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European Journal of Heart Failure 2007 9(12):1163-1171; doi:10.1016/j.ejheart.2007.10.006
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© 2007 European Society of Cardiology

Sepsis is associated with an upregulation of functional β3 adrenoceptors in the myocardium

S. Moniotte1, C. Belge1, B. Sekkali, P.B. Massion2, B. Rozec, C. Dessy and J.-L. Balligand*

Unit of Pharmacology and Therapeutics FATH 5349, Université catholique de Louvain Brussels — Belgium

* Corresponding author. Unit of Pharmacology and Therapeutics FATH 5349, Université catholique de Louvain 52 avenue Mounier, B1200 Brussels, Belgium. Tel.: +32 2 764 5260; fax: +32 2 764 5269. Balligand{at}mint.ucl.ac.be (J.-L. Balligand)


   Abstract
Top
 Notes
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 
Objective: To analyze the implication of the β3-adrenoceptor (β3-AR) pathway in human septic myocardium and a murine model of sepsis, a condition associated with myocardial depression.

Methods and results: β3-AR and eNOS protein abundance were increased (332±66.4% and 218±39.3; P<0.05) in hearts from septic patients. The effect of BRL37344, a β3-AR-preferential agonist, was analyzed by videomicroscopy on the contractility of neonatal mouse ventricular myocytes (NMVM) incubated with conditioned medium from LPS-stimulated cultured macrophages (Mc-LPS+ medium). Stimulation of untreated NMVM with BRL37344 dose-dependently decreased the amplitude of contractile shortening (P<0.05). This response was abolished by L-NAME (NOS inhibitor). Incubation in Mc-LPS+ medium potentiated the depressing effect of BRL37344 (P<0.05) as well as of SR58611A (P<0.05) in wild-type myocytes. Importantly, the contractile depression was abrogated in cardiomyocytes from β3-AR KO mice.

Conclusions: β3-AR are upregulated during sepsis in the human myocardium and by cytokines in murine cardiomyocytes, where they mediate an increased negative inotropic response to β3 agonists. Activation of the β3-AR pathway by catecholamines may contribute to the myocardial dysfunction in sepsis.

Key Words: Receptors • Adrenergic • Beta • Cytokines • Pharmacology • Nitric oxide synthase • Myocardial contraction

Received March 8, 2007; Revised September 21, 2007; Accepted October 18, 2007


   1. Introduction
Top
 Notes
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 
Sepsis represents the systemic response to infection and is initiated through the effects of one or more components of the invading microorganisms, including structural elements like endotoxin from Gram-negative bacteria, or secreted exotoxins like the toxic shock toxin from some staphylococcal and streptococcal strains. When this inflammatory stimulus is particularly intense, effects on the cardiovascular system may dominate the clinical presentation, with a sepsis-associated myocardial depression. In humans, this myocardial depression is characterized by reversible biventricular dilatation, decreased ejection fraction and profound systemic vasodilation with decreased response to fluid resuscitation [1].

A hallmark of the early myocardial depression is the resistance to catecholamine stimulation, although its underlying mechanism remains poorly defined. Endotoxins are thought to trigger the local and systemic release of endogenous inflammatory mediators (cytokines) such as tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-1β (IL-1β). Further amplification of the inflammatory response through the stimulation of polymorphonuclear leukocytes, tissue macrophages and monocytes, platelets, and endothelial cells leads to the release of additional biologically active mediators, including platelet activating factor and nitric oxide. Accordingly, exposure of cardiac myocytes to TNF-{alpha} results in a decreased responsiveness to epinephrine and isoproterenol, even at maximally tolerated catecholamine concentrations [2].

A number of studies provided evidence for both NO-independent [3-7] and NO-dependent [8-13] mechanisms of contractile depression following cytokines exposure in vitro and in vivo; NO was similarly involved in the depressed myocardial responsiveness to catecholamines observed in patients with heart failure, a condition also associated with systemic inflammation [14-17]. Although much of the evidence points to the inducible isoform of nitric oxide synthase (or iNOS) as the source of myocardial or systemic NO [9-13] it may not be the only isoform involved. Indeed, early studies suggested that cytokines may acutely depress cardiomyocyte contraction through activation of a constitutively expressed NOS [18,19], although the identity of the isoform was not resolved. Among the constitutive enzymes, the direct implication of eNOS in sepsis has received little attention and remains unclear so far, with both systemic pro-inflammatory [20] and anti-inflammatory [21] roles. A recent study [22] showed that mice with cardiomyocyte-specific overexpression of NOS3 (or eNOS) are even protected from myocardial dysfunction and death associated with endotoxemia.

Aside from the NOS isoform involved, the identity of the β-adrenergic receptor isotype(s) implicated in the catecholamine resistance is poorly defined. We previously demonstrated the presence of β3-adrenoceptors in human ventricle both at the mRNA level by the amplification of specific transcripts by RT-PCR and at the protein level with a specific monoclonal antibody [23,24]. The expression of this receptor is clearly upregulated in cardiac tissue from dilated and ischaemic failing patients [24]. Of note, we and others have described the coupling of this receptor to the production of NO, and eNOS was previously shown to mediate the blunted inotropic effect of catecholamines in response to β3-adrenoceptor stimulation in mice [25]. Indeed, contrary to β1- and β2-adrenoceptors, β3-adrenoceptors mediate a negative inotropic effect in most mammalian, including human heart preparations, an effect that is more resistant to desensitization than the β1 and β2 responses [24]. Altogether, these data suggest that the high adrenergic tone characteristic of heart failure, but also sepsis, might alter cardiac contractile activity as a result of unmasked β3-adrenergic stimulation together with reduced β1- and β2-adrenoceptor function.

Therefore, in the present study, we analyzed expressional changes of the β-1, -2 and -3-adrenergic receptors in human myocardial tissue from septic compared with non-septic patients. We also assessed the effect of cytokine exposure on the specific modulation of cardiomyocyte contractility by the β3-adrenergic pathway using β3-preferential agonists/antagonists, as well as the impact of genetic deletion of the β3-adrenoceptor on cardiomyocyte function.


   2. Methods
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 
2.1. Selection of patients
All investigations were performed with the approval of the local Ethics Committees and conformed with the principles outlined in the Declaration of Helsinki. Non-septic heart tissues were obtained from normal, innervated myocardium from donor hearts which were not transplanted for technical reasons (n=15). Six out of 15 donors had received synthetic inotropic amines in the 48 h preceding explantation. Samples of septic myocardium were collected from hearts of patients who died from severe sepsis (n=7). Details regarding their drug regimens and clinical characteristics are shown in Table 1.


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  		Table 1 Patient characteristics

 
2.2. Isolation and preparation of rat alveolar macrophages
Alveolar macrophages were obtained by tracheal lavage of anesthetized male Wistar rats (250 to 275 g) by a previously described technique [8,26]. After several washes, the cell pellet was re-suspended at a concentration of 0.5x106 cells/ml in endotoxin-free, L-arginine-free DMEM medium containing 0.1% BSA with 100 U/mL penicillin and 100 U/mL streptomycin and cultured at a density of 2.5x106 cells per 60-mm culture dish in a 95% O2/5% CO2 atmosphere at 37 °C. One hour after plating, cells were washed three times with DMEM to remove nonadherent cells. Macrophages were then exposed to endotoxin (LPS component of Salmonella typhosa, Sigma L6386) at a concentration of 10 µg/mL for 24 h. LPS-treated macrophage-conditioned medium (referred to as Mc-LPS+) was harvested, centrifuged at 1500 g for 10 min to remove cell debris, and then stored at –80 °C for further use.

2.3. Cardiomyocytes culture
Primary cardiac myocytes were isolated and cultured as previously described [27]. Briefly, 8-10 newborn (2 to 3 days old) mouse pups (C57Bl6 or FVB strains) were sacrificed under aseptic conditions, the ventricles were removed, placed in a dish containing Hanks' Balanced Salt Solution (HBSS) and cut into 4 pieces. Fragments were transferred to a 50-ml flask containing 30 ml of 2.5 g/L trypsin solution (GibcoBRL) and incubated 4-5 h at 4 °C under constant agitation. The hearts were then digested by a 1 mg/ml collagenase solution (GibcoBRL) in Dulbecco's modified Eagle's medium (DMEM). Ventricular myocytes were then collected by centrifugation (1000 rpm for 5 min at 4 °C), re-suspended in cold DMEM, pooled and disposed on top of a 39% Percoll isotonic solution. After centrifugation, pelleted myocytes were re-suspended by pipetting in DMEM containing 10% FBS and antibiotics and preplated 1 h at 37 °C. Myocytes were finally seeded in plastic 35 mm2 tissue culture dishes until videomicroscopic analysis. One day after plating, the medium was replaced and the cells began to beat spontaneously after 1-2 days in culture.

For the experiments using macrophage-conditioned medium, cultured cardiomyocytes were treated with a 50% dilution of Mc-LPS+ in DMEM for a minimum of 6 h before contractility measurements.

2.4. Contractility measurements
For the measurement of contractility, cultured neonatal mouse ventricular myocytes were mounted on the stage of an inverted fluorescence microscope (Zeiss, Axiovert S 100) equipped with a 40x oil-immersion objective in a circular temperature-controlled chamber (Intracell, Cambridge, UK) at 37 °C, pH 7.4. Light-dark contrast at the cell edges provided a marker for measurement of amplitude of cell shortening monitored with a video motion analyzer. All measurements were processed by the SoftEdge software (IonOptix, Milton, MA, USA).

2.5. Quantification of mRNA by reverse transcription and polymerase chain reaction
Left ventricular tissues collected from human hearts were homogenized in a GTC extraction buffer. 1 µg total RNA was reverse transcribed with random hexamers in a total volume of 50 µl. Primers and probes were designed to selectively amplify the human β1-adrenoceptor (β1-AR), β2-adrenoceptor (β2-AR), β3-adrenoceptor (β3-AR) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs (see Table 2). Polymerase chain reactions were performed in parallel from each sample in the ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems) with 0.05 unit/ml of AmpliTaq Gold and the appropriate reaction mix containing 2.5 pmol of the FAM-TAMRA probes and ~100 ng of ventricular cDNA. PCR cycle conditions were: 50 °C for 2 min, 95 °C for 10 min followed by 40 cycles of 15 s at 95 °C, 1 min at 60 °C.


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  		Table 2 Gene-specific oligonucleotide probes sequences (human)

 
For mRNA quantitation from cardiomyocytes, cells were washed with cold PBS and total RNA was isolated using TRIzol (Invitrogen). RNA was then treated with DNase to eliminate any genomic DNA contamination. After reverse transcription, real-Time quantitative RT-PCR, according to the SYBER Green principle, was performed in 96-well plates in a final reaction volume of 25 µl, composed of 12.5 µl of IQ– SYBRR Green supermix (BIO-RAD), 0.5 µl of each primer (500 nM final concentration; for sequences, see Table 3), 10.5 µl Milli Q and 1 µl cDNA, using an IQ–5 multicolor Real-Time PCR detection system (BIO-RAD). Amplifications were performed starting with a 3 min polymerase activation step at 95 °C, followed by 40 cycles of denaturation at 95 °C for 15 s and combined primer annealing/extension at 60 °C for 30 s. Fluorescence increase was automatically measured during PCR. The thermal denaturation protocol was run at the end of PCR to determine the number of products. All reactions were run in triplicate. As negative controls, PCR was performed on water and on RNA without reverse transcription.


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  		Table 3 Gene-specific oligonucleotide probes sequences (murine)

 
Results were expressed as Ct (number of cycles needed to generate a fluorescent signal above a pre-defined threshold), {Delta}Ct (corresponding to the difference between the Ct of the gene of interest and the Ct of the gene used to normalize the results) or 2–{Delta}{Delta}C{tau} (for cell experiments). Relative gene expression was normalized to GAPDH (human samples) or HPRT (cell experiments), which we previously validated as housekeeping genes in cardiac tissue (including human) in previous studies [28]. Since the {Delta}Ct cannot predict the absolute differences in copy numbers between target and reference genes, we also measured the expression of target gene mRNAs against standard curves previously established by us for the quantitation of β-adrenoceptors and GAPDH mRNA molecules [28] to measure the number of β-adrenoceptor mRNA normalized per number of GAPDH mRNA molecules.

2.6. Western blotting experiments
Denatured proteins from human ventricular tissues were separated on 10% SDS-PAGE gels and transferred on nitrocellulose. Membranes were incubated for 1 to 4 h with the primary antibody [i.e. anti-eNOS (Transduction Labs, Lexington, KY, USA.), anti-β3-adrenoceptors (generously provided by Dr. J. Arch, SmithKline Beecham Pharmaceuticals, Harlow, UK), anti-GAPDH(Cell Signaling)] washed in TBST (Tris-buffered saline containing 0.1% Tween 20), incubated with the appropriate secondary antibody diluted at 1:10.000 and revealed by chemiluminescence. Densitometric values for each sample were expressed as a percentage of the mean value obtained for the corresponding control (non-septic) samples analyzed on the same gel.

2.7. Statistical analysis
Data are presented as mean±SEM. Statistical significance of the drug effect was assessed using one-way ANOVA followed by a Dunnett test. Comparison of the concentration-response curves was performed by two-way ANOVA. Statistically significant differences between groups in terms of mRNA or protein expression were calculated by a Student's t test for unpaired data. Statistical significance was accepted at the level of P<0.05.


   3. Results
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Appendix A. Supplementary data
 References
 
3.1. Patient characteristics
The age and disease severity of the patients are shown in Table 1. Septic patients had a mean SOFA (Sepsis-related Organ Failure Assessment) score [29] of 8.2±1.3. Heart donors were significantly younger than patients with septic shock (39±5.2 vs 62±4.6 years, P=0.02). A minor proportion of septic patients had received drugs commonly used for heart failure treatment (Table 1).

3.2. β-adrenoceptors mRNA expression in human septic hearts
We applied a real-time RT-PCR assay to quantitate β-adrenergic mRNAs in samples of ventricular myocardium from control and septic patients. We found that the abundance of the messenger RNAs for β3-adrenoceptors, normalized to that of the housekeeping GAPDH, was higher in septic than in non-septic hearts, with mean {Delta}Ct's of 8.1±0.92 (reflecting higher relative expression) and 11.6±0.29, respectively (P=0.0001; n=7 and 15 patients). When the number of specific mRNA molecules was quantitated using standard curves assessed for each gene, and again normalized to that of the housekeeping GAPDH (Fig. 1A), the relative abundance of the β3-adrenoceptors was 6.0x10–4±3.96x10–4 and 1.5x10–5±0.33x10–5 mRNA molecules of β3-adrenoceptors/mRNA molecules of GAPDH in septic and in non-septic hearts, respectively (P=0.038; n=7 and 15 patients).


Figure 01
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  		Fig. 1 Expression of β1-, β2- and β3-adrenoceptors mRNA and proteins in human left ventricular muscle from non-septic and septic patients. A. Shown are the absolute numbers of β-adrenoceptors mRNA molecules normalized to that of the housekeeping GAPDH calculated following the standard curves assessed for each gene (see methods) and compared between non-septic (white bars) and septic hearts (black bars); the abundance of the β1- and β2-adrenoceptors was unchanged in septic hearts, while the β3-adrenoceptor mRNAs were increased, with a mean ratio of 6.0 10–4±3.96 10–4 in septic versus 1.5 10–5±0.33 10–5 in non-septic hearts, respectively (n=7-15; P=0.04). B. Shown are immunoblotted β3-adrenoceptor (upper), GAPDH (lower) proteins in cardiac extracts from representative non-septic and septic patients. β3-adrenoceptors (normalized to GAPDH) were strongly upregulated in myocardium from septic compared with non-septic patients, as shown from the mean densitometric data for the comparative expression of β3-adrenoceptors normalized to expression levels in non-septic hearts (n=5).

 
For β2-adrenergic receptor expression, we obtained mean {Delta}Ct values of 7.8±1.01 and 11.3±0.31, in septic and non-septic hearts, and the quantitation of relative abundance of the β2-adrenoceptors showed a trend towards an increase (albeit not significant) in sepsis (22.9x10–2±17.4x10–2 in septic hearts and 3.5x10–3±0.84x10–3 in non-septic hearts; Fig. 1A; P=0.062; n=7 and 15 patients).

Similarly, when the same analysis was repeated for the β1-adrenoceptor mRNA, we did not find any significant difference between the two groups of patients (Mean {Delta}Ct: 5.6±0.56 and 5.9±0.22 in septic and non-septic hearts; P>0.05, corresponding to 16.4x10–3±9.07x10–3 mRNA molecules of β1-adrenoceptors/mRNA molecules of GAPDH and 9.0x10–3±1.11x10–3 mRNA molecules in septic hearts and non-septic hearts, respectively) (Fig. 1A).

3.3. β3-adrenoceptor protein expression in human septic hearts
Using a rat monoclonal antibody specific for the human β3-adrenoceptor in extracts of non-septic hearts, we found a single immunodetected band of a size compatible with a glycosylated form of the receptor, as observed by us in human heart [24] and in other human tissues. Moreover, compared to levels observed in control, non-septic hearts, β3 adrenoceptors (normalized to GAPDH proteins) were significantly increased in cardiac tissues from septic hearts(332±66.4%) (Fig. 1B). Similarly, the abundance of eNOS proteins was increased in septic human hearts compared with non-septic hearts (218±39.3%, n=7 and 15 patients). Subsequent analysis of extracts from epicardial, mid-myocardial and sub-endocardial layers from both ventricules showed no transmural difference in β3-adrenoceptor abundance (data not shown).

3.4. Functional responses of isolated mouse cardiomyocytes to β3-adrenergic stimulation
To assess the functional impact of β3-adrenoceptor stimulation and of its expressional changes on myocardial function, we used an in vitro model of purified mouse cardiomyocytes that were exposed (or not) to LPS-activated macrophage-conditioned medium, containing the inflammatory mediators released during sepsis, as previously used by us and others [8,30].

3.5. Effect of the preferential β3-adrenoceptor agonist, BRL 37344, on shortening amplitude
To validate the existence of a functional β3-adrenoceptor pathway in this in vitro model, the effects of BRL 37344, a β3-preferential agonist, were first analyzed by videomicroscopy on the contractility of untreated (i.e. without Mc-LPS+ pre-treatment) cardiomyocytes. In the absence of drug treatment, contractile shortening was very stable over time (up to 15 min; see Fig. 2A and legend; also up to 30 min not shown) in these preparations. Upon incubation with a single dose of BRL 37344 (0.1 µM), we observed a decreased amplitude of contractile shortening to –19.1±5.66% of baseline at 15 min (n=23; P<0.05). Notably, after incubation with the NOS inhibitor, L-NAME, this effect was abolished, with a shortening amplitude of 103.7±6.37% of baseline (n=9; P>0.05 vs untreated cells) (Fig. 2A).


Figure 02
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  		Fig. 2 Contractile shortening of isolated mouse cardiomyocytes in response to the β3-preferential agonist, BRL 37344. A. BRL 37344 induces a NOS-dependent decrease in contractile shortening. The contractile response of isolated neonatal mouse cardiomyocytes was recorded by videomicroscopy on an inverted microscope and reported as % of basal shortening amplitude at different times after exposure to the β3-preferential agonist, BRL37344 (0.1 µM). In the absence of drug (filled squares), shortening amplitude remained constant over time, at 99.0±6.28% of baseline after 15 min (and 104.7±5.28% after 30 min; not shown) (n=6). Upon exposure to BRL 37344 (filled triangles), the amplitude of contractile shortening progressively decreased (–19.1±5.6% of baseline at 15 min; n=23; P<0.05). After pre-incubation with the NOS inhibitor, L-NAME (10 µM; 30 min; filled circles), the effect of BRL 37344 was abolished, with a mean contractile amplitude of 103.7±6.4% of baseline (n=9). B. The potentiation of the decrease in shortening amplitude to BRL 37344 upon pre-treatment with LPS-activated macrophages-conditioned medium is partly NOS-dependent. Pre-incubation of neonatal cardiomyocytes for 6 h with the LPS-treated macrophages-conditioned medium (Mc-LPS+; see text) potentiated the depressing effect of BRL 37344 to –45.3±4.8% (open squares; n=6; P<0.05 vs BRL 37344 on cells not cultured in Mc-LPS+, see A). After pre-incubation with the NOS inhibitor, L-NAME (10 µM; 30 min), this effect was blunted, with a maximal decrease in shortening amplitude of –23.7±10.83% (filled circles; n=3; P=NS versus cells not cultured in Mc-LPS+ medium). C. Incubation of cardiomyocytes with LPS-activated macrophages-conditioned medium upregulates their expression of β3-adrenoceptors mRNA. RNA from cultured cardiomyocytes incubated as above was processed for real-time quantitative PCR using probes specific for the murine β1-, β2-, or β3-adrenoceptors (see Methods and Table 3 for probes sequences). Shown are mean values (from 3 different cultures) of each β-adrenoceptor mRNA levels in Mc-LPS+-treated cells (hatched bars) relative to control, untreated cardiomyocytes (white bars).

 
Cumulative dose-response curves obtained with increasing concentrations of BRL 37344 from 1 nM to 1 µM showed a decrease of contractile shortening to –30.9±6.59% of baseline at 0.1 µM. This response was unchanged and even potentiated by nadolol, a β1-β2-blocker, with a maximum decrease of contractile shortening to –47.8±7.36% at 0.1 µM, but abolished by bupranolol, a β1-β2-β3-blocker, with a modest, transient increase of contractile shortening to 117.4±8.76% of baseline at 0.1 µM (n=3-4 in each group) (Suppl. Fig. 1A). The selective β3-antagonist L-748,337 also abolished the negative inotropic effect of cumulative doses of BRL 37344, with a contractile amplitude of 106.7±8.30% after 0.1 µM BRL 37344 (n=5; P>0.05 vs BRL 37344 only; Suppl. Fig. B). Nadolol, bupranolol, or L-748,337 alone had no effect on contractile shortening of wild-type myocytes (91±4.8%, 89.3±3.8% and 99±2.9% of baseline at 10 min, respectively; P>0.05).

3.6. Potentiation of the effect of BRL 37344 by incubation with activated macrophage-conditioned medium (Mc-LPS+)
Pre-incubation for 6 h with the Mc-LPS+ medium potentiated the depressing effect of BRL 37344 (0.1 µM) to –45.3±4.8% at 15 min (n=6; P<0.05; Fig. 2B). After pre-incubation with 10 µM L-NAME for 30 min, this effect was blunted, with a maximal negative inotropic effect of –23.7±10.8% (n=3; P>0.05 versus cells not cultured in Mc-LPS+) after 15 min.

3.7. Upregulation of β3-adrenoceptors upon exposure of cardiomyocytes to activated macrophage-conditioned medium (Mc-LPS+)
The potentiation of the contractile depression by BRL 37344 in cytokine-exposed cardiomyocytes was paralleled with an upregulation of the expression of β3-adrenoceptors mRNA, as measured by RT-quantitative PCR (257.6±25.3% of control, n=6; P=0.0002 Fig. 2C). β2-adrenoceptors mRNA were also marginally increased (133.2±21.2%), whereas β1-adrenoceptors were unchanged.

3.8. The potentiating effect of Mc-LPS+ pre-incubation is lost in myocytes from β3-AR KO mice
To ascertain the involvement of β3-adrenoceptors in the potentiating effect of Mc-LPS+, the response to β3-agonists was also examined in cardiomyocytes from mice homozygous deficient for the β3 adrenoceptor (Ardb3 tm1Low1 [31], kindly provided by Drs. P. Valet [INSERM, Hop.Rangeuil, Toulouse, France] and B.B. Lowell [Harvard Med. School, Boston, MA] and their wild-type, FVB littermate). Since in this FVB background, BRL 37344 evoked a slight, unspecific depression of contractility even in cardiomyocytes from the β3-AR KO (not shown), we used a combination of the β3 agonist, SR58611A with the β1-2 blocker, nadolol, that reproduced the typical β3-mediated depression in wild-type, FVB cardiomyocytes, but not in myocytes from littermate β3-AR KO (see Supplemental Fig. 2). SR58611A produced a prominent depression of contractile shortening in wild-type (FVB) cardiomyocytes that had been pre-incubated with Mc-LPS+ medium (–49±9.1%; P<0.05; Fig. 3; comparable to the effect of BRL37344 in Mc-LPS+-treated C57Bl6 myocytes, Fig. 2B). However, SR58611A failed to produce any contractile depression in myocytes from β3-AR KO even after incubation with Mc-LPS+ medium (P>0.05 compared with baseline; n=4).


Figure 03
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  		Fig. 3 The decrease in shortening amplitude upon pre-treatment with LPS-activated macrophages-conditioned medium is abrogated in cardiomyocytes from mice genetically deficient in β3-adrenoceptor. The β3-adrenocetor agonist, SR58611A (1 µM) decreased the shortening amptitude of cardiomyocytes from FVB (wild-type; filled squares; –49±9.1%; P <0.05) mice incubated in Mc-LPS+ medium to the same extent as BRL37344 (in C57Bl6 cardiomyocytes; see Fig. 2B; note that nadolol alone had no effect, see text). However, this effect of SR58611A and nadolol was totally abrogated in cardiomyocytes from β3-AR KO despite similar pre-incubation in Mc-LPS+ medium (filled triangles; n=5; P>0.05).

 

   4. Discussion
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Appendix A. Supplementary data
 References
 
We demonstrate that, as previously described in the chronically failing human heart [24], β3-adrenoceptor proteins and mRNAs are overexpressed in the myocardium of patients who died from sepsis. Using a model of isolated mouse ventricular cells, we observed that agonists of the β3-adrenoceptor decreased cell shortening, confirming the existence of a functional β3-adrenoceptor pathway in these cells; moreover, upon treatment with macrophage-conditioned medium to mimic exposure to sepsis-related cytokines in vitro, the decrease in cell shortening with β3-adrenoceptor agonists was potentiated, whereas such contractile depression was absent in cells from mice with genetic deletion of β3-adrenoceptors, suggesting a functionally upregulated β3-adrenoceptor pathway upon cytokine exposure that may contribute to the cardiac depression observed during sepsis. Although a similar upregulation of β3-adrenoceptors has already been identified in cardiac samples during heart failure [24] and diabetes [32], also associated with systemic or local inflammation, this is the first report of increased myocardial expression in human sepsis and upregulation of a functional β3-AR pathway upon cytokines exposure of cardiomyocytes in vitro. In human heart failure, we previously found a 2- to 3-fold increase in β3-adrenoceptor proteins in ischaemic and dilated cardiomyopathies compared to healthy patients [24]. Similarly, in hearts samples from rats with streptozotocin-induced diabetes [32], Dinçer et al. found a 97% increase in mRNA encoding β3-adrenoceptors and a 100% increase in plasma membrane density of β3-adrenoceptors after 14 weeks. A potential limitation is our use of hearts from brain-dead patients as controls, due to prevailing hyperadrenergism in this condition; nevertheless, since norepinephrine was shown to upregulate β3-adrenoceptors in cardiomyocytes [33], this would result in underestimation of the difference with septic hearts.

Also, we did not strictly compare hearts similarly sampled from patients who died from causes other than sepsis.

In human ventricular endomyocardial biopsies, the preferential β3-adrenoceptor agonist, BRL 37344, or the non-specific beta-agonist, isoprenaline in the presence of β1- and β2-AR blockade with nadolol, produced negative inotropic effects [23]. Accordingly, in the present study on isolated murine cardiomyocytes, the negative inotropic effect of BRL 37344 was antagonized by the nonselective β-adrenergic antagonist bupranolol or by the selective β3-AR antagonist L-748,337, but preserved in the presence of nadolol, confirming the existence of a functional β3-adrenoceptor pathway in these cells. In line with the previous demonstration of the NO-dependence of the cardiodepressant effects of β3-adrenergic stimulation in human myocardium [34], we also found that the effect of BRL 37344 was abrogated by NOS inhibition with L-NAME in uninduced murine cardiomyocytes. In our cytokine-treated isolated cardiomyocytes, the incomplete reversal of the β3-adrenergic effect with L-NAME may reflect incomplete NOS inhibition or the recruitment of additional, NOS-independent downstream signaling mechanisms. Of note, the expression of eNOS was increased in the hearts of patients who died from sepsis, which would support a reinforced β3-adrenergic cardiodepression in this condition. Such upregulation of eNOS in acute inflammatory conditions was previously observed in other human tissues, such as the inflamed peritoneum [35].

The mechanism of the β3-adrenergic depression of cardiomyocyte contractility remains incompletely characterized. In addition to NOS activation, we and others have found that β3-adrenergic activation increased intracellular levels of cGMP, which is known to modulate several aspects of excitation-contraction coupling [36-41]. In a previous study using a similar model of cytokine-treated cardiomyocytes, we have demonstrated that, together with cGMP increases, endogenous NO attenuated the β-adrenergic increase in intracellular cAMP and contractility, in part through phosphodiesterase activation [42]. The receptor pathway mediating this increased cGMP generation with isoproterenol, however, remained unknown. The present data, recapitulating similar functional observations using a preferential β3-adrenoceptor agonist suggest the specific involvement of this β3-adrenoceptor isotype. This is further supported from our demonstration of the strong upregulation of its mRNA in cytokine-exposed cells, paralleled with a potentiation of the contractile depression with β3-adrenoceptor agonists. Notably, the abrogation of this effect in cytokine-treated cardiomyocytes from mice with genetic deletion of the β3-adrenoceptor both reinforces the proof of causality for this pathway and excludes non-specific effects of the Mc-LPS+medium. The identity of the cytokine(s) responsible for the upregulation of this β3-adrenoceptor effect equally deserves further study, although our previous studies had shown that TNF {alpha} and IL-1 β, both present in the macrophage-conditioned medium, were critical for the contractile dysfunction in our in vitro model [43].

Although the density of cardiac β3-adrenoceptors is far lower than the other two isotypes, as confirmed at the mRNA level in our human samples, β3-adrenoceptors differ from β1- and β2-ARs in that they lack the phosphorylation sites for the β-adrenergic receptor kinases and the cAMP-dependent protein kinase [44], and may not be downregulated in pathophysiological conditions associated with an excessive adrenergic drive such as heart failure and sepsis. Because the contribution of β3-adrenoceptors to the cardiac responses of β-adrenergic agonists in the human heart in vivo is still not clear at present, questions arise as to the pathophysiological or clinical implications of our data. We proposed that the β3-adrenoceptor pathway may subserve different functions in the heart depending on the pathophysiologic context. In the normal heart, its activation may exert a negative counterregulation against catecholamine-mediated toxicity, thereby moderating oxygen consumption, preventing calcium overload and ultimately cardiomyocyte toxicity, as exemplified by the phenotype of β1-adrenoceptor-overexpressing mice [45]. At early stages of cardiac dysfunction, endogenous NO production may, in addition to attenuating β1-β2-adrenergic inotropic responses, improve diastolic relaxation [46], thereby compensating systolic dysfunction by increasing diastolic reserve. In support of the latter, a recent study in a murine model of sepsis [22] reported that the impairment of relaxation parameters induced by endotoxemia in WT mice was prevented in mice with cardiomyocyte-specific overexpression of eNOS (NOS3 tg). β3-adrenoceptor activation of upregulated eNOS, as observed in our human samples, may subserve a similar adaptative role in early sepsis, perhaps evolving into a maladaptative, cardiodepressant effect at later stages. If so, administration of specific β3-antagonists may be useful to counteract the contractile depression in sepsis, provided the optimal time window can be defined. More insights into these potential roles of the β3-adrenoceptors and their impact on the clinical use of β-blockers with differential affinity for the three β-adrenoceptor isotypes in the treatment of acute cardiac dysfunction should be gained from ongoing studies of animal models with cardiac-specific deletion or overexpression of the β3-adrenoceptor.


   Appendix A. Supplementary data
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
References
 
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ejheart.2007.10.006.


   Acknowledgements
 
This work has been supported by EU FP6 grant LSHM-CT-2005-018833, EUGeneHeart, grants from the Politique Scientifique Fédérale (PAI-P6/55, Belgium), the French Community of Belgium (Action de Recherche Concertée 06/11-338) and FRSM (3.4603.01). S.M. and C.B. are recipients of a fellowship of the ‘Fonds National de la Recherche Scientifique' (FNRS). CD is a FNRS Research Associate. The authors wish to thank the Cardiovascular Surgery Division and the Department of Pathology, Saint-Luc University Hospital (Brussels) for collecting human tissues.


   Notes
Top
 Notes
Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 
1 These authors equally contributed to this work. Back

2 Present address: Department of General Intensive Care, University Hospital Center, Sart-Tilman, B-4000 Liège, Belgium. Back


   References
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 

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beta(3)-Adrenoceptor stimulation on top of beta(1)-adrenoceptor blockade "Stop or Encore?".
J. Am. Coll. Cardiol., April 28, 2009; 53(17): 1539 - 1542.
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