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Cardiovascular Research 2007 75(4):728-737; doi:10.1016/j.cardiores.2007.05.017
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Copyright © 2007, European Society of Cardiology

Heterozygous {alpha}2C-adrenoceptor-deficient mice develop heart failure after transverse aortic constriction

Ralf Gilsbacha,1, Marc Bredeb,c,1, Nadine Beetza, Eduardo Mouraa,d, Verena Muthiga, Carolin Gerstnerb, Frederico Barretoa, Stefan Neubauere, Maria Augusta Vieira-Coelhod and Lutz Heina,*

aInstitute of Experimental and Clinical Pharmacology, University of Freiburg, Germany
bDepartment of Anesthesiology, University of Würzburg, Germany
cDepartment of Pharmacology, University of Würzburg, Germany
dInstituto de Farmacologia e Terapêutica, Faculdade de Medicina, Universidade do Porto, Portugal
eDepartment of Cardiovascular Medicine, University of Oxford, UK

* Corresponding author. Institute of Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, Albertstrasse 25, 79104 Freiburg, Germany. Tel.: +49 761 2035314; fax: +49 761 2035318. lutz.hein{at}pharmakol.uni-freiburg.de

Received 5 February 2007; revised 28 April 2007; accepted 15 May 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgment
 References
 
Objective Feedback regulation of norepinephrine release from sympathetic nerves is essential to control blood pressure, heart rate and contractility. Recent experiments in gene-targeted mice have suggested that {alpha}2C-adrenoceptors may operate in a similar feedback mechanism to control the release of epinephrine from the adrenal medulla. As heterozygous polymorphisms in the human {alpha}2C-adrenoceptor gene have been associated with cardiovascular disease including hypertension and chronic heart failure, we have sought to characterize the relevance of {alpha}2C-gene copy number for feedback control of epinephrine release in gene-targeted mice.

Methods Adrenal catecholamine release, basal hemodynamics and susceptibility to develop heart failure after transverse aortic constriction were tested in mice with two copies (+/+), one copy (+/–) or no functional {alpha}2C-adrenoceptor gene ({alpha}2C–/–).

Results Heterozygous {alpha}2C-receptor deletion ({alpha}2C+/–) resulted in a 43% reduction of adrenal {alpha}2C mRNA copy number and in a similar decrease in {alpha}2-receptor-mediated inhibition of catecholamine release from isolated adrenal glands in vitro. Urinary excretion of epinephrine was increased by 74±15% in {alpha}2C+/– and by 142±23% in {alpha}2C–/– mice as compared with wild-type control mice. Telemetric determination of cardiovascular function revealed significant tachycardia but no hypertension in {alpha}2C-adrenoceptor-deficient mice. {alpha}2C+/– mice were more susceptible to develop cardiac hypertrophy, failure and mortality after left-ventricular pressure overload than {alpha}2C+/+ mice.

Conclusion Adrenal {alpha}2-mediated feedback regulation of epinephrine secretion differs fundamentally from sympathetic feedback control. A single adrenoceptor subtype, {alpha}2C, operates without a significant receptor reserve to prevent elevation of circulating epinephrine levels. This genetic model may provide an experimental basis to study the pathophysiology of {alpha}2C-adrenoceptor dysfunction in humans.

KEYWORDS Neurotransmitters; Transgenic animal models; Hypertrophy; Autonomic nervous system; Adrenergic system

This article is referred to in the Editorial by Petrashevskaya and Liggett (pages 631–633) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgment
 References
 
The sympathetic nervous system is an essential regulator of cardiovascular function, including control of heart rate, contractility and vascular tone. Inhibition of sympathetic activity represents an important pharmacological strategy in the treatment of hypertension, arrhythmias, coronary heart disease and chronic heart failure. However, the complexity of adrenergic receptor subtypes which are involved in sympathetic signaling is not yet matched by an equal number of pharmacological agents which can be applied for human therapy. Part of the discrepancy between the number of adrenergic receptor subtypes and the lack of sufficiently subtype selective ligands may result from the fact that the physiological relevance of subtype diversity in the adrenergic system is only partly understood [1,2].

Molecular cloning has led to the identification of three {alpha}2-adrenoceptor subtypes, {alpha}2A, {alpha}2B, and {alpha}2C [3,4]. A fourth subtype, {alpha}2D, has been suggested from pharmacological studies but later turned out to be the rodent orthologue of the human {alpha}2A-adrenoceptor [3,4]. Transgenic mouse models with targeted deletions in individual {alpha}2-adrenoceptor genes have been developed to assign physiological and pharmacological functions to specific {alpha}2-receptor subtypes (for reviews, see Refs. [2,5–8]). From these mouse studies, it was concluded that all three {alpha}2-receptor subtypes may serve as presynaptic feedback regulators of catecholamine release from sympathetic neurons with {alpha}2A being the functionally predominant subtype [9,10]. We have previously shown that {alpha}2C-adrenoceptors in the murine adrenal medulla may operate as inhibitory feedback regulators to control the release of epinephrine from chromaffin cells [11].

Genomic sequencing of human {alpha}2-adrenoceptor genes has led to the identification of a large number of genetic variants in all three receptors (for overview, see Refs. [12–16]). In particular, association studies have suggested that a deletion of four amino acids in the third intracellular loop of the {alpha}2C-adrenoceptor ({alpha}2C-DEL322–325) may be involved in the development of chronic heart failure and hypertension [17–19]. Direct evidence for a functional role of {alpha}2C-adrenoceptors in heart failure is still lacking. Thus, we have further investigated the relationship between {alpha}2C-receptor density, adrenal epinephrine secretion and cardiovascular function in mice with targeted deletion of {alpha}2C-adrenoceptors. As an extension to previous investigations, this study primarily focuses on mice lacking a single {alpha}2C-adrenoceptor gene copy. The results demonstrate that {alpha}2C controls adrenal catecholamine secretion in a gene dosage-dependent manner with little or no receptor reserve.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Acknowledgment
 References
 
2.1 Generation and genotyping of {alpha}2C-adrenoceptor-deficient mice
The generation of mouse lines lacking {alpha}2-adrenoceptor subtypes has been described in detail [20–22]. The targeted {alpha}2C-adrenoceptor allele was crossed back onto a C57BL/6J background for >12 generations. Mice used in this study were generated from intercrosses of heterozygous congenic {alpha}2C+/– mice. Genotypes were confirmed by polymerase chain reactions (Fig. 1a) performed with genomic DNA isolated from tail biopsies as described in detail [23]. Mice were maintained in a specified pathogen-free facility. All animal procedures were approved by the responsible animal care committees of the Universities of Freiburg and Würzburg. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).


Figure 1
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  		Fig. 1 Targeted deletion of the {alpha}2C-adrenoceptor gene and adrenal histology in mice. (a) Schematic representation of the murine {alpha}2C-receptor allele that was targeted for deletion by insertion of a neomycin cassette as described previously [22]. Gray areas indicate location of the sequences encoding the transmembrane regions of the {alpha}2C-receptor (transmembrane domains six and seven were deleted by gene targeting [21]). Arrowheads show location of polymerase chain reaction primers used for genotyping. Lower panel: representative polymerase chain reactions to detect wild-type ({alpha}2C+/+) and {alpha}2C-deficient alleles of the {alpha}2C-receptor gene. (b, c and d) Histological structure of the adrenal gland is not altered by deletion of the {alpha}2C-adrenoceptor gene. Left panels: low magnification, bar 100 µm; c, cortex; m, medulla. Right panels: higher magnification of the adrenal medulla, bar 20 µm. 0.5 µm araldite sections stained with methylene blue.

 
2.2 Quantitative real-time PCR
Quantitative real-time polymerase chain reaction (qPCR) was carried out as previously described [24,25]. In brief, adrenal glands were rapidly removed from decapitated mice. RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Total RNA (1 µg per sample) was DNase treated and reverse transcribed according to the manufacturer's instructions (QuantiTect Rev. Transcription Kit, Qiagen). For qPCR 35 µl of amplification mixture (Qiagen, Quantitect SYBR Green Kit) was used containing 20 ng of reverse transcribed RNA and 300 nM primers (MWG, Ebersberg, Germany) (Table 1). Reactions were run in triplicates (10 µl) on a MX3000P detector (Stratagene, Amsterdam, Netherlands). The cycling conditions were: 15 min polymerase activation at 95 °C and 40 cycles at 95 °C for 15 s, at 58 °C for 30 s and at 72 °C for 30 s. Absolute copy numbers were determined using standard curves of corresponding linear DNA-fragments (7 points from 109–102 copies diluted in 10 µg/ml carrier RNA (Qiagen)). Results were normalized to β-actin values.


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  		Table 1 Sequences of primers used for quantitative real-time PCR

 
2.3 Catecholamine release from isolated adrenal glands
Adrenal glands were prepared from mice after cervical dislocation and placed into modified Krebs–Henseleit solution of the following composition (in mM): NaCl 118, KCl 4.8, CaCl2 2.5, MgSO4 1.2, NaHCO3 25, KH2PO4 1.2, glucose 11, ascorbic acid 0.57, disodium EDTA 0.03, oxygenated with a mixture of 95% O2 and 5% CO2 in the presence of a monoamine oxidase inhibitor (pargyline, 100 µM) and a catechol-O-methyltransferase inhibitor (tolcapone, 1 µM). The adrenal medullae were isolated from the glands and then placed in superfusion chambers, one per chamber, and were continuously superfused with Krebs–Henseleit solution [26]. After a 90-min period of stabilization, successive 5-min samples of the superfusate were collected into tubes containing 0.3 ml of 2 M perchloric acid. At the end of the experiments, the adrenal medullae were placed in 1 ml of 0.2 M perchloric acid and catecholamines were determined in superfusates and tissues. The effect of the nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) on catecholamine release was determined by addition of 500 µM of DMPP in a single 5-min period. The inhibitory effect of {alpha}2-adrenoceptor activation on catecholamine release was determined by addition of 100 nM medetomidine 15 min before nicotinic stimulation. The spontaneous outflow of norepinephrine and epinephrine was calculated as a fraction of the norepinephrine or epinephrine content of the tissue at the onset of the respective collection period (fractional rate; min–1). The overflow elicited by nicotinic stimulation was calculated as the difference ‘total norepinephrine or epinephrine outflow during and after stimulation’ minus ‘basal outflow’, and was then expressed as a percentage of the norepinephrine or epinephrine content of the tissue at the onset of stimulation [27].

2.4 [3H]norepinephrine release from isolated atria
Release experiments were carried out as previously described [10,30]. Mice were killed by cervical dislocation and left and right atria were removed and preincubated in 2 ml medium containing 0.1 mM [3H]norepinephrine (Amersham, Freiburg, Germany) for 45 min at 37 °C. They were transferred to 12 superfusion chambers equipped with platinum electrodes, one atrium per chamber, where they were superfused at 37 °C at a rate of 1.2 mlxmin–1. After 45 min of superfusion successive 2-min superfusate samples were collected. The preincubation medium consisted of (mM): NaCl 118, KCl 4.8, CaCl2 0.2, MgSO4 1.2, NaHCO3 25, KH2PO4 1.2, glucose 11, ascorbic acid 0.57, Na2EDTA 0.03. The superfusion medium was the same but contained 2.5 mM CaCl2 and 1 mM desipramine. The medium was saturated with 5% CO2 in O2. Six periods of electrical stimulation (20 pulses/50 Hz, 1 ms pulse width, 80 mA) were applied at 16 min intervals. At the end of the experiments, tissues were solubilized and tritium was determined in superfusate samples and tissues. The electrically evoked overflow of total tritium reflects exocytotic release of [3H]norepinephrine and is termed thus in this paper.

2.5 Catecholamine determination
The assay of the catecholamines norepinephrine and epinephrine in tissues, urine and superfusate samples was performed by high performance liquid chromatography with electrochemical detection as previously described [28]. The lower limit of detection of catecholamines ranged from 350 to 1000 fmol.

2.6 Transverse aortic constriction
Male mice (8–10 weeks old) were anesthetized with 2.0% isoflurane [vol/vol] in 45 ml/min oxygen flow. After thoracotomy, a 7.0 nylon suture was placed around a 27 G hypodermic needle to constrict the aortic arch. The degree of aortic stenosis was assessed by morphometric analysis of paraffin sections of the aortic arch as described [19].

2.7 Hemodynamic measurements
For measurements in conscious, unrestrained mice, blood pressure and electrocardiograms were recorded by telemetry (DSI, Transoma Medical, USA, TA11-PAC10 for aortic pressure) 10–20 days after implantation of telemetry devices during day (7 a.m.–7 p.m.) and night (7 p.m.–7 a.m.). Mice which had been subjected to transverse aortic constriction were catheterized at the end of the respective observation period (Fig. 6). For left-ventricular catheterization with a 1.4 F pressure–volume catheter, mice were anesthetized with isoflurane (2 vol.% in O2) and placed on a 37 °C table [19]. The microtip catheter was inserted into the right carotid artery and the pressure tip was advanced into the left ventricle [19].


Figure 6
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  		Fig. 6 Increased mortality of {alpha}2C-adrenoceptor-deficient mice after transverse aortic constriction (TAC). (a) Survival of mice was followed for up to 7 months after TAC. Mortality was significantly increased in {alpha}2C+/– mice and even more so in {alpha}2C–/– mice (30% mortality: 32 days for {alpha}2C–/– and 102 days for {alpha}2C+/– mice). Kaplan–Meier plots, *p<0.01, log-rank test, n=15 mice per genotype. (b) Sirius red staining of midventricular sections from sham-operated or aortic-constricted (TAC) mice 7 months after the operation. Interstitial fibrosis after TAC was apparent by abundant red staining in {alpha}2C+/+ and {alpha}2C+/– mice (lower panels). Bar, 50 µm. Inserts: Hematoxylin-eosin stained midventricular cardiac sections. Bar, 1 mm. (c and d) Heart weight/body weight and heart weight/tibia length ratios were significantly increased in {alpha}2C+/+ and {alpha}2C+/– mice after TAC (n=5–9 mice per genotype, *p<0.05, TAC vs. sham). (e and f) Left-ventricular contractility (dp/dtmax) as well as left-ventricular relaxation (dp/dtmin) were reduced in {alpha}2C+/– mice after TAC. All data are means±SEM, n=5–9 mice per genotype, *p<0.05, TAC vs. sham.

 
2.8 Histology
After left-ventricular catheterization, hearts were fixed with 4% paraformaldehyde in phosphate-buffered saline, embedded in paraffin and stained with hematoxylin-eosin. Left-ventricular myocyte cross-sectional areas were analyzed by computer-assisted morphometry [19]. Adrenal glands were embedded in araldite and processed as described [29].

2.9 Statistical analysis
Hemodynamic and histological data as well as urinary catecholamine values were analyzed using ANOVA followed by Bonferroni post-hoc tests or Student t test, respectively. Survival curves were created by Kaplan–Meier plots and tested via log-rank test. A p value of less than 0.05 was considered as statistically significant. Results are displayed as means±SEM.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Acknowledgment
 References
 
3.1 Generation of {alpha}2C-adrenoceptor-deficient mice
Mice with intact ({alpha}2C+/+) or deleted {alpha}2C-adrenoceptor genes ({alpha}2C+/– and {alpha}2C–/–) were born at the expected Mendelian ratios and did not show any signs of developmental or structural defects (Fig. 1a) (Ref. [22] and data not shown). The size and microscopical structure of the adrenal gland did not differ between genotypes (Fig. 1). In order to determine the expression of the three {alpha}2-adrenoceptor subtypes and catecholamine synthesizing enzymes in the adrenal medulla, quantitative RT-PCR was performed (Fig. 2). In heterozygous {alpha}2C+/– mice, {alpha}2C mRNA copy numbers were reduced from 30±1 copies per 105 β-actin mRNA copies in wild-type specimens to 17±2 copies per 105 β-actin copies in {alpha}2C+/– adrenal glands (p<0.001, n=5 per genotype). In specimens from {alpha}2C–/– mice, no {alpha}2C mRNA was detected (Fig. 2a). Expression levels of {alpha}2A- or {alpha}2B-adrenoceptor mRNA or catecholamine synthesizing enzymes were not significantly affected by deletion of the {alpha}2C-adrenoceptor gene (Fig. 2a,b).


Figure 2
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  		Fig. 2 Expression of {alpha}2-adrenoceptor subtypes and catecholamine synthesis enzymes in the adrenal gland after targeted deletion of the {alpha}2C-adrenoceptor gene in mice. (a) {alpha}2C mRNA showed the highest expression of all three {alpha}2-subtypes in adrenal glands from wild-type mice. Levels of {alpha}2C mRNA were significantly reduced in {alpha}2C+/– adrenal glands. (b) mRNA expression of catecholamine synthesizing enzymes in the adrenal gland. Abbreviations: DBH, dopamine β-hydroxylase; TH, tyrosine hydroxylase; PNMT phenylethanolamine N-methyltransferase. Means±SEM, n=5 per genotype. ***p<0.001 vs. {alpha}2C+/+.

 
3.2 Regulation of adrenal catecholamine secretion by {alpha}2C-adrenoceptors
Feedback regulation of adrenal catecholamine secretion was investigated in adrenal tissue slices incubated in vitro in superfusion chambers (Fig. 3). Basal release of catecholamines was significantly higher in adrenal glands from {alpha}2C+/– and {alpha}2C–/– mice than from wild-type control mice (Fig. 3a,b). Upon stimulation with the nicotinic receptor agonist, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), release increased 2–3 folds without differences between genotypes (Fig. 3a,b). Activation of {alpha}2-adrenoceptors by medetomidine reduced DMPP-stimulated release of epinephrine by 88±4% (n=4) and release of norepinephrine by 92±5% (n=4), respectively (Fig. 3c). In {alpha}2C+/– adrenal glands, the inhibitory effect of medetomidine was significantly blunted to 54±7% (p<0.01, n=4) for epinephrine release and to 57±5% (p<0.05, n=4) for norepinephrine overflow, respectively. In {alpha}2C–/– adrenal glands, {alpha}2-mediated inhibition of epinephrine secretion was almost completely absent (7±2%, p<0.001, n=4). In contrast, 28±6% (p<0.001, n=4) of the stimulated norepinephrine release could not be inhibited in {alpha}2C–/– adrenal glands. Taken together, these results demonstrate that step-wise reduction of {alpha}2C-adrenoceptors is paralleled by a reduction of the feedback inhibition.


Figure 3
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  		Fig. 3 Feedback control of catecholamine secretion from isolated adrenal glands and atria in vitro. Secretion of epinephrine (a) and norepinephrine (b) from slices of adrenal gland tissue was measured in superfusion chambers in vitro. Basal release was significantly higher in specimens from {alpha}2C+/– and {alpha}2C–/– mice. Catecholamine secretion stimulated by the nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) did not differ between genotypes. (c) Inhibition of DMPP-stimulated catecholamine release by the {alpha}2-adrenoceptor agonist, medetomidine (100 nM) from slices of adrenal glands. {alpha}2-mediated inhibition was significantly blunted in {alpha}2C+/– and {alpha}2C–/– adrenal glands. Means±SEM, n=4 per genotype, *p<0.05 vs. {alpha}2C+/+, #p<0.05 {alpha}2C–/– vs. {alpha}2C+/–. (d) Effect of medetomidine on electrically evoked overflow of [3H]norepinephrine from atria. *p<0.05, **p<0.01 vs. {alpha}2C+/+ means±SEM, n=12.

 
3.3 Atrial feedback inhibition by {alpha}2C-adrenoceptors
To examine whether inactivation of one allele or two alleles of the {alpha}2C-adrenoceptor gene affects feedback inhibition at peripheral sympathetic nerve terminals, the effect of medetomidine on electrically stimulated norepinephrine outflow from atria was determined. The {alpha}2-agonist medetomidine inhibited norepinephrine release in a concentration-dependent manner in atria of all three genotypes (Fig. 3d). The resulting pKD values from atria of wild-type and heterozygous {alpha}2C-deficient mice did not differ significantly ({alpha}2C+/+ 9.3±0.1 vs. {alpha}2C–/– 9.3±0.1, n=12). The concentration response curve for medetomidine was significantly (p<0.001 vs. {alpha}2C+/+ and {alpha}2C+/–) shifted to the right in atria from {alpha}2C–/– mice ({alpha}2C+/– 8.9±0.1, p<0.001 vs. {alpha}2C+/+, n=12). In contrast to the results obtained from adrenal glands these results indicate that inactivation of a single allele of the {alpha}2C-receptor gene did not affect the potency of feedback inhibition from atria.

3.4 Renal catecholamine secretion
In order to assess the in vivo relevance of {alpha}2C-adrenoceptors for adrenal catecholamine regulation, renal excretion of catecholamines was determined in 24 h urine samples (Fig. 4a,b). Consistent with the in vitro data, urine epinephrine levels were increased by 74±15% (p<0.01, n=4) in heterozygous and by 142±23% (p<0.001, n=4) in homozygous {alpha}2C-deficient mice (Fig. 4a). Urine norepinephrine excretion was not significantly affected by deletion of the {alpha}2C-adrenoceptor gene (Fig. 4b).


Figure 4
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  		Fig. 4 Urine catecholamine levels in wild-type and {alpha}2C-adrenoceptor-deficient mice. (a) 24 h epinephrine excretion was significantly increased in {alpha}2C+/– and {alpha}2C–/– mice as compared with wild-type littermates. (b) Norepinephrine excretion was not altered in {alpha}2C+/– and {alpha}2C–/– mice as compared with wild-type ({alpha}2C+/+) mice. *p<0.05, **p<0.01, {alpha}2C+/– vs. {alpha}2C+/+, means±SEM, n=6–8 mice per genotype.

 
3.5 Cardiovascular consequences of {alpha}2C-adrenoceptor deletion
As the long-term consequences of {alpha}2-adrenoceptor deletion for resting blood pressure in mice are unknown, telemetric pressure transducers were inserted into the left carotid artery and implanted subcutaneously. Two weeks after surgery, blood pressure and heart rate were determined for five consecutive days in 2-min intervals (Fig. 5). In all genotypes, nighttime systolic and diastolic blood pressure and heart rate were significantly higher than daytime values (Fig. 5a,b,d). At night, heart rate was 4.9% higher in {alpha}2C–/– mice than in {alpha}2C+/+ mice (p<0.05, n=5 mice per genotype) (Fig. 5a). When the effect of locomotor activity on heart rate was assessed, increased heart rate correlated significantly with the degree of activity. In addition, {alpha}2C genotype significantly affected heart rate at rest and at all levels of locomotor activity (Fig. 5c).


Figure 5
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  		Fig. 5 Effect of {alpha}2C-adrenoceptor deletion on hemodynamic parameters. Heart rate (a), systolic (b) and diastolic pressure (c) were assessed in mice 2 weeks after implantation of a telemetric pressure transducer. {alpha}2C–/– mice showed significant tachycardia during the day and also at night (a). *p<0.05 vs. {alpha}2C+/+. (c) Heart rate increased significantly with locomotor activity. Genotype was significantly associated with increased heart rate at all activity levels. 2-way ANOVA, ***p<0.01. Means±SEM, n=5 mice per genotype.

 
As increased sympathetic activity is a well-known risk factor of adverse cardiovascular outcome, we tested whether mice with targeted deletion of the {alpha}2C-adrenoceptor gene were more prone to develop heart failure after left-ventricular pressure overload as compared to wild-type mice. Cardiac pressure overload was induced by transverse aortic constriction (TAC) under anesthesia. Perioperative mortality did not differ between genotypes ({alpha}2C+/+ 20%, {alpha}2C+/– 17%, {alpha}2C–/– 26%). The aortic banding operation resulted in similar degrees of aortic stenosis in WT, {alpha}2C+/– and {alpha}2C–/– animals (aortic stenosis lumen area: {alpha}2C+/+ 17.0±2.5%, {alpha}2C+/– 12.1±1.5%, {alpha}2C–/– 14.3±2.7% of sham control, n=5–9 mice per genotype group). Postoperative survival, hemodynamic parameters and left-ventricular hypertrophy were followed for up to 7 months after aortic constriction (Fig. 6a). None of the {alpha}2C+/+ mice died until the end of the observation period whereas survival was significantly reduced in {alpha}2C+/– and even more in {alpha}2C–/– mice after aortic banding (Fig. 6a). The survival curve of {alpha}2C–/– mice after TAC did not differ significantly from our previous report [19]. None of the sham-operated control animals died postoperatively until the end of the observation period.

We have previously reported that increased mortality of {alpha}2C–/– mice after TAC resulted from chronic heart failure [19]. Thus, in this study we further focused on the characterization of the {alpha}2C+/– mice after aortic constriction (Fig. 6). In response to TAC, wild-type and {alpha}2C+/– mice developed cardiac fibrosis and hypertrophy (Fig. 6b,c). Heart weight to body weight ratio increased by 59% in {alpha}2C+/– mice after aortic constriction as opposed to 23% in {alpha}2C+/+ animals (Fig. 6c). Similar increases were identified for heart weight to tibia length ratios after TAC ({alpha}2C+/– +32% vs. {alpha}2C+/+ +51%, Fig. 6d). Functional analysis with a microtip catheter revealed a decrease in left-ventricular contractility (dp/dtmax) by 40.6% in {alpha}2C+/– mice after TAC but no significant change in {alpha}2C+/+ mice (Fig. 6e). Similarly, left-ventricular relaxation (dp/dtmin) was reduced by 44% in {alpha}2C+/– mice after TAC without a significant reduction in {alpha}2C+/+ mice (Fig. 6f).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Acknowledgment
 References
 
The present study provides novel insight into the feedback regulation of catecholamine release from the adrenal medulla. Heterozygous deletion of the {alpha}2C-adrenoceptor in mice reduced the efficacy of an {alpha}2-agonist to inhibit adrenal catecholamine secretion by approximately 50%. As a consequence of partial disruption of adrenomedullary feedback inhibition, urine epinephrine excretion was elevated in heterozygous {alpha}2C+/– mice and these mice were more susceptible to develop heart failure after transverse aortic constriction. Thus, in contrast to feedback control in sympathetic nerves, adrenal feedback regulation lacks a significant receptor reserve.

Previous studies in mice deficient in {alpha}2-adrenoceptor subtypes have mostly focused on the role of the three {alpha}2-receptor subtypes, {alpha}2A, {alpha}2B, and {alpha}2C, in sympathetic and central adrenergic feedback inhibition. In vitro evidence suggested that all three {alpha}2-adrenoceptor subtypes can inhibit release of norepinephrine from sympathetically innervated tissues [9,10]. In isolated mouse atria, {alpha}2A-receptors were identified to serve as feedback regulators at high neuronal action potential frequencies whereas the {alpha}2C-subtype inhibited norepinephrine release at lower levels of neuronal activity [10]. In contrast, in vivo {alpha}2A- and {alpha}2C-receptors were found to differentially control sympathetic and adrenomedullary catecholamine secretion, respectively [11].

In the present study, heterozygous deletion of {alpha}2C-adrenoceptors resulted in diminished adrenomedullary feedback control of epinephrine release, indicating low or absent receptor reserve in the adrenal medulla. This finding indicates that adrenal feedback control differs fundamentally from sympathetic feedback control. In peripheral sympathetic or central adrenergic neurons, {alpha}2-adrenergic receptors are expressed at very high levels, reaching 200–300 fmol/mg of membrane protein in the brain [30,31]. In this situation, activation of only a small percentage of receptors (<20%) elicits full inhibition of transmitter release [32,33]. The remaining 80% of receptors which are not necessary to achieve maximal inhibition of neurotransmitter secretion constitute a "receptor reserve" [32,33].

These observations are also consistent with results of experiments performed with gene-targeted mice with dysfunctional {alpha}2A-receptors. Upon heterozygous deletion of the {alpha}2A-adrenoceptor in mice, a significant phenotype became apparent only after activation of the receptor with a partial agonist [34]. Secondly, in a gene-targeted mouse expressing an {alpha}2A-receptor variant ({alpha}2A-D79N) at 20% of the density of the wild-type {alpha}2A-receptor, presynaptic feedback inhibition in sympathetic nerves was not altered [20], suggesting that 20% of functional {alpha}2A-receptors are required for presynaptic feedback control in adrenergic neurons.

In contrast, sequential deletion of {alpha}2C-adrenoceptor genes resulted in a step-wise reduction in adrenal feedback control. Heterozygous deletion of {alpha}2C-receptors caused a reduction of {alpha}2C-mRNA copy numbers by 43% (Fig. 2) which was accompanied by a concomitant increase in whole body epinephrine spillover. Protein levels of {alpha}2-adrenoceptors in the adrenal medulla were below the level of detection by standard radioligand binding (i.e. <10 fmol/mg membrane protein, data not shown). Thus, adrenal chromaffin cells contain {alpha}2-adrenoceptors at a density which is one order of magnitude lower than receptor density in adrenergic neurons in the CNS (200–300 fmol/mg) [30]. These findings are consistent with a recent report in which the density of adrenal {alpha}2-adrenoceptors was estimated to be 30±5 fmol/mg membrane protein [35]. Thus, in the adrenal medulla feedback regulation operates without "spare" or "reserve" {alpha}2-receptors. The presence of a high number of spare receptors sensitizes receptor signal transduction such that lower concentrations of receptor agonists are sufficient to elicit full responses. Teleological, feedback control without spare receptors may be better suited to regulate the high concentrations of the adrenal hormone epinephrine directly at the site of its release.

An important question would be why only epinephrine and not norepinephrine levels were elevated in the urine of {alpha}2C+/– and {alpha}C–/– mice despite the fact that {alpha}2-agonist can inhibit the release of both catecholamines from adrenal glands in vitro? The answer may lie in the different amounts of epinephrine and norepinephrine stored and released from the adrenal medulla. The quantity of norepinephrine stored and released from the adrenal medulla may not be sufficient to result in a detectable elevation of plasma or urine norepinephrine concentrations. In the mouse adrenal gland, norepinephrine represents 25% and epinephrine 75% of the total catecholamine content [36]. In isolated adrenal glands, basal or DMPP-stimulated release of epinephrine (in % of stored epinephrine) was 3-fold higher than norepinephrine release (see Fig. 3a,b). The total amounts of epinephrine (approximately 900 pmol/mg adrenal weight) and norepinephrine (approximately 100 pmol/mg adrenal weight) released from the adrenal gland in vitro differ by a factor of 9–10. Thus, in the mouse in vivo adrenal norepinephrine may represent only a small percentage of plasma and urine norepinephrine with the majority being released from sympathetic nerves.

In chronic heart failure, catecholamine release from sympathetic nerve and adrenal medulla is activated [19,35]. Once cardiac dysfunction reaches a critical point, the degree of sympathetic activation correlates strongly with the severity of failure and overrides the effects of presynaptic inhibitory receptors. Thus, presynaptic/adrenal {alpha}2-receptors may be more important to control catecholamines in a healthy situation rather than providing a brake during maximal activation of sympathetic tone during cardiac disease. Dysfunction of this feedback control may thus predispose patients to develop or accelerate cardiac hypertrophy and failure.

The findings of the present study may be relevant to evaluate the significance of polymorphisms in the human {alpha}2C-adrenoceptor gene. Recently, a number of genetic polymorphisms have been identified in human adrenergic receptor genes [15,17,37–40]. While the pathophysiological mechanism of some of these genetic variants still remains unknown, several observations suggest that a deletion of four amino acids in the intracellular region which is involved in G-protein coupling of the {alpha}2C-adrenoceptor ({alpha}2C-Del322–325) may be of particular relevance [17]. When expressed at low densities in Chinese hamster ovary cells, the human {alpha}2C-Del receptor was 90% deficient in coupling to several intracellular second messenger systems including inhibition of adenylyl cyclase [17]. However, as 8% of Caucasians and 60% of the African-American population carry this polymorphism, most people who have been identified to date are heterozygous carriers, i.e. they have one intact {alpha}2C-receptor gene and one dysfunctional copy.

The {alpha}2C-Del allele has been associated with elevated arterial blood pressure and exaggerated yohimbine-induced increases in blood pressure and heart rate in healthy adults [18]. In addition, two studies have demonstrated that the {alpha}2C-Del allele was associated with the development of heart failure in African-Americans as well as in Caucasians [19,39]. In a retrospective analysis of patients with dilated cardiomyopathy, {alpha}2C-Del genotype was associated with lower mortality [41]. However, a recent study in African-Americans carrying the {alpha}2C-Del allele did not identify an association with increased blood pressure, heart rate or incidence of hypertension [42]. Until now, none of the clinical studies has included a haplotype analysis to search for a possible association between {alpha}2C-adrenoceptor genotype and cardiovascular disease. Interestingly, Small et al. have identified 24 haplotypes of the human {alpha}2C-adrenoceptor gene which resulted in significant expression differences between haplotypes in recombinant cell lines in vitro [38].

In the present study, diminished feedback control of adrenal epinephrine secretion resulted in tachycardia and increased susceptibility to develop heart failure after left-ventricular pressure overload. Patients with decompensated heart failure show increases in both plasma epinephrine and plasma norepinephrine, whereas patients with chronic stable heart failure usually have increases in norepinephrine only [43–46]. It has been suggested that increased epinephrine levels are related to catabolic metabolic balance and cachexia in patients with chronic heart failure [47].

The pathophysiological importance of adrenal feedback control has been emphasized by a recent report demonstrating an essential role of upregulation of the receptor kinase GRK2 in chromaffin cells in animal models of chronic heart failure [35]. However, the treatment of patients with chronic heart failure with the {alpha}2-agonist moxonidine has unexpectedly resulted in increased mortality rates [48].

The present study demonstrates that feedback inhibition of adrenal catecholamines by {alpha}2C-adrenoceptors operates without a detectable receptor reserve. Thus, alterations in {alpha}2C-receptor density and/or coupling efficiency to intracellular G-protein signaling pathways may directly affect the release of epinephrine from the adrenal gland. These findings may help to identify the pathophysiological relevance of genetic variants of the human {alpha}2C-adrenoceptor and devise new strategies for pharmacological control of diseases with increased adrenomedullary activity.

Time for primary review 22 days


   Acknowledgment
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgment
References
 
This study was supported by the Deutsche Forschungsgemeinschaft (SFB487 "Regulatory Membrane Proteins"; TP A2, in support of L.H.).


   Notes
 
1 Both authors contributed equally. Back


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgment
 References
 

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