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Cardiovascular Research 2002 55(4):778-786; doi:10.1016/S0008-6363(02)00459-5
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Copyright © 2002, European Society of Cardiology

Expression of ten RGS proteins in human myocardium: functional characterization of an upregulation of RGS4 in heart failure

Clemens Mittmanna,*, Chin Hee Chunga, Grit Höppnera, Christina Michalek, Monika Nosea, Christian Schülera, Antje Schuha, Thomas Eschenhagenb, Joachim Weilc, Burkert Piesked, Stephan Hirte and Thomas Wielanda

aInstitut für Experimentelle und Klinische Pharmakologie und Toxikologie, Abteilung für Pharmakologie, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany
bInstitut für Experimentelle und Klinische Pharmakologie und Toxikologie, Universität Erlangen-Nürnberg, Abt. für Klinische Pharmakologie und Toxikologie, Nürnberg, Germany
cMedizinische Klinik und Poliklinik, Universitätsklinikum Regensburg, Regensburg, Germany
dMedizinische Klinik, Universitätsklinikum Göttingen, Göttingen, Germany
eAbteilung für Herz- und Gefässchirurgie, Universität zu Kiel, Kiel, Germany

* Corresponding author. Tel.: +49-40-42803-4707; fax: +49-40-42803-4876

Received 17 January 2002; accepted 25 April 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 
Objective: RGS proteins (regulators of G protein signalling) negatively regulate G protein function as GTPase activating proteins. By controlling heterotrimeric G proteins they may regulate myocardial hypertrophy and contractility. We investigated the expression of RGS proteins in the human heart and whether they take part in the pathophysiological changes of heart failure. Methods and results: Using RNase protection assays (RPAs) RGS2, 3L, 3S, 4, 5 and 6 were identified in the myocardium from terminally failing human hearts with dilated (DCM, n = 22) or ischemic (ICM, n = 18) cardiomyopathy and from nonfailing donor hearts (NF, n = 9). With reverse transcriptase polymerase chain reaction in addition mRNA of RGS1, 9, 12, 14 and 16 were detectable. Compared to NF in failing LV myocardium RGS4 mRNA and protein was upregulated 2–3-fold (mRNA, 10–21 mol/µg±S.E.M.: NF: 22±5§, DCM: 51±10*, ICM: 37±8; §P<0.05 vs. DCM+ICM, *P<0.05 vs. NF, § P<0.05 vs. DCM+ICM; protein, % of NF±S.E.M.: NF: 100±35, DCM 266±60*, ICM: 205±64, n = 5, *P<0.05 vs. NF). In contrast, RGS2, 3L, 3S, 5, 6, and 16 protein and mRNA levels did not vary between failing and NF hearts. In order to investigate the impact of RGS4 on Gq/11 mediated signalling, PLC activity was measured in human LV membranes. Recombinant RGS4 blunted the endothelin-1 (ET-1) stimulated PLC activity. When overexpressed by adenoviral mediated gene transfer in rabbit ventricular myocytes RGS4 abolished the inotropic effect of ET-1. Conclusion: The upregulation of RGS4 in failing human myocardium diminishes Gq/11-mediated signalling and can be involved in the desensitization of Gq/11-mediated positive inotropic effects.

KEYWORDS Contractile function; Endothelins; G-proteins; Heart failure; Myocytes


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 
The regulators of G protein signalling (RGS proteins) negatively regulate G protein function as GTPase activating proteins (GAPs) [1,2]. More than 20 different mammalian RGS proteins have been identified [1]. Most of them regulate G proteins of the Gi/Go- and Gq/11-families although a subset of RGS proteins are specific for the G12-family [1,2]. The rat heart expresses multiple RGS proteins [3,4]. mRNA transcripts for RGS1, 3, 4, 5, and 6 are highly expressed while mRNAs for at least five others are detectable by polymerase chain reaction (PCR). The functional significance of the abundance of RGS proteins remains enigmatic, particularly so since most RGS proteins possess in vitro GAP activity for both Gi/Go and Gq proteins. Recently it has been demonstrated that some RGS proteins may exhibit G protein coupled receptor (GPCR) selectivity [5]. Thus, it is feasible that myocardial cells alter their levels of particular RGS proteins to regulate signals from specific GPCRs. Such a regulation of the expression of RGS3 and RGS4 in the myocardium and myocardial cells has been described for models of hypertrophy or failure [4]. By transgenic myocardial overexpression of RGS4 in mice it has been suggested that RGS proteins may counterregulate Gq mediated hypertrophic signalling [33,34]. This was suggested as a beneficial response in the hypertrophied heart since a fourfold overexpression of RGS4 was able to alleviate cardiac dysfunction and LV chamber diameters in mice overexpressing Gq [34,37].

In the rat heart the expression of 10 different RGS proteins has been demonstrated [3]. Little systematic data exist about the expression of RGS proteins in the human heart. RGS2, 3, 4, 5, 11 and 12 mRNAs and/or proteins have been detected by immunoblot, Northern blot or PCR [1–10]. It is largely unknown, however, which of these RGS proteins are actually expressed at a relevant level and whether additional RGS proteins are present in the human myocardium.

Therefore, we investigated the expression of RGS proteins in human left ventricular myocardium addressing the following questions: (a) which of the RGS proteins that regulate Gi/Go- and Gq/11-mediated pathways are detectable at the mRNA and protein level, (b) what is their level of expression, (c) are there differences in the expression of RGS proteins between failing and nonfailing human myocardium from dilated or ischemic myocardium and (d) are changes in the expression of RGS proteins of functional significance for myocardial contractility?


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusion
 References
 
2.1 Procurement of tissue
Ventricular myocardium was obtained from patients with terminal heart failure resulting from idiopathic dilated cardiomyopathy (DCM) or ischemic cardiomyopathy (ICM), who underwent orthotopic heart transplantation. Clinical data are reported in Table 1. Nonfailing hearts were obtained from prospective organ donors whose hearts could not be transplanted due to technical or clinical reasons (mean age 47±6 years). Diagnosis leading to clinical death were polytrauma in one, intracerebral bleeding in five, polytrauma and intracerebral bleeding in one, subarachnoidal bleeding in one, and cerebral venous thrombosis in one patient. On inspection, these hearts appeared to have normal ventricles. Procedures for obtaining human tissue complied with the Helsinki Declaration. Permission for these studies was obtained from the local Ethics Committee. Written informed consent was taken from all patients or the family of prospective heart donors before cardiectomy. The investigation conforms with the principles outlined in the Declaration of Helsinki.


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  		Table 1 Clinical and hemodynamic data from patients with heart failure

 
2.2 Preparation of RNA
Total RNA was extracted as described previously [11,12]. The concentration was determined photometrically at 260 nm and the quality of the RNA was checked on an ethidium bromide stained 1% agarose gel.

2.3 Reverse transcriptase polymerase chain reaction (RT-PCR)
Reverse transcription was performed with 1 µg of myocardial total RNA for 1 h at 42 °C following a standard protocol using superscript II 200 U (Gibco BRL). PCR conditions were as follows (25 µl): primer 0.4–0.8 µM each, dNTP 0.25 mM, 1x PCR buffer, 1.25 U Taq-DNATM-Polymerase (Ambion, Austin, TX, USA), MgCl2 1–1.5 mM, 2 µl of cDNA, annealing 60°C, 60 s, synthesis at 72 °C for 30 s, denaturing at 95 °C for 30 s, 35 cycles. The following primer pairs were used (forward, backward, corresponding to nt of the coding sequence): RGS1: 119–139, 411–431; RGS3: 652–672, 1111–1131; RGS5: 206–226, 444–464; RGS6: 350–370, 611–631; RGS7: 570–590, 850–870; RGS9: 1730–1750 1952–1972; RGS12: 1563–1583, 1779–1799; RGS14: 159–179, 445–465; RGS–16: 109–129417–437. The specificity of the PCR products was established by automated sequencing after subcloning in the pGEM-T® vector.

2.4 RNase protection assay (RPA)
The specific cDNA fragments obtained by RT-PCR were subcloned in pT7-Blue® (Novagene, Bad Soden, Germany). A 247 base pair (bp) EcoRI/EcoRI fragment (nt 331–577 of the cds) of human RGS2 and a 700 bp BamHI/XbaI fragment of human RGS4 containing the complete cds and a part of the 3'-non-cds were subcloned in pBluescript® SK (Stratagene, Heidelberg, Germany). A 677 bp PstI/PstI fragment of human atrial natriuretic peptide (hANP) cDNA (kind gift of Dr. M. Böhm, Cologne, Germany) was subcloned in pBluescript® SK. cDNA of the {alpha}-subunit of the rat stimulatory G protein (G{alpha}s) was subcloned in PGEM-2 as described previously [13]. 32P labelled antisense RNA probes were transcribed with linearized plasmids as follows (specific nucleotides: antisense (as)/sense): RGS1: 312 (as); RGS2: 247/247; RGS3: 480/480; RGS4: 275/624; RGS5: 258/258; RGS6: 281/281; RGS9: 242 (as); RGS12: 236 (as); RGS14: 306 (as); RGS16: 328 (as); hANP: 202/677; G{alpha}s: 93/615.

RPAs were performed with the RPA II® kit (Ambion) as described [13] using 5–10 µg of total RNA. For absolute quantification sense-cRNAs were produced by in vitro transcription of linearized plasmids and the concentration was calculated as described [13]. Values for RGS proteins and ANP were normalized to the expression of G{alpha}s [14].

2.5 Preparation of myocardial membranes
Preparation of membranes for immunoblots and PLC assays were performed as described [14]. Protein concentration was determined according to Bradford [15] using IgG as a standard.

2.6 Immunoblotting
Immunologic identification and quantification was achieved by quantitative Western blotting as described [12] with minor modifications. A 50-µg amount of myocardial membranes was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE; 6%/15% stacking/running gel). Four different concentrations of the respective recombinant RGS proteins served as a standard curve for all RGS proteins except RGS3L. Specific bands were detected with ECL® (Amersham, Buckinghamshire, UK) after exposition to X-Ray films (X-OMAT® AR, Kodak). Data were standardized to the expression of G{alpha}s. The following first antibodies were used: anti-RGS1 1:2000 (Santa Cruz SC), anti-RGS2 1:2000 [14], anti-RGS3 1:2000 [14,16], anti-RGS4 1:2000 [22] and anti-RGS5 1:2000 were kind gifts from J.H. Kehrl, NIH, Bethesda, MD, USA, anti-RGS16 1:2000 kind gift of Dr. C.K. Chen [17]. anti-G{alpha}s: 1:20 000, 3A-150 Gramsch Laboratories, Schwabhausen, Germany.

2.7 Purification of recombinant RGS proteins
The coding sequences of human (h) RGS1, hRGS2, hRGS3S, hRGS4, hRGS5 and mouse RGS16 were subcloned into the bacterial expression vector pET15b (Novagen, Germany) and recombinant His6-tagged proteins were expressed and purified as described before [17].

2.8 Measurement of PLC activity in human ventricular myocardium
PLC activity in purified left ventricular myocardium was measured exactly as described previously [14].

2.9 Generation of recombinant adenovirus
Recombinant adenovirus for RGS4 (Ad-RGS4) and as a control empty virus coding only for GFP (Ad-GFP) was generated as described [19,20]. The vectors were kindly provided by B. Vogelstein (Baltimore, MD, USA).

2.10 Primary culture of rabbit ventricular myocytes and adenoviral gene transfer
Female bastard New Zealand/Chinchilla rabbits (Charles River, Sulzfeld, Germany, 2–3 kg) were heparinized (1000 units) and anaesthetized (sodium thiopental 50 mg/kg, i.v.). Ventricular myocytes were isolated as described [21]. After sedimentation the myocytes were plated at a density of 50 000 cells/dish on laminin (20 µg/ml)-coated 35 mm culture dishes with M199 medium (M199 medium-sigma, 5 mM taurine, 5 mM D,L-carnitine, 5 mM D,L-creatine, 100 U/ml penicillin, 0.1 mM streptomycin). Cardiomyocytes were infected with adenoviral constructs (multiplicity of infection, MOI 1) at a minimal volume of culture medium. Two hours after infection the cells were washed and cultured with M199 medium for 48 h. The overexpression of RGS4 was confirmed after subjecting 200 µg of crude homogenates of cardiomyocytes to PAGE and immunoblotting as described above.

2.11 Measurement of single-cell shortening
In cardiomyocytes showing fluorescence for GFP single-cell shortening was measured by an edge detection system (Crescent Electronics) at a stimulation frequency of 1 Hz at 37 °C and a sampling rate of 240 Hz as described [21].

2.12 Statistics
For comparisons between two groups Student's t-test, for more than two groups analysis of variance (ANOVA) was calculated using the Newman–Keuls multiple comparison test as a post test. A P value ≤0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusion
 References
 
3.1 mRNA expression of RGS proteins in human myocardium
In order to identify which RGS proteins are present in human left ventricular (LV) myocardium on the mRNA level we performed RPAs and RT-PCR for RGS1, 2, 3, 4, 5, 6, 7, 9, 12, 14, and 16 as described in the Methods section. Using 5–15 µg of total RNA we detected with the RPA specific signals for RGS2, 4, 5, 6, and for RGS3S and 3L [14] (Fig. 1). RGS1, 9, 12, 14, and 16 were detectable by RT-PCR, but no specific signals were seen by RPA (Fig. 2). Amplificates were subcloned in the pGEM-T® vector and the identity was verified by automated sequencing.


Figure 1
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  		Fig. 1 Phosphoimager images of representative RNase protection assays for (A) RGS2, (B) RGS3, (C) RGS4, (D) RGS5, and (E) RGS6 in 5–10 µg of total RNA (duplicates) from left ventricular (LV) myocardium from patients with dilated (DCM) or ischemic (ICM) cardiomyopathy or from nonfailing controls (NF). Expression of G{alpha}s was used as an internal standard in all samples as shown in (A). In the assays with RGS3 (C) the G{alpha}s probe was added only in every third lane of a triplicate to avoid interference with RGS3S. Expression of ANP mRNA (A) was determined in addition as a marker for heart failure. Regional expression of ANP was determined for comparison as a control in right and left atrial (RA, LA) and right ventricular myocardium (RV). u/d: Undigested/digested antisense riboprobe. STD: Pooled myocardial RNA at 1, 2, 3, 5, 7, and 10 µg in duplicate was used as a standard. sgl: Myocardial RNA hybridized with ANP alone (left lane) or with RGS2 and G{alpha}s (right lane). Note that the no signal of the undigested ANP riboprobe was detectable (sgl) (A), which excludes an interference with the specific RGS2 signal. Position of the antisense probes is depicted at the left and of the protected fragment at the right side.

 

Figure 2
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  		Fig. 2 RT-PCR for RGS1, RGS9, RGS12, RGS14, and RGS16 with 1 µg total pooled RNA from left ventricular myocardium from patients with dilated and ischemic (n = 3) cardiomyopathy (1% ethidium bromide stained agarose gel). Std: DNA molecular weight standard. PCR was conducted with cDNA after reverse transcription (RT) or as a negative control in total RNA treated identically but omitting reverse transcriptase (–).

 
3.2 mRNA expression of RGS proteins in failing and nonfailing myocardium
We compared the mRNA and protein levels of RGS proteins in left ventricular myocardium from terminally failing hearts with ICM, DCM and from nonfailing donor hearts. ANP mRNA, a well described marker which expression is upregulated in heart failure, was significantly increased in LV failing myocardium (Fig. 1) from ICM (n = 8, 14±8 amol/µg) and DCM (n = 8, 100±68 amol/µg) as compared to NF (n = 7: 0.4±0.1 amol/µg). In contrast, mRNA content of G{alpha}s (NF (n = 7): 1.3±0.3, DCM (8): 1.0±0.08; ICM (8) 1.3±0.2 amol/µg total RNA) was not different between the groups (Fig. 1) and therefore was used as a standard. RGS2 mRNA levels showed high interindividual differences but no significant differences between nonfailing and failing myocardium (Table 2). Expression of both variants of RGS 3 [14], RGS5 and RGS6 mRNA was also not different between the groups (Table 2). In contrast, RGS4 mRNA showed a significantly (P<0.05) increased expression in failing LV from DCM and DCM+ICM reaching 170–240% of the level in the nonfailing controls (Fig. 3A).


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  		Table 2 mRNA expression of RGS2, 3S, 3L, 4, 5, and 6 in human left ventricular myocardium

 

Figure 3
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  		Fig. 3 Quantification of RGS4 mRNA (A) by RNase protection assays with total RNA from terminally failing left ventricular myocardium from patients with dilated (DCM) or ischemic cardiomyopathy (ICM) or from nonfailing controls (NF). The number of hearts is shown in the columns. (B) Original immunoblot and quantification of RGS4 protein by immunoblots. rec: Recombinant RGS4; *: P<0.05 vs. NF; §: P<0.05 vs. DCM+ICM.

 
3.3 Protein expression of RGS proteins
We investigated by quantitative immunoblots, which RGS proteins could be detected in myocardial membranes with the available antibodies against RGS1, 2, 3, 4, 5, and 16. 50 µg of myocardial membranes from LV were analyzed in parallel to the recombinant RGS proteins (Table 3, Fig. 3B). For control the expression of G{alpha}s was investigated (not shown). The antibody against G{alpha}s detected both specific bands at 42 and the 47 kDa [18].


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  		Table 3 Protein content of RGS2, 3S, 3L, 4, and 16 in human left ventricular myocardium

 
We did not consistently detect specific signals for RGS1 and RGS5 in LV myocardium, whereas the recombinant proteins were detected at 27 and 28 kDa (not shown). RGS2, 3S, and 3L were detected at 30, 26, and 67 kDa, respectively, with the antibody directed originally against RGS2 as described previously [14].

Using the anti RGS4 antibody [22], we detected a 30 kDa band which migrated slightly slower than the recombinant protein (Fig. 3B). In addition, preincubation of the antibody with recombinant RGS4 blocked this band which therefore was regarded to be specific for RGS4 (not shown). The antibody against RGS16 [17] detected a band at 31 kDa which could be blocked by preincubation with recombinant RGS16. As estimated in comparison to recombinant proteins the amount of RGS2, RGS3S, RGS4, and RGS16 were in the range of 0.5–5 ng/µg membrane protein. The expression of RGS4 was significantly (P<0.05) increased in membranes from failing left ventricular myocardium from DCM and from DCM+ICM to 205–265% (Fig. 3B, Table 3). This increase paralleled that detected on the mRNA level. Expression of RGS2, RGS3S, RGS3L, and of RGS16 was not significantly different between the groups (Table 3).

3.4 Effect of RGS4 on ET-1 stimulated PLC activity
In order to assess whether RGS4 regulates Gq mediated signalling in human ventricular myocardium we investigated GTP (100 µM) and GTP+ET-1 (10 µM) stimulated PLC activity in human left ventricular membranes. Recombinant RGS4 (4 µM) blunted the ET-1 mediated stimulation of PLC activity to baseline levels (Fig. 4).


Figure 4
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  		Fig. 4 PLC activity in left ventricular myocardial membranes from three failing hearts (DCM+ICM) in the presence or absence of recombinant RGS4 (4 µM) stimulated with GTP (100 µM) or GTP+ET-1 (10 µM). * P<0.05 vs. GTP, § P<0.05 vs. control.

 
3.5 Effect of RGS4 on contractility
Since preparations from myocardial tissue contain both cardiomyocytes and non cardiomyocytes we further characterized the effects of overexpressed RGS4 in isolated cardiomyocytes from rabbits. Cardiomyocytes were infected with Ad-RGS4 or Ad-GFP as a control (MOI 1) for 48 h. About 30% of cells showed fluorescence by GFP and only GFP positive cells were used. When compared to recombinant proteins homogenates from Ad-RGS4 infected cells contained about 3.5–7 ng RGS4/µg protein (not shown). The average overexpression was estimated to reach about 3–6-fold. This cannot be extrapolated to single cells, however, since GFP-overexpressing cells were selected for the experiments. Fractional shortening was investigated as described previously [21]. A cumulative concentration response curve (0.0001–0.1 µM) was investigated for ET-1 in both groups. ET-1 increased concentration dependently fractional shortening by about 60–70% in Ad-GFP infected cells. No effect was seen in Ad-RGS4 infected cells (Fig. 5). In contrast, isoprenaline (10 µM), given as a control, increased fractional shortening in both groups without significant difference: baseline: Ad-GFP: 4.1±0.7%, Ad-RGS4: 3.6±0.4%, isoprenaline: Ad-GFP: 11.1±2.0 vs. Ad-RGS4: 8.0±1.1%, P>0.05, n = 5.


Figure 5
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  		Fig. 5 Fractional shortening of rabbit ventricular cardiomyocytes infected with Ad-GFP or Ad-RGS4 at increasing concentrations of ET-1 (0.0001–0.1 µM). * P<0.05 vs. Ad-GFP. Fractional shortening at baseline or after isoprenaline (10 µM) at the end of the experiment was not different: Baseline: Ad-GFP: 4.1±0.7%, Ad-RGS4: 3.6±0.4%, isoprenaline: Ad-GFP: 11.1±2.0 vs. Ad-RGS4: 8.0±1.1%, P>0.05.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusion
 References
 
Little is known about the specific function of the different RGS proteins present in the myocardium [3]. Presumably, they are involved in the regulation of different effects mediated via Gi/Go, Gq/11 and G12/13 proteins such as contractility, growth and hypertrophy. In isolated cardiomyocytes, overexpressed RGS4 antagonized growth promoting effects mediated via endothelin- and {alpha}1-adrenecoptors as well as the stimulation of the transcription of atrial natriuretic peptide and myosin light chain [22]. It is unknown, albeit conceivable, that other RGS proteins also mediate growth inhibition and antihypertrophic effects in the myocardium. Therefore, we investigated, which RGS proteins are present in the human myocardium and whether a regulation of the expression of RGS proteins participates in the pathophysiology of heart failure. In summary, we identified 11 different RGS proteins and subtypes in left ventricular human myocardium, including the two different forms of RGS3 described previously [14]. On the mRNA level six forms showed a high (RGS2, RGS5, RGS6) or intermediate expression (RGS3S, 3L, RGS4) and five forms (RGS1, RGS9, RGS12, RGS14, RGS16) were detectable by RT-PCR, but below the detection limit of the RPA. This variety of RGS protein expression is in accordance with findings in the rat heart [3], where the mRNAs of 10 different RGS proteins were described. On the protein level, only RGS2, 3S, 3L, 4, and 16 were detectable. We suppose that the expression of RGS1, 5, and RGS6 protein in human LV myocardium is too low to be detected by the antibodies used.

4.1 Upregulation of RGS4 in heart failure
In order to assess the role of RGS proteins in the pathophysiology of heart failure the multiplicity of G-protein mediated intracellular signalling pathways and their changes in the failing heart must be considered (for reviews, see Refs. [23,24]). Increased expression of inhibitory G proteins in the failing myocardium may participate in the desensitization of β-adrenoceptor mediated positive inotropic effects [25–27] and thereby aggravate heart failure. In addition, an increased Gi mediated activation of the MAPK pathway in cardiac fibroblasts [28] may be relevant for myocardial remodelling. On the other hand, upregulated Gi proteins could promote antiapoptotic [29] or antiarrhythmic effects [30]. G-proteins of the Gq-family likely mediate growth signals for myocytes and can promote myocardial hypertrophy, apoptosis and failure [31,32].

An increased expression of RGS4 in the failing heart, as found in this study, likely counterregulates these Gi/Go- and Gq/11-mediated functions. Especially, the influence of RGS proteins on Gq/11 mediated effects on myocardial growth and hypertrophy has been a matter of debate. In an animal model of heart failure levels of RGS3 and 4 decreased, possibly promoting myocardial growth [4]. In contrast, the myocardial expression of RGS4 mRNA increased by about 400% in mice with right ventricular hypertrophy after pulmonary artery banding [4]. In cell culture experiments cardiomyocytes upregulate both RGS3 and 4 following a hypertrophic stimulus like bFGF [4]. Taken together with the growth inhibiting effects of RGS4 in myocytes [22] an increased myocardial expression of RGS4 [4] may represent a counterregulatory antihypertrophic mechanism. Such an anti-hypertrophic action of RGS4 was confirmed by transgenic overexpression of RGS4 and both, RGS4 and Gq in mice [33,34]. Recently a moderate increase in RGS3 and an increase in RGS4 expression was described in terminally failing human myocardium as estimated by quantitative PCR and immunoblot analysis [35]. Whereas our data support the upregulation of RGS4 in heart failure they are in obvious discordance regarding RGS3. The authors, however, did not differentiate between RGS3S and RGS3L. Using the RPA which is regarded a standard for RNA quantification we clearly did not see an increased mRNA expression of either form. Similarly the protein expression of RGS3S was not increased. Even though there was no significant difference in the RGS3L protein expression (P = 0.35) we cannot completely exclude that with a substantial higher number of hearts an increase up to about 50% might have been detected. Whether the discrepancies are due to differences in the assay system or the sample acquisition remains to be elucidated, however.

We only can speculate about the mechanisms of the upregulation of RGS4 in failing human hearts and the contribution of neurohumoral stimulation and the abnormal mechanical load. Different stimuli such as pulmonary banding, growth factors [4] or bacterial endotoxin (LPS) [36] promote an overexpression of RGS4 in rodent hearts. When analyzing the influence of the medication within the failing group (DCM+ICM), we did not find a significant impact of diuretics, cardiac glycosides, nitrates, sympathomimetic and antiarrhythmic drugs, or ACE inhibitors. We cannot definitely exclude drug effects, however. E.g., the three patients with heart failure that did not receive ACE inhibitors tended to have lower levels of RGS4 mRNA [0.41 (n = 3) vs. 0.73 (n = 19) amol/µg, P>0.05].

Irrespective of the mechanisms of the gene regulation we hypothesized that the upregulation of RGS4 to 200–300% in failing human myocardium inhibits Gq/11 mediated signalling and thereby negatively modulates stimulation of growth and hypertrophy and contractility. This seems likely since the increase in the expression in this study was comparable to the increase observed and described as functionally active in the transgenic mice model. We tested the effect of an upregulated RGS4 expression on Gq/11 mediated signal transduction in the human heart and the influence on contractility in rabbit cardiomyocytes. RGS4 was able to inhibit ET-1 mediated activation of PLC in left ventricular myocardial membranes. Therefore, we hypothesised that an upregulated RGS4 also reduces Gq/11 mediated positive inotropic effects in cardiomyocytes. In the failing myocardium the positive inotropic effects of {alpha}1-adrenoceptors or endothelin receptors are reduced [38,39]. This desensitization was poorly understood as neither the {alpha}1 nor the total endothelin receptors are downregulated [38–44] and the mRNA and protein expression of Gq/G11 is unaltered [42]. A negative regulation of these pathways by an increased expression of RGS4 may account for such a desensitization. By means of adenoviral overexpression of RGS4 in isolated rabbit ventricular myocytes we were also able to show such an inhibitory action on the ET-1 mediated positive inotropic effect. Albeit we do not know the actual amount of RGS4 overexpression in a specific cardiomyocyte the expression of GFP enabled us to select only cardiomyocytes containing the adenoviral construct and expressing the encoded proteins. Since overexpression of RGS4 in isolated cardiomyocytes completely abolished ET-1 mediated inotropic effects we conclude that the upregulation of RGS4 in failing myocardium sufficiently explains the observed desensitization of Gq/11 mediated inotropic effects in failing human myocardium.


   5. Conclusion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
References
 
In summary, we found an expression of 11 members including two variants of the RGS protein family in the failing and nonfailing human heart. The upregulation of RGS4 in failing left ventricular myocardium may be involved in the regulation of growth and hypertrophy and can explain some of the desensitization processes in failing human hearts.

Time for primary review 25 days.


   Acknowledgements
 
This study was supported by the Deutsche Forschungsgemeinschaft (DFG).


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

  1. Wieland T., Chen C.K. Regulators of G protein signalling: a novel protein family involved in timely deactivation and desensitisation of signalling via heterotrimeric G proteins. Naunyn-Schmiedeberg's Arch Pharmacol (1999) 360:14–26.[CrossRef][Web of Science][Medline]
  2. Watson N., Linder M.E., Druey K.M., Kehrl J.K., Blumer K.J. RGS family members: GTPase-activating proteins for heterotrimeric G protein {alpha}-subunits. Nature (1996) 383:172–175.[CrossRef][Medline]
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