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Cardiovascular Research 2002 53(1):156-164; doi:10.1016/S0008-6363(01)00443-6
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Copyright © 2001, European Society of Cardiology

Endotoxin induces desensitization of cardiac endothelin-1 receptor signaling by increased expression of RGS4 and RGS16

Monica Pattena,*, Jan Bünemanna, Bryan Thomaa, Elisabeth Krämera, Martin Thoenesa, Sabine Stübea, Clemens Mittmannb and Thomas Wielandb

aMedizinische Klinik, Abteilung für Kardiologie, Universitäts-Krankenhaus Hamburg Eppendorf, Martinistr. 52, 20246 Hamburg, FRG
bInstitut für Experimentelle und Klinische Pharmakologie und Toxikologie, Universitäts-Krankenhaus Hamburg Eppendorf, Martinistr. 52, 20246 Hamburg, FRG

* Corresponding author. Tel.: +49-40-42803-5989; fax: +49-40-42803-4884 patten{at}uke.uni-hamburg.de

Received 9 January 2001; accepted 24 August 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Endotoxin (LPS)-induced acute cardiac failure during sepsis is associated with alterations in G protein mediated signal transduction. We therefore examined the expression of the G proteins Gi, Gq, and Gs and of four ‘regulators of G protein signaling’ (RGS) proteins, RGS1, RGS4, RGS5, and RGS16 in rat hearts. Methods: For in vivo experiments, Wistar rats were treated with 600 µg/day E. coli LPS, intravenously) and hearts were excised after 6, 24 and 72 h. Cultured neonatal rat cardiomyocytes were treated with 4 µg/ml LPS for 24 and 72 h. Isolated membrane proteins were used for Western blot analysis and for evaluation of phospholipase C (PLC) activity. RGS16 mRNA was detected by RNAse protection. Results: LPS induced Gi protein 1.4-fold 72 h after in vivo administration of LPS, whereas expression of Gs and Gq was unaltered. After 6 h of LPS treatment, RGS16 mRNA was transiently up-regulated 3.7-fold, followed by transient protein induction (24 h: 2.5-fold; 72 h: 1.5-fold). Similarly, RGS4 protein was transiently induced (24 h: 3.1-fold; 72 h: 1.5-fold), whereas expression of RGS1 and RGS5 was not altered. Similar to the LPS-treated rat hearts, RGS16 expression was transiently induced by LPS in cultured neonatal rat cardiomyocytes (24 h: 1.6-fold, 72 h: 0.9-fold). To determine the functional consequences of the RGS protein induction phospholipase C (PLC) activity was analyzed in membranes obtained from solvent and LPS-treated hearts. Basal and endothelin-1-stimulated PLC activity was transiently repressed by LPS with a maximum after 24 h although no apparent changes in PLCβ1 or endothelin receptor expression could be detected. Conclusion: These data suggest that the rapid up-regulation of cardiac RGS4 and RGS16 is associated with a desensitization of endothelin-1 receptor signaling. Up-regulation of these RGS proteins may thus be involved in the early onset of cardiac failure during sepsis.

KEYWORDS Endotoxins; Heart failure; G-Proteins; Sepsis; Signal transduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Septic shock induced by bacterial endotoxin (LPS) is a predominant course of death in intensive care units [1] and often associated with acute heart failure, characterized by an impairment of myocardial contractility. In patients with chronic heart failure, catecholamine refractoriness accompanied with a down-regulation of β-adrenoceptors and induction of the inhibitory G protein subunit G{alpha}i has been reported to play an important role in the development of contractile dysfunction [2–4]. It was shown that also in patients with catecholamine-refractory septic shock syndrome an increase in myocardial G{alpha}i is associated with acute cardiac failure [5]. Most likely, LPS-induced cardiodepression is mediated by cytokines, e.g. tumor necrosis factor {alpha} (TNF{alpha}) in vivo [6]. Moreover, in a cell culture model of neonatal rat cardiomyocytes (NRCM) TNF{alpha} triggers the above described increase in G{alpha}i protein expression [7]. These observations indicate that endotoxin and cytokine-induced acute heart failure is, at least in part, associated with similar molecular alterations as described for the so far better investigated chronic heart failure models.

Recently, a novel family of intracellular signaling molecules, named regulators of G protein signaling (RGS) proteins, has been identified. They act as GTPase activating proteins (GAP) for G{alpha} subunits and thus are potent inhibitors of G protein mediated signaling. Most of the RGS proteins interact with G{alpha}i- and G{alpha}q-subfamily members but have no influence on the G{alpha}s-subfamily [8–10]. At least 10 different RGS proteins are expressed in the heart, of which one, i.e. RGS16, was found to become rapidly induced in cardiac myocytes during isolation procedures [11]. In addition, it has been reported that its close relative RGS4 is up-regulated on the mRNA and protein level in patients with chronic heart failure [12,13].

We therefore analyzed possible changes in the cardiac expression of RGS1, RGS4, RGS5 and RGS16 after in vivo administration of LPS in an adult rat model. To verify LPS-induced changes in G protein expression, the expressions of G{alpha}i, G{alpha}q, and G{alpha}s were also determined in this model. We report herein that both RGS16 mRNA and protein expression, are transiently increased in the adult rat heart and in isolated neonatal rat cardiac myocytes after LPS treatment. A similar transient increase in expression was observed for RGS4 but not for RGS1 and RGS5. Concomitantly to the changes in RGS protein expression, the Gq mediated stimulation of phospholipase C (PLC) by endothelin-1 (ET-1) receptors was repressed in membranes of LPS-treated hearts.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 In vivo treatment with LPS
Adult male Wistar rats (300–350 g) were treated with 600 µg/day E. coli LPS (Difco, 026:B6) or saline into a tail vein and whole hearts were excised after 6, 24 or 72 h. Aorta and atria were cut off and ventricles were shock frozen in liquid nitrogen. All animal experiments were performed in accordance to the ‘Guide for Care and Use of Laboratory Animals’ as published by the NIH (No. 85-28, revised 1996).

2.2 Cell culture experiments
Neonatal rat cardiomyocytes (NCRM) were isolated as described before [14]. Cells were allowed to settle for 4 days prior to treatment with 4 µg/ml LPS or diluent (0.9% NaCl) for 24 and 72 h.

2.3 Preparation of membranes
Membranes were prepared from frozen ventricles exactly as described [15]. NCRM were washed with phosphate buffered saline, scraped off the dishes and homogenized in 2 ml KHCO3 buffer (1 mM, 2 µg/µl aprotinine). After centrifugation (30 min at 14 600xg) the crude membranes were resuspended in KHCO3 buffer.

2.4 Western blot analysis
Membrane proteins (50 µg) were separated on a sodium dodecyl sulfate–polyacrylamide (15%) gel (SDS–PAGE) and electrophoretically transferred to nitro-cellulose. Filters were blocked in TTBS, pH 7,4 (10 mM Tris, 154 mM NaCl, 0,05% Tween 20) with 3% skimmed milk and washed in TBS (10 mM Tris, 154 mM NaCl). Incubation with specific antibodies for β-tubulin (1:750, SIGMA), G{alpha}i (1:500, sc-386, Santa Cruz Biotechnology), G{alpha}s (3A-150; 1: 75 000, Gramsch Laboratories), G{alpha}q (1:1000, sc-392, Santa Cruz Biotechnology), RGS1 (1:500, sc-6209, Santa Cruz Biotechnology) RGS4, RGS5 (1:1000, J. H. Kehrl, Bethesda, MD), RGS16 (1:1000, C.-K. Chen, Pasadena, CA), PLCβ1 (1:1000, sc-205, Santa Cruz Biotechnology), ETAr and ETBr (1:1000, #AER-001, #AER-002, Alomone) was performed overnight. Filters were again washed in TTBS prior to incubation with a species-specific anti-IgG-HRP-conjugated secondary antibody (dilution 1:2000) for 2 h. Detection was performed with ECL system (Amersham–Pharmacia). Signals were normalised to β-tubulin within the same sample.

2.5 RNA analysis
Total myocyte mRNA was isolated by guanidinium thiocyanate phenol–chloroform precipitation [16]. RGS16 mRNA was quantified by RNAse protection assay (RPA) (RPAII®, Ambion) as described [17]. Total mRNA (10–15 µg) was hybridized with [{alpha}32P]UTP-labelled antisense probes complementary to 276 bp of the rat RGS16 (subcloned in pBluescript SK, Stratagene) and 80 bp of the last coding exon of the 18S gene (Ambion). Protected fragments were separated on a 6% denaturing polyacrylamide gel and quantified by phosphoimaging (BAS 2000, Fuji, Japan). Signals were normalised to 18S within the same sample [17].

2.6 Measurement of phospholipase C activity
Phospholipid vesicles were prepared as described [18]. The assay was started by adding 20 µg of the myocardial membranes to a reaction mixture (70 µl) containing 35 µl phospholipid vesicles, Hepes 10 mM, KCl 67.5 mM, NaHCO3 2.5 mM, EGTA 2.5 mM, glucose 2.8 mM, ATP 1 mM, LiCl 10 mM, CaCl2 0.75 mM, MgCl2 1 mM, pH 7.2 and the indicated stimuli. After 60 min, the reaction was stopped by adding 100 µl of 1 M HCl/5 mM EGTA plus 250 µl CHCl3/MeOH/HCl (100:100:6). Inositol phosphate production was analyzed as described [18]. All values were determined in duplicates.

2.7 Statistics
Statistical significance was estimated by Student's t-test for unpaired observations. A P-value of less than 0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Influence of LPS administration in vivo on G{alpha}i, G{alpha}s, and G{alpha}q protein expression in the rat heart
Chronic heart failure induced by pressure overload or ischemia in humans is associated with an increased expression of G{alpha}i subunits, whereas the expression of G{alpha}s subunits remains unaltered [3,4,19]. During sepsis, acute heart failure in humans is also associated with an increase in G{alpha}i [5]. To evaluate our rat model, we thus examined G{alpha}i, G{alpha}s, and G{alpha}q protein expression after systemic application of LPS in vivo. G{alpha}i protein expression was induced in the presence of LPS after 72 h (Fig. 1), whereas there was no difference in G{alpha}i expression compared to the saline-treated control group after 6 and 24 h of LPS treatment. In contrast, expression of G{alpha}s and G{alpha}q was not different in LPS-treated animals compared to the saline-treated control group (Fig. 1 and Table 1).


Figure 1
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  		Fig. 1 Influence of LPS on the expression of G{alpha}i, G{alpha}s, and G{alpha}q in the rat heart. Adult Wistar rats were treated with 600 µg/day LPS or diluent (0.9% NaCl) and whole hearts were excised after 6, 24 or 72 h. Membrane proteins were isolated and G{alpha}i, G{alpha}s, and G{alpha}q protein expression was detected by Western Blot analysis. Panels A, B, and C show characteristic blots for G{alpha}i, G{alpha}s, and G{alpha}q, respectively.

 

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  		Table 1 G protein expression in rat hearts 6, 24 and 72 h after treatment with LPS or NaCl

 
3.2 LPS induces RGS16 mRNA and protein expression in vivo
Using the same preparations from saline- or LPS-treated animals, we determined cardiac mRNA and protein expression of RGS16. The RGS16 specific riboprobe resulted in a single 276 nucleotide protected fragment in the RPA (Fig. 2). The expression of RGS16 mRNA was rapidly induced 6 h after LPS treatment. Induction of RGS16 mRNA however, was transient as there was no more significant difference detectable after 24 and 72 h compared to the saline-treated control group.


Figure 2
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  		Fig. 2 Transient induction of RGS16 mRNA by LPS. RNA was isolated from hearts of adult Wistar rats treated with 600 µg/day LPS (grey bars) or diluent (0.9% NaCl, open bars) 6, 24 or 72 h post treatment. RGS16 mRNA levels were detected by RNAse protection (Panel A). Autoradiographs were analyzed by densitometry and RGS16 mRNA expression was normalised to the expression of 18S RNA within the same sample. Data are expressed as the ratio of RGS16 mRNA over 18S expression±S.D. The numbers of animals per treatment group are indicated at the bottom of each column (*P<0.05; Panel B).

 
To evaluate whether these transient changes in RGS16 mRNA expression are of biological relevance, RGS16 protein expression was measured in heart preparations from the same animals using the RGS16 specific antiserum CT-265 [20]. As described before [21], a specific signal of about 31 kDa was recognised and could be blocked by pre-incubation of the antiserum with recombinant RGS16 (data not shown). As shown in Fig. 3D, RGS16 protein expression was increased in LPS-treated animals. Compared with the induction of RGS16 mRNA, up-regulation of RGS16 protein was delayed but similarly transient. There was no significant difference in RGS16 protein expression 6 h after LPS treatment, but expression increased about 2.5-fold 24 h post treatment. Up-regulation was still detectable after 72 h, however at lower levels (Table 2).


Figure 3
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  		Fig. 3 LPS induces RGS16 and RGS4 but has no effect on RGS1 and RGS5 protein expression in vivo. Adult Wistar rats were treated with 600 µg/day LPS or diluent (0.9% NaCl) and whole hearts were excised after 6, 24 or 72 h. Membrane proteins were isolated and RGS1 (Panel A), RGS4 (Panel B), RGS5 (Panel C) or RGS16 (Panel D) protein expression was detected by Western blot analysis.

 

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  		Table 2 RGS protein expression in rat hearts 6, 24 and 72 h after treatment with LPS or NaCl

 
3.3 LPS induces RGS4 but has no influence on RGS1 and RGS5 protein expression in vivo
In addition to RGS16, at least 10 other RGS proteins are expressed in the rat heart [11]. The different functions of these proteins are poorly understood. At least on the mRNA level three structurally related RGS proteins, RGS1, RGS4 and RGS5 are highly abundant in rat ventricular myocytes [11].

We therefore studied cardiac expression of RGS1, RGS4, and RGS5 protein in the presence of LPS in vivo. As described before, RGS4 protein was detected by the antiserum as a 30-kDa protein [22,23], whereas RGS1 and RGS5 were detected at 27–28 kDa [21]. All signals could be blocked by pre-treatment with the respective antigen (data not shown).

As shown in Fig. 3B, similar to RGS16, RGS4 expression was transiently induced by LPS. The maximum of induction (about 3.1-fold) was reached after 24 h and increased expression was still significant after 72 h (about 1.5-fold; Table 2).

In contrast, no influence of LPS treatment on the expression of RGS1 and RGS5 proteins could be detected. (Fig. 3A,C, Table 2).

3.4 Transient induction of RGS16 by LPS in isolated cardiac myocytes
To test whether the induction of RGS proteins by LPS detected in whole heart preparations is due to alterations in cardiac myocytes, RGS16 expression was measured in isolated neonatal cardiac myocytes in culture treated with LPS for 24 and 72 h. As shown in Fig. 4, LPS transiently induces RGS16 protein in cardiac myocytes after 24 h similar to what was observed in whole heart, suggesting that changes of RGS expression in the heart are due to a direct effect of LPS on cardiac myocytes.


Figure 4
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  		Fig. 4 RGS16 protein is induced by LPS in cultured neonatal rat cardiac myocytes. Neonatal rat cardiac myocytes were isolated and cultured for 4 days prior to treatment with 4 µg/ml LPS for 24 and 72 h. Membrane proteins were isolated and RGS16 protein expression was detected by Western blot analysis (Panel A). Expression was analyzed by densitometry and normalised to β-tubulin expression within the same sample. Data are expressed as the ratio of RGS16 protein over β-tubulin expression±S.D. Data were obtained from four independent preparations (*P<0.05; Panel B).

 
3.5 LPS transiently represses basal and ET-1 induced phospholipase C activity
Overexpression of RGS4 in cultured cardiomyocytes inhibits Gq-mediated phenylephrine and ET-1-induced cardiomyocyte hypertrophy [24]. In addition, RGS4 overexpressing animals show significantly reduced ventricular hypertrophy in response to pressure overload and blunted responses to phenylephrine [25]. Thus, to investigate whether the induction of RGS proteins by LPS results in a reduced activity of PLC, PIP2 hydrolysis was determined in cardiac membranes from LPS- and saline-treated animals under basal conditions and after stimulation with ET-1. In the presence of 100 µM GTP the mean basal PIP2 hydrolysis in the saline-treated control group (n=13) was 109.8±17 pmol/mg per min and the mean ET-1-stimulated PIP2 hydrolysis was 150±17 pmol/mg per min. As shown in Table 3, LPS significantly represses PLC activity 24 h post treatment in the presence and absence of ET-1. In contrast to the saline-treated rats, no significant stimulation by ET-1 was observed in the LPS-treated animals after 24 h (Fig. 5). In membranes obtained after 6 and 72 h of LPS treatment no significant alterations on basal as well as on ET-1-induced PLC activity was detected compared to saline-treated control animals (Table 3).


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  		Table 3 Basal (GTP) and endothelin-1-stimulated PIP2 hydrolysis (GTP+ET-1) in rat hearts 6, 24 and 72 h after treatment with LPS or NaCl

 

Figure 5
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  		Fig. 5 Inhibition of ET-1-stimulated PLC activity by LPS. PLC activity was determined in membranes obtained from hearts of adult Wistar rats 6, 24 or 72 h after treatment with 600 µg/day LPS or diluent (0.9% NaCl). Basal (GTP) and endothelin-1-stimulated PIP2 hydrolysis (GTP+ET-1) was determined. The data shown give the relative PLC activity after 24 h of treatment with saline or LPS as percent of the basal PIP2 hydrolysis (GTP)±S.D.

 
3.6 LPS has no effect on PLC or ET-1 receptor protein expression
Changes in the amount of PLC or endothelin receptor expression might attenuate ET-1 signal transduction processes. Therefore, we investigated whether LPS influences the expression of these proteins, which could account for the observed reduction in ET-1-induced PLC activity by LPS. The expression of PLCβ1 as well as of the two ET-1 receptor subtypes ETAr and ETBr, shown to coexist in the human heart [26], were determined in the in vivo model. LPS had no influence on PLCβ1 protein expression 6, 24 and 72 h post treatment (Fig. 6, Table 4). In addition, no alterations in protein expression of the two ET-1 receptor subtypes could be detected between the LPS- and the diluent-treated groups (Fig. 6, Table 4).


Figure 6
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  		Fig. 6 LPS does not alter PLCβ1 and endothelin receptor expression. Adult Wistar rats were treated with 600 µg/day LPS or diluent (0.9% NaCl) and whole hearts were excised after 6 24 or 72 h. Membrane proteins were isolated and PLCβ1 (Panel A), ETAr (Panel B) and ETBr (Panel C) protein expression was detected by Western blot analysis.

 

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  		Table 4 PLCβ1, ETAr and ETBr expression in rat hearts 6, 24 and 72 h after treatment with LPS or NaCl

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In the present study, possible alterations in the cardiac expression of RGS1, RGS4, RGS5 and RGS16 were analyzed in adult Wistar rats after in vivo application of endotoxin as a model for septic shock. Similar to patients with catecholamine-refractory shock syndrome [5], the expression of G{alpha}i was up-regulated 72 h post LPS treatment. In accordance with other rat models for heart failure [19] there was no detectable alteration of G{alpha}s protein expression.

We demonstrate that the cardiac expression of RGS4 and RGS16 rapidly increases after LPS challenge in this model. For RGS16 we provide evidence that the induction of RGS16 protein is associated with a transient up-regulation of RGS16 mRNA. An increase in RGS16 mRNA expression was observed 6 h post treatment followed by an up-regulation of protein expression after 24 h. These data are in line with the reported increase in RGS16 mRNA expression in porcine hearts after treatment with LPS [27]. Moreover, RGS16 protein expression was also induced by LPS in isolated NRCM in cell culture, suggesting that the induction of RGS16 can be attributed to cardiac myocytes. RGS4 protein expression paralleled the changes in RGS16 protein expression with regard to time dependence and the extent of protein increase after treatment with LPS. Recently, RGS4 expression has been shown to be transcriptionally regulated in cardiac myocytes [22]. These data may suggest that both RGS4 and RGS16 expression are regulated via the same LPS-induced signaling cascade in cardiac myocytes. The observed phenomenon of a transient up-regulation of RGS proteins is of particular interest. Possible explanations such as inactivation or excretion of LPS can be excluded. The in vivo half time of the LPS preparations used in our study was 16 h (according to the manufacturer's protocol). Therefore LPS was administered every 24 h until the animals were sacrificed. In addition, the experiments with cultured NRCM revealed a similar transient expression profile. Taking into account that the up-regulation of G{alpha}i expression is more delayed, one may conclude that the transient up-regulation of RGS4 and RGS16 is part of a co-ordinated response to LPS challenge of cardiac myocytes.

Stimulation of Gq-coupled receptors, such as ET-1 receptor or {alpha}1-adrenoceptor agonists, is involved in the development of cardiac hypertrophy and failure [28–31] through activation of PIP2 hydrolysis by phospholipase C (PLC). Stimulation of Gq protein-coupled receptors also induces a positive inotropic effect in cardiomyocytes which, at least in part, is mediated by an inositol 1,4,5 triphosphate-dependent release of calcium from the sarcoplasmatic reticulum [32]. A decrease in basal and receptor-stimulated inositol phosphate formation has been reported in aortic preparations of septic rats [33], and LPS treatment for 18 h in vivo was shown to induce a decrease in basal and {alpha}1-adrenoceptor stimulated inositol phosphate formation concomitant with a decrease in {alpha}1-adrenoceptor-induced vasoconstriction [34]. To analyse the functional consequences of LPS on Gq-mediated signaling in the heart, we thus studied whether LPS affects basal and endothelin-1 (ET-1)-stimulated PLC activity in cardiac membranes from LPS-treated animals. We detected a transient repression of PLC activity in LPS-treated hearts paralleling the time dependence of RGS protein expression. Both basal and endothelin-1-induced PLC activities were significantly reduced within 24 h, whereas no changes were observed 6 or 72 h after treatment with LPS. As we could not detect any changes in the expression of G{alpha}q, PLCβ1 protein or the ET-1 receptor subtypes ETAr and ETBr during LPS treatment, and R6S4 and R6S16 have been shown to negatively regulate G{alpha}q activation (for review see Ref. [10]), we conclude that the increased expression of these RGS proteins is responsible for the loss in PLC activity. This interpretation is further corroborated by several reports. Overexpression of RGS4 in cultured cardiomyocytes and transgenic animals inhibits phenylephrine and endothelin-induced responses involved in cardiomyocyte hypertrophy [24,25]. Recombinant RGS16 inhibits ET-1 stimulated PLC activity in human ventricular membranes [35]. Finally, it has been demonstrated that TNF{alpha}, which is known to play an important role in endotoxin-induced sepsis, decreases basal and {alpha}1-adrenoceptor stimulated inositol phosphate formation in cultured neonatal cardiac myocytes [36].

Taken together, our data demonstrate that RGS4 and RGS16 are rapidly and transiently induced in rat heart by endotoxin in vivo due to a direct effect of LPS on cardiac myocytes. The changes in RGS protein expression are likely responsible for the observed changes of basal and ET-1-stimulated PLC activity, which may be one important step involved in the early onset of cardiac failure during endotoxemia.

Time for primary review 27 days.


   Acknowledgements
 
This study was supported by grants to M. Patten and T. Wieland by the ‘Deutsche Forschungs-Gemeinschaft’ (DFG).


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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