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Biomedical subjects

Stephen B Liggett

Publications and source records attributed to Stephen B Liggett.

At least 19 recordsLinked to original sources
PubMed2025

β1- and β2-adrenergic Receptor Haplotypes Regulate Therapeutic Responses to Placebo and the Biased Ligand β-blocker Bucindolol.

BACKGROUND: ADRB1 and ADRB2, encoding cardiac myocyte &#x3b2;1- and &#x3b2;2-adrenergic receptors (ARs) that mediate pathologic myocardial remodeling in response to chronically increased signaling, contain N-terminus haplotype variants capable of influencing agonist- or biased ligand-induced receptor internalization that uncouples canonical signaling and initiates EGFR/ERK1/2 cardioprotection. METHODS: In two heart failure (HF) clinical trial genetic substudies we investigated effects of internalizing vs. internalization-resistant ADRB1/ADRB2 haplotypes on clinical or biomarker responses to the biased ligand &#x3b2;-blocker bucindolol vs. placebo or vs. the nonbiased &#x3b2;1-antagonist metoprolol, and in haplotyped isolated human heart preparations we measured ERK1/2 activation in response to these same interventions. RESULTS: In subjects with &#x2265;3 internalizing ADRB1+ADRB2 haplotypes (6.7% subcohort) placebo treatment was associated with fewer clinical events compared to subjects with internalization-resistant haplotypes (Odds Ratio (OR) 0.28, 95% CI (0.10, 0.82)). In contrast, placebo treatment in subjects with &#x2265;3 internalization-resistant haplotypes (70% subcohort) was associated with more clinical events in comparison to subjects with internalizing haplotype counterparts (OR 1.64 (1.46, 1.84)). Bucindolol treatment was equal to placebo in the &#x2265;3 internalizing subcohort, but was superior to placebo in the internalization-resistant subcohort (bucindolol vs. placebo OR 0.49 (0.41, 0.58)). In subjects with all 4 haplotypes internalization-resistant (25% subcohort), bucindolol vs. placebo reduced time to first event rates by 62.3&#xb1;17.5% (P <0.01, 1.68&#xb1;0.34 fold > the all-haplotypes parent population and additive to 1.92&#xb1;0.58 fold when the ADRB1 haplotype contained Arg389 rather than Gly389). The same bucindolol vs. placebo pattern was observed for NT-proBNP or norepinephrine reduction vs. metoprolol. In these comparisons ADRB2 and ADRB1 haplotypes behaved similarly, and although the haplotypes differed in frequency between Black and non-Black subjects, within haplotypes there were no by-race differences in therapeutic effects. Bucindolol but not metoprolol activated ERK1/2 signaling in isolated ventricular preparations with &#x2265;3 internalization-resistant haplotypes. CONCLUSIONS: 1) Both &#x3b2;1- and &#x3b2;2-AR haplotypes regulate therapeutic responses in HF; internalizing species confer protection against clinical events in placebo-treated subjects, while in internalization-resistant haplotypes the biased ligand &#x3b2;-blocker bucindolol but not the non-biased ligand metoprolol is associated with favorable effects. 2) The biased ligand cardioprotective effect may be related to internalization-dependent or -independent ERK1/2 activation.

Beta Adrenergic Receptors↗
PubMed2004

Polymorphisms of cardiac presynaptic alpha2C adrenergic receptors: Diverse intragenic variability with haplotype-specific functional effects.

The presynaptic alpha2C adrenergic receptors (AR) act to inhibit norepinephrine release in cardiac and other presynaptic nerves. We have recently shown that a genetic variant in the alpha2CAR coding region (Del322-325), which renders the receptor partially uncoupled from Gi, is a risk factor for heart failure. However, variability of heart failure phenotypes and a dominance of Del322-325 in those of African descent led us to hypothesize that other regions of this gene have functional polymorphisms. In a multiethnic population, we found 20 polymorphisms within 4,625 bp of contiguous sequence of this intronless gene encompassing the promoter, 5' UTR, coding, and 3' UTR. These polymorphisms occur in 24 distinct haplotypes with complex organizations, including multiple 5'-upstream polymorphisms in regions known to direct expression, a 3' UTR substitution polymorphism within an insertion/deletion sequence, and the radical coding polymorphism that deletes four amino acids. Relatively low linkage disequilibrium between many polymorphisms, few cosmopolitan haplotypes, prevalent ethnic-specific haplotypes, and substantial genetic divergence among haplotypes was noted. The dysfunctional Del322-325 allele was partitioned into multiple haplotypes, with frequencies of 48% to 2%. The functional implications of the haplotypes were ascertained by whole-gene transfections of human neuronal cells, where haplotype was significantly related (P < 0.001) to expression levels of receptor transcript and protein. Expression varied by as much as approximately 50% by haplotype, and such studies enabled haplotype clustering by phenotypic, rather than genotypic, similarities. Thus, depending on phenotype, expression-specific haplotypes may amplify, attenuate, or dominate the cardiomyopathic effect attributed to the alpha2CDel322-325 marker.

Haplotypes↗
PubMed2004

A primate-dominant third glycosylation site of the beta2-adrenergic receptor routes receptors to degradation during agonist regulation.

beta(2)-adrenergic receptors (beta(2)AR) of all species are N-linked glycosylated at amino terminus residues approximately 6 and approximately 15. However, the human beta(2)AR has a potential third N-glycosylation site at ECL2 residue 187. To determine whether this residue is glycosylated and to ascertain function, all possible single/multiple Asn --> Gln mutations were made in the human beta(2) AR at positions 6, 15, and 187 and were expressed in Chinese hamster fibroblast cells. Substitution of Asn-187 alone or with Asn-6 or Asn-15 decreased the apparent molecular mass of the receptor on SDS-PAGE in a manner consistent with Asn-187 glycosylation. All receptors bound the agonist isoproterenol and functionally coupled to adenylyl cyclase. However, receptors without 187 glycosylation failed to display long term agonist-promoted down-regulation. In contrast, loss of Asn-6/Asn-15 glycosylation did not alter down-regulation. Cell surface distribution and agonist-promoted internalization of receptors and recruitment of beta-arrestin 2 were unaffected by the loss of 187 glycosylation. Furthermore, acutely internalized wild-type and Gln-187 receptors were both localized by confocal microscopy to early endosomes. During prolonged agonist exposure, wild-type beta(2)AR co-localized with lysosomes, consistent with trafficking to a degradation compartment. However, Gln-187 beta(2)AR failed to co-localize with lysosomes despite agonist treatments up to 18 h. Phylogenetic analysis revealed that this third glycosylation site is found in humans and other higher order primates but not in lower order primates such as the monkey. Nor is this third site found in rodents, which are frequently utilized as animal models. These data thus reveal a previously unrecognized beta(2)AR regulatory motif that appeared late in primate evolution and serves to direct internalized receptors to lysosomal degradation during long term agonist exposure.

Adenylyl Cyclases↗
PubMed2004

Corticosteroid pharmacogenetics: association of sequence variants in CRHR1 with improved lung function in asthmatics treated with inhaled corticosteroids.

Corticosteroids mediate a variety of immunological actions and are commonly utilized in the treatment of a wide range of diseases. Unfortunately, therapy with this class of medications is associated with a large proportion of non-responders and significant side effects. Inhaled corticosteroids are the most commonly used asthma controller therapy. However, asthmatic response to corticosteroids also varies widely between individuals. We investigated the genetic contribution to the variation in response to inhaled corticosteroid therapy in asthma. The association of longitudinal change in lung function and single nucleotide polymorphisms from candidate genes crucial to the biologic actions of corticosteroids were evaluated in three independent asthmatic clinical trial populations utilizing inhaled corticosteroids as the primary therapy in at least one treatment arm. Variation in one gene, corticotropin-releasing hormone receptor 1 (CRHR1) was consistently associated with enhanced response to therapy in each of our three populations. Individuals homozygous for the variants of interest manifested a doubling to quadrupling of the lung function response to corticosteroids compared with lack of the variants (P-values ranging from 0.006 to 0.025 for our three asthmatic populations). As the primary receptor mediating the release of adrenocorticotropic hormone, which regulates endogenous cortisol levels, CRHR1 plays a pivotal, pleiotropic role in steroid biology. These data indicate that genetic variants in CRHR1 have pharmacogenetic effects influencing asthmatic response to corticosteroids, provide a rationale for predicting therapeutic response in asthma and other corticosteroid-treated diseases, and suggests this gene pathway as a potential novel therapeutic target.

Administration, Inhalation↗
PubMed2004

PKC-alpha 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-alpha as a fundamental regulator of cardiac contractility and Ca(2+) 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-alpha into cardiac myocytes enhances or reduces contractility, respectively. Mechanistically, modulation of PKC-alpha activity affects dephosphorylation of the sarcoplasmic reticulum Ca(2+) ATPase-2 (SERCA-2) pump inhibitory protein phospholamban (PLB), and alters sarcoplasmic reticulum Ca(2+) loading and the Ca(2+) transient. PKC-alpha 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-alpha 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-alpha functions as a nodal integrator of cardiac contractility by sensing intracellular Ca(2+) and signal transduction events, which can profoundly affect propensity toward heart failure.

Animals↗
PubMed2004

Medetomidine analogs as selective agonists for the human alpha2-adrenoceptors.

alpha(2)-Adrenoceptor (AR) agonists have therapeutic applications in a variety of diseases. Medetomidine, an alpha(2)-AR agonist, belongs to 4-substituted imidazole class of compounds and is highly selective for the alpha(2)-AR. The benzylic methyl group of medetomidine and naphthalene imidazole was proposed to interact with rat brain alpha(2)-ARs via a methyl binding pocket in a manner analogous to its presence in alpha-methyl norepinephrine. A series of derivatives containing hydrophilic and hydrophobic substituents, as well as chiral and conformationally rigid analogs were used. In current binding and functional studies using human alpha(2)-AR subtypes expressed in Chinese hamster ovary cells, optimal interactions were observed with the presence of the methyl group on the benzylic carbon atom of naphthyl imidazole. Data obtained with various analogs have demonstrated that size, electronegativity, lipophilicity, chirality and conformational flexibility of the substituents at the carbon bridge of naphthyl imidazole are important factors for interaction of the imidazole class of ligands with these alpha(2)-AR subtypes. Taken collectively, the results obtained support the existence of the methyl binding pocket for optimal ligand receptor binding interactions in human alpha(2)-AR subtypes. Further, the results also suggest that, additional modifications of medetomidine and naphthyl methyl imidazole at the benzylic carbon atom, and/or on the aromatic and imidazole ring systems could provide insights into the chemical requirements for optimizing alpha(2)-AR subtype selectivity. This could eventually lead to the discovery of promising compounds for the evaluation of the physiological importance of the three alpha(2)-AR subtypes.

Adrenergic alpha-2 Receptor Agonists↗
PubMed2004

Genetically modified mouse models for pharmacogenomic research.

It is now evident that differences in the DNA sequence of genes involved with drug action can lead to interindividual differences in effectiveness and adverse reactions to therapeutic drugs. Pharmacogenomics raises the possibility that drug discovery and patient management could move from a 'one drug fits all' approach to one in which therapy is tailored to patients' genomes. Genetically modified mice that mimic human variation in drug response can provide one of the tools to move the field towards these goals.

Animals↗
PubMed2003

Common genomic response in different mouse models of beta-adrenergic-induced cardiomyopathy.

BACKGROUND: Although beta-adrenergic receptor (AR) blockade therapy is beneficial in the treatment of heart failure, little is known regarding the transcriptional mechanisms underlying this salutary action. METHODS AND RESULTS: In the present study, we screened mice overexpressing Gsalpha, beta1AR, beta2AR, or protein kinase A to test if a common genomic pathway exists in different models with enhanced beta-adrenergic signaling. In mice overexpressing Gsalpha, differentially expressed genes were identified by mRNA profiling. In addition to well-known markers of cardiac hypertrophy (atrial natriuretic factor, CARP, and beta-myosin heavy chain), uncoupling protein 2 (UCP2), a protein involved in the control of mitochondrial membrane potential, and four-and-a-half LIM domain protein-1 (FHL1), a member of the LIM protein family, were predicted to be upregulated. Upregulation of these genes was confirmed by quantitative reverse transcriptase-polymerase chain reaction at all time points tested during the development of cardiomyopathy in mice overexpressing Gsalpha. In mice overexpressing beta1AR, beta2AR, or protein kinase A, increased UCP2 and FHL1 expression was also observed at the onset of cardiomyopathy. BetaAR blockade treatment reversed the cardiomyopathy and suppressed the increased expression of UCP2 and FHL1 in mice overexpressing Gsalpha. CONCLUSIONS: UCP2 and FHL1 are important candidate genes that correlate with the development of betaAR-induced cardiomyopathy in different mouse models with enhanced betaAR signaling. In addition to preserving cardiac function, betaAR blockade treatment also prevents the genomic regulation that correlates with the onset of heart failure.

Adrenergic beta-Antagonists↗
PubMed2003

Protein kinase Calpha negatively regulates systolic and diastolic function in pathological hypertrophy.

The protein kinase C (PKC) family is implicated in cardiac hypertrophy, contractile failure, and beta-adrenergic receptor (betaAR) dysfunction. Herein, we describe the effects of gain- and loss-of-PKCalpha function using transgenic expression of conventional PKC isoform translocation modifiers. In contrast to previously studied PKC isoforms, activation of PKCalpha failed to induce cardiac hypertrophy, but instead caused betaAR insensitivity and ventricular dysfunction. PKCalpha inhibition had opposite effects. Because PKCalpha is upregulated in human and experimental cardiac hypertrophy and failure, its effects were also assessed in the context of the Galphaq overexpression model (in which PKCalpha is transcriptionally upregulated). Normalization (inhibition) of PKCalpha activity in Galpha(q) hearts improved systolic and diastolic function, whereas further activation of PKCalpha caused a lethal restrictive cardiomyopathy with marked interstitial fibrosis. These results define pathological roles for PKCalpha as a negative regulator of ventricular systolic and diastolic function.

Adrenergic beta-Agonists↗
PubMed2003

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

Catecholamines stimulate cardiac contractility through beta(1)-adrenergic receptors (beta(1)-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 beta-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 G alpha(s) expression, and fibrosis with heart failure This phenotype was recapitulated in homozygous, end-stage, failing human hearts. In addition, hemodynamic responses to beta-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 beta-receptor blockade.

Adrenergic beta-Antagonists↗
PubMed2003

Hierarchy of polymorphic variation and desensitization permutations relative to beta 1- and beta 2-adrenergic receptor signaling.

Agonist-promoted desensitization of G-protein-coupled receptors results in partial uncoupling of receptor from cognate G-protein, a process that provides for rapid adaptation to the signaling environment. This property plays important roles in physiologic and pathologic processes as well as therapeutic efficacy. However, coupling is also influenced by polymorphic variation, but the relative impact of these two mechanisms on signal transduction is not known. To determine this we utilized recombinant cells expressing the human beta(1)-adrenergic receptor (beta(1)AR) or a gain-of-function polymorphic variant (beta(1)AR-Arg(389)), and the beta(2)-adrenergic receptor (beta(2)AR) or a loss-of-function polymorphic receptor (beta(2)AR-Ile(164)). Adenylyl cyclase activities were determined with multiple permutations of the possible states of the receptor: genotype, basal, or agonist stimulated and with or without agonist pre-exposure. For the beta(1)AR, the enhanced function of the Arg(389) receptor underwent less agonist-promoted desensitization compared with its allelic counterpart. Indeed, the effect of polymorphic variation on absolute adenylyl cyclase activities was such that desensitized beta(1)AR-Arg(389) signaling was equivalent to non-desensitized wild-type beta(1)AR; that is, the genetic component had as much impact as desensitization on receptor coupling. In contrast, the enhanced signaling of wild-type beta(2)AR underwent less desensitization compared with beta(2)AR-Ile(164), thus the heterogeneity in absolute signaling was markedly broadened by this polymorphism. Inverse agonist function was not affected by polymorphisms of either subtype. A general model is proposed whereby up to 10 levels of signaling by G-protein-coupled receptors can be present based on the influences of desensitization and genetic variation on coupling.

Adenylyl Cyclases↗
PubMed2003

Hereditary dysautonomias: current knowledge and collaborations for the future.

The hereditary dysautonomias (H-Dys) are a large group of disorders that affect the autonomic nervous system. Research in the field of H-Dys is very challenging, because the disorders involve interdisciplinary, integrative, and "mind-body" connections. Recently, medical scientists, NIH/NINDS representatives, and several patient support groups gathered for the first time in order to discuss recent findings and future directions in the H-Dys field. The H-Dys workshop was instrumental in promoting interactions between basic science and clinical investigators. It also allowed attendees to have an opportunity to meet each other, understand the similarities between the various forms of dysautonomia, and experience the unique perspective offered by patients and their families. Future advances in H-Dys research will depend on a novel multi-system approach by investigators from different medical disciplines, and it is hoped that towards a common goal, novel "bench-to-bedside" therapeutics will be developed to improve the lives of, or even cure, patients suffering from dysautonomic syndromes.

Animals↗
PubMed2003

Polymorphisms of adrenergic receptors: variations on a theme.

Recent studies have revealed that most of the adrenergic receptor genes are polymorphic, leading to changes in the amino sequence of the encoded receptor. The variations occur in multiple functional regions of the receptors, and appear as haplotypes with other coding and noncoding polymorphisms in their genes. The consequences of such genetic variability have been explored in recombinant cell-based systems and in human studies. Adrenergic receptor polymorphisms have been shown to alter receptor binding, G-protein coupling, regulation, and expression compared with their allelic counterparts. Here, the genetic and molecular characterization of these polymorphisms is reviewed, as well as their potential impact on pharmacogenetics, disease risk, and disease modification.

Humans↗
PubMed2003

An Ile to Met polymorphism in the catalytic domain of adenylyl cyclase type 9 confers reduced beta2-adrenergic receptor stimulation.

Adenylyl cyclase (AC) mediates signalling following activation of G(alphas)-coupled receptors such as the beta2-adrenergic receptor (beta2AR). Genetic variation in the receptor component of this pathway can alter signal transduction and the response to beta-agonists in asthma, but little is known about downstream effectors. Here, we characterize the population genomics and signalling effects of a polymorphism within the coding region of the AC9 gene that results in an Ile to Met substitution at amino acid 772 within the C1b region of the enzyme. Allele frequencies were 0.300 and 0.375 in Caucasians and Asians but were lower in African-Americans (0.163). The functional effects were studied in stably transfected HEK293 cells recombinantly expressing equivalent levels of wild-type (Ile772) and polymorphic (Met772) AC9. The polymorphic substitution results in a loss of function compared to wild-type AC9. Met772 AC9 has lower basal and beta2AR-mediated adenylyl cyclase activities compared to Ile772 AC9, as well as reduced activity following stimulation of G(alphas) by NaF. Direct stimulation of AC9 activity by Mn2+/- was also depressed in Met772 membranes, indicating decreased catalytic function, consistent with the location of residue 772. AC9 mRNA and protein were expressed in multiple human lung cell-types, including airway smooth muscle and airway epithelium. In the treatment of asthma, there is marked heterogeneity in the response to inhaled beta-agonists which is associated with polymorphisms of the beta2AR. Identification of a common AC9 variant that confers reduced enzyme activity reveals an additional polymorphism that should be considered in pharmacogenetic studies of beta-agonist therapy of asthma.

Adenylyl Cyclases↗
PubMed2003

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.

Animals↗
PubMed2003

Antithetic regulation by beta-adrenergic receptors of Gq receptor signaling via phospholipase C underlies the airway beta-agonist paradox.

beta-adrenergic receptors (betaARs) relax airway smooth muscle and bronchodilate, but chronic beta-agonist treatment in asthma causes increased sensitivity to airway constriction (hyperreactivity) and is associated with exacerbations. This paradox was explored using mice with ablated betaAR genes (betaAR-/-) and transgenic mice overexpressing airway smooth muscle beta2AR (beta2AR-OE) representing two extremes: absence and persistent activity of airway betaAR. Unexpectedly, betaAR-/- mice, lacking these bronchodilating receptors, had markedly decreased bronchoconstrictive responses to methacholine and other Gq-coupled receptor agonists. In contrast, beta2AR-OE mice had enhanced constrictive responses. Contraction to permeabilization with beta-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 betaAR-/- mice and sensitized in cells from beta2AR-OE mice. Thus, betaAR 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-beta1, whose expression was altered by betaAR in a direction and magnitude consistent with the physiologic and cellular responses. These results establish a mechanism of the beta-agonist paradox and identify a potential asthma modifier gene (phospholipase C-beta1), which may also be a therapeutic target in asthma when chronic beta-agonists are required.

Animals↗