Cardiovascular Research

Cardiovascular Research 2003 60(3):478-487; doi:10.1016/j.cardiores.2003.09.014
© 2003 by European Society of Cardiology

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Copyright © 2003, European Society of Cardiology

Inhibitory G-proteins and their role in desensitization of the adenylyl cyclase pathway in heart failure

Ali El-Armouche^a, Oliver Zolk^b, Thomas Rau^a and Thomas Eschenhagen^*,a

^aInstitut für Experimentelle und Klinische Pharmakologie, Universitätsklinikum Hamburg–Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany
^bInstitut für Experimentelle und Klinische Pharmakologie und Toxikologie, Friedrich-Alexander-Universität Erlangen, Germany

*Corresponding author. Tel.: +49-40-428032180; fax: +49-40-428034876. Email address: t.eschenhagen{at}uke.uni-hamburg.de

Received 24 July 2003; revised 3 September 2003; accepted 10 September 2003

	Abstract

Top Abstract 1. Introduction 2. G-proteins implicated in... 3. Heart failure and... 4. Does desensitization of... 5. What is the... 6. How does an... 7. Is desensitization of... 8. Clinical consequences References

 
Heart failure is accompanied by stereotypic alterations in cardiac gene expression. These changes are most likely secondary in the pathogenesis and can be viewed as protective, e.g. as energy-saving mechanisms, but at the same time, they aggravate contractile dysfunction and the deficit of failing cardiac myocytes to respond to altered hemodynamic needs. One of the best-studied, paradigmatic examples of this dichotomy is heterologous desensitization of the cardiac adenylyl cyclase (AC) signaling pathway. It protects against detrimental consequences of the excessive adrenergic drive, but it also blunts the most powerful inotropic support of the heart. Desensitization is associated with downregulation of β-adrenergic receptors, increased β-adrenoceptor kinases and increased inhibitory G protein -subunits, G_i. Whereas a causative role of the former is generally accepted, the role of the increase in G_i has remained controversial for many years. The present article summarizes early and novel findings that, in the view of the authors, provide solid evidence for G_i to play an important role in the adaptation of cardiac AC to various pathophysiological conditions.

KEYWORDS G-proteins; Beta-adrenergic desensitization; Heart failure; Adenovirus; Betablocker

	1. Introduction

Top Abstract 1. Introduction 2. G-proteins implicated in... 3. Heart failure and... 4. Does desensitization of... 5. What is the... 6. How does an... 7. Is desensitization of... 8. Clinical consequences References

 
The cardiac β-adrenergic signaling system mediates most effects of neuronally released and circulating catecholamines and represents the most powerful regulatory input into the heart. It mediates acute effects such as the increase in force of contraction (inotropism), rate of cardiac relaxation (lusitropism), heart rate (chronotropism), and impulse conduction through the AV node (dromotropism). However, chronic β-adrenoceptor-mediated hypertrophic and proapoptotic actions of catecholamines are being increasingly recognized and may be of similar importance. The β-adrenergic signaling system is a paradigmatic example of a G protein-coupled receptor system, where the biological response is initiated by binding of a ligand to a specific receptor, which as a consequence of ligand binding, undergoes a conformational change that, in case of an agonist, triggers activation of guanine nucleotide-binding proteins (G-proteins) and finally ACs to synthesize the second messenger cAMP from ATP. Increased intracellular cAMP in turn activates cAMP-dependent protein kinase (PKA), which then phosphorylates target proteins such as cardiac Ca²⁺ channels, the Ca²⁺-release channel, phospholamban, and troponin I whose change in activity cause the physiological responses mentioned above.

Patients with terminal heart failure exhibit a relative or absolute refractoriness to catecholamines in vitro and in vivo. Similar phenomena has been observed at the level of isolated muscle preparations or isolated cells from patients with heart failure [1,2]. The reduction in β-adrenergic contractile responsiveness is reflected by reduced stimulation of cAMP synthesis in broken heart membranes and has been termed desensitization of β-adrenergic receptor-mediated activation of AC [3]. It is associated with a decrease in β-adrenergic receptor density [1], uncoupling of β-adrenergic receptors from G_s as a consequence of increased β-adrenergic receptor kinase (β-ARK=GRK 2) activity [4,5], as well as increased G_i protein and transcript levels [6–9]. In concert, these molecular alterations are assumed to account for desensitization, and thereby, for the blunted contractile response to catecholamines in heart failure, but the role of each alteration alone is difficult to evaluate since they occur simultaneously. The present article will discuss established mechanisms with a focus on G_i.

	2. G-proteins implicated in AC regulation in the heart

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The mammalian heart expresses the long and the short isoform of G_s, as well as the structurally and functionally closely related G_i-2, G_i-3 and two or three G_o subunits (for review, see Ref. [10]) with G_i-2 being the quantitatively prevailing G_i/o isoform in human atrium and ventricle [9,11–13]. When the G_i/o family has been collectively inactivated by pertussis toxin (PTX), the main consequences of muscarinic and adenosine receptor stimulation in the heart are lost. These include the inhibition of heart rate (likely involving inhibition of the hyperpolarization-activated cyclic nucleotide gated cation channel, HCN4, and L-type calcium channels), inhibition of atrial force (via activation of I_KACh) and the accentuated antagonism of β-adrenergic positive inotropy in the ventricle (likely via inhibition of AC and subsequently L-type calcium channels). Experiments in knockout mice extended these conclusions and suggest that G_o is essential for muscarinic inhibition of L-type calcium currents in ventricular myocytes, but not in muscarinic activation of I_KACh in atrial cells [14,15]. In embryonic stem cell-derived, atrial-like cardiomyocytes G_i-2 and G_i-3 appear to modulate the kinetics of muscarinic inhibition of L-type calcium currents without being necessary for full inhibition [15]. Surprisingly, the question whether or not the latter effect involves the NO/cGMP pathway remains highly controversial with several papers in favor and an even greater number against this idea (for discussion, see Ref. [16]). One reason for this confusion may be the developmental dependency of the mechanism of the muscarinic inhibition of isoprenaline-stimulated I_Ca. In one study, muscarinic inhibition of isoprenaline-stimulated I_Ca was entirely dependent on the NO/cGMP pathway at early stages of cardiac development, whereas it was dependent on G_i/AC coupling and insensitive to NOS inhibition at later stages [17]. Thus, it remains unclear whether the present conclusions with regard to G_o and G_i-2/i-3 apply to the adult cardiac myocyte. Whereas G_s directly stimulates AC, the mechanisms of AC inhibition by G_i proteins has remained somewhat obscure, mainly because it has been difficult to demonstrate direct inhibitory interaction of GTP-G_i with AC. However, in membranes from SF9 cells expressing the cardiac AC isoforms V or VI recombinant myristoylated G_i-1, G_i-2 and G_i-3, but not G_o concentration-dependently inhibited cyclase activity, both in the presence and absence of G_s, indicating that activated G_i binds directly to an inhibitory binding site on AC [18]. Additionally, the inhibitory action of G_i on AC activity may be due to the release of β subunits that either scavenge G_s or directly inhibit AC. The latter hypothesis has gained support from data showing that overexpression of β subunits suppresses AC type V and VI activity in COS-7 cells [19], but is in contrast to former data on isolated proteins showing that the cardiac AC isoforms V and VI are insensitive to β subunits [20]. The heart also expresses Gq and G11 that mediate hypertrophic actions of angiotensin II, endothelin, norepinephrine (via _1A adrenoceptors). Evidence exists for negative cross-talk between the receptor/Gq/phospholipase C and the G_s/AC pathway, e.g. at the level of phospholipase C [21]. Moreover, most Gq-coupled receptors also activate G_i pathways and thereby inhibit G_s signals (see below). Transgenic overexpression of G_q causes cardiac hypertrophy, decreased contractility, AC activity and β-adrenergic responsiveness in mice and this phenotype was partly or fully prevented by cross-bred with mice overexpressing AC type V [22] or VI [23]. The mechanisms of this unexpected interaction is unresolved, but unlikely reflects direct interaction between G_q and AC.

	3. Heart failure and sympathetic activation

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Systolic heart failure (isolated diastolic dysfunction is not discussed here) represents a clinical endpoint secondary to various cardiac diseases. Irrespective of the underlying etiology, the hallmark of all forms is the impairment of cardiac contractile function. Autocrine, paracrine, and neurohormonal mechanisms are activated to increase blood pressure and to maintain blood flow to vital organs [24]. These compensatory mechanisms include activation of the sympathetic nervous system, increased activity of the renin–angiotensin [25] and endothelin system [26,27], and increased release of vasopressin [28]. The parallel increase in natriuretic factors (ANP, BNP) on the other hand partly balances the load-increasing actions of the other systems [29].

Plasma norepinephrine concentrations are increased in patients with heart failure, at rest [24] and during exercise [30]. A substudy of the SOLVD study [31] revealed that serum norepinephrine levels were already increased in asymptomatic patients with reduced left ventricular function and, in contrast to ANP and vasopressin, were strongly predictive of outcome [32]. While the heart extracts norepinephrine from plasma in normal subjects, norepinephrine spillover from sympathetic nerve endings exceeds uptake in patients with symptomatic heart failure and makes the heart the most important source of systemic noradrenaline [33]. The increased local sympathetic activity in myocardial tissue is accompanied by a decreased number of presynaptic norepinephrine uptake sites [34], leading to an increased norepinephrine concentration in the synaptic cleft. Cardiac norepinephrine spillover is inversely correlated with the survival of the patients and the local cardiac spillover precedes the generalized sympathetic activation in patients with heart failure [34]. This and the fact that nearly all molecular alterations observed in the failing human heart can be induced experimentally by sustained β-adrenergic stimulation both in cultured cells and in whole animal experiments [11] provide strong evidence that the alterations in gene expression leading to desensitization of receptor-stimulated AC in the failing human heart are secondary to the increased sympathetic nervous activity. The impact of other neurohumoral system on the cardiac AC pathway is less well understood. Part of the actions of angiotensin II on the heart are due to the release of norepinephrine [35] and endothelin [36,37]. Endothelin, angiotensin II and natriuretic peptide type C receptors exhibit dual coupling to stimulation of phospholipase C and of G_i-mediated inhibition of AC [38–40]. Infusion of endothelin induced upregulation of β-adrenergic receptors and of G_s proteins in cardiac membranes [41], i.e. changes functionally opposite to those seen after infusion of catecholamines. Thus, there is evidence that the peptidergic systems rather counteract the actions of catecholamines on cardiac AC pathways.

	4. Does desensitization of the AC pathway explain the decreased contractile reserve in heart failure?

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In ventricular preparations from explanted human hearts with idiopathic dilated or ischemic cardiomyopathy, the positive inotropic response to catecholamines and their effect on AC activity is markedly diminished [1,42,43]. In contrast, the force-increasing effect of cAMP-independent compounds such as ouabain [42,44], sodium channel agonists [45], calcium channel agonists and calcium itself has been found to be unchanged at low frequency of stimulation [42,43,46–49]. Data with regard to the AC stimulator forskolin, whose effect is partly depending on G_s, are conflicting. Some authors found preserved positive inotropic effects in the failing human myocardium [42,43], others a significant decrease [48]. The efficacy of the membrane-permeable cAMP derivative dibutyryl-cAMP was found to be unchanged in isolated human trabeculae [48], but reduced in isolated human cardiac myocytes [49]. The key findings were confirmed at the single myocyte level [2], demonstrating that the defect is inherent to the single motor unit of the heart. Collectively, these data provided unambiguous evidence that the failing myocardium is principally able to develop normal maximal contractile force (at this point, alterations in the force–frequency relation, twitch kinetics, calcium handling and myofilament ATPase activity are not discussed (see Ref. [50]), and that blunted cAMP-generation by the receptor–G protein–AC system is of central importance for the reduced inotropic reserve of the failing human heart.

	5. What is the role of the Gi proteins increase for desensitization of the cardiac AC pathway?

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The first evidence that alterations beyond the receptor level might occur in heart failure was provided by assessing agents bypassing the β-adrenergic receptors. At that time surprisingly, the positive inotropic effect of cAMP phosphodiesterase (PDE) inhibitors was found to be diminished in isolated preparations from failing human myocardium as much as that of catecholamines [42–44]. The diminished inotropic response is not due to changes in PDE activity or its susceptibility to pharmacological inhibition because both were found to be indistinguishable in normal and failing human myocardium [51,52]. These data argued for a diminished (basal) cAMP production underlying the diminished response to PDE inhibitors [42]. Also, the heterologous nature of desensitization of receptor-stimulated AC, i.e. the blunted response to histamine, glucagon and vasointestinal peptide [42,43,53,54], cannot be explained by downregulation of β-adrenoceptors. Thus, the independent demonstration of upregulation of G_i in human heart failure by two independent groups [6,7] raised much attention and was subsequently confirmed by several groups [8,11,55,56].

As the three mentioned molecular abnormalities generally occur in parallel, the specific role of each alteration remains difficult to define. Several lines of evidence suggest that the decrease in β-adrenergic receptors accounts for an important part of the decreased inotropic response to β-adrenergic agonists [57]. The human heart contains only a small fraction of "spare receptors", which is further reduced with increasing severity of heart failure [58]. Accordingly, the density of β-adrenergic receptors correlates well with the magnitude of β-adrenergic inotropic responses in different stages of human heart failure [59,60]. The increase in βARK, a kinase that uncouples β-adrenergic receptors from AC signaling, also appears to play a role on its own, since mice overexpressing βARK showed suppressed isoprenaline-stimulation of cAMP production [61] and overexpression of a peptide inhibitor of β-ARK leads to enhanced cardiac contractility in vivo with or without isoprenaline stimulation.

In contrast, the role of G_i remained somewhat obscure. The reasons are severalfold. (1) G_i does not directly participate in the β-adrenergic signaling pathway. (2) As outlined above the exact mechanism of the G_i-mediated inhibition of AC had been a matter of debate. (3) G_i is physiologically expressed at high levels. The concentration amounts to 1–5 pmol/mg protein in human hearts [13], whereas the density of most receptors including the β-adrenergic receptor ranges between 50 and 500 fmol/mg protein, i.e. >10-fold lower. It has been argued therefore that a 40–100% increase is unlikely to have a strong impact on the system. (4) In some animal models of heart failure or hypertrophy, G_i reportedly remains unchanged and G_s is downregulated (for review, see Ref. [11]).

Yet, we believe that none of these arguments holds against an important role of G_i in heart failure and that recent findings provide strong evidence for such a role.

Early arguments for a role of the G_i increase were as follows. (1) The increase in G_i, in contrast to reduced β-adrenoceptors, can well account for the heterologous nature of desensitization of receptor-stimulated AC (e.g., reduced response to histamine) as well as the observed decrease in GTP-dependent AC stimulation [11]. (2) The increase in myocardial G_i levels occurs as one of the earliest events in animal models of hypertension [62,63] and parallels the blunted response to β-adrenergic stimulation in heart failure induced by myocardial infarction in rat [56]. Temporal inactivation of G_i/G_o proteins by pertussis toxin injection in prehypertensive SHR rats delayed the development of hypertension [64] (3) PTX catalyzed inactivation of G_i/G_o proteins restored AC stimulation by β-adrenergic agonists [7] as well as the inotropic response of isolated failing human cardiac myocytes to isoprenaline [55]. (4) The age-related decrease in β-adrenergic effects in mice overexpressing β₂-adrenergic receptors correlates with an increase in G_i and is reversible upon treatment with PTX [65]. (5) Activation of G_i opposes the pro-apoptotic action of β₁-adrenoceptor stimulation in cardiomyocytes [66–69]. (6) Clinical improvement of heart failure patients treated with β-adrenoceptor blockers is accompanied by normalization of G_i expression level [70]. Yet, all of this evidence was circumstantial.

Strong direct evidence for an important role of G_i in desensitization of receptor-stimulated AC and in the regulation of contractility and electrical conduction was provided recently by the use of recombinant adenovirus [71–73]. Rau et al. [71] tested whether overexpression of wild-type G_i-2 affects G protein-dependent AC in cardiac myocytes by the use of two replication-deficient adenoviruses encoding rat G_i-2 alone (Ad5G_i-2) and G_i-2+green fluorescent protein (bicistronic; Ad5G_i-2/GFP), as well as two control viruses (Ad5LacZ, Ad5GFP). Infection of neonatal rat cardiac myocytes with Ad5G_i-2 for two days virus dose-dependently increased G_i-2 levels by 1.5- to 7-fold. In parallel, both GTP- and isoprenaline-stimulated AC in broken membranes and isoprenaline-stimulated cAMP accumulation in intact cells were suppressed by 10–70% and 50–90%, respectively (Fig. 1A). Treatment with PTX abolished the Ad5G_i-2 effect at lower levels of overexpression (G_i-2 150% above control). At a targeted level of G_i-2 overexpression of 90% above control (i.e., levels of overexpression seen in human heart failure), isoprenaline-stimulated AC was significantly reduced by 17% and cAMP accumulation by 40% (Fig. 1B). Importantly, the levels of G_s, G_β and β-adrenoceptors were unaffected by Ad5G_i-2/GFP or Ad5GFP infection under these conditions. These data are the first direct demonstration that increases in wild-type G_i-2 suppress β-adrenergic stimulation of cardiac AC. Janssen et al. [72] and Rau et al. [71] observed that adenoviral G_i-2 overexpression in isometric contracting multicellular preparations and single myocytes from rabbit and rat, respectively, markedly attenuated the inotropic response to isoprenaline (by 60–80%, Fig. 2). These data underline that increased G_i-2 alone is sufficient to attenuate the positive inotropic effects of β-adrenergic stimulation in myocardium. Furthermore, Donahue et al. [73] showed that overexpression of wild-type G_i-2 by infusion of Ad5G_i-2 in the AV region of pig hearts reduced ventricular beating rate after the induction of atrial fibrillation (Fig. 3). A surprising aspect of these findings was the magnitude of the effect of G_i-2 overexpression on cardiac function, raising the question by which mechanism an increase of G_i-2 protein leads to desensitization of receptor-stimulated AC in the heart.

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Effect of Gi-2 overexpression on isoprenaline-stimulated AC activity and cAMP levels. (A) AC activity was determined in neonatal rat cardiac myocytes 48 h after infection upon stimulation with GTP and isoprenaline. Cells were propagated in regular medium or, 24 h after infection, treated with pertussis toxin (PTX). (B) Increasing multiplicity of infection (virus/cell) of Ad5Gi-2/GFP lowered isoprenaline-stimulated (10 µM) cAMP levels, whereas Ad5GFP had no effect. Data adapted from Ref. [71].

 

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Effect of Gi-2 overexpression on cell shortening of adult rat ventricular myocytes. Adult rat myocytes were infected with Ad5Gi-2 or left uninfected (Ctr). The isoprenaline concentration-response curve was significantly depressed and shifted to the right in Gi-2 overexpressing myocytes. Data adapted from Ref. [71].

 

View larger version (14K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Effect of Gi-2 overexpression on heart rate of pig hearts. Infusion of Ad5Gi-2 in the AV region of pig hearts reduces heart rate by 20% during acute episodes of atrial fibrillation (right panel, day 0 and day 7). No difference in heart rate was observed in controls (left panel, Adβgal). Data adapted from Ref. [73].

 

	6. How does an increase in Gi proteins translate into desensitization of the AC pathway?

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One reason may lie in the observation that G-coupled receptors exert constitutive activity, i.e. they interact with the heterotrimeric G protein in the absence of agonists. Thus, the presence of more G_i-2 may amplify the inhibitory influence of constitutively active G_i-coupled receptors. This mechanism is supported by studies demonstrating constitutive activity of muscarinic acetylcholine or A₁ adenosine receptors [74,75]. However, the inverse muscarinic agonist atropine reversed the inhibitory effect of G_i-2 overexpression on cAMP levels only partially [71], suggesting that additional mechanisms or signals from other G_i-coupled receptors explain the effect. Candidates are the A₁-adenosine receptor and, according to data of Xiao et al. [76], also the β₂-adrenergic receptor. In this respect, a recent study showed that the β-adrenoceptor agonist ICI 118,551 exerted a negative inotropic effect only in cardiac myocytes in which the concentration of G_i-2 was supranormal, e.g. in myocytes from failing human hearts (Fig. 4), β₂-adrenoceptor overexpressing TG4 mice and in normal cells overexpressing G_i-2 [77]. The hypothesis generated from these data was that binding of the inverse agonist ICI 118,551 directs the β₂-adrenoceptor to a G_i-2-coupled form producing a direct negative inotropic effect. An alternative mechanism to explain the strong effect of G_i-2 overexpression could be that G_i-2 exerts some intrinsic activity in its own, i.e. a certain fraction spontaneously releases GDP, binds GTP and exerts biological effects. This fraction would be, in absolute terms, increased by overexpression. It is difficult to separate these effects. But the common observation that preparations of recombinant and native G_i/G_o proteins (in contrast to G_s) exhibit strong basal binding activity of GTP or photoaffinity-modified GTP analogues and that G_i/G_o-coupled receptor agonists exert only a modest stimulatory effect [78,79] argue for a significant intrinsic GDP-release activity of wild-type G_i, too. The fact that the effect of high overexpression levels of G_i-2 was insensitive to PTX (Fig. 1A; Ref. [71]) also argues for some constitutive activity, because PTX-mediated ADP-ribosylation of the C-terminal cysteine residue of G_i impairs interaction with receptors, but not with effectors [80]. PTX can therefore not reverse the effect of constitutively active G_i [81]. A third possibility is that overexpression of G_i-2 acts as a scavenger of β-subunits and thereby impairs signaling through the G_s pathway. An argument that may argue against this hypothesis (but does not exclude this possibility) is that overexpression of βARKct, another scavenger of Gβ, increases AC stimulation [61].

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Contraction amplitude of myocytes from nonfailing and failing human right ventricle before and after exposure to ICI 118,551. Pertussis toxin (PTX) treatment (1.5 µg/ml) abolished the negative inotropic effect of ICI 118,551 (1 µmol/l) in failing right ventricle. Data adapted from Ref. [74].

 
In any case, the study of Rau et al. [71] directly demonstrated that the G_i-coupled signaling system as a whole exerts activity in the absence of receptor agonists and that this activity is increased with increasing concentrations of G_i-2. This strongly supports the concept of "tonic G_i activity" as deduced from several earlier observations. (a) Addition of GTP (-analogues) can inhibit forskolin-stimulated AC activity [82] and stimulate potassium channels [83] which would be difficult to explain without a significant basal exchange activity. (b) Purified membrane preparations loaded with labeled GDP spontaneously release GDP mainly from the G_i/G_o protein fraction in a temperature-dependent manner [84]. (c) Treatment of isolated cardiac myocytes or whole animals with PTX increased arrhythmogenic [85] or AC-stimulating [86] effects of β-adrenergic agonists. Furthermore, isolated murine myocytes responded to zinterol only when pre-incubated with PTX and not in the native state [87] and the age-related decrease in β-adrenergic effects in β₂-adrenergic receptor overexpressing mice (TG4) were paralleled by an increase in G_i protein levels and was reversible upon treatment with PTX [65]. (d) Overexpression of G_i-2 by infusion of Ad5G_i-2 in the AV region of pig hearts reduced ventricular beating rate after the induction of atrial fibrillation (Fig. 3, Ref. [73]).

Overexpression of G_i-2 suppressed β₁- and β₂-adrenoceptor-mediated stimulation of cAMP levels to a similar extend [71]. This was surprising because specific coupling of β₂-adrenergic receptors to G_i as proposed by Xiao et al. [87] would have predicted differential affection of β₁ and β₂ adrenoceptor effects. Yet, restoration or enhancement of β₂-adrenergic effects by PTX does not necessarily prove direct coupling because it can be as well explained by relieving the tonic inhibitory activity of G_i pathways on stimulatory AC pathways. Thus, recent experiments showed that PTX treatment sensitized the inotropic effects of β₁-adrenergic stimulation in mice where, surprisingly, no inotropic or chronotropic effects of β₂-adrenergic stimulation were observed at all [88].

	7. Is desensitization of the AC signaling pathway helpful or deleterious in heart failure?

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As discussed above, in systolic heart failure the sympathetic nervous system is activated to stimulate myocardial function and maintain circulatory needs of the body. The higher the adrenergic drive, the more profound is the degree of β-adrenergic desensitization and thereby the lesser the inotropic response and cardiac output, which in turn leads to even greater adrenergic activation. Increased G_i activity in this situation may on the one hand represent an element of this vicious cycle with detrimental effects on contractility and exercise tolerance. But it is clear that, on the other hand, increased G_i activity has also beneficial effects by protecting the heart from β-adrenergic overstimulation that would cause energy deprivation, apoptosis and potentially fatal arrhythmias.

As discussed above, an increase in G_i protein levels as seen in failing human hearts participates in desensitization of the AC signaling pathway and the reduced inotropic responses to catecholamines. Autoantibodies that activate M₂ acetylcholine receptors have been found in patients with dilated cardiomyopathy [89] and may increase the impact of G_i-mediated inhibition of receptor-stimulated AC. These findings could be interpreted as to suggest that increased G_i signaling may play a causal role in dilated cardiomyopathy. Consistent with this idea, conditional, cardiac-specific expression of a modified G_i-coupled opioid receptor with constitutive activity in transgenic mice resulted in dilated cardiomyopathy, overt heart failure, ECG changes, arrhythmia, and increased mortality [90]. This study elegantly demonstrates that overstimulation of G_i-coupled signaling pathways is sufficient to cause cardiomyopathy and thereby establishes a link between increased activity of G_i and cardiomyopathy in general. However, transgenic overexpression of a given element of a signaling cascade at unphysiologically high levels creates an artificial situation that does not allow valid conclusions as to the role of alterations in the expression of the respective signaling element under pathophysiological conditions.

In fact, the evidence for a mostly protective role is prevailing. Patients with heart failure with mild to moderate heart failure (NYHA II–III) most often die from a sudden, arrhythmogenic event, whereas patients with end-stage disease (NYHA IV) most often die from progressive heart failure [91] and bradyarrhythmic events [92]. The latter patients are expected to exhibit a high degree of β-adrenoceptor downregulation and increase in G_i. So, it was tempting to speculate that these alterations in end-stage human heart failure serve as a protective antiarrhythmic mechanism. Several animal studies support this hypothesis. For example, pacing-induced heart failure in dogs is accompanied by a reduction in β-adrenoceptor density, an increase in G_i [93], and a reduced arrhythmogenic effect of catecholamines [94]. Infusion of carbachol in rats for 4 days led to a decrease in cardiac PTX-sensitive G_i proteins and a reduction in M-cholinoceptor density. This was accompanied by a modestly reduced inotropic effects of carbachol, but a marked sensitization of isolated papillary muscles to the arrhythmogenic effects of isoprenaline or forskolin in vitro [95]. Infusion of isoprenaline, that increased Gi protein levels, decreased the incidence of isoprenaline- and forskolin-induced arrhythmias in vitro. Grimm et al. [85] found that a dose-dependent PTX-mediated inactivation of G_i in vivo closely correlated with an increase in the arrhythmogenic effects of isoprenaline or forskolin. Other groups also observed, without specifically focussing on arrhythmias, an increase in β-adrenoceptor-mediated automaticity in isolated PTX-treated rat ventricular cardiomyocytes [76]. These findings are in favour of the notion that the increase of G_i could be a protective mechanism to prevent arrhythmias during excessive sympathetic stimulation, such as in heart failure.

Recent evidence underline distinct differences between β₁- and β₂-adrenoceptors in their chronic effects on cardiac myocytes. Whereas mice overexpressing the human β₂-adrenoceptor in the heart exhibit cardiac hypercontractility and develop dysfunction only at late stages [96,97], β₁-adrenoceptor overexpressing mice quickly develop heart failure [98]. One reason may be a difference in the ability to promote apoptosis [91–93]. Stimulation of β₁-adrenoceptors increases apoptosis via a cAMP-dependent mechanism, whereas stimulation of β₂-adrenoceptors appears to inhibit apoptosis via a G_i-coupled pathway [66]. Thus, the muscarinic agonist carbachol inhibited β-adrenoceptor-mediated apoptosis and PTX, which inhibits G_i, increased the magnitude of β-adrenoceptor-stimulated apoptosis and mimicked the effect of β₂-adrenoceptor blockade. G_i might oppose the actions of G_s by inhibiting the activation of AC and/or by activating phosphatidylinositol 3'-kinase [68]. A protective role of upregulating G_i is further supported by recent data that a cross-bred of β₂-adrenoceptor transgenic and G_i-2 knockout mice showed extensive cardiomegaly and markedly decreased survival rates (S. Herzig, Cologne, personal communication).

	8. Clinical consequences

Top Abstract 1. Introduction 2. G-proteins implicated in... 3. Heart failure and... 4. Does desensitization of... 5. What is the... 6. How does an... 7. Is desensitization of... 8. Clinical consequences References

 
The increase in G_i in human heart failure can be viewed as a good example of an adaptational response of the heart: necessary as a protection against arrhythmia and apoptosis, but also detrimental in terms of contractile reserve. Therapeutic strategies to directly activate or inhibit G_i function are not in sight and, given the dichotomic character of the G_i increase, unlikely to be successful. What should be a reasonable strategy, however, is to reduce the "need for the increase in G_i" in human heart failure. Indeed, this concept is already been in clinical use and has proven highly effective in the treatment of chronic heart failure, namely the use of β-blockers. Some years ago, we could show in a small study in patients with NYHA class II–III heart failure that a 3-month treatment with metoprolol not only induces upregulation of β-adrenergic receptors but also a decrease, i.e. relative normalization of G_i-2 levels in myocardial biopsies [70]. In light of the recent findings discussed above, the decrease in G_i induced by metoprolol, even though modest, is likely to contribute to the improved exercise tolerance demonstrated in the same patients [70]. The decrease in G_i-2 upon treatment may be even more relevant than the increase in β-adrenergic receptor density given that the continued presence of the β-blocker will at least partly block the effect of endogenous catecholamines. The concept that arises from these data is that the β-blocker reduces the continuous stimulatory input exerted by increased norepinephrine and thereby reduces the signal that downregulates β-adrenergic receptors and upregulates G_i proteins. Thus, the β-blocker allows the cardiac signaling system to return towards a normal state. In contrast to any direct intervention such as overexpression of β-adrenergic receptors or inactivation of G_i proteins the normalization of cardiac signaling occurs just to an extent that corresponds to the balance between increased norepinephrine and the concentration of the β-blocker. In other words, the biological system remains in a balanced state, but at different (activity) levels of signaling components. Thus, normalization of G_i protein in patients treated with β-blockers is beneficial (and not deleterious as its acute inactivation by PTX), because the β-blocker blocks the proarrhythmic and proapoptotic action of norepinephrine. And on the other hand, the normalization of G_i proteins relieves the tonic inhibition of AC and allows stimulatory inputs from all G_s-coupled receptors (β-adrenergic receptors will participate under exercise) to increase force of contraction and cardiac output of patients with heart failure.

	Acknowledgements

 
This work was supported by the Deutsche Forschungsgemeinschaft (Es 88/8-2, GRK 750) and the Johannes und Frieda Marohn-Stiftung (Arm/00).

	Notes

 
Time for primary review 21 days

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