Genetic Variants of Adrenoceptors.

Adrenoceptors are class A G-protein-coupled receptors grouped into three families (α1-, α2-, and ß-adrenoceptors), each one including three members. All nine corresponding adrenoceptor genes display genetic variation in their coding and adjacent non-coding genomic region. Coding variants, i.e., nucleotide exchanges within the transcribed and translated receptor sequence, may result in a difference in amino acid sequence thus altering receptor function and signaling. Such variants have been intensely studied in vitro in overexpression systems and addressed in candidate-gene studies for distinct clinical parameters. In recent years, large cohorts were analyzed in genome-wide association studies (GWAS), where variants are detected as significant in context with specific traits. These studies identified two of the in-depth characterized 18 coding variants in adrenoceptors as repeatedly statistically significant genetic risk factors - p.Arg389Gly in the ß1- and p.Thr164Ile in the ß2-adrenoceptor, along with 56 variants in the non-coding regions adjacent to the adrenoceptor gene loci, the functional role of which is largely unknown at present. This chapter summarizes current knowledge on the two coding variants in adrenoceptors that have been consistently validated in GWAS and provides a prospective overview on the numerous non-coding variants more recently attributed to adrenoceptor gene loci.

Immune-mediated denervation of the pineal gland underlies sleep disturbance in cardiac disease.

Disruption of the physiologic sleep-wake cycle and low melatonin levels frequently accompany cardiac disease, yet the underlying mechanism has remained enigmatic. Immunostaining of sympathetic axons in optically cleared pineal glands from humans and mice with cardiac disease revealed their substantial denervation compared with controls. Spatial, single-cell, nuclear, and bulk RNA sequencing traced this defect back to the superior cervical ganglia (SCG), which responded to cardiac disease with accumulation of inflammatory macrophages, fibrosis, and the selective loss of pineal gland-innervating neurons. Depletion of macrophages in the SCG prevented disease-associated denervation of the pineal gland and restored physiological melatonin secretion. Our data identify the mechanism by which diurnal rhythmicity in cardiac disease is disturbed and suggest a target for therapeutic intervention.

Local sympathetic denervation attenuates myocardial inflammation and improves cardiac function after myocardial infarction in mice.

Aims: Cardiac inflammation has been suggested to be regulated by the sympathetic nervous system (SNS). However, due to the lack of methodology to surgically eliminate the myocardial SNS in mice, neuronal control of cardiac inflammation remains ill-defined. Here, we report a procedure for local cardiac sympathetic denervation in mice and tested its effect in a mouse model of heart failure post-myocardial infarction. Methods and results: Upon preparation of the carotid bifurcation, the right and the left superior cervical ganglia were localized and their pre- and postganglionic branches dissected before removal of the ganglion. Ganglionectomy led to an almost entire loss of myocardial sympathetic innervation in the left ventricular anterior wall. When applied at the time of myocardial infarction (MI), cardiac sympathetic denervation did not affect acute myocardial damage and infarct size. In contrast, cardiac sympathetic denervation significantly attenuated chronic consequences of MI, including myocardial inflammation, myocyte hypertrophy, and overall cardiac dysfunction. Conclusion: These data suggest a critical role for local sympathetic control of cardiac inflammation. Our model of myocardial sympathetic denervation in mice should prove useful to further dissect the molecular mechanisms underlying cardiac neural control.

Gαi is required for carvedilol-induced ß1 adrenergic receptor ß-arrestin biased signaling.

The ß1 adrenergic receptor (ß1AR) is recognized as a classical Gαs-coupled receptor. Agonist binding not only initiates G protein-mediated signaling but also signaling through the multifunctional adapter protein ß-arrestin. Some ßAR ligands, such as carvedilol, stimulate ßAR signaling preferentially through ß-arrestin, a concept known as ß-arrestin-biased agonism. Here, we identify a signaling mechanism, unlike that previously known for any Gαs-coupled receptor, whereby carvedilol induces the transition of the ß1AR from a classical Gαs-coupled receptor to a Gαi-coupled receptor stabilizing a distinct receptor conformation to initiate ß-arrestin-mediated signaling. Recruitment of Gαi is not induced by any other ßAR ligand screened, nor is it required for ß-arrestin-bias activated by the ß2AR subtype of the ßAR family. Our findings demonstrate a previously unrecognized role for Gαi in ß1AR signaling and suggest that the concept of ß-arrestin-bias may need to be refined to incorporate the selective bias of receptors towards distinct G protein subtypes.

Two serines in the distal C-terminus of the human ß1-adrenoceptor determine ß-arrestin2 recruitment.

G protein-coupled receptors (GPCRs) undergo phosphorylation at several intracellular residues by G protein-coupled receptor kinases. The resulting phosphorylation pattern triggers arrestin recruitment and receptor desensitization. The exact sites of phosphorylation and their function remained largely unknown for the human ß1-adrenoceptor (ADRB1), a key GPCR in adrenergic signal transduction and the target of widely used drugs such as ß-blockers. The present study aimed to identify the intracellular phosphorylation sites in the ADRB1 and to delineate their function. The human ADRB1 was expressed in HEK293 cells and its phosphorylation pattern was determined by mass spectrometric analysis before and after stimulation with a receptor agonist. We identified a total of eight phosphorylation sites in the receptor's third intracellular loop and C-terminus. Analyzing the functional relevance of individual sites using phosphosite-deficient receptor mutants we found phosphorylation of the ADRB1 at Ser461/Ser462 in the distal part of the C-terminus to determine ß-arrestin2 recruitment and receptor internalization. Our data reveal the phosphorylation pattern of the human ADRB1 and the site that mediates recruitment of ß-arrestin2.

Branch-Specific Microtubule Destabilization Mediates Axon Branch Loss during Neuromuscular Synapse Elimination.

Developmental axon remodeling is characterized by the selective removal of branches from axon arbors. The mechanisms that underlie such branch loss are largely unknown. Additionally, how neuronal resources are specifically assigned to the branches of remodeling arbors is not understood. Here we show that axon branch loss at the developing mouse neuromuscular junction is mediated by branch-specific microtubule severing, which results in local disassembly of the microtubule cytoskeleton and loss of axonal transport in branches that will subsequently dismantle. Accordingly, pharmacological microtubule stabilization delays neuromuscular synapse elimination. This branch-specific disassembly of the cytoskeleton appears to be mediated by the microtubule-severing enzyme spastin, which is dysfunctional in some forms of upper motor neuron disease. Our results demonstrate a physiological role for a neurodegeneration-associated modulator of the cytoskeleton, reveal unexpected cell biology of branch-specific axon plasticity and underscore the mechanistic similarities of axon loss in development and disease.

Peptidase inhibitor 16 is a membrane-tethered regulator of chemerin processing in the myocardium.

A key response of the myocardium to stress is the secretion of factors with paracrine or endocrine function. Intriguing in this respect is peptidase inhibitor 16 (PI16), a member of the CAP family of proteins which we found to be highly upregulated in cardiac disease. Up to this point, the mechanism of action and physiological function of PI16 remained elusive. Here, we show that PI16 is predominantly expressed by cardiac fibroblasts, which expose PI16 to the interstitium via a glycophosphatidylinositol (-GPI) membrane anchor. Based on a reported genetic association of PI16 and plasma levels of the chemokine chemerin, we investigated whether PI16 regulates post-translational processing of its precursor pro-chemerin. PI16-deficient mice were engineered and found to generate higher levels of processed chemerin than wildtype mice. Purified recombinant PI16 efficiently inhibited cathepsin K, a chemerin-activating protease, in vitro. Moreover, we show that conditioned medium from PI16-overexpressing cells impaired the activation of pro-chemerin. Together, our data indicate that PI16 suppresses chemerin activation in the myocardium and suggest that this circuit may be part of the cardiac stress response.

Interhelical interaction and receptor phosphorylation regulate the activation kinetics of different human ß1-adrenoceptor variants.

G protein-coupled receptors represent the largest class of drug targets, but genetic variation within G protein-coupled receptors leads to variable drug responses and, thereby, compromises their therapeutic application. One of the most intensely studied examples is a hyperfunctional variant of the human ß1-adrenoceptor that carries an arginine at position 389 in helix 8 (Arg-389-ADRB1). However, the mechanism underlying the higher efficacy of the Arg-389 variant remained unclear to date. Despite its hyperfunctionality, we found the Arg-389 variant of ADRB1 to be hyperphosphorylated upon continuous stimulation with norepinephrine compared with the Gly-389 variant. Using ADRB1 sensors to monitor activation kinetics by fluorescence resonance energy transfer, Arg-389-ADRB1 exerted faster activation speed and arrestin recruitment than the Gly-389 variant. Both activation speed and arrestin recruitment depended on phosphorylation of the receptor, as shown by knockdown of G protein-coupled receptor kinases and phosphorylation-deficient ADRB1 mutants. Structural modeling of the human ß1-adrenoceptor suggested interaction of the side chain of Arg-389 with opposing amino acid residues in helix 1. Site-directed mutagenesis of Lys-85 and Thr-86 in helix 1 revealed that this interaction indeed determined ADRB1 activation kinetics. Taken together, these findings indicate that differences in interhelical interaction regulate the different activation speed and efficacy of ADRB1 variants.

Cardiac myocyte-secreted cAMP exerts paracrine action via adenosine receptor activation.

Acute stimulation of cardiac ß-adrenoceptors is crucial to increasing cardiac function under stress; however, sustained ß-adrenergic stimulation has been implicated in pathological myocardial remodeling and heart failure. Here, we have demonstrated that export of cAMP from cardiac myocytes is an intrinsic cardioprotective mechanism in response to cardiac stress. We report that infusion of cAMP into mice averted myocardial hypertrophy and fibrosis in a disease model of cardiac pressure overload. The protective effect of exogenous cAMP required adenosine receptor signaling. This observation led to the identification of a potent paracrine mechanism that is dependent on secreted cAMP. Specifically, FRET-based imaging of cAMP formation in primary cells and in myocardial tissue from murine hearts revealed that cardiomyocytes depend on the transporter ABCC4 to export cAMP as an extracellular signal. Extracellular cAMP, through its metabolite adenosine, reduced cardiomyocyte cAMP formation and hypertrophy by activating A1 adenosine receptors while delivering an antifibrotic signal to cardiac fibroblasts by A2 adenosine receptor activation. Together, our data reveal a paracrine role for secreted cAMP in intercellular signaling in the myocardium, and we postulate that secreted cAMP may also constitute an important signal in other tissues.

Polymorphic variants of adrenoceptors: pharmacology, physiology, and role in disease.

The human genome encodes nine different adrenoceptor genes. These are grouped into three families, namely, the α1-, α2-, and ß-adrenoceptors, with three family members each. Adrenoceptors are expressed by most cell types of the human body and are primary targets of the catecholamines epinephrine and norepinephrine that are released from the sympathetic nervous system during its activation. Upon catecholamine binding, adrenoceptors change conformation, couple to and activate G proteins, and thereby initiate various intracellular signaling cascades. As the primary receivers and transducers of sympathetic activation, adrenoceptors have a central role in human physiology and disease and are important targets for widely used drugs. All nine adrenoceptor subtypes display substantial genetic variation, both in their coding sequence as well as in adjacent regions. Despite the fact that some of the adrenoceptor variants range among the most frequently studied genetic variants assessed in pharmacogenetics to date, their functional relevance remains ill defined in many cases. A substantial fraction of the associations reported from early candidate gene approaches have not subsequently been confirmed in different cohorts or in genome-wide association studies, which have increasingly been conducted in recent years. This review aims to provide a comprehensive overview of all adrenoceptor variants that have reproducibly been detected in the larger genome sequencing efforts. We evaluate these variants with respect to the modulation of receptor function and expression and discuss their role in physiology and disease.

MiR-378 controls cardiac hypertrophy by combined repression of mitogen-activated protein kinase pathway factors.

BACKGROUND: Several microRNAs (miRs) have been shown to regulate gene expression in the heart, and dysregulation of their expression has been linked to cardiac disease. miR-378 is strongly expressed in the mammalian heart but so far has been studied predominantly in cancer, in which it regulates cell survival and tumor growth. METHODS AND RESULTS: Here, we report tight control of cardiomyocyte hypertrophy through miR-378. In isolated primary cardiomyocytes, miR-378 was found to be both necessary and sufficient to repress cardiomyocyte hypertrophy. Bioinformatic prediction suggested that factors of the mitogen-activated protein kinase (MAPK) pathway are enriched among miR-378 targets. Using mRNA and protein expression analysis along with luciferase assays, we validated 4 key components of the MAPK pathway as targets of miR-378: MAPK1 itself, insulin-like growth factor receptor 1, growth factor receptor-bound protein 2, and kinase suppressor of ras 1. RNA interference with these targets prevented the prohypertrophic effect of antimiR-378, suggesting their functional relation with miR-378. Because miR-378 significantly decreases in cardiac disease, we sought to compensate for its loss through adeno-associated virus-mediated, cardiomyocyte-targeted expression of miR-378 in an in vivo model of cardiac hypertrophy (pressure overload by thoracic aortic constriction). Restoration of miR-378 levels significantly attenuated thoracic aortic constriction-induced cardiac hypertrophy and improved cardiac function. CONCLUSIONS: Our data identify miR-378 as a regulator of cardiomyocyte hypertrophy, which exerts its activity by suppressing the MAPK signaling pathway on several distinct levels. Restoration of disease-associated loss of miR-378 through cardiomyocyte-targeted adeno-associated virus-miR-378 may prove to be an effective therapeutic strategy in myocardial disease.

A polymorphism-specific "memory" mechanism in the ß(2)-adrenergic receptor.

Signaling through G protein (heterotrimeric guanosine triphosphate-binding protein)-coupled receptors is affected by polymorphisms in receptor-encoding genes. Using fluorescence resonance energy transfer, we found that the ß(2)-adrenergic receptor (ß(2)AR) responded to repeated activation with altered activation kinetics. Polymorphic variants of the ß(2)AR displayed divergent changes of ß(2)AR activation kinetics that closely mimicked their different efficacies to generate cyclic adenosine 3',5'-monophosphate. More efficacious variants became faster in their activation kinetics, whereas less efficacious variants became slower, compared to their initial activation. These differences depended on phosphorylation of the receptor by G protein-coupled receptor kinases. Our findings suggest an intrinsic, polymorphism-specific property of the ß(2)AR that alters activation kinetics upon continued stimulation and that may account for individual drug responses.

Polymorphisms determine beta-adrenoceptor conformation: implications for cardiovascular disease and therapy.

Beta(1)- and beta(2)-adrenoceptors are crucial regulators of cardiovascular function. Agonists and antagonists at these receptor subtypes are cornerstones in the treatment of cardiovascular disease. In humans, both of the genes encoding the beta(1)- and beta(2)-adrenoceptors carry frequent polymorphisms resulting in different variants of the receptor proteins. Whether the polymorphic nature of the receptors causes the clinically observed differences with respect to the response of the patients to therapeutic drugs is currently a matter of intense discussion. Here, we discuss recent progress regarding the determination of beta-adrenoceptor conformational changes and how these can help to clarify this issue. Specifically, novel optical methods enable us to directly assess the functional importance of beta-adrenoceptor polymorphisms on ligand-induced changes of receptor conformation. The ability to determine polymorphism-dependent differences in drug efficacy directly on the receptor level might develop into an important approach to establish individualized drug therapies based on the genetic determinants of the patients.