min/mg). However, maximal isoproterenol-stimulated levels weremarkedly higher for the Arg-389 as compared to the Gly-389 receptor (63.3 ± 6.1 versus 20.9 ± 2.0 pmol/min/mg). Agonist-promoted [35S]guanosine 5′-O-(thiotriphosphate) binding was also increased with the Arg-389 receptor consistent with enhanced coupling to Gsand increased adenylyl cyclase activation. In agonist competition studies carried out in the absence of guanosine 5′-(β,γ-imido)triphosphate, high affinity binding could not be resolved with the Gly-389 receptor, whereas Arg-389 displayed an accumulation of the agonist high affinity receptor complex (R H = 26%). Taken together, these data indicate that this polymorphic variation of the human β1AR results in alterations of receptor-Gs interaction with functional signal transduction consequences, consistent with its localization in a putative G-protein binding domain. The genetic variation of β1AR at this locus may be the basis of interindividual differences in pathophysiologic characteristics or in the response to therapeutic βAR agonists and antagonists in cardiovascular and other diseases.

PKC-α regulates cardiac contractility and propensity toward heart failure

The protein kinase C (PKC) family of serine/threonine kinases functions downstream of nearly all membrane-associated signal transduction pathways. Here we identify PKC-α as a fundamental regulator of cardiac contractility and Ca2+ handling in myocytes. Hearts of Prkca-deficient mice are hypercontractile, whereas those of transgenic mice overexpressing Prkca are hypocontractile. Adenoviral gene transfer of dominant-negative or wild-type PKC-α into cardiac myocytes enhances or reduces contractility, respectively. Mechanistically, modulation of PKC-α activity affects dephosphorylation of the sarcoplasmic reticulum Ca2+ ATPase-2 (SERCA-2) pump inhibitory protein phospholamban (PLB), and alters sarcoplasmic reticulum Ca2+ loading and the Ca2+ transient. PKC-α directly phosphorylates protein phosphatase inhibitor-1 (I-1), altering the activity of protein phosphatase-1 (PP-1), which may account for the effects of PKC-α on PLB phosphorylation. Hypercontractility caused by Prkca deletion protects against heart failure induced by pressure overload, and against dilated cardiomyopathy induced by deleting the gene encoding muscle LIM protein (Csrp3). Deletion of Prkca also rescues cardiomyopathy associated with overexpression of PP-1. Thus, PKC-α functions as a nodal integrator of cardiac contractility by sensing intracellular Ca2+ and signal transduction events, which can profoundly affect propensity toward heart failure.

Early and Delayed Consequences of β2-Adrenergic Receptor Overexpression in Mouse Hearts Critical Role for Expression Level

BACKGROUND Transgenic cardiac beta(2)-adrenergic receptor (AR) overexpression has resulted in enhanced signaling and cardiac function in mice, whereas relatively low levels of transgenically expressed G(alphas) or beta(1)AR have resulted in phenotypes of ventricular failure. Potential relationships between the levels of betaAR overexpression and biochemical, molecular, and physiological consequences have not been reported. METHODS AND RESULTS We generated transgenic mice expressing beta(2)AR at 3690, 7120, 9670, and 23 300 fmol/mg in the heart, representing 60, 100, 150, and 350 times background betaAR expression. All lines showed enhanced basal adenylyl cyclase activation but a decrease in forskolin- and NaF-stimulated adenylyl cyclase activities. Mice of the highest-expressing line developed a rapidly progressive fibrotic dilated cardiomyopathy and died of heart failure at 25+/-1 weeks of age. The 60-fold line exhibited enhanced basal cardiac function without increased mortality when followed for 1 year, whereas 100-fold overexpressors developed a fibrotic cardiomyopathy and heart failure, with death occurring at 41+/-1 weeks of age. Adenylyl cyclase activation did not correlate with early or delayed decompensation. Propranolol administration reduced baseline +dP/dt(max) to nontransgenic levels in all beta(2)AR transgenics except the 350-fold overexpressors, indicating that spontaneous activation of beta(2)AR was present at this level of expression. CONCLUSIONS These data demonstrate that the heart tolerates enhanced contractile function via 60-fold beta(2)AR overexpression without detriment for a period of >/=1 year and that higher levels of expression result in either aggressive or delayed cardiomyopathy. The consequences for enhanced betaAR function in the heart appear to be highly dependent on which signaling elements are increased and to what extent.

Genetic polymorphisms of the beta 2-adrenergic receptor in nocturnal and nonnocturnal asthma. Evidence that Gly16 correlates with the nocturnal phenotype.

Nocturnal asthma represents a unique subset of patients with asthma who experience worsening symptoms and airflow obstruction at night. The basis for this phenotype of asthma is not known, but beta 2-adrenergic receptors (beta 2AR) are known to downregulate overnight in nocturnal asthmatics but not normal subjects or nonnocturnal asthmatics. We have recently delineated three polymorphic loci within the coding block of the beta 2AR which alter amino acids at positions 16, 27, and 164 and impart specific biochemical and pharmacologic phenotypes to the receptor. In site-directed mutagenesis/recombinant expression studies we have found that glycine at position 16 (Gly16) imparts an accelerated agonist-promoted downregulation of beta 2AR as compared to arginine at this position (Arg16). We hypothesized that Gly16 might be overrepresented in nocturnal asthmatics and thus determined the beta 2AR genotypes of two well-defined asthmatic cohorts: 23 nocturnal asthmatics with 34 +/- 2% nocturnal depression of peak expiratory flow rates, and 22 nonnocturnal asthmatics with virtually no such depression (2.3 +/- 0.8%). The frequency of the Gly16 allele was 80.4% in the nocturnal group as compared to 52.2% in the nonnocturnal group, while the Arg16 allele was present in 19.6 and 47.8%, respectively. This overrepresentation of the Gly16 allele in nocturnal asthma was significant at P = 0.007 with an odds ratio of having nocturnal asthma and the Gly16 polymorphism being 3.8. Comparisons of the two cohorts as to homozygosity for Gly16, homozygosity for Arg16, or heterozygosity were also consistent with segregation of Gly16 with nocturnal asthma. There was no difference in the frequency of polymorphisms at loci 27 (Gln27 or Glu27) and 164 (Thr164 or Ile164) between the two groups. Thus the Gly16 polymorphism of the beta 2AR, which imparts an enhanced downregulation of receptor number, is overrepresented in nocturnal asthma and appears to be an important genetic factor in the expression of this asthmatic phenotype.

The Ile164 beta2-adrenergic receptor polymorphism adversely affects the outcome of congestive heart failure.

The beta2-adrenergic receptor (beta2AR), an important modulator of cardiac inotropy and chronotropy, has significant genetic heterogeneity in the population. Because dysfunctional betaARs play a role in the pathogenesis of the failing ventricle, we tested the hypothesis that beta2AR polymorphisms alter the outcome of congestive heart failure. 259 patients with NYHA functional class II-IV heart failure due to ischemic or dilated cardiomyopathy were genotyped and prospectively followed, with the endpoint defined as death or cardiac transplantation. The allele frequencies between this group and those of 212 healthy controls also were compared and did not differ between the groups. However, those with the Ile164 polymorphism displayed a striking difference in survival with a relative risk of death or cardiac transplant of 4.81 (P < 0.001) compared with those with the wild-type Thr at this position. Age, race, gender, functional class, etiology, ejection fraction, and medication use did not differ between these individuals and those with the wild-type beta2AR, and thus the beta2AR genotype at position 164 was the only clear distinguishing feature between the two groups. The 1-yr survival for Ile164 patients was 42% compared with 76% for patients harboring wild-type beta2AR. In contrast, polymorphisms at amino acid positions 16 (Arg or Gly) or 27 (Gln or Glu), which also alter receptor phenotype, did not appear to have an influence on the course of heart failure. Taken together with cell-based and transgenic mouse results, this study establishes a paradigm whereby genetic variants of key signaling elements can have pathophysiologic consequences within the context of a disease. Furthermore, patients with the Ile164 polymorphism and heart failure may be candidates for earlier aggressive intervention or cardiac transplantation.

Human phospholamban null results in lethal dilated cardiomyopathy revealing a critical difference between mouse and human

In human disease and experimental animal models, depressed Ca(2+) handling in failing cardiomyocytes is widely attributed to impaired sarcoplasmic reticulum (SR) function. In mice, disruption of the PLN gene encoding phospholamban (PLN) or expression of dominant-negative PLN mutants enhances SR and cardiac function, but effects of PLN mutations in humans are unknown. Here, a T116G point mutation, substituting a termination codon for Leu-39 (L39stop), was identified in two families with hereditary heart failure. The heterozygous individuals exhibited hypertrophy without diminished contractile performance. Strikingly, both individuals homozygous for L39stop developed dilated cardiomyopathy and heart failure, requiring cardiac transplantation at ages 16 and 27. An over 50% reduction in PLN mRNA and no detectable PLN protein were noted in one explanted heart. The expression of recombinant PLN-L39stop in human embryonic kidney (HEK) 293 cells and adult rat cardiomyocytes showed no PLN inhibition of SR Ca(2+)-ATPase and the virtual absence of stable PLN expression; where PLN was expressed, it was misrouted to the cytosol or plasma membrane. These findings describe a naturally-occurring loss-of-function human PLN mutation (PLN null). In contrast to reported benefits of PLN ablation in mouse heart failure, humans lacking PLN develop lethal dilated cardiomyopathy.

β 1 -adrenergic receptor polymorphisms confer differential function and predisposition to heart failure

Catecholamines stimulate cardiac contractility through β1-adrenergic receptors (β1-ARs), which in humans are polymorphic at amino acid residue 389 (Arg/Gly). We used cardiac-targeted transgenesis in a mouse model to delineate mechanisms accounting for the association of Arg389 with human heart failure phenotypes. Hearts from young Arg389 mice had enhanced receptor function and contractility compared with Gly389 hearts. Older Arg389 mice displayed a phenotypic switch, with decreased β-agonist signaling to adenylyl cyclase and decreased cardiac contractility compared with Gly 389 hearts. Arg389 hearts had abnormal expression of fetal and hypertrophy genes and calcium-cycling proteins, decreased adenylyl cyclase and Gαs expression, and fibrosis with heart failure This phenotype was recapitulated in homozygous, end-stage, failing human hearts. In addition, hemodynamic responses to β-receptor blockade were greater in Arg389 mice, and homozygosity for Arg389 was associated with improvement in ventricular function during carvedilol treatment in heart failure patients. Thus the human Arg389 variant predisposes to heart failure by instigating hyperactive signaling programs leading to depressed receptor coupling and ventricular dysfunction, and influences the therapeutic response to β-receptor blockade.

Bitter taste receptors on airway smooth muscle bronchodilate by localized calcium signaling and reverse obstruction

Bitter taste receptors (TAS2Rs) on the tongue probably evolved to evoke signals for avoiding ingestion of plant toxins. We found expression of TAS2Rs on human airway smooth muscle (ASM) and considered these to be avoidance receptors for inhalants that, when activated, lead to ASM contraction and bronchospasm. TAS2R agonists such as saccharin, chloroquine and denatonium evoked increased intracellular calcium ([Ca2+]i) in ASM in a Gβγ–, phospholipase Cβ (PLCβ)- and inositol trisphosphate (IP3) receptor–dependent manner, which would be expected to evoke contraction. Paradoxically, bitter tastants caused relaxation of isolated ASM and dilation of airways that was threefold greater than that elicited by β-adrenergic receptor agonists. The relaxation induced by TAS2Rs is associated with a localized [Ca2+]i response at the cell membrane, which opens large-conductance Ca2+-activated K+ (BKCa) channels, leading to ASM membrane hyperpolarization. Inhaled bitter tastants decreased airway obstruction in a mouse model of asthma. Given the need for efficacious bronchodilators for treating obstructive lung diseases, this pathway can be exploited for therapy with the thousands of known synthetic and naturally occurring bitter tastants.

Sequencing and Analyses of All Known Human Rhinovirus Genomes Reveal Structure and Evolution

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.